3 mabne ROPER P: = “—— pesiggoe ~ nace ¥ + OB ¢ - ee ha Digitized by the Internet Archive in 2010 with funding from University of Toronto Sia) a http://www.archive.org/details/annalsoftropical0/live A aug f Oo re > | Laie enti rai ANNALS OF EROPIGAL MEDICINE AND PARASITOLOGY a7 iM AJADITONT FAO 241448 tee WoOIOTIAABAD (AA et Z i? aT aT ee ee A plese bare jie aes nme (‘THE UNIVERSITY OF LIVERPOOL )} ANNALS ”) OF TROPICAL MEDICINE AND PARASITOLOGY ISSUED BY THE LIVERPOOL SCHOOL OF TROPICAL MEDICINE Edited by Professor J. W. W. STEPHENS, M.D.Cantab., Pee. Prormsom R NEWSTEAD, M.Sc. J.P. F-R.S., A.L.S.,. F.E.S.,-Hon. F.R.H.S. AND Proressor Sir RONALD ROSS, K-C.B., F.R.S., M.D.,. F-R.C.S. Major I.M.S. (Rert.) Editorial Secretary Dr. H. B. FANTHAM VOLUME VII (March 31, to December 30, 1913) With forty-two plates, twenty-two figures in text, and seventeen charts AT THE UNIVERSITY PRESS, 57 ASHTON STREET. |> ‘eC ae y 5 LIVERPOOL : \o- “ / 0 oe Poa Editorial Secretary Dr. H. B. FANTHAM, School of Tropical Medicine, The University, Liverpool. CONTENTS Nor. March 31, 1913 Macrik, J. W. Scorr. ‘Trypanosomiasis of Domestic Animals in Northern Nigeria. Plates I—II1... Srepuens, J. W. W.; and Fantuam, H. B. Further Measurements of Trypanosoma rhodesiense and T. gambiense Mayer, T. F. G. A New Mosquito-proof and Storm-proof House for the Tropics. Plate IV... CurisropHers, Major S. R. Contributions to the Study of Colour Marking and other Variable Characters of Anophelinae with Special Reference to the Systematic and Phylogenetic Grouping of Species. Plates V—VIII Biacktock, B. A Study of the Posterior Nuclear Forms of Trypanosoma rhodesiense (Stephens and Fantham) in Rats ... BaLrour, ANDREW. Animal Trypanosomiasis in the Lado (Western Mongalla) and Notes on Tsetse Fly ‘Traps and on an Alleged Immune Breed of Cattle in Southern Kordofan. Plates IX, X ‘THomson, Davin. Sanitation on the Panama Canal Zone, ‘Trinidad and British Guiana. Plates XI, XII, XIII ‘THomson, JoHN Gorvon ; and ‘Homson, Davin. The Cultivation of One Generation of Benign Tertian Malarial Parasites (Plasmodium vivax) in Vitro, by Bass’s Method. Plate XIV Scott, H. Haroxp. Fulminating Cerebro-Spinal Meningitis in Jamaica PAGE 27 41 IOI r25 zh Pf .. Pe ae = rae 3 ti) - iy am 1 «AE . ; a a 8 va : ah ro ‘Qs a - : ps: perce en gael ra titnionitt ae i te), Volume VII March, 1913 No. ANNALS OF TROPICAL MEDICINE AND PAR ASP PrOpoG Y ISSUED BY THE LIVERPOOL SCHOOL OF TROPICAL MEDICINE Editor Prorrssor Sir RONALD ROSS, K.C.B., F.R.S., Mayor I.M.S. (Ret.), WED., D.P.H, FACS. Disc. LL.D: In Collaboration with Prorrssor J. W. W. STEPHENS, M.D. Cantab., D.P.H. Proresson R. NEWSTEAD, M.Sc., J.P., F.R.S., A.L.S., F.E.S., Hon. F.R.H.S. Proressor J. L. TODD, B.A., M.D., C.M. McGill, D.Sc. Liv. H. WOLFERSTAN THOMAS, M.D., C.M. McGill. ANTON BREINL, M.U.Dr. H. B. FANTHAM, .D.Sc.Lond., B.A. Cantab., A.R.C.S., F.Z.5. Editorial Secretary Dr. H. B. FANTHAM, School of Tropical Medicine, The University, Liverpool. C. Tinling & Co., Ltd. Printers to the University Press of Liverpool 53 Victoria Street THE INCORPORATED LIVERFOGE sCHOOL OFd ERGPICAL, MEDICINE (Affiliated with the University of Liverpool) Hon. President: Her Royal Highness PrincESS CHRISTIAN Hon. Vice-Presidents: ‘THE DUKE oF NORTHUMBERLAND, K.G. THE EARL oF DeErRpBy, G.C.V.O. EARL CROMER, G.C.B. VIScouN IT MILNER. UrC.b. LorpD.-PIRRIE, K.P: SIR OWEN PHILipps, K.C.M.G. SIR EDWIN DURNING LAWRENCE, Bart. Mr. O. HARRISON WILLIAMS COMMITTEE Chairman: SIR WILLIAM H. LEVER, Bart. Vice-Chairman: Mr. F. C. DANSON MMi. Et. [. READ; C.M.G. Colonial Office Sir ALFRED DALE University of Liverpool Sir W. B. BowRIna, Bart. | | Council of University of Liverpool Professor CATON j if ty of P Bee es PE RINGION | Senate of University of Liverpool Professor HERDMAN i Dr. C. J. MACALISTER Mr. GEORGE P. NEWBOLT Mr. C. BOOTH (Jun.) era Mr. T. F. HARRISON f Steamship Owners’ Association Mr. A. R. MARSHALL Mr. W. ROBERTS Mr. CHARLES LIVINGSTON Mr. GEORGE BROCKLEHURST Mr. J. W. W. DANSON Mr. J. A. TINNE Mr. ROBERT RANKIN Professor E. W. HOPE Mr. ].-W- ALsop Mr. ALEC L. REA, Hon. Treasurer Mr. A. H. MILNE, C.M.G., Secretary. ; B 10 Exchange Buildings, Liverpool Royal Southern Hospital Shipowners’ Association Staff 1, At the University of Liverpool Propessors » JOHN WILLIAM WATSON STEPHENS, M.D,, Cantab,, DPA, Sir Alfred Yones Professor of Tivo “i al Medi ine ROBERT NEWSTEAD, M.Se, J.P. F.R.S., ALLS, FAS, Dutton Memorial Professor of Entomology Major Stk KONALD ROSS, K.C.B,, FRCS, D.P.H,, M.D,, D.Se, LL.D, F\R.S:i | Nobel Laureate 1902, (Indian Medical Service, retired), Professor of Tropical Sanitation Assistant Leciwvers « HAROLD BENJAMIN FANTHAM, D,Se,, BA, Assistant Lecturer in Parasitology HAROLD WOLFERSTAN THOMAS, M.D., CM, Asvistant Leetuver in Entomology HENRY FRANCIS CARTER, S.ELA.C, Dipr,, FES, J.O, WAKELIN BARRATT, M.D, D,Se,, (Lond,) Major JOSEPH FITZGERALD BLOOD, M,D,, M.Ch, (Indian Medical Service, retired Prov, ERNEST EDWARD GLYNN, M,A,, M.D,, (Cantab.), M.R.C.P,, M.R.C.S, Prov, E, W, HOPE, M.D,, D,Se, WILLIAM THOMAS PROUT, M,B,, C.M.G, ffonoraey Lecturers Assintant in Cryotherapy JOUN GORDON THOMSON, M,A,, M.B,, Ch,B Honorary Siatistician WALTER STOTT Bibliogvapher « » WALTER DRAWZ Clerk to the Laboratory NORA MURPHY 2 At the Runcorn Research Laboratories Divector WARRINGTON YORKE, M.D, ASSESIANE « B, BLACKLOCGK, M.D., D.P.H: 3 At the Royal Southern Hospital, Liverpool Physicians CHARLES JOHN MACALISTER, M,D,, ER. E.P; JOHN LLOYD ROBERTS, M.D, M.R.C.P, Surgeons - D, DOUGLAS CRAWFORD, F.R.C.S., ROBERT JONES, F.R.C.S, GEORGE PALMERSTON NEWBOLT, M.B,, F.R,G.S, Tropical Pathologist» JOHN WILLIAM WATSON STEPHENS, M.D,, Cantab, Clinical Pathological dsvistané » DAVID THOMSON, M.B. Ch.B., D.P.H, 4. On Expedition HAROLD WOLFERSTAN THOMAS, M.D,, C.M, HARALD SEIDELIN, M.D, Laboratory JOHNSTON LABORATORY, UNIVERSITY OF LIVERPOOL Researek Laboratory : RUNCORN Hospital : ROYAL SOUTHERN HOSPITAL, LIVERPOOL Secretary's Office: Bre, EXCHANGE BUILDINGS, LIVERPOOL NOTICE - The following courses of instruction will be given by the Liverpool School of Tropical Medicine during 1913 :— Full Course begins 6 January. Advanced Course begins 2 June. Diploma Examination, 7 April. Certificate Examination, 30 June. Full Course begins 15 September. Diploma Examination, 15 December. The full Course of Instruction is open to all qualified medical men, and the Examination to all students who have taken out this full course. Fee for the full Course of Instruction—Thirteen Guineas. Fee for the Diploma Examination—Five Guineas. Fee for the Short Course of Instruction—Four Guineas. Fee for the use of a School microscope during one term—Ten shillings and sixpence. For prospectus and further information, application should be made to the Dean of the Medical Faculty, University of Liverpool. The following have obtained the Diploma in Tropical Medicine of the University of Liverpool : — Diploma in Tropical Medicine Date of Date of Diploma Diploma 1904 Augustine, Henry Joshua 1905 Macfarlane, Robert Maxwell 1904 Bennett, Arthur King 1905 Maddock, Edward Cecil Gordon 1904 Bruce, William James 1905 Moore, James Jackson 1904 Byrne, John Scott 1905 Nightingale, Samuel Shore 1904 Clayton, Thomas Morrison 1905 Radcliffe, Percy Alexander Hurst 1904 Dalziel, John McEwen 1905 Young, John Cameron 1904 Dee, Peter : 1906 Adie, Joseph Rosamond 1904 Greenidge, Oliver Campbell av reek Frank otitis 1904 Hehir, Patrick 1906 Bate, John Brabant 1994 Khan, Saiduzzafor 1906 Bennetts, Harold Graves 1904 Laurie, Robert 1906 Carter, Robert Markham 1904 Maclurkin, Alfred Robert 1906 Chisholm, James Alexander 1904 McConnell, Robert Ernest 1906 Clements, Robert William 1904 Nicholson, James Edward 1906 Dundas, James 1904 Philipson, Nicholas : 1906 T’aichnie, Norman 1904 Sharman, Eric Harding 1906 Jeffreys, Herbert Castelman 1904 Thomson, Frank Wyville 1906 Mackenzie, Donald Francis 1904 Walker, George Francis Clegg 1906 Pailthorpe, Mary l‘lizabeth 1g05 Anderson, Catherine Elmslie 1906 Palmer, Harold Thornbury 1905 Brown, Alexander 1906 Pearse, Albert ves 1905 Caldwell, Thomas Cathcart 1906 Sampey, Alexander William 1905 Critien, Attilio 1906 Smithson, Arthur Ernest 1905 Hooton, Alfred 1006 Taylor, Joseph van Someron 1905 Hudson, Charles Tilson 1906 Taylor, William Irwin tg05_ Illington, Edmund Moritz 1906 Tynan, Edward Joseph Date of Diploma 1900 Watson, Cecil Francis 1906 ©=Willcocks, Roger Durant rg06 ©6Williamson, George Alexander 1907 Allan, Alexander Smith 1907. Allwood, James Aldred 1907. Bond, Ashton 1907. Branch, Stanley 1907. Collinson, Walter Julius 1907. Davey, John Bernard 1907. Donaldson, Anson Scott 1907. Fell, Matthew Henry Gregson 1907. Gann, Thomas William Francis 1907. Graham, James Drummond 1907. Hiscock, Robert Carroll 1907 Keane, Joseph Gerald 1907. Kennan, Richard Henry 1907. Kenrick, William Hamilton 1907. Le Fanu, George Ernest Hugh 1907. Mackey, Charles 1907. Maddox, Ralph Henry 1907. McCarthy, John McDonald 1907. Raikes, Cuthbert Taunton 1907 Ryan, Joseph Charles 1907. Vallance, Hugh 1908 Caverhill, Austin Mack 1908 Crawford, Gilbert Stewart 1908 Dalal, Kaikhusroo Rustomji 1908 Dansey-Browning, George 1908 Davidson, James 1g08_ Dickson, John Rhodes 1g08 Dowdall, Arthur Melville 1908 Glover, Henry Joseph 1908 Greaves, Francis Wood 1908 Goodbody, Cecil Maurice 1g08 Harrison, James Herbert Hugh 1908 Joshi, Lemuel Lucas 1g08 Le Fanu, Cecil Vivian 1908 Luethgen, Carl Wilhelm Ludwig 1908 Mama, Jamshed Byram}i 1908 McCay, Frederick William 1g08 McLellan, Samuel Wilson 1908 Pearce, Charles Ross 1908 Schoorel, Alexander Frederik 1908 Smith, John Macgregor 1908 Stewart, George Edward 1908 Tate, Gerald William 1908 Whyte, Robert 190g Abercrombie, Rudolph George 1g0g Allin, John Richard Percy 1909 Armstrong, Edward Randolph 1909 Barrow, Harold Percy Waller 1909 Beatty, Guy 1909 Carr-White, Percy 1909 Chevallier, Claude Lionel 1909 Clark, William Scott 1909 Cope, Ricardo 1909 Fleming, William 1909. Hanschell, Hother McCormick 1909 Hayward, William Davey 1909 Henry, Sydney Alexander 1909 Innes, Francis Alexander Date of Diploma 1909 Jackson, Arthur Frame 1909 Kaka, Sorabji Manekji 1909 McCabe-Dallas, Alfred Alexander Donald 1909 Meldrum, William Percy 190g Murphy, John Cullinan 1909 Samuel, Mysore Gnananandaraju 1909 Shroff, Kawasjee Byramiee 1909 Thornely, Michael Harris 1909 Turkhud, Violet Ackroyd 190g Webb, William Spinks 1909 Yen, Fu-Chun I910 Brabazon, Edward 1910 Castellino, Louis 1910 Caulerick, James Akilade 1910 Dowden, Richard 1910 Haigh, William Edwin 1910 Hamilton, Henry Fleming 1910 Hefferman, William St. Michael 1910 ©Hipwell, Abraham 1910 Homer, Jonathan 1910 Houston, William Mitchell 1910 James, William Robert Wallace 1910 Johnstone, David Patrick 1910 Korke, Vishnu Tatyaji 1910 Macdonald, Angus Graham 1910 Macfie, John Wm. Scott 1910 Manuk, Mack Walter 1910 Murison, Cecil Charles 1910 Nanavati, Kishavlal Balabhai 1910 Nauss, Ralph Welty 1910 Oakley, Philip Douglas 1910 Pratt, Ishmael Charles 1910 Sabastian, Thiruchelvam 1910 Shaw, Hugh Thomas 1910 Sieger, Edward Louis 1910 Sousa, Pascal John de 1910 Souza, Antonio Bernardo de 1910 Waterhouse, John Howard 1910 White, Maurice Forbes 1911 Blacklock, Breadalbane 1911 Brown, Frederick Forrest 1911 Chand, Diwan Jai tgtt Holmes, John Morgan Igtt ITevers, Charles Langley 1911 Iles, Charles Cochrane I9gtIt Ingram, Alexander 1911 Kirkwood, Thomas 191l Knowles, Benjamin 1911 Liddle, George Marcus Berkeley 1911 Lomas, Emanuel Kenworthy io1r Mackarell, William Wright 1911 MacKnight, Dundas Simpson 1g11 Mascarenhas, Joseph Victor 1911 Murray, Ronald Roderick -Igtt Oluwole, Akidiya Ladapo 1g1I Rao, Koka Ahobala 1911 Sinton, John Alexander 1911 Tarapurvalla, Byramji Shavakshah 1g11 Taylor, John Archibald 1911 Woods, William Medlicott Date of Date of Diploma Diploma Igt2 Aeria, Joseph Reginald 1912 Myles, Charles Duncan 1912 Anderson, Edmund Litchfield 1912 Pelly, Huntly Nevins 1912 Borle, James 1912 Prasad, Bindeshwari 1912 Bowie, John Tait 1912 Prentice, George 1912 Brassey, Laurence Percival 1912 Ross, Frank Ig12 Christie, David 1912 Russell, Alexander James 1912 Dillon, Henry de Courcy Hutchison 1912 Dunn, Lillie Eleanor 1912 Ruthven, Morton Wood 1912 Hardwicke, Charles 1912 Sandilands, John 1912 Jagose, Jamshed Rustomji 1912 Seddon, Harold 1912 Kochhar, Mela Ram 1912 Smalley, James 1912 McGusty, Victor William Tighe {912 Strickland, Percy Charles 1912 Milne, Arthur James Hutchison 1912 Mitra, Manmatha Nath 1912 Watson, William Russell EDITORIAL NOTICE By order of the Committee of the Incorporated Liverpool Schoo! of Tropical Medicine, the series of the Reports of the School, which had been issued since 1899, were followed, from January I, 1907, by the Annals of Tropical Medicine and Parasitology, of which this is the first number of the seventh volume. Altogether twenty-one Memoirs, besides other works, were published by the School since 1899, and of these ten, containing 519 quarto cr octavo pages and gs plates and figures, were published during the two years 1904 and 1905. The Annais are issued by the Committee of the School, and will contain all such matter as was formerly printed in the Reports—that is to say, accounts of the various expeditions cf the School and of the scientific work done in its laboratories at the University of Liverpool and at Runcorn. In addition, however, to School work, original articles from outside on any subject connected with Tropical Medicine and Hygiene, and Parasitology may be published 1f found suitable (see notice on back of cover); so that, in all probability, not less than four numbers of the Annals will be issued annually. Each number will be brought out when material sufficient for it has been accumulated. rita sit Say ah \ert Y faalviwt mt ci ne a ; Wiacbhde segs } Sy imoita badedre ie). vata’. s9ot =f ir ou sie ta F fmitgoree flee oifgtl” vet neers Fa) . A 7 . a EI . hi L M ale 4 : 7 rine) 4 > \ 3 : ——— 3 . 7 hed | CVA =, ay: ~ i ; ips re) vie Salutea aon! 2 i 1 selegea oD ES PEs .n88 i raat 5} 10 2 > “¢ J otal mab .pawollnt stv OGek. ‘yout E ae ‘ ; 5 ke J eet Pantene Kt Tt 7 CBG tte aL Dts no ses aoe ay ws t We ile - : : ripley iis ¢S > 5 it fot’ ssdintudh: ee ; ne D> ' , i ] 4 : v s ' T= } Lier € t-te Ore BIO" vo" e Re ee 20 | 7 : i te j 4 ng ‘alee is ; - 7 20 of bar ROOT — ona : } 2 : A Piet Sh : x M 7 7 | | a tO Pa LPeRAET beh : bontaet 4 2 sis = zen +f 3 § lineman: 8 Rn Pe Lz s~ Ce a Be 5 ISB - Tha site waa at me sit} Th eronrhbsqye 2dr a sup 16 io BiG eee ON ae ~ aes 2 ww \ a ahd oda ssincinsod een aes yoiubs shows Setparens - ¢ oa, “heat an = > dior. leaatie sovswod gerlibeeet erin i . ue ET alte oh : é fe =! ; - bor ‘ . be > : ak bsneat a 4 rT ei | : ‘ Fe .AaY LRT ee bith. ple ) ff he e 7 f ti y; sere 5 I is anatltivny. ined? fol TRYPANOSOMIASIS OF DOMESTIC ANIMALS IN NORTHERN NIGERIA BY ee SOOT ft MAe rE MAC. MB CHB. WEST AFRICAN MEDICAL STAFF (Recezved for publication 19 December, 1912) PEATES: =i Trypanosomiasis is very prevalent amongst domestic animals in Northern Nigeria, and, at any rate in the south-western portions of the Protectorate, accounts not only for a heavy mortality, but also for serious financial losses, due to sickness, forced sales and the slaughtering of stock in unsuitable markets. It is difficult to give any adequate idea of the grave nature of the losses, but some conception of their extent may be gathered from the two following instances:—During the greater part of 1912 I was stationed in Ilorin, a province which, with the exception of one division, is considered not unsuitable for horses. | Nevertheless, during the eight completed months of my residence, ten out of the fifteen horses in the possession of Europeans cont-acted trypanosomiasis, and six died of the disease. An experi™ent was also made with a view to introducing animal transport into the province. Ten healthy donkeys were selected for this purpose, and sent to carry loads from Ilorin town to Agugi, a village some thirty miles to the east. Within three months all had died of trypanosomiasis. At Zungeru, during the year 1911, twenty-five horses were treated for this disease in the isolation camp, and of these 40 % either died or were so seriously affected that their owners thought it wise to part with them at the small price a native is always willing to pay for a sick animal. At Lokoja the disease is even more serious, and Dr. C. F. Watson has estimated that among the horses treated for tsetse disease there, the rate of dead and incapacitated horses is Oven 73> %. e here can, moreover, be little doubt that. the disease is spreading; a fact proved by the additions made from time to time to the list of localities in which the Government declines to compensate officers for the loss of horses which have died from trypanosomiasis contracted in the discharge of official duties. 2 Difficult as it is to estimate the losses among horses belonging to the small number of Europeans stationed in the Protectorate, it is altogether impossible to ascertain those of the natives. They must, however, be very great, and their magnitude may be gauged by the experiences of the Emirs who attended the Coronation Durbar at Zaria in June, 1911, one of whom is said to have lost 70 per cent. of his horses. Before the British undertook the administration of Nigeria, certain tracts of country known to be unsuitable for horses were sedulously avoided. Europeans, however, are compelled to penetrate these districts in the discharge of their duties, and are followed by the native officials, not only at the sacrifice of their own animals, but also greatly to the detriment of the whole country, since the infected horses returning home, or passing through other districts, spread the disease in every direction. Besides horses, the cattle, sheep, goats and dogs are also affected. In their long trek to the coast, the herds of cattle from the north carry trypanosomiasis with them, maintaining the infection in old localities and introducing it into new. The herdsmen are quick to detect the symptoms of the disease, and, if they appear in one of their animals, are accustomed to slaughter the beast wherever they may happen to be. The fate of those that sicken is to be butchered by the roadside, and it is a common sight to see a carcase cut up and laid out for sale far away from any native town. Ilorin is situated at a point on the main caravan road where the routes from Kano and Sokoto converge, and the majority of the animals slaughtered there for the market were found on examination to be suffering from trypanosomiasis. In Table I the results of the examinations of thirty-five of these animals is shown : — Taste I.—Trypanosomes found in the blood of thirty-five sick animals being slaughtered for the Tlorin market Host I. brucei | T. vivax | T. nanum or Double pecorum infection Cattle—Fulani breed ... a ” I ast 6 I I ub Sheep and rams A Asha I 8 I Goats 4 =e ie otalse =: ie oad oe 30 x zs 35 I 3 In November, 1911, Sir David Bruce published in No. 31 of the Sleeping Sickness bulletin’ a list of identifications of fifteen cases collected by me in Northern Nigeria of trypanosomiasis in horses. Since then I have added to these a number of fresh cases, both in horses and other domestic animals, bringing the number up to eighty-eight. An analysis of eighty-six of these cases, in which the infection was natural, is given below (Table II). Taste II1.—Showing the infecting agent in eighty-six cases of naturally acquired trypanosomiasis in domestic animals in Northern Nigeria Host | . brucei T. vivax | T.nanum or| T. theilert Double | pecorum infections ae | 7 as, Horse ive — al 14 18 8 | Z. 3 Donkey __... ae Bt. 2 2 Cattle— Fulani breed... ie I 18 I Dwarf breed _... oe so 2) noe I Sheep and ram... af I 8 I Goat a J. ve ge 4 Dog ae 53 a 555 I Totals eel 18 53 | 10 I 4 series of experiments on animals or to attempt cross immunity tests, the morphological characters of the trypanosomes found in blood- films had to be relied on for identification. By this means it was, of course, impossible to distinguish between the more closely allied species. TZ. thezlevi was observed once, but with this exception the trypanosomes detected fell naturally into three main groups, and, as individuals of each type had previously been examined by Sir David Bruce, his identifications have been adopted.? T. brucez.* In twenty cases of natural infection trypanosomes were found which closely corresponded with the type described and figured by Sir David Bruce as 7. bvucez.3. In four cases (three horses and one donkey) posterior nuclear forms were detected, and * This trypanosome is probably that for which Prof. Stephens and Dr. Blacklock have recently proposed the name 7. ugandae. (Proc. Roy. Soc., B, Vol. LXXXVI, pp. 187-191). 4 in guinea-pigs and rats inoculated from three of these cases they appeared at certain stages of the disease in relatively large numbers. Posterior nuclear forms have been described in 7. eguzperdum, T. pecaudi and T. brucei, as well as in T. rhodeszense. Their occurrence in these cases of trypanosomiasis in Nigeria does not therefore assist in precise identification. Sleeping sickness, how- ever, is either altogether absent or quite uncommon at Zungeru and Ilorin, where the majority of my cases were found. In a number of films the long forms of the parasite were observed to have their posterior extremities peculiarly blunt and almost rectangular. No attempt was made to plot a curve to represent the variation in size of this trypanosome. Great variations were observed from day to day in the relative percentages of short and long forms, as 1s indicated in the tables given below, where the different forms were enumerated in the case of a horse, and in those of a rat and a euinea-pig inoculated with his blood. (See table on page 5.) The disease set up by these trypanosomes appears to be extremely fatal. Of the eleven horses and two donkeys suffering from this form of trypanosomiasis of which I have complete records none recovered. In horses the disease seems to occur in two forms, some cases end fatally in two to four weeks, others linger on for as many months. All the cases in which posterior nuclear forms were observed were of the acute type, death occurring within a month of the onset of the disease. The general symptoms in horses were fever, emaciation, and more or less pronounced oedema of the legs, belly and scrotum. The appetite was irregular, but was often ravenous almost up to the hour of death. In the later stages restlessness was a distressing feature. Inflammation of the eyes was observed in two cases out of fourteen, and a greenish discharge from the nose, coughing up of mucus, and a herpetiform eruption on the upper lip occurred each in one case. The animals presented a dejected appearance in the early stages of the disease, their heads drooped, they dragged their hind legs in walking, moved slowly and with difficulty and were apt to stumble. They were notably insensitive to pain, as for instance, to the prick on the ear necessary to obtain blood for examination. Finally they became so weak and wasted that they could not rise. 5 Natural infections with trypanosomes of this type were found in the horse, donkey, Fulani cattle, and sheep. The parasite was also successfully transmitted by inoculation to a cow of the dwarf breed found in pagan districts of Nigeria. (See page 10.) GUINEA-PIG Forms with | Stumpy and long, free | intermediate flagella | forms Guinea-pig injected from horse. (Trypanosomes first appeared in the blood on July 18) 15% 85% 40% 60% 56% | 44% 16°5 % | 835% Died July 21 | Horse (No. 35) Rar Dates 190s es a Forms with | Stumpy and | Forms with | Stumpy and long, free intermediate! long, free | intermediate flagella | forms flagella forms July 8 24% | 76% » 9 42% | 58% fe) | Rat injected from horse. (Trypanosomes first appeared in the blood on July 16) » 13 29% 71% s, 16...) Trypanosomes very scanty 89 % 11% a mehe _ 8% | 2% » «18 52% 48% 19 15% 85% io 20 74% 26% 10% 90% » 21 8% 92% kz? 6% | 4% 88% L2G Sf eas Died ‘a 2G) | alga 58% 42% Aug. I 85% 15% stl iod 69% 31% 5 Died T. vivax. By far the most common form of trypanosome in my series of cases was one presenting the morphological features of It occurred in fifty-six of my eighty-six cases, and in fifty-three cases was the only trypanosome present, being but once associated with 7. bvucez and twice with 7. nanum or pecerum. T. vivax. 6 This parasite was found in the following hosts:—Horse, donkey, Fulani cattle, dwarf cattle of Nigeria, sheep, goat and dog. From the fact that it was found in the dog, it is probable that this trypanosome is the parasite to which Ziemann* gave the name T. vivax, and not the allied 7. cazgalboui described by Pecaud as occurring in Dahomey in horses. For Laveran and Mesnil say: ‘Le fait que 77. Cazalbouz ne peut étre inoculé ni au singe, ni au chien, ni aux petits rongeurs, permet de distinguer facilement ce trypan. des especes qui s’en rapprochent au point de vue morpho- logique, mais qui sont pathogénes pour ces animaux.’> In horses the disease set up by this parasite seems to be mild in Northern Nigeria. Of the fifteen cases of which I have complete records only one died, whereas fourteen recovered. The general symptoms were fever, emaciation and oedema of the legs, belly and scrotum, sometimes well marked, at other times scarcely perceptible. The animals presented a dejected appearance, moved awkwardly and reluctantly, and appeared as though tired. In severe cases they were somewhat insensitive to pain. The appetite was always good. In one case cough was a symptom, and in another nasal catarrh. No affections of the eyes were observed. Three cases were met with in donkeys, and of these two died and one recovered. The case of one of those that died was, however, complicated by a concurrent infection with 7. d7ucez. Eighteen cases were found amongst Fulani cattle. Most of them were animals treking down towards Lagos from Kano or Sokoto which had sickened on the road, and were therefore about to be butchered for the Ilorin market. Their native owners believed them to be dying, and were therefore having them slaughtered so as to anticipate death; but, beyond this assertion, I have no data from which to gauge the mortality from the disease in this breed of cattle. Both the dwarf cattle which came under my notice suffering from 7. vivax, however, recovered. Similarly the eight sheep and rams I studied were animals about to be slaughtered so as to anticipate death, and the same remark applies to the four goats. The one dog in which 7. vzvax was found died. T. nanum or pecorum. In a few cases small trypanosomes g-12 in length without free flagella were found in blood-films, and these have been identified as 7. xanum or pecorum. ‘It will be : remembered the name 7. fecorum was introduced by Bruce to cover the group of trypanosomes of which the 7. dzmorphon of Laveran and Mesnil and 7. congolense are members, and that xanum is only distinguishable from pecorum by animal experiments.”¢ Trypanosomes of this type were met with in thirteen cases, in ten alone and in three associated with 7. vivax or T. brucet. Ten of these cases were horses, two Fulani cows, and onearam. Of the ten cases in horses one had a concurrent infection with 7. vivax and another with 7. d7zcez, leaving eight uncomplicated cases, of which one died, three recovered, and the remaining four had unknown issues. The number of cases of this variety of trypanosomiasis that came under my notice does not, therefore, enable me to give any general account of the symptoms of the disease in Northern Nigeria. It may, however, be noted that oedema was present only to a slight extent in the cases seen, although lachrymal and nasal discharges were relatively more frequent than in infections with either ZT. vivax or T. brucet. T. theilertz. T. theilert was observed once in a cow of the dwarf breed found in Nigeria. The animal was feverish for a few days and wasted considerably, but soon recovered from the infection. The period when trypanosomiasis is most common is undoubtedly the rainy season, which corresponds to the time during which the tsetse flies, which in the dry season retire to the banks of the streams and rivers, are most widely distributed over the country. The accompanying chart of the rainfall and the number of admissions of horses to the isolation camp at Zungeru for the year IQII illustrates this point, although the number of cases dealt with is too small to form a reliable index. A better proof is afforded by the fact that the natives congratulate themselves at the end of the rainy season that those of their horses which have withstood the disease so far will be safe until the rains recommence. The dry season is also the time of year selected by the herdsmen of the north to undertake their long trek south with their cattle and sheep. The province of Ilorin in Northern Nigeria is peculiar, inasmuch as, whilst G. palpalis and G. tachinoides are distributed all over it, tsetse flies of the morsttans group—G. submorsitans and G. longz- palpis—are restricted to one, the Patigi or eastern, division. In 8 my series of cases of trypanosomiasis collected at Ilorin, twenty- four were animals that had never been into the Patigi division, and of these fourteen were infected with TJ. vzvax, six with T. brucez and four with 7. nanum or pecorum. The Patigi division is shunned by all herdsmen, and it is generally recognised that horses can neither live there nor be taken into the district without contracting tsetse disease. Two horses that had lived for years in Tlorin town were taken to Patigi during my last tour of service. - > . w > = 3 = - SS MOISY BBS ELT Rh Sl 9e Sr nis ace ly 4 & = < = > > < a ° Zz e Chart showing the rainfall in inches (continuous line) and the number of cases of trypanosomiasis of horses admitted to the isolation camp (dotted line) at Zungeru during IgIt. Both returned infected with 7. d7ucez, and both died shortly after wards. In this connexion it should be mentioned that Bruce concludes that, in Uganda, ‘ The carrier of T7ypanosoma vivax 1s probably Glossina palpalis,7 and that G. morsitans is known to transmit 7. drucez. It is possible that in the native towns and European stations flies of the genus Stomoxys, which abound in these places, may play a part in the transmission of trypano- somiasis. The horse (No. 42), for instance, had not been within 9 two miles of any spot known to be haunted by tsetse flies for five weeks previous to the onset of his symptoms, and it is practically certain that during this period he could not have been bitten by tsetse flies. He was, however, tormented by Stomoxys flies, which were exceedingly common at the time. Both S. xzgra and S. calcitrans have been taken at Llorin. Treatment. The treatment usually adopted in Northern Nigeria for all cases of trypanosomiasis in horses is the administration of arsenious acid and perchloride of mercury by the mouth. This is the routine treatment adopted at the isolation camps at Zungeru and Lokoja. The doses usually employed are nine grains of the arsenic and three ounces of a I in 1,000 solution of the perchloride of mercury daily, divided into three doses; but I have also tried much larger doses, administered for a few days at a time only, in the hope that the sudden shock of arsenic might eliminate the trypanosomes before they had time to react to the drug, and with a view to avoiding the cumulative action on the host that has certainly sometimes proved fatal. It is doubtful whether this treatment has any beneficial results. In my experience it has not saved a single animal infected with 7. d7ucez, and it is impossible to judge of its action on 7. vivax, as this form of trypanosomiasis is as a rule not fatal, the animals recovering even when untreated. At one time it was asserted that such treatment accounted for the cure of 50 % of the cases at Zungeru. This, however, was before it was known that half the trypanosomiasis there was due to T. vzvax, and was capable of spontaneous cure. The higher mortality recorded at Lokoja is probably accounted for by the larger percentage of cases of trypanosomiasis due to 7. O7ucez which occur at that station. Intravenous injections of antimony have met with no greater success in trypanosomiasis due to 7. b7ucez. Organic arsenical compounds have been used in only a few cases, the high cost of the injections and the technical difficulties of administration standing in the way of the general use of these preparations until it is certain, not only that a cure will result, but also that immunity against future attacks will be established. Arsenic and mercury have been used as prophylactics also, but without success. 10 No immunity follows an attack of trypanosomiasis, reinfections with the same or a different species of trypanosome being met with. The dwarf cow (No. 48) in my series was infected successively with T. theileri, T. vivax and T. brucet, and finally died of the last form of trypanosomiasis. The donkey (No. 47), after recovering from an infection with 7. vivax, was infected by injection with T. brucez, and succumbed to the disease. Treatment of any sort is, however, applicable only to Govern- ment stock and horses in the possession of Europeans. The great majority of cases, occurring in animals which are the property of natives, cannot be brought under treatment. It might be possible, nevertheless, to limit the spread of the disease by the establishment of isolation camps, and much might be effected by systematic clearing along the roads. The main caravan routes especially should be cleared, and inducement offered to the natives to farm the land on either side of the roads. Dwarf cattle. In certain districts in which the Fulani cattle do not live a few dwarf cattle are found, which, because they occur in pagan country, are often referred to as ‘pagan cattle.’ In Ilorin they are kept in all the districts occupying the south-eastern corner of the province, from Ofa Ora to Eyjiba on the north, and extending west along the southern boundary as far as Oke Awra. Dr. Foy, who made some interesting observations and experiments on a bull of this breed, thus described his physical characters. ‘The body frame,’ he says, ‘ was thick-set and broad and compara- tively long for its height, the legs thick and short, the neck thick and short, the head short, and the horns short.’® The accom- panying photograph of a bull and cow brought from Awtun will help to give an accurate idea of the appearance of these singular animals (P]. III, fig. 5). The most remarkable point about these dwarf cattle, which are bred for slaughter only and are not used for milking, is that they live in districts in which the Fulani cattle cannot exist. For this reason it has been conjectured that they have acquired a high degree of immunity to trypanosome infections. Dr. Foy, who, as already stated, experimented on a bull of this breed, concluded :— “(1) That a certain breed of cattle found in pagan districts possess a high degree of natural immunity in that they may harbour the trypanosome in the blood and yet keep in good condition and i i i ee Il show no signs of the disease, nor do they die from the infection so long as their environments are favourable. These environments are a free life, with ample food, especially plenty of green grass. Confinement, poor feeding, and hard exercise, tend to make the disease manifest itself clinically. ‘(2) That such domesticated cattle may act as a reservoir of infection since the blood may prove infective at such times when clinical symptoms manifest themselves, although the trypanosome may not be found on making a microscopical examination. *(3) That the subcutaneous injection of 26 c.c. of serum obtained from the blood of one of these cattle when manifesting no clinical symptoms of the disease did not prove infective when inoculated into a calf, nor did it act as a prophylactic when the calf was exposed to natural infection subsequently.’ He adds, in another part of the same paper, ‘all strains of trypano- somes used or met with in the work were of the 7. brvucez type.’ At Ilorin I was able to carry out only two experiments with these cattle, but as the subject requires careful investigation, and promises to provide results of practical importance, I should, perhaps, place them on record here. On May 11th, 1912, two of these dwarf cattle (a cow and a bull) arrived at Ilorin from Awtun. As they had treked up all the way by road, they arrived in rather poor condition, but seemed, on the whole, to have stood the unusual exertion wonderfully well. On May 13th I made a thorough examination of the blood of each without detecting any trypanosomes, and four days later injected a rat and a guinea-pig each with 1 c.c. of blood taken from the cow. Neither of these animals developed any symptoms of disease, and, although they were kept under close and constant observation for four weeks, trypanosomes were never seen in their blood. The subsequent history of the cattle is given below :— Dwarr Butt (No. 50) May 11th, 1912.—Arrived at Ilorin. May 13th, 1912.—Blood examination—negative. August 30th, 1912.—Trypanosomes (J. vivax) in the blood. Bull looks well. Untreated. September 15th, 1912.—Blood examination—negative. Bull, however, looks ill. September 19th, 1912.—Bull died of anthrax. No trypanosomes found in the blood nor in smears made from the organs after death. Dwarr Cow (No. 48) May 11th, 1912.—Arrived at Tlorin. May 13th, 1912.—Blood examination—negative. 12 May 17th, 1912.—Blood examination—negative. Subcutaneous injections of the cow’s blood made into a rat and a guinea-pig. Rat under observation twenty-eight days, but never showed trypanosomes. Guinea-pig under observation twenty-eight days but never showed trypanosomes. August 30th, 1912.—Cow feverish and emaciated, no oedemas. Trypanosomes (I. theilert and JT. vivax) present in the blood. Untreated. September 7th, 1912.—Trypanosomes still present in the blood. Subcutaneous injection of two c.c of the cow’s blood into a guinea-pig. Guinea-pig under observation nineteen days. Accidentally killed. Never showed trypanosomes. September 18th, 1912.—Blood examination—negative. One c.c. of the cow’s blood injected subcutaneously into a rat. Rat under observation twenty-two days but never showed trypanosomes. September 27th, 1912.—Cow looks well, and her blood has been free from trypanosomes since September rsth. Two-c.c. of blood from donkey (No. 45), heavily infected with T. brucet, injected into cow subcutaneously. October 8th, 1912.—Trypanosomes numerous (T. brucet). The blood was negative up to October sth. November 3rd, 1912.—Cow died. Trypanosomes still present in the blood but scanty. Both animals developed a natural infection with 7. vzvax, and apparently recovered, for they ceased to show trypanosomes in films made from the peripheral blood. The cow in addition had a concurrent infection with 7. ¢hezlevz. The cow was then successfully infected by injection of blood from a donkey heavily infected with T. brucez, and, although the parasites soon became scarce in her blood, she died five weeks after the injection still showing a few trypanosomes in the peripheral blood. It would be rash to draw definite conclusions from such experi- ments, but so far as they go they do tend to show that this breed of cattle, like horses, possesses an immunity to 7. vzvax, inasmuch as although these parasites may appear in their blood they do not produce a rapidly fatal disease. The immunity does not appear, however, to extend to ZT. bvucez. The question then arises how do these cattle live in districts where the Fulani herdsmen dare not take their cattle to graze? The dwarf cattle are generally to be found in the immediate vicinity of villages, and unlike the Fulani cattle do not wander widely over the country in search of pastures. It was thought possible, therefore, that they might thus escape the attacks of G. swbmorsitans, the species of tsetse fly whose distribu- 13 tion (as already pointed out) is coextensive with the areas habitually shunned by the Fulani herdsmen. In consequence, collections of flies were obtained at Odo Okeri, Eri, Oro and Oke Onio, from spots where the cattle were actually grazing. In none of these collections was G. submorsitans present, although both G. falpalis and G. tachinoides were taken. I have to thank Mr. T. A. G. Budgen for very kindly superintending the work of a collector who was sent with him for this purpose. I can only hope that at some future date, and under more favourable conditions, it may be possible to make a thorough investigation of this interesting subject. In conclusion, I would like to express my thanks to Mr. E. C. Duff for affording me an opportunity of examining the dwarf breed of cattle; to Dr. C. F. Watson and Dr. W. Morrison for very kindly sending me blood-films from Lokoja; to Dr. G. R. Twomey, who relieved me at Ilorin, for following the last stages of my experiments; and to Serjt. Moore for the careful record kept by him of the cases of trypanosomiasis occurring at Zungeru. A tabular synopsis of the cases of trypanosomiasis in domestic animals, collected in Northern Nigeria, is appended. ‘oondg] PlALC| arg Aq suorjvoyyuopy , | ce a xvata Lif, te ce ve | ce Fe XDA or ce ce oe PoLOA0I0% y XDA1d "Ff, | 1161 ‘Avy Ch xy ‘syjuour $ |p caetarpyosotur ospy | ped » WIndg "ZT, | 1101 “any ae Ab | | ce NPA 7 ce cb ee | ” { | P2.LAOIODY » Moan 7, |} 1161 “gow be ’ ce | ‘syoom Z IIT | prc » WONG * T, me Ltd oid | PoAOIO y MAID * 7, es be niosun7, *piny Arie yo sonimurnh oFsvy posavyostp pur ‘shup | Moy v UL yang voIY 9YI JOpuN powsoy Surjoms oFavp y ‘asvd osoaos y | (2) y KPa * 7, | 116 “unl i vdyuy | Bysreuy et Avou 7 | 6c (2) y 100Ndg * J, ) O161 “9Ddq “yy onuog] *punols dy} OF UMOP SOLUTE JUOG pYoY OY) YA o[o410 | [’us PUL punos pue punod Suryyem Ayjurjsuos pure ysox oF ofqeun | ‘197UM puL pooy Rursnyoa Spayriovia Ajourosyxo Apsoyqeryy “syyuoW z@ YLT perc » tondg “7, | 1161 “url e ee “OSLOSIP JFUDLINIAVIUL JO poly | (2) 4 XVOD * 7, se Ch pe | Ke 4 oa "7, | o161 “AON ks nioduny | ylinsorad a 10 ninupu "7, | o161 %4dog ie Upte | “RUT pao OU Sosrd PITA | PILDAODO , vain *Z, | o161 “ny LOT Buu | | SAVIO uOlLoyjuop| | owed WOT OF wm a | VRIADINE NUAMNLUOKN NI GaLOIVIOC) STIVININV OLLSHINOG HO SISVIINOSONVdAUL HO SUSVO FO SISHONAS 15 poeqafur 8td-roumny *OATZVU B 03 pfOS *q[nso1 dATIESOU UTM ‘soBreyostp jeseu pur yeurdiyor’y "CE “ON 99s Jng “8a[o1qsa] pue uawopaqe ‘s8aj ‘syurof yo ewaps0 poyswur {19 , “QATZVU BP OF PlOS (SYIUOUL IOJ IT, , squour Jo} TIT ; *stserumosourd 414 yo yov}9e snotAdid v wWOIy poIDAODAI pr}Y SYIVWIAYL *panutquoI——VYAADIN] NYAHLAON NI GAaALIATIO,) | *AIOJLIOGL'T YyIavasay UO SUNY IY, “YOopprig (2) Poet9AO0I IY (2) P2TIAOIOY ce ce Poe9AODOY (2) pad P2I9AOIIY BerG INsoy 199N4Q *Z, uinaooad 10 mnuvu *T, ninsorad. 10 mnuvu * fT, uinsorad. 10 wnuvu ‘Tf, | XDAt2 * J, xDAIA *T, 1a9NAq * I, XDAID *T, unsorad 10 mnuvu *T, unsorad 10 unuvu * fT, 4 xpate “7, 4 109n4q “1, 422949 * 5, |. «vate *7, | 4 299M49 +L UOlTLOYWUopy] ‘aq Aq suorjroyrquepy 4 zor “gay ce “ce ae z161 “urf | 1161 ‘d0q | | 1161 ‘Aon, | | zi6r “qoy | zi61 ‘sry ins iad 1161 ‘AON 21° qd “ ce ce ce a3 ce ce uc ce ce ‘as ce oe cc ce ce ‘a3 “cc oe niosunz ce ad ay ce ce ee ce vfoxory tT 1 ley d gt JsO}T “SIVWINY OLLSAWO(] JO SISYIINOSONVGANT, JO SUSVD 41O SISdGNAG DVI ‘ON yal ‘oO _ “syjuour @ ynoge II] “Auod Sunod Aran y *Aqueas AroA 199mg “7, “YAOI 19q0}9Q UO TI TJS *yuasoid suu0ydurds akg shep ez qT “pady asey pooyq s,Auod ur paqoajap DVIILYOIO YA] ‘shep I []] ‘juasaid surzoy avayonu s0119380g “juasaid ovriepyoso1ur ospy “uorjoofur Jayje Avp YI1I uo yQvap ‘skep g uoeqnoul Std-voums uy ‘uorsafur saqye Aep Y39z UO qaeap ‘sdepg UoryeqnouTyeI UY *7Y3Is vUTapao ‘yUasa.d Sud0} IvapNUIOIIaysog ‘skep Of T]~ “quasaad osyr aeLE[youNA, “1°34 “T ‘Iq “UONIDOzUT PITAL “RoOYIIeIp asnjorg “sauog jo Svq osaurt v Auod ay} Butavay AqI099r] Surpisqns ewapao payseur fraA ‘skep 61 yy “Zz ‘on se asioy aur *yuasoid dvIILTYOINIU OsTy syiewoy eS ee Ml a ee a “SIVWINY OILSAWO(] JO SISVINOSONVdAU,], AO SASVQ dO SISAONAS *PanttzuoI—VWASIN, NYGHLYON NI GaALOATIO’) pad pr19A0d0y (2) pad peroA0s0xf pd PrVAOIIY pd PpoA0doxy INsoy 1a9ndq * I, pue xvaia *7, xDO12 * 7, 1a9NAg *T, pure wnsorad 10 wnuvu *T, 199N4q * I, 199Ndq * J, mn.sorad 10 mnuvu *T, 1a9n4q * I, XDOIA * 7, xvA1e *T, pure wnsosad 10 wnuvu *T, XDQI2 *T, uiniorad 10 unuvu “7, uot LIHIVUepyT z161 ‘4dag ee oe oe z161 “3ny z161 ‘Aqnf zi6r ‘aunf z161 ‘judy zi61 “qay avd ce cc ce ce oe ee oe ee ae ce as10F] SOF] “c “ce uLO]] sie uyoT] "yO uL0]] ase uo} niasun7 | | | | | 90eld ib ob St bf £¢ zt 1 ‘ON ™ ac te +e wt ‘ynog Suryory, ‘uorqsafur soqye sAvp ZE paid ‘Sp ‘on AoyUop worz poqoofuy "S$ -3y ‘TIT ‘Id *eulapao ou JO ay ‘worsefur s9jye skep Fz potc] Sh ‘on Aayuop wosy poqooluy "syooa. g ynoqe IT “F “8y “IT ‘Id ‘Aep yags uo parp ‘sdep or uorQeqnour sid-voutns uy ‘dep yst uo aaye yas ‘skep or woryeqnour yr uy ‘syvom f noge ]]T ‘vuepr0 IySrg “quasar sursoy avapInU JOTIaIsOg = *E “BY “TI ‘Id *syoom £ qnoqr [IT ‘kep puch ypeap ‘skep £z-61 uoreqnour sid-raurns uy *syvoM 7 [I] ‘vuapso payseu TJaA, “Juasard sursof avapnu sotsaysog “Lb ON 10 S$ on Aayuop woz shxow0yg Aq Apoastp paqoayur Ayqissog = *z “BY ‘TF ‘Id syIvUloy *panutquor—WIAAOIN, NYAHLUON NI GaILoaAT1O() ac | ae peraqysnrys P2DA ODIO Pg ae peteA0I9y PKG poroAosoy | (a3 ue oe ce seal { WNsay 10 XVAIA "TZ, xvate * 7, xvA1a *T, mindsorad mnuvu *T, xvn1A *T, 109NLQ * T, NVA *T, Lapega *L 1IINAG * xDAI2 * a 1dINAG * 129NLQ “L 199NdQ * I, tang * T, | UOTeIYyTIUApT ee cc “ z161 ‘4das zi161 “3ny ZOMG) z161 ‘4dasg z161 “Sny z161 ‘390 oe at z161 ‘4das z161 “sny oe z161 ‘390 aq Inq warjng ce ce MOD TURIN] 184 JaeMGg ce ce MOD FILA] ce “e oe ae Aayuo ce ds1O0 FY Js0}] “SIVWINY SILSAINO(, 410 SISVINOSONVdAY [, JO SUS¥) AO SISHONAS ee ce ee oe ce (as ce e ee ce ce ue urz0}] 18req uy0]] +S €s vwl£b tr th ZV 18 ce “ce ce *(aqnoz eqqef) Aaqunos esneyy WoIF *(aqnor 181y¥q) vftopeyy w0Iy 4c *yuasoid surs0j avaponu ioTIaysog = *(29n01 eqqaf) “ce ce ce ce ce cc oe ee ‘oursy WOIF YING Suryo.1y, ae ae ce ‘skep OF [I] ce ce ce “ce ce ce ‘oury WOIZ YANO Zuryery, | SyIVUIOY * panutyuorI—* VINADIN) NUGHLUON NI aILIATIOZ+ "MOD UTIOT] ce ce ce ce ce ce ac oe ce oe ‘oury WO, YING Suryasy, ae oe ae “eo ry: cc e a3 ec ce a9 pereqysnrys porozysners aq 0} urpeqy 0} 3UaS oe ce ee ce 4c oe ce ce ce 66 pa1e7ysnrys yNsoy xva1a *T, xVAA2 * 7, xDAID * TZ, 1a9NAQ * J, xDQ1A * Tf, xDAIA "TZ, pure mnsosad 10 mnuvu *Y, xvA1a OS BOW xDa1a xDA10 xDQ1a xvatn xvata Bb WwW WB Ww xva10 xva1a bs i xpata xDa1a xva1D uoljzeoyTyUopy cc ce ce z161 “390 ce ae 73 oe oe 66 oe oe “6 73 ce z161 ‘das avg ce “ce ee 73 73 MOD TURN ce cs oe ve ce ce | [eq urn MOD TULTN ce 1Nq rarng MOD TURIN, 380; “SIVWINY SILSANOGd AO SISVINOSONVdAU J, JO SSVI) AO SISHONAS “cc ce ce ee ce oe ia ns “e ce ce cc “cs ce ee “ce wy0}] | | | | ” “ “ee "3203 UlIO]] ‘(aqnox eqqof) oury wrio1y YNOg Zuryary, *(aynor 1319eq) sur0q] ‘edepy wor “ce ce ‘OUR WOIF “YON UlOIt ce ce “ce “cc ce ce ce oe ce ‘ouryy WOIZ YINOS Buryary, a b1-£1 Adummys ‘Wf 1z suri0y SuoT --——— = = SyIVUIOY *panu1qu0I—VIXIOIN] NUIHLAON NI GILIATIOD ped ce iss “ce ce (73 cc oe ce ce ia9 “ce ce ce po1e7 Yysurys yNsoyy xDa10 * XDAL2L * xDQI1a * xData * ish lest ist lst tse NVA * mnaorad 10 wnuvu * TZ, xvate by XDQAI2 xvA10 XDA12 xDQ10 xpata BW KR WA BH WB WB OW XDA 1a9Ndg xa uorTyPoOytyuapy z161 ‘490 z161 ‘4dag ce z161 *490 ce ae “6 “e ce “ec z161 ‘4dag a1eq *"SIVIWINY OILSANO(] JO SISVINOSONVdAUT, JO SISVD &6 ce ce qwoy wey daays oe a3 wey daays ae ce wey daays ysO}] IO SISHONAS ac ee oe oe oe oe “ ae ce “ee “ce oe ce ce urtoyy aR ‘ON, 20 SUMMARY 1. Trypanosomes presenting the morphological characters of T. brucez, T. vivax, T. nanum or pecorum, and T. theilerz have been found in Northern Nigeria in the blood of domestic animals ; T. brucez in the horse, donkey, Fulani cattle, dwarf cattle and sheep; 7. vivax in the horse, donkey, Fulani cattle, dwarf cattle, sheep, goat and dog; 7. nanum or pecorum in the horse, Fulani cattle and sheep; and 7. ¢hezleri in the dwarf cattle. 2. ZT. vivax is the most common form at any rate in the south- western portions of the Protectorate, being present in fifty-five out of eighty-four cases collected in Ilorin province, the Niger province, and at Lokoja. 3. Of twenty Fulani cattle treking down towards the coast from the provinces of the north, and found to be suffering from trypanosomiasis, eighteen harboured 7. vzvax. 4. In horses 7. vivax produces a much less serious disease than T. brucez. Of fifteen cases infected with 7. vzvar, of which the records are complete, fourteen recovered. Of eleven cases infected with 7. drucez none recovered. 5. The dwarf breed of cattle found in certain tsetse-haunted districts of Northern Nigeria, and credited with a natural immunity to trypanosomiasis, while apparently recovering from infections with 7. vivax, succumb to T. bruce. REFERENCES 1. Sleeping Sickness Bureau Bulletin, Vol. 3, No. 31, p. 422, 1911. 2. Ibid. 3. Reports of the Sleeping Sickness Commission of the Royal Society, No. XI. 4. H. Ziemann. Centralbl. f. Bakter., 1, Orig., t. XX XVIII, 1905. 5. A. Laveran et F. Mesnit, Trypanosomes et Trypanosomiases, p. 552, 1912. 6. Sleeping Sickness Bureau Bulletin, Vol. 3, No. 31, p. 423, 1911. 7- Reports of the Sleeping Sickness Commission of the Royal Society, No. XI. 8. ‘A third report on experimental work on animal trypanosomiasis,’ by H. Andrew Foy, D.P.H., in The Journal of Tropical Medicine and Hygiene, October 16th, 1911. a Paral 40 ae IES = of) gerolt stor Aur. i | ~~ ¥ 5 bd pti a at rh) 22 EXPLANATION GPPLATE @ Fig. 1. Horse (No. 35). Trypanosomiasis (7. b7ucez). Ilorin, July, 1912. Fig. 2. Horse (No. 42). Trypanosomiasis (TZ. d7ucez). Ilorin, October, 1912. il ATAMd JO worn AV ATS F a i - 77S nino 1 Ht bond NS dlaktonoe meqy? | t olf} yolnod 3 1 ¥- 2 .* . ‘ ; . | | = aa | _ JesguA wHiTOK.. .Garwd . Vp alasuttoeonngy | (te 0%) yatta, 2 spi hor sdesiqee 24 EXPLANADION, OF PLATE 1 Fig. 3. Donkey (No. 44). Trypanosomiasis (Z. d7ucez). lorin, August, IQ12. Fig. 4. Donkey (No. 45). ‘Irypanosomiasis (7. 47ucez). Ilorin, September, 1912. pe! Per BANOS. hi a “ OL EN i. oe _ | Tae o> AND i, = is ne Fag ANS ia ms ; , Z a Es : | . _ — = ‘e 7 : “Fi ? € rete od eames Ps | aa Pe yang ot “s ae imc | Soishl pater Naty. : + ot, ee x | cpacinsyn' ct Saal ade asec Cir reuslne ci iecty eee othe ae = hy = af . ee oe. + : it Fase fie fy pau ™24n exciew ae ite’. i tat BPP : tite asl ae ete : ee al toss: «| cide the daiaes: Sau Gee sible” Soe ot Way: ih: STAID 40 “OTS MAN AA or prey . 2 ot ‘ =f \erel (~ L 2 2 P spelen INR i Eile i es e745. . “T= ud ‘ eS « > Te Ebjenas. (0.8 = i= We pee ke, fey t- ie FTrare’ Ulemis Peete»: = is = 4 ; i fies bey abiected Tan <> f Ales; *Yinl ge ew as cs ’ ve t Pi shake oa | ac aghsite> > ie aie! y* ae or aryet) 2 or a af a te i | SRNR RREENES aie Srypasecoret by acai OF fe 2 Pa am eueiiins,) Ve te rS.e : ris. 104 . FE hapeinws ao Greil se . ‘ sea S ’ : . . = phate, | Wistedie | ni Moai i ; > Severs Py sti = : = SAT : : arly eoithate a ; = at'g ; = ; ; fete.) -% -. 43 co HAAS : : en 4 : ' eprwert r , [a $ z Me prensbicorne r «ke eT : “ ~~ c ren eee: J _ z ere grpersiety 1- = Ae Sy : riod ae" > ee “Hest: 4 ep -1hv (unesner ; 2h ee : ar E : = = pel Bhed. voalee? Bint. earare? 5 : : =a == = 77 Saas We shall sybeaquentiy anak rat poise of T = aeetg pects of fagures | et a i 26 -EXPLANATION OF PLATE III Fig. 5. Dwarf cattle. Ilorin, May, 1912. 27 FURTHER MEASUREMENTS OF TRYPANOSOMA RHODESIENSE ANB TGA MBIENSE BY Je We W- STEPHENS, “M.D; *CANTAB:,” DvP: A. AND H. B. FANTHAM, D.Sc. LOND., B.A. CANTAB. (Received for publication 21 December, 1912) In our former paper (May, 1912) on this subject, we came to the conclusion that it was advisable in measuring trypanosomes to confine our observations to those from a single animal, for example, a rat, as although it could not be definitely proved that the size of the trypanosome varied in different animals, yet it appeared likely from the general consensus of opinion that this might be so. From the statistical side the criticism has also been brought that samples of twenty at any particular time are too small. In our present series of measurements therefore, we have, as far as possible, met these objections by measuring always from a single animal, a rat, and by measuring one hundred trypanosomes each day for the first ten days of the infection. We may here briefly repeat our method, as it has been subjected to some criticism. 1. We project the trypanosomes on a screen in a dark room and trace them, instead of drawing them with a camera lucida. It has been objected that this method cannot be used in the wilds of Africa, but we never stated that it could or should, and that is no reason why we should not use it in a laboratory. Our critics might as well object to our using electric ght. 2. We measure the trypanosomes by means of the ‘tangent line’ method. We believe that as this method is the most accurate known—our critics have not attempted to deny this—we are again amply justified in preferring it to the less accurate compass method, even though the difference may be only I or 2. It seems to us hardly a matter for argument that if it is worth measuring 1,000 trypanosomes at all, it is worth doing so accurately, especially if length is to be considered a criterion of specificity. We believe that it is important to give the actual data as to measurements as Bruce does, and not simply averages, as the actual data are necessary for a closer analysis than averages permit. We give first, then, the fundamental data for each trypanosome (TL. rhodesiense and T. gambiense) and the tables compiled from them. We shall subsequently make a comparative analysis of the two sets of figures. 28 |] | |] | |) — | | J} -— ]} — — —_] —_—] —— gt SL.bz 20 3 lise Ad ; Elie ie feel erion| lee ly laely le dn la |e ie. ase fer fer fer fof feet tebe fs le fe fe Geeta wien ltrscliera (ps dhe Flan Pieseal ime sil goice A ash (ne aioe | eeeliolegs pe Blanton res vcd Be eee Tioe rll em ea) tere agp le eeaal ae Hey AL. -e. [Rp Me Venn dees | Ge Le Mllinee Wipe ore otal ee eng Glee P| oe | el eicemte| taetdbe-ael wey (\- fla da fe lemme tailed ar foe foe fee fester fer forfee fe fete [2 [s [2 le |e fe fella fw le le fo | aro deg ipat - ee Ee le ae i Me eels ie Fa | 2 | eee lomee || les | e- | i | on Zoe | [ee fee feet [oe (ar Vr fap cle [a (g lm [a fa font eo | oe [ite [on [oe [oe P16 $9.97 $6.92 eden gecMh ph cee lee Hebe pel ce lu. \ep | peers aT Ae Be: a 8 Po istoe | Mae tlie leclea eT Te See pe ge ee, |) seated rt] eeerliay eee Nal Z L.9z Pct ee es Pie (ae IS IAG Pe Peed Gal oF A RIE ae i EE Oe ee ea 9 ° Avq puz we Tee ea ETE TS Ta Te ye la fe fe fe de fede fete |= lo ar J [mag ti) Movll Pom floss [eerec|fetalices [once fube lox EP Eenl ge) Be a|Eb | eerie altdosd| oneal lave eseacdaeaalctealape ear gi-bz 3 Sz.zz ote Wee Sheol galt eda) deel |g. ieee [epee Peogtee arn tlesalaee exe: re £ Bie ee ie Re Sle lice Weps (eel mealies Me ine debe ce | a * - ~~ a > as £ G3 cy charset Es a = 72 _amentensht 7 ” nf = * iv 3 1 ‘ é 4 Py J Sis a - a _) = i <5 aa 4 r : 7 _ 7 — e - _ + -e ¥ mo: a, 41 A NEW MOSQUITO-PROOF AND STORM- PROOF HOUSE FOR THE TROPICS BY T; F.--G.— MAYER, WEST AFRICAN MEDICAL STAFF (Received for publication 16 January, 1913) PLATE IV There are all kinds of houses in the tropics. There is the old house built on enormous piles so that its occupants may be well away from the damp of the ground; there is the house built on the ground; and there is the house that is raised on short pillars only three or four feet high. Provision for ventilation is equally varied. In olden days the houses were designed in England, and the window area had the same relation to the floor area as obtains here; then the window area was increased to one-tenth that of the floor area; this proportion was again gradually increased, until now in some places a window area equal to one-half of the floor area is regarded as the minimum necessary for comfort. The discovery of the transmission of malarial fever by mosquitos was followed by the grafting on of mosquito-proof wire gauze to the windows and doors of existing houses, with the result that in those in which the ventilation space was small, the rooms were made insufferably hot and stuffy; consequently, the mosquito- proofed window was made to open and shut and the house was turned into a mosquito trap. With the closing in of the house by mosquito-proofing came the growth of the verandah and the diminution in size of the rooms, the verandah now being used as a living room except during cold or stormy weather. From these few remarks it is clear that, even taking into account the demands of local conditions, house construction in many parts of the tropics is not based on such fixed principles to guide architects as obtain, for instance, in England. 42 The importance of efficient mosquito protection is well known. Its value as compared with quinine prophylaxis against malaria may be gauged from the following figures of Celli :— Treatment Infected with Malaria Mosquito protection plus quinine ... Sor oes aide 175% Mosquito protection alone... ob EAD a a 2 Ee Quinine alone ... si cs aoe “ac ee ee 20:0) 9, No protection ... Sct ans 50 ae ee oon aR The difficulty of completely protecting a house raised on pillars from mosquitos may also be noted. Faults in the flooring nearly always exist which allow of the entry of mosquitos. Dr. Leonard Hill states that to render a room uncomfortably hot and stuffy, the air in it must be hot, damp and still. In many parts of the tropics the first two of these conditions are almost constant, the last only therefore concerns us here. Three factors tend to keep still the air enclosed in a mosquito- proofed house. 1. The mosquito proofing itself, by shutting off more than 30 % of the space to which it is applied. 2. The loss of air movement owing to friction with the wires and to the interference with the air particles which impinge on the netting. 3. The division of the house by walls into rooms and passages hindering through ventilation. Factors 1 and 2 should strictly be considered together, as a certain amount of the air impinging on the wires will ultimately pass through the netting. But without attempting the general consideration of the motion of the air, it is clear that for a given uncovered area the loss of movement will be greater for a small mesh than for a large one, on account of the greater length of edge of wire. If the uncovered area be equal to an open square of side a and consist of z small squares each way, then, as each side of a wire has its effect on the air movement, the total length of edge is 2? X 4 | = 4an. 43 Messrs. Humphreys, Ltd., of Knightsbridge—the makers of the first mosquito-proofed house used in Sir Patrick Manson’s classical experiment—have designed for me a house for use in the tropics embodying certain principles which I believe are new. The house here illustrated (Plate IV) is constructed almost entirely of steel; wood-work is reduced to a minimum, and, where its use is unavoidable, is specially treated to withstand the attacks of insects, especially white ants. The house is built on a plinth of concrete and has a floor and a low wall of similar material. Their surfaces are cement-rendered. The walls are continuous with a steel framing, filled in completely with mosquito netting made of a specially woven composite materia] proved to be stronger and more durable than any other. This netting is sandwiched between perforated metal sheets, which prevent bulging and render entry impossible except by the spring doors at each end of the lobby-entrances. The roof is covered with non-conducting fibro-cement slates and is ventilated by means of louvre windows at each end. It is completely shut off from the room below by an asbestos ceiling. There are no walls or partitions within the mosquito proofing, so that whatever breeze there may be, blows straight through the house from one side to the other. Should this wind be too cold or too strong, as for instance during storms, it can be cut off at will, by shutters which are provided all round the house and which are capable of closing the whole of it. These are manipulated from within by turning a handle, and can be made either to come down from above, as illustrated, or to move upwards from below. The division of the interior of the house is left to the occupant, the ideal being the minimum required for privacy, and it is suggested that by the use of sun blinds, screens and curtains, the open character of the house may be preserved, but permanent brick or stone walls may be built if desired. It is possible to make the house of any number of stories, and to build it on piles if desired. In the house we have been considering that the terms wall area and available ventilation space are almost synonymous; it must not be forgotten, however, that the proportion between wall space and cubic content vary greatly according to the size and shape of the building. 44 In buildings of the same shape the cubic content varies as the square of the wall area, provided that the height is constant; while a square house has a greater cubic content for a given wall area than an oblong one. In conclusion I have to thank Sir Ronald Ross and Professor Simpson for their interest, and for many useful criticisms and suggestions. I have also to thank Messrs. Humphreys, Ltd., not only for the amount of work they have done, but also for their courtesy. . | . 45 CONTRIBUTIONS TO THE STUDY OF COLOUR MARKING AND OTHER VARI- ABLE CHARACTERS OFANOPHELINAE WITH “SPECIAL REFERENCE TO THE SYSTEMATIC AND PHYLOGENETIC GROUPING OF SPECIES BY Major S. R. CHRISTOPHERS, I.M.S. (Recetved for publication 30 January, 1913) PLATES V-VIII CONTENTS PAGE I. Vartation ty Cotrour Marxine or Anopbelinae os oct se ae SG II. ORNAMENTATION OF THE WINGS IN Anopbhelinae 52 Nodal Leucogenetic Centres of the Wing ... a 5 Internodal Leucogenetic Centres of the Wing a at - aon) (G3 Variation in Colour Marking shown by Individuals of the same a 65 III. VariaTIoN IN STRUCTURE OTHER THAN THAT CONNECTED WITH SCALES ae aay TV. SrrucruraL VARIATION IN THE IMMATURE STAGES... Bee — “ec SME V. Variation as DisPLayeD By SCALES... ah 24 a a Fe say 5 VI. CtassiricaTion, PHyLoGeny aND GroGRaPHICAL DisTRIBUTION OF THE Anophelinae 81 VII. Gernerat CONCLUSIONS ... “i =2: aa: se ee sr a Bree VIII. Tasurar SUMMARY 28 = a aoe is oi: oer see sang 87) REFERENCES 93 EXPLANATION OF PLATES ... I. VARIATION IN COLOUR MARKING OF ANOPHELINAE Nature of colour marking. With few exceptions, the markings of Anophelines are due to the varied distribution of light and dark scales. A mixture of light and dark scales may occur, e.g., on the wing veins of certain species or on the legs when these show mottling ; but the most striking feature of the ornamentation of the sub-family is the occurrence of patches of either dark or light scales forming dark or light spots respectively. Where the dark scales predominate the appearance is that of light spots on a dark surface; where, on the other hand, the extent of light scaling is greater, an appearance of dark spots on a light ground is produced. 46 Leucogenetic centres and residual pigment areas. In a great many species the scheme of coloration is similar, the actual markings being brought about merely by different degrees of development of the scheme. The process of advancing ornamentation is one of increased whitening, and consists in the progressive appearance and spread of pale areas which have their origin at certain fixed points (leucogenetic centres). Although the pale areas developing from these centres may spread until they coalesce with neighbouring pale areas, they do not, as a rule, do this, and there is a distinct hesitation shown at the final extinction of intervening dark areas. Also extension of the pale areas does not necessarily encroach equally on both sides of a dark area, and the behaviour of markings in this respect suggests that there exist something very like actual pigment centres having a definitely fixed position. Points at which the pigment seems thus to make a last stand will be referred to as veszdual pigment areas, or simply as pigment centres. In some cases centres become quite obliterated, some being apparently less resistant than others, but even in a high degree of whitening, such as frequently occurs in the case of the wing, where many of the pigment areas are displayed as mere points, the number actually present is not much reduced. A row of these pigment centres lying near the ends of the longitudinal veins of the wing constitute the marginal spots of Donitz, and others have an equally fixed and characteristic nature. By referring markings to the leucogenetic centres which produce them (and so far as one can see, these centres of whitening are a very real phenomenon), and to the position of the pigment centres, the study of colour markings is greatly simplified. Position of colour markings. The most important colour markings are those of the palps, legs and wings. There are, of course, variations in the coloration of other parts of the body which may assume importance in particular species. Almost all Anophelines have a head covered with dark scales behind, and with a ‘ forehead’ patch of pale white or creamy scales prolonged forward as a tuft between the eyes. The absence of this marking would be an important feature in any species. In the case of the proboscis, only a few species show any ornamentation other than the light tip due to the fact that the 47 labellae are not dark scaled like the rest of the proboscis. But a very striking variation is seen in a few species where the outer portion of the proboscis is pale or snowy white. Markings of the palps. A certain number of species show palps without any markings (unbanded palps). In some cases there is a faint indication of bands, due very often to the chitin showing at the joints, but there is no actual pale ornamentation. This is the condition which is sometimes referred to as palpal banding in A. nigripes, Staeger. A. immaculatus, Theo., also stated to have banded palps, shows very indefinite and indistinct ones, not at all sharply marked as in ordinary Anophelines. Entirely unornamented palps are present in all Stethomyzas, including S. corethroides, Theo. (vide section on scale structure), and S. aitkeni, James and Liston. Unbanded palps are also commonly present in Auopheles, and occur in Myzorhynchus and Cycloleppteron. Otherwise the palps are usually ornamented. In Anopheles the unspotted wing species also have unbanded palps (except A. (?) smztht, Theo.*). The species A. gzgas, Giles, and A. simlensis, James, with banded palps have been separated by James! as Patagiamyia. A. formosus, Ludlow, since it is a hill species whose description closely resembles that of gzgas, would seem also to be a Patagiamyia. A. punctipennis, Say., which is a Patagiamyia (vide section on scale structure), has banded palps. It would be very interesting to know what was the condition of the prothoracic lobes in A. ezsenz, A. crucians, etc. A. welcomei, Theo., is a peculiar species; it has no prothoracic tuft, but I am doubtful if it should be considered as an Anopheles. When the palps are ornamented, this is in the form of bands, supplemented in some cases by extra spots of white (speckling) on certain segments. It is usual to talk of four or three-banded palps, etc., some of the bands being described as broad or narrow, as the case may be. The only accurate method is to refer to the position of the bands in respect to the palpal segments. This relation is most easily expressed in terms of leucogenetic centres. In spite of considerable variety of appearance, a comparatively few general schemes of coloration associated with different relative lengths of the palpal _ * Notr.—This species has a scale tuft on the prothorax and is therefore not an Anopheles in the sense of James. 48 segments (vide section on structural variation) can be made to account for the markings. SCHEME A. Palps with leucogenetic centres at each end of the segments, but none of these actively spreading. The effect of the appearance of the leucogenetic areas (following upon complete absence of banding) in this scheme is the production of four narrow pale bands, including the pale apex. If the species be orthodactyloust, a somewhat different appearance is given to that produced in the case of a markedly heterodactylous* species (compare C. pulcherrima with the variety of P. nursez showing a dark apical band. Plate V, figs. 8 and 10). This degree of extension of whitening is not exceeded in Myzorhynchus, and is typical of palpal ornamentation seen in Myzorhynchus, Cycloleppteron, Arribalzagia, Patagiamyia and Cellza. It seems to be a stable condition, and is but little liable to variation in individuals belonging to these groups. In Myzomyza, Pyretophorus and Nyssorhynchus this stage is unusual, and when it does occur it is most often as an individual variation, e.g., the four-banded palp varieties of N. fulzgznosus, Ne. fowlert and P. nurset. A permanent four-banded condition in this group is seen in P. cinereus, Theo. SCHEME B. Leucogenetic centres at the ends of the segments, with that at the end of the apical segment active. In this scheme the apical centre extends, making the apical segment entirely pale. This results in the ordinary type of three- banded palp. Considerable difference in general appearance again depends on whether the palp is orthodactylous or heterodactylous (Plate V, figs. 11-13). The narrow apical band in Myzomyzat (culicefacies, funestus, etc.) is homologous in regard to extent of palp involved with the broad apical band of Pm. vossz, but not with the narrow apical band in Pt. szmlensis or C. pulcherrima. The broad band in * Vide Section III. Tt Nore.—As pointed out by Edwards®, Nyssorbynchus, Blanchard (= Laverania, Theo.) having as type species argyrotarsis, Rob-Desy., must take precedence over Cellia, Theo., a new name being required for the group generally known as Vyssorbynchus. Again rossi, Giles, is the type species of Myzomyza, Blanchard (= Grassia, Theo.) so that the name Pseudomyzomyia, Theo., is incorrect, whilst a new name is required for what is now generally known as Myzomyia. In the present paper, however, the names in general use by Theobald are retained as being less confusing than new ones or old ones applied in a new sense. 49 Pm. rossi again is not comparable with the broad apical band in N. maculatus. (Plate V, fig. 14.) Certain species, as previously mentioned, which normally or sometimes exhibit this condition (i.e., whitening of the whole apical segment) may at other times show a black band, due to incomplete extension of the apical pale area. The liability to this variation is not merely accidental. In the case of Nyssorhynchus, those species liable to the variation are distinctly related and exhibit affinities to Cellza. In Myzomyza incomplete extension of the apical white area is rare. It is more common in the heterodactylous Pyretophorus, e.g., P. cinereus, as a variation in P. nursez, etc. A few species, all apparently related, show a peculiar variant of palpal banding, due to the absence of development of the apical pale area. These dark-tipped species which show three bands are M. turkhudz, Liston, M. hispaniola, Theo., P. myzomifacies, Theo., and P. chaudoyer, Theo. In M. turkhudi this character is liable to some variation, the dark tip not being always very clearly shown. SCHEME C. So far, the only leucogenetic centre that has shown active spreading properties is that at the apex of the terminal segment. Another type of palp is seen in which the centres at both ends of the penultimate segment and at the apex of the second segment also become active. In this case whitening spreads up and down the shafts of the segments to produce the appearance seen in N. maculatus (Plate V, fig. 14). Here the residual pigment area is displayed as occupying almost the exact middle of the segment. The apical white band in such a case is not comparable with what looks like a very similar band in the case of Pm. rossz, etc. Variation here usually consists in a partial, or more rarely in a complete effacement of the pigment centre on the penultimate segment. SCHEME D. What seems to be essentially a distinct scheme of ornamentation to any of the above, is one which may be described as showing in each segment only one apically situated leucogenetic centre and a basally situated residual pigment area. Species showing this type of palpal ornamentation are chiefly included under the many-spotted-winged species of Donitz, 1.e., M. punctulata, Nm. elegans, N. annulifes, etc. 50 The scheme is beautifully demonstrated in Ch. kochz, Donitz, and it is apparently that on which the palpal ornamentation of N. karwari, James and Liston, is based (vide Plate V, fig. 15). The condition is very distinct from that ordinarily seen in Nyssor- hynchus. Possibly other variations or schemes of palpal ornamentation exist, but enough has been said to show the nature of the condition present in the great majority of species. Markings of the legs. |The occurrence of pale areas at the terminations of the femur, tibia and first tarsus is usual, and such may be conveniently termed ‘kuee spots. Except as an unusual variation, knee spots appear to be absent only in Ste¢homyza. The occurrence of pale areas at the termination of the tarsal segments gives rise to an appearance which may be called /arsal banding. Tarsal banding may be of several kinds, depending upon whether the banding is a mere trace not very definite and confined to the actual joint, or whether it is broad and distinct involving an appreciable extent of the ends of the segments. In some cases, though banding is narrow, it is (as in P. jeyporensis) very distinct. In the case of broad banding, there are again two conditions depending upon whether the light area is merely somewhat paler than the rest of the segment or is pure white. The latter condition is, of course, much more conspicuous. In some species the tips of the hind legs (and very rarely those of other legs) are white. This may be a condition in which there is a large continuous white area (as in NV. fulzginosus, My. paludis, etc.), or one in which the whole tarsus shows alternate dark and light bands, one of which includes the tip. In the former case it will be found that two or three segments are completely devoid of any dark scales. In the latter case, though the terminal segment may be all white, the other segments still have some portion of their extent dark. In some cases, though the tip is white, not even one segment is altogether white, the terminal portion of white consisting in this case, as a rule, of a half or less, of the last tarsal segment. In separation of species the exact details regarding the number of segments white is important, but in the present paper, dealing somewhat broadly with the subject, I shall refer to two conditions only, i.e., (@) a continuous area of white involving at 51 least two segments; (4) an interrupted black and white appearance of the tarsus in which the tip is white (i.e., as in N. maculatus, Theo.). In the case of the tarsus of certain Culicinae there may be.a development of pale areas at both ends of the segment or at one or other end only, so that there results apical and basal, apical, or basal banding as the case may be. Seeing how important the coloration scheme appears to be, such distinctions may have a good deal more systematic importance than one might be ready to grant at first sight. So far I have not sufficiently studied the nature of broad tarsal banding, and for the present shall not lay any stress on differences in this respect. In many species, however, the banding is purely apical, whilst in Pm. vossi, for example, it is distinctly apical and basal. The condition called speckling is one in which distinct spots or bands are produced on the tibia or femur. Under an objective a certain amount of admixture of dark and light scales is often seen, and this may lead to a ‘mottled’ appearance under a lens. This is quite distinct from the defined spots and bands referred to as speckling. Speckling signifies a high degree of ornamentation and is usually associated with tarsal banding and, very frequently, with a white tipped tarsus. An absence of speckling is characteristic of Myzomyza and of the saperpictus group of Pyretophorus. Speckled species of Pyretophorus seem to be in many ways distinct from the latter group. Note, for example, P. avdensis, Theo., with ‘furry’ palps resembling those of a Nyssorhynchus, and the highly ornamented P. aureosquamiger, Theo. Vzde also remarks on the scale structure and palpal characters of P. costalis. In Nyssorhynchus speckling is the rule, and reaches a high degree of perfection. In Axopheles, Myzorhynchus and Patagiamyta, speckling is absent, but it is developed in Cycloleppteron, evidently a specialised but closely related genus to Myzorhynchus. Ornamentation of the wing in Anopheles. The study of wing markings is complicated by the number of veins which may bear spots, the fact that the arrangement of spots on each vein may vary and that combinations are possible. 52 II. ORNAMENTATION OF THE WING IN ANOPHELINAE As is well known, only the veins in mosquitos carry scales, and any spots that may be present are, therefore, restricted to these structures, and are linear in shape. In this respect the spots on Anopheles differ from those in butterflies, not only in being linear, but in the fact that the spots in butterflies, as has been shown by Mayer,? originate on the wing between the veins, and only involve the veins by extension. In the Nematocera two types of spots are seen (a) dark spots situated around vein junctions, and (6) dark wavy bands with serpiginous edges which extend across the wing. These often look as if their wavy margins had been caused by encroachment of pale areas developed between the veins. The former type of spots are constantly encountered. On page 92, for example, of Theobald’s Monograph, Vol. I, is figured a wing of Rhyphus in which dark patches are seen (1) at the junction of the subcosta with the costa, (2) at the junction of the first and upper branch of the second with the wing edge, (3) at the termination of the lower branch of the second, (4) at the cross vein linking the second with the next vein, and at two other junctions. The most striking development of pale spots forming between the veins, and dark spots in connection with vein junctions I have seen has been in the case of certain species of spotted-winged Chirvonomus. In these species both wing membrane and vein is covered with minute hairs not unlike scales, and so the condition approaches that seen in Anophelines, whilst it seems to show what the primitive condition may have been. In these wings the two conditions seen are (1) aggregations of the hairs (or scales) in dense patches in the neighbourhood of the main cross veins and at two areas which seem to represent the bifurcation of the second and fourth longitudinal veins, and (2) distinct pale eye-like spots which occur on the wing membrane and have the minute scales covering them also pale (Plate VI, fig. 4). In one species examined there was a complete series of such spots occupying the cells and ranging along the whole outer and posterior aspect of the wing. I have described these conditions at some length because 53 I believe they help one to understand certain of the facts to be described in the case of Anophelines, and because they seem to show so distinctly the primitive character of scale aggregation at the cross veins, and the actual existence of something very like my leucogenetic centres. Primitive spotting in Anophelinae. The spotting seen on the wings of Anopheles is of two kinds: (2) Spots due to special aggregations of scales; (6) Spots due to alternate areas of light and dark scales. The first type of spotting is very familiar in the case of the wings of A. maculipennis, Meigen. In this species no pale scales are present on the wing veins, but there are aggregations of scales forming dark spots. Such aggregations occur at the origin of the second longitudinal, at the origin of the third longitudinal, to a less extent on the neighbouring parts of the fourth and fifth veins (1.e., in the neighbourhood of the cross veins), and at the bifurcation of the second and fourth veins. This type of spotting is conspicuous in A. maculipennis, because the wings are devoid of other markings; but it is by no means confined to this species or to species with unspotted wings, and it occurs through a wide series of Anophelines, often along with considerable ornamentation of the second type of spotting. As it is seen chiefly (if not entirely) in what I shall show reason to believe are certain primitive groups, and as it is quite distinct from the spotting due to pale and dark scales, and as something very like it occurs in other groups than the Culicidae, I have called this przmztzve spotting. All species of Myzorhynchus which I have examined show it, also Cycloleppteron grabhamiu, Theo. It is very plainly to be seen in Pt. lindesayi, Giles, and is prominent in L. aszatica, Leicester. The species P. atratzpes, Skuse, has an unspotted costa, its palps are unbanded, and it shows very marked and prominent primitive spotting. It clearly cannot be a Pyretophorus, and the certainty with which one can speak is a good example of the value of colour markings in systematic work upon the Anophelinae. Scale clusters. In Myzorhynchus and Cycloleppteron there is a condition allied to this primitive spotting, in which dark spots on the veins are formed by scales larger than those on the light 54 areas. Such areas of large scales are seen, especially on the sixth vein, forming the two dark spots characteristic of these genera. Representation of spots on the lower surface of the wing. The veins of Anophelines carry scales on both surfaces. The character of the scales carried on each depends, however, on the particular vein concerned. In all Anophelines that I have examined, the upper surface of the first, third, fifth and sixth longitudinal veins carries blunt appressed, and what may be described as fla¢ scales. On the under surface, all these veins (where they have scales at all) carry quite a different kind of scale, which in most Anophelines 1s pointed, and projects freely from the vein. The arrangement of scales carried by the second and fourth vein is reversed, flat scales being present on the under and projecting ones on the upper surface. What have been called lateral wing scales are, for the most part, simply the projecting scales on one or other aspect of the vein, as the case may be. As the end of the wing is approached, 1.e., about the level of the forks of the second and fourth, the flat scales become increasingly long and projecting, so that in this position both surfaces of the wing carry projecting scales. This accounts for the appearance of the spots in this region which have a blurred look. Except for a patch of scales towards its free end, or at most, over half its length, the sixth vein is devoid of scales on the lower surface of the wing. The main fifth and the inner part of the branch are similarly bare underneath. The result is that spots on these veins are very distinct and, under an objective, appear to be formed of special small flattened scales. The remaining veins carry scales on both surfaces. On the whole, the upper surface of the wing is the more heavily scaled, and partly on this account, and partly owing to the preponderance of flat scales, the colour markings are, as a rule, more intensely represented on the upper than on the under surface. In most Anophelines, allowing for the somewhat more intense coloration of the upper surface and the scale characters noted, the spots on the upper and under surface correspond. But in certain groups there is a marked lack of pale areas on the under surface corresponding with those on the upper, which may reach such an extent that the wzder surface of the wing -is practically dark scaled 55 throughout. S Myzxorbynchus sinensis ... ae Sat B oA barbirostris ab ae B 4 paludis ... a pit B Cycloleppteron grabhamit ? ae S Chagasia fajardot ae + Myzxorbynchus (?) natalensis O ob b Neomyzomyia elegans O ae S Cellia squamosa var. arnoldi O SUSE bb ss) Squamosa ... O ie bb ,, albimana O ++ » albipes O ge ak b 35 pulcherrima O + bb Pyretophorus ardensts O abs Myxorbynchella nigra at re parva ... fe Nyssorbynchus fuliginosus O + bb 5 N1VIPeS ... O = bb a jamest oO + b ie maculipalpis oO te b “4 pretoriensis O S - maculatus O +5 S 4. theobaldi Oo at S % karwart O * willmori O + S Christophersta kocht Oo + S Myzxomyia culictfactes ... O 4 S a listont O =F S of funesta O + S ‘ turkhudt O a S 55 albirostris O b Pyretophorus cinereus O + S FF nurset 306 O + Ss Pseudomyzomyta rosst ... O + S + ludlowt O o S Pyretophorus jeyporensis O a b costalis O + S Cellia jacobi O + ) 75 belonging to the genera Myzomyia, Nyssorhynchus, etc., those ot the former being more triangular in shape and less scoup-like than those of the latter. (Vzde Plate V, figs. 1 and 2.) V. VARIATION AS DISPLAYED BY SCALES A detailed account of scale structure would be foreign to the purpose of this paper. But it is necessary in connection with other variable features to summarise briefly the main facts regarding scale variations in Anophelines. This is the more necessary in that it seems to be taken for granted that the only possible classification of the Anophelinae based on scales must be that actually employed by Theobald. That this is a very limited view to take must be granted by all who have paid close attention to scale characters. It may be found that other scale characters not perhaps so conspicuous as those so far employed may be of much greater use than has been suspected. It will be useful therefore, even if the subject is only rapidly passed in review, to indicate the broader principles of scale structure variation. In doing so I shall take the opportunity of making certain revisions in regard to the position certain species now occupy, positions which it would be unfair later on to consider as showing a discrepancy between the results of scale character and colour marking classifications. Head Scales. Marked variations in the head scaling are restricted to a very few groups. The occurrence of linear head scales has been used by James! to differentiate the genus Neoste- thopheles (type N. aitkeniz, James). This genus is clearly the same as Theobald’s Stethomyza (type S. nzmba, Theo.), in which the head scales are also linear. An examination of specimens of A. corethroides, Theo., in the British Museum shows that this unspotted winged species from Australia has linear head scales, and should come in the genus Stethomyza and not in Anopheles (vide also palpal characters). A. zmmaculatus, Theo., and A. smithi, Theo., have head scales of the ordinary expanding type. Scaling of the palps. In some species the scales over most of the palps are of small size and relatively appressed. In such 76 species the palp has a smooth thin appearance. In others the scales are long and outstanding, giving to the palp a more or less shaggy look. Difference in this respect, though not used in the definition of generic groups, is by no means unimportant. One would view with suspicion any supposed Cedlza having smooth thin palps, or a supposed Myzomyza with shaggy palps. Pm. rossz, Giles, a species with shaggy palps until recently termed a Myzomyia, is now known not to conform in scale characters with the definition of this genus; presumably the related species M. indefinata, Ludlow, M. ludlowi, Theo., M. mangyana, Banks, are also rightly excluded. The species M. lutzi, Theo., at present the only South American representative of Myzomyza, has shaggy palps with a quite different type of ornamentation to that seen in any other Myzomyza, and the correctness of the present position of this species must be considered as problematical. Between what may be called the sawperfectus group of Pyre- tophorus, which has very long thin palps, and P. costalzs, Loew., there is also a very marked difference in this respect, which is accompanied by marked structural as well as important scale distinctions. Antennal Scales. One of the characters of the genus Calvertina (C. lineata, Ludlow) is the presence of outstanding scales on the second segment of the antenna. I have not seen a specimen, but otherwise the scale characters from the description seem very like those of Nyssorhynchus. The markings also read very like those of N. fuliginosus, James and Liston. Prothoracic Lobes. James (1) has called attention to the importance of the fatagza or prothoracic lobes from the point of view of classification, and his separation of Patagzamyia (type Pt. gigas, Giles) from Anopheles is based on the presence in the former of a well-marked tuft of outstanding scales on these organs. Using this feature one can distinguish as Patagzamyztas, in addition to those noted as belonging to the genus by James, A. punctipennis, Say., and A. smithz, Theo., both of which have prothoracic tufts. Judging from the light thrown by colour markings, a tuft of scales on the prothorax is an extremely important character. Unfortunately it is only in a proportion’ of species that the 77 description given notes the presence or absence of this character. A prothoracic tuft appears to be of general occurrence in Myzo- rhynchus (barbirostris, umbrosus, sinensis, mauritianus), there is a tuft in Cycloleppteron grabhamit, in Arribalzagia maculipes and in Myzorhynchella nigra. The tuft characteristic of Patagzamyza is present also in Lophoschelomyia asiatica, Leicester. Myzorhynchus wellingtonianus, Alcock, and Feltinella pallidopalpi, Theo., all of which species would appear from their colour markings to be related to Pt. lindesayz, Giles. A tuft is present in CAristophersia kochi = (Cellia kochi, Donitz.), Neomyzomya elegans, James, Cellia squamosa, Theo., and curiously enough in P. costéalzs, Loew. On the other hand, a large series of species do not show this structure. At present, information is too scanty to enable any hard and fast use to be made of the prothoracic scaling in classification, but the table accompanying this section indicates approximately and very tentatively the possible significance of the occurrence of a tuft in this situation. Mesothoracic Scaling. ‘The presence or absence of broad scales on the thorax has been used along with other characters to differentiate a number of genera. Restricting observations to certain groups, the presence or absence of broad mesothoracic scales seems to serve the purpose of defining genera fairly well. By its aid Myzomyza is fairly clearly marked off from Pyretophorus, etc., and if the shape and character of the scales is also taken into account, this latter genus can be quite well differentiated from Nyssorhynchus. Extending our observations, a good many reasons for restricting the significance of thoracic scaling become apparent, and any groups in which primary divisions were formed on this character would be very heterogeneous. The great difficulty in scale structure is to know what significance can be attached to particular variations. Thus in comparing Pm. ludlowz, Theo., and P. costalis, Loew., which differ very slightly in markings, character of the palps, etc., one does not know whether the presence in the latter of mesothoracic scaling is to be taken as sufficient proof that they are unrelated or not. The same difficulty occurs in the case of abdominal scaling *peoig AIBA sapeos SuIAA A3Seys sdieg ee oq baw} no ooh O38 B28 alia} a, 4 ST One 2 S08. aap oe 5 =| lon Le | ° > Pu. —_— Ss Se (satoads auros) etyag PITJO209 NN snyoudyrossh Ny (urya sdjeq) (dno13 1asunu) snioydoyaikg a vrAwmozAul-opnasg (uryy sdyeg) sajoaydouy BIAWOZATAT (uty sdqed) eTAUOYIIIS XBIOYZOSOUT uo So]BOS proig Xe1OYJOSOUT uo SayvoOs pRroIq ON xvioyjoid uo 4ynq oN (xeioyzo1d UO soTvOs pdAIND MOIIeU snuiasd Ivijndeg) eAqstIyD visesey) eyoyoudAysrozATA, vsoupnbs *) (snzvzaundotpamu *7)) eIsezjeqiiy eisroydoqsty9 uosayddajopoda snyoudyrozAyy (¢) (erzsaqs0yy) (¢) (vtsournSue yy) erAuropayosoydorT (peosq viAuOZAUIOIN AJ9A JOU safeos Sur) S1]D4809 “q sisuaqojou “Ay AEM ERL (uryy sdjeg) etAurersejeg xe1OYJOSOUL uo SoTRIS peolg ON xe1IOYJOSOUT uo SoTBIS peolg s3jn4 [eiaqey] suo] yi uawopqy SoTBOs JnNoyyIM UIWOpqy $]ynq [e10qe'T s]Jnj Jeaquaa Auryy sayeos 19430 qnoyyiIM Jo YIM juaseid uajjo nq TeIQUaA y $]jn} ou ‘squaursas [eIaAes UO saTeIg sqjnj ou ‘quaUIsos ysv] UO saTeos MOF YIIM usWOpgYy SoTeos jo proAsp usWOpqgy So]BIS JO ploAsp usWOpqgy sa]vas jo quaw -as uly paaind | ivtjpnoad MOIICTU p,ssoidde pio jo Zurpeos peozy Ivaut] sayvos pvaH sayeos Pe3H addy Areur xeioyjoid uo yyny, *sdaqovIvYS ayes [e1sued uo Surdnoi3 9AT}e}U9} ZUIMIYS ATAV], Fis in the species Ne. wéllmori, James, and N. maculatus, Theo., which it is practically impossible or very difficult indeed to distinguish by any other characters than that one has a very much more scaly abdomen than the other. Metathoracitc Scaling. Limited observations only, which have not shown much promise, have been made in this respect. It was thought that since the metathorax represented a primary division of the thorax such scaling as was present might have more significance than its mere extent or conspicuousness might at first suggest. The halteres which spring from the metathorax, as one might expect from their representing an undeveloped pair of wings, carry scales. Usually the halteres are quite ball like, but in certain species (e.g., Avrzbalzagia maculipes) the wing-like character is much more apparent (Plate VIII, fig. 39). Wing Scales. Considerable differences occur in the shape of the wing scales. The most noticeable variation is in the breadth of the scales. There is also a difference pointed out by James, in that whilst in some species the scales are broadest in the middle (elliptical), in others they are broadest towards the free end (oblanceolate). This character serves, according to James, to distinguish between the genera Anopheles and Myzomyia. Leg Scales. The genus Lophoschelomyia has been formed for L. asiatica, Leicester, which has outstanding scales on the femur. Recently another species showing the same appearance, My. wellingtontanus, Alcock, has been described. Examination of specimens in the British Museum shows that both these species are not only probably related to one another, but have affinities with Pt. lindesayt, Giles. Pt. lindesayi is at once differentiated by possessing a very striking white band about the middle of the femur. So far as one could see, this colour peculiarity was a pure idiosyncrasy of the species. But the presence of a white band in connection with the structural peculiarity of a prominent scale tuft on the femur in L. asiatica and My. wellingtonianus suggests that the band in dzzdesayi was once also accompanied by scale tufts. The band is not quite in the same position in ZL. aszatica as in My. wellingtontanus but its occurrence along with a scale tuft is unlikely to be a mere coincidence. The three species in question in any case appear to be closely related, and if the genus Lopho- 80 schelomyia is retained, it should probably include Pr. lindesayi (vide diagrams of wing markings, Pl. VIII, figs. 1, 3). Abdominal Scales. Abdominal scaling, as pointed out by James, shows two distinct characters depending upon whether the scales project to form tufts or not. Tufts are usually, but not always, associated with ordinary scaling. They may be lateral or ventral. The genus Celdza, as is well known, shows a marked development of lateral tufts; the same condition is shown in Arribalzagia maculipes, Theo. The peculiar species Ch. kochi, Donitz., shows a marked development of ventral tufts, whilst the (apparently) totally unrelated Myzorhynchus group has one such tuft (not, however, present in all species). Pyretophorus is defined as having no scales on the abdomen. On close inspection I find that there are a few scales in P. costalis, Loew., especially in the male, the appearances being much like those seen in Pm. rossi, Giles. P. costalis, Loew., is the type of the genus, so that the definition of Pyretophorus (abdomen without scales) now held will not serve. The group of mosquitos like P. nursei seem to have a quite scaleless abdomen. A common condition is that in which the last two or three segments only carry scales. This is best known in Nyssorhynchus, but it also occurs in Lophoschelomyia, Manguinosia, and Kerteszia. In Cellia, Neocellia, Christophersia, and Arribalzagia the abdomen carries large numbers of scales. In the table I give a quite tentative and provisional grouping based on scale structure, adopting as broad an outlook as possible. It will be noticed that I have given great prominence to the condition of the prothoracic lobes. It is possible that this may require modification, but at present all my observations have gone to show the extreme significance of the presence or absence of a tuft of scales on the prothorax. 81 VI. CLASSIFICATION, PHYLOGENY AND GEOGRAPHICAL DISTRIBUTION OF THE ANOPHELINAE A tabular statement of groups as characterised, and to some extent defined, by structural characters and colour markings accompanies this paper. Though scale characters are given in this, they only enter very occasionally into the formation of the groups, and are purposely placed in parenthesis in order that their close agreement with the general scheme of classification given may be apparent. It will be seen that in the main Theobald’s genera stand out clearly. Consideration of other characters, however, modifies Theobald’s divisions as follows :— (1) It shows certain genera to be composite. (2) It groups the genera together so that one has some realisation of their zoological value. (3) It shows that it is not impossible that a system of quite good genera, more or less on Theobald’s line, could be with advantage retained, and that it is premature to resort as yet to the abolition of this grouping. I do not propose, in the present paper at least, to offer any new system of nomenclature, which would require, in order not to add to to the confusion already too apparent, the most careful and detailed systematic study of the subfamily. It will be sufficient for the present if the quite natural character of Theobald’s genera, taken as a whole, is emphasized. What seems mostly now required is accurate and detailed descriptions of species, with a_ really comprehensive and authoritative revision of the Anophelinae. But a list of the groups as defined by structural characters and colour markings, and a tentative arrangement of these as they appear to be phylogenetically related, should not be open to any objection. Retaining Theobald’s nomenclature, but noting the necessity of a number of emendations as regards the species at present contained in these genera, and subdividing the genera where necessary, the following represents, as far as one can judge, the phylogenetic relationship of these groups. Cee ee ra) S vo S| ow ee [-B) a (dnoss_ snyoiduadns) snioydoihg ‘ei F: sopaydouy ae vidwozk — vrAuopayasoydoy — ; ow § vikuvisvieg — 3 1SS0L “We TS) be |e = oy a & Vee Nabe ty S$Yv1509 “_ fore) I | " : 5 snyoukyrossAN = 6 — a ZS BPD 4G: — =| ‘3 eyppES WS ees vyjoyoudysoz Ay 5 OBO a wm i sisugvqvu “hy —— == * vo “G sajaydouroany — is] é snyoudys0zAyA,. — 7 uoiajyddajop>fq§ ——______—— visezeq ity ——— 2h. eee a Pe AVNITIHAONY FHL JO LNINAONVALV DILANTDO TAH G TAILVLNGT, 83 In this arrangement, phylogenetic advance is associated with both an increase in colour markings (1i.e., the development of white) and in scaling. It is not difficult to arrange a correlation table in which increasing degrees of whitening and increased degrees of scaling form the ordinates and abscissae respectively. The striking nature of the correlation between the two series of characters is then made very apparent by the grouping of species about the diagonal line. The same correlation is also very evident in the phylogenetic scheme given. Considering the fact that the Culicinae are all scaly, one might think it probable that Anophelines were a branch in which scales were disappearing. But my own observations on this point make it more probable that it is the other way. The more scaly an Anopheline is the more advanced phylogenetically it would appear to be. Certain of the groups, it will be seen, occupy a much more primitive position in the scheme than others, and as this seems to be demonstrated by whatever character they are considered, and as there are definite characters by which one can distinguish these apparently old type species, I have named them Protoanopheles. The peculiar Australasian type I have called Neoanopheles. The ordinary Anophelines exhibiting the regular colour scheme I have outlined may approximately be termed the Deuteroanopheles. The nature of these groups, and the characters on which they are based, can be obtained from the large table accompanying this paper. Briefly stated, the constitution of the main groups noted is as follows :— PROTOANOPHELES. Stethomyia. Anopheles. Patagiamyia and Lophoschelomyza. Myzorhynchus, Cycloleppteron, Arribalzagia, etc. DEUTEROANOPHELES. Myzomyia. Branch A. Pyretophorus. Pseudomyzomyia, etc. Branch B. Nyssorhynchus. Neocellia. Branch C. Cellza. 84 NEOANOPHELES. Many spotted winged species and their related types. Between branches A and C of the Deuteroanopheles there are distinct differences in ornamentation, such as, for example, a much greater tendency to a completely broken third costal spot and narrow costal interruptions, but the most striking difference is in the much greater intensity of the black and white ornamentation. The origin of the branch B 1s difficult to determine. It is especially interesting as including some of the most dominant species, 1.e., Pm. rosst, P. costalis, Ne. stephensi, Pm. ludlowiz. GEOGRAPHICAL DISTRIBUTION Having on structural grounds arrived at certain groupings, it 1s interesting to note thé geographical distribution of these groups. Stethomyia, though it contains very few species, has a distribution area which includes South America and Australia, as well as India and Malay. Myzorhynchus, if we consider the closely-related genus Cyclo- leppteron, also has a very extended distribution, which includes South America and Australia. Anopheles and Patagiamyia are the dominant genera of Europe and North Amerca. There is, however, what appears to be an Australian representative of this branch, as well as hill species in the East. In the case of the Deuteroanopheles, certain of the genera have a much more restricted distribution. Thus Pyretophorus, in the restricted sense in which I have been using the term, is dominant in Africa (North), occurs in South Europe and in the extreme North-west of India. It does not occur in South America or Australia, and it is scarcely represented in the Anopheline fauna of India and Malay. Myzomyiza is essentially African, Indian and Malayan. Nyssorhynchus is also African and Malayan, with a distinct dominance in the latter area and in India. The most widely distributed of the genera of the Deutero- 85 anopheles is Cedlza, and this fact is not out of keeping with the position assigned to this genus in the scheme of phylogeny. The Neoanopheles are distinctly Australasian and Malayan. More than the above outline of the main features of geographical distribution of species cannot be attempted here, but it will be seen that to a large degree consideration of the geographical distribution of species gives distinct support to the ideas respecting phylogeny which I have put forward in this paper. VII. GENERAL CONCLUSIONS Colour markings, equally with structural characters, can be utilised in natural classification of the Anophelinae and in the placing of species in groups to which they have affinities. A classification based on colour marking, supported by structural differences in the palps and larval characters, approximates very closely in regard to the groups formed with one based on scale structure. It shows, however, the relation and affinities between the groups much more clearly than does scale structure classification as at present employed in the distinction of genera. Colour markings, and general characters as a whole, seem to point to there being a group of more primitive forms (PROTO- ANOPHELES) occurring as old world species (Anopheles, Myzo- rhynchus and Patagiamyia), and as new world representatives of this group Cycloleppteron, Arribalzagia and Myzorhynchella. There is also a distinct group corresponding to Donitz’s Australasian species with more than three spots on the sixth vein (NEOANOPHELES). Their area of special prevalence is Australasia and Malaya. The majority of Anophelines belong to a group the colour markings of which are all on a given colour scheme (DEUTERO- ANOPHELES). This group shows two main divisions, characterised by a notable difference in the intensity of their coloration and the ‘effectiveness’ of ornamentation, possibly arising from two different lines of evolution. Their area of special prevalence is an area including Africa, South Asia and Malay. I am very greatly indebted to Prof. R. Newstead and to Mr. H. F. Carter for their great kindness in furnishing me with 86 material for studying a number of African and South American species, including specimens of Cy. grabhamiz, without which I could not have carried out many of my observations. I am also indebted to Prof. Newstead and Mr. Carter, as well as to Mr. F. W. Edwards, for their kindness in giving me every facility to make use of the collections at Liverpool and the British Museum respectively. I also wish to acknowledge my indebtedness to Prof. G. H. F. Nuttall and to Mr. H. Scott, at Cambridge, for help in regard to literature and in other respects. 87 VIII. TABLE SHEWING GROUPING OF SPECIES OF ANOPHELINAE ACCORDING TO COLOUR MARKINGS AND OTHER VARIABLE CHARACTERS A. NODAL CENTRES AT CROSS VEINS AND BIFURCATIONS DARK. (PROTOANOPHELES.) A’. CosTA DEVOID OF ANY LIGHT AREAS EVEN AT APEX. WINGS USUALLY WITHOUT ANY PALE MARKINGS. 1. Palps markedly heterodactylous with marked relative elongation of the second segment. Attitude cwlex-like. Wings entirely unspotted. Palps unbanded. Legs entirely without markings even knee spots being absent. Small mosquitos. (Larval antenna without branched hair. Palmate leaflets lanceo- late.) Head scales linear. No scales on prothorax, mesothorax or abdomen. Corresponds exactly with Stethomyia, Theo.; Neostethopheles. James. S. nimba, Theo. S. aitkeni, James and Liston. S. culiciformis, James and Liston. S. corethroides, Theo. Probably synonyms—S. treachert, Leicester; S. fragilis, Theo. ; S. pallida, Ludlow. tN Palps orthodactylous. Attitude anopbeles-like. Palps_ thin, unbanded. Primitive spotting may be present. No admixture of light and dark scales. Knee spots usually present otherwise legs are quite unornamented. (Larval antenna with branched hair (small), palm-leaflets lanceolate and serrated). Head scales expanded. No prothoracic tuft. No scales on mesothorax or abdomen. Corresponds with Anopheles sense of James. A. maculipennis, Meigen. A. bifurcatus, Linn. A. nigripes, Staeger. A. algeriensis, Theo. A. barianensts, James. A. barberi, Coq. A. immaculatus, James. (?). A. eiseni, Cog. (?). A. crucians, Wied. (?). 88 A’. CosTA WITH AT LEAST ONE PALE INTERRUPTION WHICH MAY BE AT THE APEX. (b') Wings without prominent admixture of pale and dark scales. 3. Costa dark broken only at apex or at subcosta with or without small basal interruption. Palps usually unbanded. Primitive spotting prominent. Tarsus unbanded. (Larva with branched hair on antenna. Palmate leaflets with filament but showing approach to lanceolate type.) Head scales expanded. Tuft on prothorax. No broad scales on mesothorax. May or may not be scales on last segments of abdomen. 3a. With white band on femur which may be associated with tufts of outstanding scales in this situa- tion. Lophoschelomyia. L. asiatica, Leicester. L. lindesayi, Giles. L. wellingtonianus, Alcock. 36. Without white band or femur. L. (?) atratipes, Skuse. 4. Costa prominently spotted. (Usually two, not more than three, main spots). Palps banded. Primitive spotting may be present (but not conspicuous). White spots on wings for most part represented on lower surface. Large mosquitos. (Larval antenna carries a branched hair. Palmate leaflets with filament.) Head scales expanded. Tuft on prothorax. No scales on meso- thorax (or abdomen). Corresponds to Patagiamyia, James. Pt. gigas, Giles. Pt. simlensis, James. Pt. punctipennis, Say. Pt. smithi, Theo. (Unspotted wings.) Probably A. formosus, Ludlow. Perhaps A. franciscanus, McCracken. A. perplexans, Ludlow. A. pseudopunctipennis, Theo. Would probably include also Feltinella pallidopalpi, Theo. 5. Costa dark with narrow interruptions but with four main costal spots. Wings markedly dark beneath. Palps shaggy. (Larval antenna without branched hair. Palmate leaflets lanceolate.) Head scales flattish. Broad scales on mesothorax. No scales on abdomen. Myzorbynchella. 89 (b°) Wings with prominent admixture of dark scales. Pale spots on the wing deficient as a rule on under surface. Hence, when third spot is light 1t shows dark border of projecting dark scales seen through wing membrane. Primitive spotting the rule. Sixth vein with at least one scale cluster of dark scales. (Larval antennae with branched hair. Palmate leaflets lanceolate and serrated. Frontal hairs markedly branched.) Prothoracic lobes with tuft. 6. Legs not speckled. Costal edge dark with two (at most three) minute pale interruptions. Basal portion of costa unbroken by pale areas. Palps markedly orthodactylous, shaggy, un- banded or with scheme A ornamentation. Large mosquitos Old World species. No broad scales on mesothorax. Some ordinary scales present or absent on abdomen. A ventral tuft of dark scales usually present on penultimate segment of abdomen. Corresponds to Myzorbynchus. My. barbirostris, Van der Wulp. My. pseudobarbirostris, Ludlow. My. bancroftit, Giles. My. umbrosus, Theo. My. strachani, Theo. My. sinensis, Wied. My. pseudopictus, Grassi. My. paludis, Theo. My. mauritianus, Grandpre. 7. Legs markedly speckled. Costal edge may be dark as in last group or broken into distinct spots. Accessory spot often present. Palps shaggy with scheme A ornamentation. Large mosquitos. New World species. 2a. Sixth vein with two dark spots. Wing with inflated scales but resembling that of Myzorhynchus in general arrangement. No broad scales on mesothorax. Cycloleppteron Cycloleppteron grabhami, Theo. 26. Sixth vein with many spots. Broad scales on mesothorax. Abdomen with many scales and lateral tufts. Arribalzagia. Ar. maculipes, Theo. Cy. mediopunctatus, Theo. (Ar. pseudomaculipes). (Ar. malefactor). (Cy. intermedium). go B. NODAL CENTRES AT CROSS VEINS AND BIFURCATIONS PALE. COSTA WITH FOUR DISTINCTLY MARKED MAIN COSTAL SPOTS. B’. Patps ORNAMENTED ON Scueme. .A—C. Costa wIiTHOUT Accessory SPOTS OTHER THAN THE BASAL ONES. Nor MORE THAN THREE DARK SPOTS ON SIXTH LONGITUDINAL Vein. (DEUTEROANOPHELES.) (a2) Tips of hind tarsi not white. a’. Palps markedly heterodactylous. Tups of hind tarsi never white. Banding of front tarsal joints never broad. Legs never mar species on fixed are present on s ‘kedly speckled. Wing markings of scheme (Scheme A). If three spots ixth vein there 1s a pale interruption on upper branch of second longitudinal. Completely broken third costal spot unusual. (Larval antenna without branched hair. Palmate leaflets with filament. Frontal hairs usually simple unbranched.) No prothoracic tuft. No scales on abdomen, even in male. 1. Sixth vein with two or less dark areas. No pale internodal spot on branch of fifth. Branches of second long, rarely with pale interruption. Still more rarely interruptions on _ branches of fourth. Fift h vein dark at junction of branch. Mesothorax without broad scales. Corresponds to Myzomyia in restricted sense (i.e., not including Pm. rossi, M. 1 M. M. M. M. M. M. M Hae utzt, etc.). culictfactes, Giles. nil1, ‘Theo. rhodesiensis, Theo. hebes, Donitz. umbrosa, Theo. listont, Liston. . funesta, Giles. sergentti, according to wing. 2. Sixth vein with three dark spots or two. Pale internodal spot on branch of fifth. Branches of two and four veins with pale interruptions. Fifth vein usually pale at junction of branch. Palps peculiarly dark band (four long and thin. Not infrequently show apical palpal bands) or apex dark. Broad scales on Mesothorax. Pyretophorus 1 (superpictus group). aa Baa aa) hs hy . superpicius, Grassi. . nursei, Theo. . nigrifasciatus, Theo. . cleopatrae, Willcocks (MSS.) . cardamatisi, Newstead and Carter. distinctus, Newstead and Carter. Costa unusual but wing otherwise showing typical Pyretopborus condition. . palestinensis, Theo. . cinereus, Theo. gI a’’. Palps not markedly heterodactylous. 3. Upper branch of second vein with pale interruption. Tip of hind tarsus not white. Palps destinctly orthodactylous but with whole of apical segment pale. Banding of front tarsus broad. Apical segment of palps in spite of length pale through- out. Hind tarsi not white but speckling usual. Whitening of wing considerable with completely broken third costal spot usual. All internodal pale spots developed and some residual pigment areas usually obliterated. A composite group according to scale structure. Prothoracic tuft. Preapical costal spot broken. Scales on mesothorax. A few scales on last segment of abdomen especially in male. Pyretophorus 11. P. costalis, Loew. (P. pseudocostalis, Theo. ?). (P. merus, Donitz ?). (P. marshallit, Theo. ?). No prothoracic tuft. Preapical costal spot not broken. A few broad scales on mesothorax. A few scales on last abdominal segment especially in male. Pseudomyzomyia, Theo. = Nyssomyzomyia, James. Pm. rossi, Giles. Pm. indefinata, Ludlow. Pm. ludlowi, Theo. No prothoracic tuft. Nyssorhynchus-like scales on thorax. Ne. stepbenst. 4. Upper branch of second without pale interruption. Palps ortho- dactylous and with scheme A pattern. Bridging of pale spots on costa by dark areas on first long vein marked feature. Prothoracic tuft. Broad scales on mesothorax. Abdomen with lateral tufts. O22 squamosa, Theo. _ Related to Myzxorbynchus and Cellia. A peculiar species. Isolated position in this table due to fact it does not possess white tips to tarsus. Note prothoracic tuft. (6) Tips of hind tarsi white. Ornamentation very black and white. Speckling and banding usual. Full development of internodal pale areas of wing, but delayed appearance of pale area on upper branch of second vein. (Larval antenna without branched hair. Palmate leaflets with filament. Frontal hairs often show some degree of branching.) Characteristic thorax. Q2 broad white creamy scales on meso- 5. Palps ordinarily three banded (scheme B). Orthodactylous or with some tendency in light forms to be slightly heterodactylous (N. maculatus). Abdomen with scales on last few segments (Nyssorbynchus) or on many segments (Neocellia) but without lateral tufts. No prothoracic tuft. Dark group. Upper branch of second long vein without interruption. Hind tarsi usually show un- interrupted white area extending over several segments. Palps destinctly orthodactylous, ornamented scheme B, with tendency in individuals to revert to A. N. fuliginosus, Giles. N. nivipes, Theo. (NV. freerae, Banks.) (N. phillipinensis, Ludlow.) Ne. fowlert, Christophers. N. jamesi, Theo. N. pretoriensis, Theo. N. maculipalpis, James and Liston. Light group. Upper branch of second long vein with interruption. Hind tarsi usually with alternate black and white areas. Palps with shorter terminal segment than last group and with scheme C ornamentation (two broad apical bands). N. maculatus, Theo. N. theobaldi, Giles. Ne. willmori, James. 6. Palps ordinarily four banded (scheme A) and markedly orthodac- tylous. Abdomen with lateral tufts. Prothoracic tuft in some species. Cellia. Dark group. Bridging of pale spots on costa common. Light Also absence of interruption on upper branch second. C. argyrotarsis, Desvoidy. C. albimana, Wied. C. jacobi, Hill and Haydon. C. cincta, Newstead and Carter. (C. squamosa, Theo. Hind legs not white, vide above.) (C. squamosa, var. arnoldt. Hind legs not white, vide above.) group. Upper branch of second interrupted. Costal spots not bridged. C. pulcherrima, Theo. C. pharoensis, Theo. oe) B”’. Patrps witH ScHEME D oRNAMENTATION. More THAN THREE DARK SPOTS ON THE S1xTH VEIN. ACCESSORY COSTAL SPOTS OFTEN PRESENT IN ADDITION TO BASAL ONES. (NEOANOPHELES.) With prothoracic tuft. My. natalensis, Hill and Haydon. * P. watsontt, Leicester. * Nm. elegans, James. * (Nm. leucosphyrus, Donitz.) * N. annulipes, Walker. * N. masteri, Skuse. * Ch. kochi, Donitz. Query any prothoracic tuft. M. punctulata, Donitz. * N. deceptor, Donitz. * N. thorntonti, Ludlow.* N. karwart, James and Liston. All species show same character of palpal ornamentation. * Shows more than three dark spots on sixth. REFERENCES James, S. P. (1g10). A new arrangement of the Indian Anophelines. Records of the Indian Museum, Vol. IV, No. s. Mayer (1897). On the color and color patterns of moths and butterflies. Proc. Bost. Soc. Nat. Hist., Vol. XXVII, pp. 243-330. Hixt and Haypon (1907). A contribution to the study of the characteristics of larvae of species of Anophelina in South Africa. Annals of the Natal Gov. Museum, Vol. I, Part 2. Donrrz (1902, 1903). Beitrage zur Kenntnis der Anopheles. Zeit. f. Hygiene, Bd. XLI, p- 15, and Bd. XLII], p. 215. Nurratt and SuipLey (igor, 1902, 1903). The structure and biology of Anopheles. Jour. of Hygiene, Vol. I, Nos. 1, 2 and 4, pp. 45, 269, 451; Vol. II, No. 1, p. 58; Vol. III, No. 2, p. 166. Epwarps, F. W. (1911). Some new West African species of Anopheles (sensu lato), with notes on nomenclature. Bull. Ent. Res., Vol. II, Part 2, p. 141. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. SP WwW nN mS 94 EXPLANATION OF PLATES PLATE, VY. Nymphal trumpets of A. bzfurcatus, Linn. Nymphal trumpet of Ne. wzllmori, James. Leaflet of palmate hair of My. senenszs, Wied. Leaflets of palmate hairs. (a) C. squamosa var. arnoldi, after Newstead and Carter. (06) N. annulifes, Walker, after Theobald. (c) A. dzfurcatus. (d) Cyclo- leppteron grabhamu, Theo., after Theobald. (e) Myzo- rhynchus (?) natalensis, Hill and Haydon, after these authors. (f) P. ardensis, Theo., after Hill and Haydon. Antenna of larva. (a) A. bifurcatus. (6) A. punctz- pennis, Say., after Smith. Female palp of My. barbirostris, Van der Wulp, showing orthodactylous character and unbanded condition. Female palp of C. pulcherrima, Theo. Orthodactylous with scheme A ornamentation. Female palp of Pt. lindesayz, Giles. Orthodactylous and unbanded. . Female palp of P. nursez, Theo. Heterodactylous with appressed scales and scheme A ornamentation. . Female palp of P. jeyporensis, James. Heterodactylous with appressed scales and ordinary Deuteroanopheles type of ornamentation, 1.e., scheme B, with involvement of the whole of the apical segment. . Female palp of P. costalis, Loew. Orthodactylous with scheme B ornamentation. . Female palp of M. funesta, Giles. Heterodactylous with appressed scales and scheme B ornamentation. . Female palp of N. maculatus, Theo. Scheme C ornamenta- tion. . Female palp of WN. maculatus contrasted with palps ornamented on scheme D. (a) NV. maculatus. (6) M. punctulata, Donitz. (¢) Ch. kochi, Donitz. (d) N. karwart, James. After James. . Sketch of palp of S. corethroides, Theo., showing remark- able modification in the relative lengths of the different segments. Heterodactylous. Type 2. . Palp of S. nzmba, Theo., after Theobald. e ia Sprite ry atart | Fy ae. 0 ee ee piv lo extn) Lavntounté paiwadg para dats Sif —. ob aiken labo.) evita Pome a) car! . ean irsgis! qd inbormige eulaits . pro swore ; f artas weal A E Aame aodnig yaeeconen Lsebll "5A .. ee nN fogqes! al#on asi) asaal soma & feo tate knteon nism oibbits “MK ast aug Suge leteoo neal Leagepnclt ee? i DAM So oe Preattns toge: dejaooentam denigés ee Se E10 denete Ne emiosyissiqe bas tsesd 7 am) ’ f is p 5 ; Seas e: astiqu 16 cotharre wud ‘Fotes eonith: pate Ai? lentbuitgnol hionse 15 Rolbibur: iti = Stead aya? spat aR AS atew dycvese 419 soon Y “sil = snot i id to Arona, inoin tg lanbiasd os = er e* dearbud . é 7S ibe He ehisne! jeratioiv mu orf of at al,,30 dt we vothire) 6% py" arcu : ckamebas! Lelie Sp eittntisukwtsid ao coli ) a 7 —— Ey cerita® oilaristieis’ } * ati woe fF ; | pres WH a pe Stet o7 cxltnser aad eotdcis:)) i O43” itate Ge foqe ) rey ra) asic. iowa let” ae. didi AinAy een (eromulH ithe (3) ae itieondud, whe: i ind wi brows -¢ Sasaqhy pit fs gore aise; route: wit 0 ae iteboV. (6) MA esaotose gioeemr due Wii bar p xtoion bso “a yeh boba ey aryier Pag. 11. 96 PLATE VI Diagram showing structural features of wing and leuco- genetic centres. Nodal points are shown by small circles, internodal by larger ones. Pigment centres are shown dark. Pigment centres. Ac'., Ac’. Basal accessory pigment areas. MLAB Inner main costal spot centre. m.m.C. Middle main costal spot centre. s.a.m.c. | Subapical main costal spot centre. a.m.c. Apical main costal spot centre. 27., 2a. Basal and apical centres of stem of second vein. 2'b., 2'a. Inner and outer centres of upper branch of second longitudinal vein. 2"a., 2"b., 2"c. Outer, inner and middle centres of lower branch of second vein. 3a., 3. Residual pigment centres of third longi- tudinal. Ar., 4a. Centres on stem of fourth longitudinal. 4'a., 4'b., 4"a., 4"6. Centres on fork of fourth longi- tudinal. s'a., 5'6., 5’c. Centres on branch of fifth longitudinal. 57., 5"a. Characteristic centres of fifth longitudinal vein. 67, 6m, 6a. Centres for sixth vein. 67. is Donitz’s root spot of sixth. Cre Oe Ed Ope Oe Nodal Nodal points. points in connection with h. Humeral. cross veins. Sc. Subcostal. C.1'. (?)- Nodal point con- Ap. Apical. cerned in the formation of Ds Sector. the inner pale spot on costa. S'. Accessory sector. C.1”. (?) Nodal point con- cerned with subapical spot. t, 2/, 2", 3,4’, 4", 55 5", O-- Nodal” pomts— on swine, margin. Fig. Fig. Fig. Fig. 97 Internodal points. 2!, 22, 23. Internodal points on branches of second vein. The relatively late appearance of 2! 1s characteristic of Nyssorhynchus and dark Cellzas. ay 3: One or two internodal points of third vein. 4, 4!, 42. Internodal points of stem and branches of fourth vein. 51, 52, 53. Points on main vein and branch of fifth vein. Gt .67: Points on sixth vein. 2. Portion of the wing of a species of ¢zpulidae, showing venation homologies and extra cross veins. h. Humeral cross vein. C.1'. Cross vein joining subcosta to first longitudinal. Cink. Ditto upper branch of second to first longitudinal. R,, R., Ry, Ry.5, Radial system. R.S. Radio-sector. Si: As in figure 1. Si: Ditto. 3. Showing area of cross veins, and relation to nodal point S. Gos. Gu,,.Cv,, Cv.. Crosstveing22F: S. = Sector, S’. = Accessory sector nodal point. sc. = Subcosta. 4. Wing of a species of Chzronomus, showing pale spots on wing membrane involving the hair-like scales covering the wing and aggregation of hair-like scales to form dark spots at the area of the cross veins. 5 to Fig. 13. Diagrams of representative wings, showing arrangement of nodal and internodal points as developed in different groups. Dark spots represent nodal points which remain pigment areas. Circles half dark represent pale spots not developed on under surface of the wing. Small black dots in circles mean admixture of dark and light scales present. Dark stars mean scale aggregations. Fig. Fig. Fig. 98 PLATE VII Wing of A. maculipennis, showing primitive spotting. Wing of My. umbrosus, showing primitive spotting, want of representation of pale spots on under surface of wing, and admixture of dark and light scales. A scale cluster on the sixth vein is also shown. 2a. Under surface. Wing of M. funesta var., showing first appearance of nodal points with few internodal centres. Pale scaling not intensely white. Wing of N. maculatus, showing extreme development of nodal and internodal centres, with residual pigment areas well displayed. White scaling intensely white. S ao away. i= Pye... eu ra “Oo 45 " ia ee: wil) .aueshihal suc gein et Ts ae aeeey TAS9ys ak al —— Pah WARS TLORNG, Lee (AER ey ee a. Dee ERT Ree “ie > SmeceAT ordi aaa Wiyaie Vh tis SMiwaee: Aagbize: ely tet om. rhs Seah ae rary Ace eH Watiwa? AMesWT Oey —_ S | : a oe es oP os ned Car ; ASST BA LSI HeLa? - - 48 sa i an is VV eee “at hier aT Pais AA PANS ser bas ey RTE The patiigit, amok soduer baw orn en 2 gation t- = 2} sha eal ie eae Avast ASIA a wd. aln’s ome Pe tey Sian s\ Ps ORR in. /: bape onet reat picks W, parca. Lipa; 48.) Swi TGA 2090S) WHE NPE EMO *h a a Staph! 2) stiiniyedt Wh front 4-ceee eho As WAQSHAD a HEH Sap ttivnts (95 cahontheclig *, Pe ee te -. (anirotle rs AA Srakioy Ppaclens Shap GH tak WA Roya WONT Rien. Re Hoses: osevissalay Laity Mh RAT: a! Pie ee eile ae ee vena a Bly: | piGee! ° ae es ‘ es mee ws BT Fh SD wer oR ae = hall or NG AGIAN - een) BEA) Shir [> a ce ; — is | ony pie peat tin. ~oyntw Pe. os By fe ee = ot Lg pfltaaniad gailnoe amb uiagnstni ae Ee ct Panthsst Re ee Ce aan 100 PLATE VIII Diagrams of the spotting of the wings in a number of species of Anophelinae, especially to show arrangement of costal spots. Figs. 1-38. Fig. 39. Halter of Cy. mediopunctata, showing resemblance to “ unexpanded wing, with light scaling above and intensely dark scaling beneath. Patagtamyta lindesay1, Giles. My. wellingtonitanus, Alcock. Lophoschelomyta astatica, Leicester. Patagtamyta gigas, Giles. Patagtamyta simlensis, James, var. Myzorhynchus umbrosus, Theo. Ditto under surface. Myzorhynchus mauritianus, Grandpré. Myzorhynchus stnensts, Wied. Cycloleppteron grabhamu, Theo. Ditto under surface. 12. Cycloleppteron mediopunctatus, Theo. 13. Arrtbalzagia maculipes, Theo. 14. Myszorhynchella nigra, Theo. 15. Ditto under surface. 16. Cellza sguamosa, Theo. 17. Cellia squamosa, Theo. | 18. Nyssorhynchus fultginosus, Giles. | 19. Mysorhynchus (?) natalensis, Hill and Haydon. . 20. Pyretophorus (?) watsonz, Leicester. | Neomyzomyta leucosphyrus, Donitz. | Myzomyta (?) punctulata, Donitz. | Christophersia kocht, Domitz. Nyssorhynchus (?) annulipes, Walk. (Costa only shown. ) Pyretophorus costalis, Loew. Cellia jacobt, Hill and Haydon. Myszomyta culictfactes, Giles. Myzomyta listont, Liston. Pyretophorus cinereus, Theo. Pyretophorus nurset, Theo. Pyretophorus cardamitist, Newstead and Carter. Pyretophorus chaudoyet, Theo. Pyretophorus jeyporensis, James. Nyssorhynchus theobaldt, Guiles. Neocellia stephenst, Liston. Pseudomyzomyia rosst, Giles. Pyretophorus ardensts, Theo. Pyretophorus (?) atratipes, Skuse. eet mM OW) ON Du W N NNN WN pe OO Ie WwW Wo CO™1 O*VUI & Go NW = OW) on™ Own On Gd Wo Go Go Go Ga Go Go WO WO IO [ol A SLUDY OF. THE POSTERIOR NUCLEAR FORMS OF TRYPANOSOMA RHODESIENSE (STEPHENS AND FantHaM) IN RATS BY be DEAK LOOK. M. Diy 1) /P. iH. From the Runcorn Research Laboratories (Recezved for publication 5 February, 1913) Trypanosomes, obtained from a human source, presenting a posterior position of the macronucleus were first described by Stephens and Fantham.! Upon this morphological peculiarity they founded their species 77ypanosoma rhodesiense. Since the publica- tion of their paper, a similar condition has been described in trypanosomes from human sources by various observers in different regions. The patient whose strain they described contracted the disease in a portion of Rhodesia in which the absence of Glossina palpalis rendered it necessary to seek a different carrier. Stannus and Yorke? discovered posterior nuclear forms in a strain taken from a case of human trypanosomiasis in Nyasaland. More recently Stannus? has noted the presence of these forms in a strain obtained from a case of human trypanosomiasis from Portuguese East Africa, while the Royal Society’s Commission? confirms the observation of Stannus and Yorke with regard to the presence of posterior nuclear forms in strains from human trypanosomiasis in Nyasaland. The Commission bears out their conclusion that the trypanosome of the human trypanosome disease of Nyasaland is probably identical with 7. rhodesiense. The occurrence of posterior nuclear forms is not, however, confined to strains of human trypanosomiasis. Such forms have also been recorded among animal trypanosomiases, for example in IT. pecaudi by Wenyon,° in a strain of 7. eguzperdum by Yorke and Blacklock,® and in a strain of 7. drucez from Uganda by Blacklock.’ 102 THE INCIDENCE OF POSTERIOR NUCLEAR FORMS IN T. RHODESIENSE Stephens and Fantham (loc. cit.) observed that the forms of trypanosome which presented a posterior position of the nucleus were the short and stumpy ones, and that in rats forms having this peculiarity made their appearance in the peripheral blood about the fifth or sixth day of the disease, and increased in number to the seventh or eleventh day, when they formed about 6 % of the parasites present. SHORT AND STUMPY The term ‘short and stumpy’ appears to be used in somewhat different senses by different observers, in relation to trypanosomes. For example, Bruce (loc. cit.) says ‘ There is no free flagellum in the short and stumpy forms.’ In a table attached, however, he shows that the amount of free flagellum in the ‘short and stumpy’ forms (620 individuals) averages 0°6. Stephens and Fantham (loc. cit.) say of the stumpy forms of 7. rhodesztense which have a posterior nucleus that there is a well-marked blepharoplast and a very short free flagellum. It appears, therefore, to be not quite decided what constitutes a ‘short and stumpy’ form. Is it a trypanosome which measures less than a given number of microns, e.g., 21; 1s it a trypanosome which has absolutely no free flagellum; or is it a trypanosome which is not only less than a certain length, but also has absolutely no free flagellum? In the experiments given below the large majority of forms which had a posterior nucleus measured Jess than 21“, but numbers were found which exceeded this length. Again, of the parasites which conformed to the definition as regards shortness, numbers had absolutely no free flagellum, while numbers had a very definite portion of flagellum free. In some cases this portion was of considerable length. It was not impossible, for example, to discover forms which while they measured only 19 in length, yet possessed a free portion of flagellum amounting to 4p. One finds thus that when studying the posterior nuclear forms in these experiments, one is not dealing with short forms in the strict sense that they measure in all cases less than 21m nor with 103 stumpy forms in the sense that they possess absolutely no free flagellum. There are overlapping forms which do no come under both categories, in fact there are a few which come under neither, yet present the posterior nucleus. For practical purposes, however, it may be said that the posterior nuclear forms, whether free- flagellated or aflagellar, which exceed the measurement of ‘short forms,’ were few in number. The experiments detailed below were undertaken in order to determine in a given infection in rats the time of appearance of posterior nuclear forms in the peripheral blood, and the numerical relationship which these forms bear to other forms of trypanosome present from day to day. METHOD OF CARRYING OUT THE EXPERIMENTS A guinea-pig infected with 7. rhodeszense and having numerous parasites in its blood was utilized as the source of infective material. Four groups (A, B, C, D) of rats, each group containing six rats, were selected, the rats being as nearly as possible of the same weight. The parasites per cubic millimetre in the blood of the guinea-pig were estimated by means of the Thoma Zeiss haemo- cytometer, and dilutions made in a warmed mixture of 1 % sodium citrate and 0°85 % sodium chloride. The dilutions were made so that the rats of the first two groups received 1,000,000 trypano- somes, the rats of the third group 4,000,000, and the rats of the fourth group 8,000,000. The animals were all inoculated intra- peritoneally, the amount of the injection in each case being 0'5 c.c. INCUBATION AND DURATION The average incubation period of Group A (1,000,000 trypano- somes) was 4’8 days, the average duration of the disease 13°6 days. Group D (8,000,000 trypanosomes) had an average incubation period of 4°5 days and an average duration of 15°5 days. Table | gives details as to the incubation and duration of the disease in the individuals of each group. From this table it appears that no definite variation in either incubation or duration could be attributed to the relative numbers of trypanosomes injected. 104 Taste I.—Giving incubation and duration of infection with T. rbodesiense in 24 rats. Group No. of Experiment | Incubation in days Duration A | Rat 2231 A n- aA 5 13 1,000,000 | = B aos vt) 4 13 Trypanosomes 2» | Cc | 5 | 14 inoculated ~- D 6 14 pas 4 14 1 Ie 5 14 | B Rat 2232 A 5 | 13 1,000,000 B 6 | 13 (e 5 | 16 D 5 | 16 * E = 4 | 13 F | 6 13 | } Cc | Rat 2229 A 5 | 13 4,000,000 F Sdt .| 4 12 - Cc 4 | II i D -| 4 | 10 ae E a 4 | 13 ee F | 6 13 D Rat 2230 A | 4 17 8,000,000 B 5 4 17 * Se | 4 15 ’ D 5 I4 > E 5 15 aoe 3 ! "5 Average .. ‘ 4:7 13°7 105 ENUMERATIONS From the day on which parasites first appeared in the peripheral blood until the death of the animal, a film was examined daily in the fresh state, and a thin film dried and stained. The dried films were fixed in absolute alcohol and stained with Giemsa’s stain. 1. Preliminary count. In each stained film a count of 200 parasites was made wherever possible. Every trypanosome met with was counted, whether long, intermediate or short, dividing or non-dividing. The number of posterior nuclears was noted, non-dividing forms only being chosen. The result, therefore, gave the number of non-dividing posterior nuclear forms per 200 of all forms. The posterior nuclear forms were classified carefully according to the position of the nucleus. Those forms were classified as A in which the nucleus, although definitely posterior to the centre of the parasite (excluding free flagellum) still lay close to the centre. C forms were those in which the nucleus lay adjacent to the blepharoplast, while B forms were intermediate in position. The drawings (Text-fig. 1) give the positions indicated for each group. A B Cc Text-Fic. 1. Showing various positions of nucleus. A. B. C. RESULT OF THE PRELIMINARY COUNT The first trypanosomes which appeared in the peripheral blood of each rat were of the long and slender, or intermediate types. Subsequently, short forms made their appearance, and after a few days posterior nuclear forms. In no case was a posterior nucleated 106 form found in the first day’s count of 200 in any rat. The posterior nuclear forms made their appearance in the peripheral blood with considerable regularity, in the following order :— first A forms, next B forms, and lastly C forms. Thus of the twenty-four rats, A forms appeared first in thirteen, B forms first in six. In four, A and B forms appeared together, while in one, A and C forms appeared together. In none of the rats was a C form the first type of posterior nuclear to appear. DAY ON WHICH POSTERIOR NUCLEAR FORMS FIRST APPEARED The earliest day of the disease on which posterior nuclear forms were first seen in the peripheral blood in this count was the seventh, the latest on which they first appeared was the thirteenth. The average day of their appearance in the twenty-four rats was the ninth day of the disease. From the time of their first appearing, posterior nuclear forms were usually to be found up to the death of the animal. It was observed that the posterior nuclears increased not only actually as the disease progressed, but also relatively to other forms of parasite. The greatest number of posterior nuclear forms in any 200 counted occurred on the day of death in fourteen out of the twenty-four rats, on the day preceding the day of death in seven rats, two days before the day of death in two rats and previous to this day in the remaining rat. From the enumeration of the parasites in films from the infected animals by this preliminary count, the results obtained were as follows :— (1) The first forms of parasite found present in the peripheral blood were long and intermediate free flagellated forms. (2) Short forms appeared later. (3) Posterior nucleated forms only appeared after the disease had developed somewhat. (4) Posterior nucleated forms increased in numbers from the time of their appearance, both actually and relatively to other forms of trypanosome. (5) Of the posterior nuclear forms those with the nucleus near the centre of the trypanosome appeared first, those with the nucleus 107 near the blepharoplast last, while the forms with the nucleus between those two extremes made their appearance at an intermediate stage of the disease. THE CONSTANCY OF APPEARANCE OF POSTERIOR NUCLEAR FORMS IN THESE EXPERIMENTS Laveran® states that the morphological peculiarity of T. rhodesiense (posterior nuclears) in rats and mice is not constant. In these experiments, comprising twenty-four rats, there was no exception. Posterior nuclear forms were found in all cases in the preliminary count. Two rats only did not present the advanced form (C form) during this count, but this form was easily found on further search. 2. Second count. Two rats out of each group, each having a good infection, were chosen for the purposes of this count. A thousand trypanosomes were enumerated whenever possible in each daily film, and the posterior nuclear forms noted as before. The results of this more laborious procedure confirmed the conclusions arrived at as the result of the first examination, the earliest day on which posterior nuclears were first found being the sixth to the tenth. It appeared also that C forms occurred in greater proportion in those animals which lived the longest time, and that within the group of posterior nuclear forms the C forms generally increased relatively to the A and B forms towards the end of the disease. Table II gives an illustration of some of these points; it gives the number of posterior nuclear forms of the three kinds per thousand of trypanosomes during the course of the disease in two rats. POST-MORTEM RELATIVE INCREASE OF POSTERIOR NUCLEAR FORMS In several of the rats from which films were taken after death it was observed that the proportion of posterior nuclear to other forms increased considerably. This phenomenon appears to be related to the observation that frequently after death the ‘short stumpy’ forms of parasite in 7. rhodeszense resist the processes of disintegration in the blood of the dead host better than the long forms. Bevan and MacGregor? drew attention to this fact. It has also been recorded by Swellengrebel!® in the mouse, and by Blacklock!! in the rat. 108 Tasie II.—To show the dates of appearance of posterior nuclear forms of various grades in the peripheral bood of two rats infected with T. rhodesiense, — per 1000 trypanosomes. Rat 2230 C Rat 2231 A Date | } A B cul anda aie [7 o%e Sie mien oe rink sane Oct?'-*4 hal _ | — a | — = = (Inoculation) | ee pe rll Ace fatairaptcit = - 3 Gases — — | — = = = = (are —_ | — = = 22 = : 8 see —— | —— — —s — —— ” 9 I me 7} arg nee: he | se, TOUS: 6 3 I = = Se ELM. 2 2 = I == = | | | | ie ee ae — -— 4 2 — ” 13 I ie I 7 i | = iT ’ I4 I | ire 2 4 7k ei > 5 se == I I 6 2 I | - 16 ...| 4 I 3 | 3 fe) 5 4 sees 2 _ | I 8 } I I | { | rhea {ee Pes 14 12 6 7 I 5 ox og ES) 6 27 | 21 — == a | | 5 oF ad 32 31 22 = =F i * Less than 1000 counted on film. + After death of rat. THE SIGNIFICANCE OF POSTERIOR NUCLEAR FORMS IN T. RHODESIENSE Many explanations have already been advanced to account for these forms of parasite. Bevan!? states that the presence of short forms of trypanosome with the macronucleus slightly posterior to the centre is a common feature in many species, and especially in trypanosomes undergoing degeneration or taking on the resistant form in the presence of adverse or unusual conditions. This 109 explanation does not suffice to explain the typical posterior nuclear forms in 7. rhodesiense, because in this species it is not a case of the nucleus being slightly posterior to the centre merely. There are certainly many forms which conform to this description, but the typical posterior nuelear forms do not fall under this category ; they are something much more definite. As regards the suggestion that they are the product of degeneration, it is difficult to explain why they are not found when 7. gambiense degenerates. As previously stated, there is some evidence that such forms are capable of great resistance to the processes of disintegration in the cadaver. It appears improbable that the same phenomenon, ‘posterior nuclears,’ could be evidence at one and the same time of degeneration and resistance: it is possible, of course, that the trypanosomes may assume this arrangement of the nucleus in response to the demands of an unsuitable environment, and that subsequently if the environment continues to be unsuitable they degenerate, still retaining this arrangement of the nucleus. But the study of 7. gambzense under conditions where the parasites are obviously degenerating has not, up to the present, led to the discovery of such forms. ABERRANT FORMS Bruce (loc. cit.) refers to the posterior nuclear forms seen by him in the 7. rhodestense of Nyasaland as ‘aberrant’ forms. In view, however, of the fact that they are so constant in their appearance in this strain and form an integral part of it, it seems hardly justifiable to treat them merely as ‘ aberrant’ forms. DUE TO TECHNIQUE It has been stated that they may be due to methods of taking films, fixing or staining, but so many observers have noted their appearance, using different methods of treatment for the films, that it seems reasonable to discard this view. In seeking for an explanation of their presence, it is essential to discover, if possible, whether or not they ever occur in T. gambiense. Up to the present, so far as one is aware, they have not been described in this trypanosome. It may be argued 1@ te) that the number of 7. gambiense strains which has been examined is small. If, however, it can be established that they are entirely absent from 7. gambiense, it will be difficult to attribute their presence in 7. rhodesiense to such changes as degeneration, or to resistance, or to relegate them to the class of ‘aberrant’ forms. If further examination of strains of 7. gambiense fails to reveal their presence, it must be concluded that these forms constitute an important distinction between the two parasites 7. gambzense and T. rhodesiense. The fact that such forms are found in animal strains does not diminish their claim to attention, since animals are known to be infected with human trypanosomes. The argument, that if so many animal strains were capable of infecting human beings there would be a great amount of human trypanosomiasis in regions where it does not at present abound, is not conclusive. It has to be definitely proved that there is actuaily no human trypanosomiasis in such animal-infected regions. The experience in Nyasaland, and in Rhodesia, Northern and Southern, renders it necessary to be cautious in considering a region free from sleeping sickness. Even if human cases were rare, it may fairly be argued that we do not by any means fully understand the factors that govern the successful infection of human beings. FLY VARIATION If we regard 7. rhodesiense simply as a variation of the human parasite of the Gambia, it is possible that the explanation of their occurrence is to be sought, not so much in the conditions of their environment in the blood of human beings and in that of inoculated animals, as in other factors, for example, the transmitting agent, the fly. Against this hypothesis we have the facts that although T. vivax and T. pecorum are each known to be transmitted by both G. palpalis'3 and G. morsitans,\* they do not appear to be modified morphologically by the difference of carrier. It would be of considerable interest to ascertain whether in cases where 7. gambzense is transmitted by G. morsiztans such posterior nuclear forms are to be found in the blood of infected animals. ill MULTIPLE INFECTION The possibility of there being an infection in human beings with more than one species of trypanosome suggests itself. In favour of this one might adduce the facts that not only do animals in nature frequently suffer from mixed infections, but also that certain species of fly have proved themselves capable of transmitting more than one species of trypanosome. It seems probable that human beings are susceptible to mixed infection. The proof and demonstration of a double infection, given that the human being harboured species which were each _ universally pathogenic to laboratory animals would, with the means at present at our disposal, be practically impossible. In this connection one might refer to the original Gambian Horse Trypanosome described by Dutton and Todd"; in this case infection was considered to be caused by a single species of trypanosome, a view which is not now generally accepted. It is only where the pathogenicity of the trypanosomes taking part in a mixed infection is widely divergent as regards laboratory animals that proof of the presence of more than one sepcies of parasite can be given. Against the hypothesis of mixed infection in the case of T. rhodesiense is the evidence derived from transmission experi- ments. Kinghorn and Yorke,!® transmitting 7. rhodeszense by means of Glossina morsitans bred in the laboratory, found posterior nuclear forms in all animals which became infected. This implies, accepting for a moment the mixed infection hypothesis, that the flies which became infected acquired and passed on the mixed infection in each case. This is, perhaps, the strongest argument against the hypothesis. . iy eer - iret aes (nit Pt or T24. ' Ate & Je@ - 7 7. pe ee ee _ he a terre re TROSUCTION . : “— wer eed Tal wh migeettsies ty Tie therrverit : 7 ete lier dil ve, scat Mth ioe te eat) sith: +! lnm fSyueF * Waciat gts . x an Ti] 1 hye 20a vy Cascad teem pine “¥ wy the sade Teepitel, did (h o%—, Cine - ei hed Pidanigt eta Por’ the Thies? Res 2 | wag revert ene Shien ‘daring eg. these * tle Lariary Wark Pong cp, | nary = ; +. . vin? (re The “aeiicel fet 30 “The * )2 A ae 3 ivy a as - = 124 PLATE CX Fig. 6. Tsetse fly trap. Fig. 7. Pigmy bull of black breed from Southern Kordofan, said to be immune to ‘ fly.’ 125 SANITATION ON THE PANAMA CANAL ZONE, TRINIDAD AND BRITISH GUIANA BY DAVID° THOMSON, M.B., CH.B. Epin., D.P.H. CANTAB. (Beng part of the Report on the Malaria Expedition to Panama, 1912) (Recewved for publication 1 March, 1913) PLATES XI, XII, XIII CONTENTS PAGE INTRODUCTION Sn8 aco 586 as ass = “ee Act “of 125 COMMENCEMENT OF SANITATION ON THE ISTHMUS ... sic 5c mo! 128 SANITATION IN COLON... ae s- ae Bae Rt bes Bor 128 SANITATION IN PANAMA Town ... za oe eee ack ae os 129 SANITATION ON THE ZONE OF THE CANAL—AnTI-Mararia Work oee 130 AntI-YELLow Fever Work a ee aR ads “oe Be ss 136 SANITARY MEASURES ADOPTED TO SAFEGUARD AGAINST PLAGUE as ae 137 MEASURES AGAINST ANKYLOSTOMIASIS... dc Ai re ad hc 137 AnTI-TyPHOID AND ANTI-DyseNTERY Work AND GENERAL SANITATION ... 138 Tue HosPirats oN THE CaNAL ZONE SHE de ae =i ee 138 RESULTS OF SANITATION ON THE CANAL ZONE aoe 5 ase ade 140 SANITATION IN TRINIDAD 55 sa Re Ea see aie S07 141 SANITATION IN British GUIANA shi 3350 ee 290 Bae Soe 143 REFERENCES Spe we -- aa oF sc Sic oe Sc 146 EXPLANATION OF PLATES oe oe “6: re “es re Sc 148 INTRODUCTION In September, 1912, I was sent on an expedition to the Panama Canal Zone by the Liverpool School of Tropical Medicine to study certain malarial problems with Dr. James, Chief Assistant Physician to the Ancon Hospital. The American Canal Commission kindly allowed me to stay in the Ancon Hospital, and it was there that we conducted our researches. Although occupied for the most part with special research, I was nevertheless able, during my three months’ stay, to see most of the sanitary work going on. I received much information on the subject from the medical men in the 126 hospital, and Colonel Gorgas, Chief Sanitary Officer, Colonel Phillips and Mr. Le Prince, Chief Sanitary Inspector, were always willing to give me all the information that | required regarding their organisation and the methods employed. Moreover, I walked over most of the line of the Canal Zone with Dr. Orenstein, Chief Assistant Sanitary Inspector, who was most kind in showing me everything that he possibly could regarding the anti-malaria work. After all that I have seen there, I feel compelled to express much admiration regarding the great system of sanitation, and nothing was apparently left undone in the eradication of malaria and other diseases in general. There was no apparent defect in Colonel Gorgas’s sanitary administration. All trusted him in his work with implicit confidence, and the highest authorities gave him power to do what he considered best in great as well as in small measures. The money required was granted with the full belief that it was necessary for the good health of the American inhabitants, and for all workers on the Canal. They realised that good health was to be obtained at all costs, not only for its own sake, but in order to enable them to finish the great undertaking which they had commenced. The Americans, in the Canal Zone at least, are convinced that sanitation ‘pays.’ They had before them the great lesson of the disastrous failure of the French Canal diggers, and they have learnt that lesson well. No one who had not lived on the Canal Zone before the Americans took it over could possibly realise fully the great changes that have been brought.about. The Ancon cemetery, which is a veritable forest of tombstones erected chiefly to French victims (Pl. XII, fig. 9), helps one to realise that in bygone times the risk of death was very high. In an unhealthy tropical country like West Africa, there is always a feeling of insecurity. People are constantly sick, many die, and one feels that perhaps one’s own turn is coming next. Mosquitos abound, one is constantly bitten; mosquito nets may be used, quinine may be taken every day, and even though one 1s careful to drink only filtered or boiled water, yet a feeling of security against malaria and dysentery is never felt. So it was in Panama less than ten years ago. Formerly mosquitos were a plague on board vessels lying in Colon harbour, but since the American occupation that is all changed, and now no mosquitos come aboard. I 127 arrived on the Canal Zone fully equipped, among other things, with a mosquito net and plenty of quinine which are always so necessary in tropical expeditions, but soon realised that both the mosquito net and the quinine were not needed. There were no mosquitos about, and every American house was mosquito-proof. My three months’ stay covered the latter part of the rainy season and the commencement of the dry season, yet during all that time I feel sure that 1 was not bitten by a single mosquito,* and in consequence | did not take one single grain of quinine. The water supply was good, and was frequently examined bacteriologically. Healthy European women and children were met everywhere, no one seemed to be ill, and one felt as secure from disease as anywhere in temperate climates. I have been in Sierra Leone, Forcados, Bonny and Old Calabar on the West Coast of Africa, also in Trinidad, British Guiana and Jamaica. In all of these places I was bitten by mosquitos, even though my stay had been only for a few days. In all I had to take quinine as a prophylactic against malaria. In these places, moreover, there are not so many white women and children seen as on the Canal Zone at Panama. In West Africa there are no white children and few white women, for the simple reason that it is too unhealthy. I think I have said enough to indicate, without going into the statistics of death-rate, etc., that Panama, once notorious as a white man’s grave, is now as healthy as most temperate countries, and the favourable reports which may be read concerning this matter are in no way exaggerated. I have known several traders whose occupation has brought them many times to the Canal Zone during the last ten to fifteen years, and all alike marvel at the great changes which have occurred for the better in so short a time. Most of the authentic reports on the improved health in Panama have been gleaned from the statements made by Colonel Gorgas. He has in no way exaggerated the results of his sanitary work. Again, it must be remembered that this greatest achievement in tropical sanitation, which the world has yet seen, reflects great credit, not only on Colonel Gorgas and his staff, but also on the highest officials on the Canal Zone and on the American Government who have supplied the money without * Mosquitos are fairly common still at Frijoles and Monte Lirio. 128 complaint and without putting obstructions in the way, actively or passively, and who have been among the first to realise that thorough sanitation in the tropics ‘ pays financially.’ 3 COMMENCEMENT OF SANITATION ON THE ISTHMUS Plans for the sanitation of Colon and Panama formed part of the discussion at the American Commission of 1899-1901. These towns, although situated on the Canal Zone, belong to the Republic of Panama. In January, 1904, the quarantine of Colon and Panama was turned over to the United States, and in June of that year the permanent sanitary organisation was established with Colonel W. C. Gorgas as head and Dr. H. R. Carter, a yellow fever expert, as the Director of Hospitals. The work was hampered by scarcity of supplies, notably copper wire screening, which could not be purchased in the United States in large quantities. An epidemic of yellow fever, lasting from July, 1904, to December, 1905, accelerated the delivery of supplies, and made it necessary to expedite the sanitation work, lest the force slowly organising be depleted. There were 246 cases and 84 deaths, all of which were among the non-immunes who had come to the Isthmus on account of the Canal work. The Americans commenced sanitation, therefore, before attempting any operations on the Canal. They realised that it would be advantageous to make the place healthy before bringing the white population there. They were pioneers in that they built every house mosquito-proof. SANITATION IN COLON This town is situated at the Atlantic end of the Canal. It has a population of 18,000. In 1904 it had a population of 10,000, and about 9,000 of these lived in shanties built on piles. At high tide the houses were surrounded with water, so that no one could walk along the streets without. danger of falling into the mire (Pl. XI, figs. 1, 3). Behind the town was a large mangrove swamp. There was no proper water supply, and no sewage system. Mosquitos abounded, and malaria and other diseases ran riot. Since then the town site has been filled in by the Americans. It has been raised several feet, so that it is now dry, well paved (fig. 2), 129 clean, well drained and healthy, with a modern sewage system and water supply. The Panama Republic and the Panama Railway Company are paying for the work. SANITATION IN PANAMA TOWN Panama is situated near the Pacific end of the Canal, and has a population of about 37,000. As already stated, there was a yellow fever epidemic raging there from July, 1904, to December, 1905. This, however, was the last of the disease in that town. The Americans in a little over a year eradicated it entirely, and the conditions are now such that it could hardly exist there again. This alone is a triumph which must have already paid financially many fold. Panama Town had previously been an endemic yellow fever centre for centuries, and in a little over one year the disease was stamped out and not a single case has occurred there since. The methods adopted to control the epidemic were isolation of all cases as well as suspected cases, in mosquito-proof quarters, and thorough fumigation of all houses which had cases or suspected cases, in order to kill the mosquitos in them. At the same time a campaign was started to destroy the breeding places of the Stegomyia fasciata. At that time every house in Panama had roof gutters and water tanks or barrels, this being almost the sole water- supply of the town. There was no proper drainage or sewage system. The streets were unpaved, without proper gutters, and in the rainy season became a hopeless quagmire (fig. 4). The conditions, therefore, favoured greatly the breeding of the Stegomyia fasciata. The Americans employed no makeshift methods in remedying these defects. They brought in a proper pipe-borne water-supply from a reservoir. They installed into every house modern closets, with a pipe-borne sewage system. They paved and guttered every street (fig. 5), and removed as far as possible all the roof gutters from the houses along with the water barrels. They enforced strict sanitary regulations on the inhabitants of the town, and established a health department to see that these regulations were carried out. In addition, the ground floors of the houses were rendered uninhabitable to rats by cementing, and cement gutters were constructed alongside the pave- 130 ments in order to catch the rain from the gutterless eaves of the houses. These were the drastic measures enforced on _ the inhabitants of Colon and Panama. The Americans lent the money and supplied the labour. It cost the two towns about £455,000. They are paying it back with interest through water rates which are collected directly by the United States. The inhabitants who had lived there in a slovenly way without interference, as_ their forebears had done for centuries, were naturally very angry at this apparent tyranny, but already they are becoming satisfied. Those who have families have already noticed an unprecedented freedom from disease among their children. Plague, typhoid fever, dysentery and malaria are no longer to be feared, and now they realise that what was done was good. The sanitation of Colon and Panama is to-day as up-to-date as in any town in Europe. The water-supply is examined very frequently by expert bacteriologists. The quarantine at Panama and Colon is-most rigid. Guayaquil in Ecuador is an endemic yellow fever centre, and is only about four days’ sail from Panama, hence a very strict quarantine is necessary. In fact, if the Ecuadorians do not soon take steps to make their town sanitary, it is highly probable that the Americans will force them to do it, as it is a constant menace to the health of Panama. Trinidad and all Venezuelan and Colombian ports are under quarantine for yellow fever, though I believe that since my return Trinidad has been removed from the list. SANITATION ON THE ZONE OF THE CANAL—ANTI-MALARIA WORK On the Canal Zone the most prevalent disease has been malaria, and much of it is pernicious malaria. The sanitary measures adopted were, therefore, principally anti-mosquito measures. Countless swamps and Anopheles breeding places existed along the line of the Canal. A huge swamp lay behind Colon, and extensive swamps also surrounded the town of Panama. After enormous labour, these have been filled up by material excavated from the Canal. Two methods have been adopted in filling the swamps. One consists in dumping material (rock and earth) into them from railway waggons. The other method, known as a ‘hydraulic fill’ (Pl. XII, fig. 8), is to force into them a liquid stream of mud 131 and sand through pipes leading from large suction dredgers employed in deepening the Canal. In many cases these pipes are laid long distances, even a mile or more distant from the Canal. I have seen swamps filled in this way to the depth of many feet, so that only the tops of the trees which grew in these swamps are now visible (fig. 8). Large unhealthy areas have been obliterated in this way. The present town of Balboa on the Pacific entrance to the Canal is being built on one of these filled-in swamps. Strictly speaking, however, these huge undertakings can hardly be considered as anti-malaria work. In describing this, I cannot do better than give an abstract of a paper by Colonel Gorgas (1909) on the subject. He states that the anti-malaria work on the Isthmian Canal cannot be well understood unless preceded by a brief description of the sanitary organisation as it bears upon this work. The bulk of the activities of the Department of Sanitation on the Isthmus has no immediate bearing upon sanitation, namely, that which deals with religious instruction, care of the sick, care of the insane, of lepers, street cleaning, garbage collection, etc. The Department of Sanitation spent in 1908 some two million dollars, but only five hundred thousand dollars (£100,000) of this was spent on pure sanitation. The American Canal Zone consists of a strip of land ten miles wide, of which the Canal is the centre. This strip extends about forty-five miles in length from North to South. The population to be protected against malaria consists of about 50,000 labourers and their families, and is scattered all over this five hundred square miles, though they are principally collected along the line of the Canal, more particularly into some forty camps and villages near this line. The temperature, rainfall, and character of the terrain are all excellently suited for the breeding of Anophelines all over this territory (fig. 7). The rainfall averages over 100 inches yearly, and though there are four months in which there is practically no rainfall, there is enough water for the Anophelines to breed freely during these four months. During the five years of occupancy of the Isthmus up till 1909, 250,000 people have resided in this zone, and these have been located principally in places formerly unoccupied along the line of the Canal, and as the villages are intended for only temporary 132 occupancy, the conditions are a good deal like those of an army going into a new country, and increase the difficulty of sanitation enormously. The anti-malarial measures consist : — 1. In destroying the habitat of the Anophelines, during their larval stage, within a hundred yards of dwellings. 2. Destroying within the same area all protection for the adult mosquito. 3. Screening all habitations so that mosquitos cannot have access. 4. Where breeding places cannot be destroyed by draining, use is made of crude oil and larvicide. These measures are given in the order in which they are considered important. For the purpose of carrying into effect these measures, the five hundred square miles of territory have been divided into seventeen districts. These districts are under the charge of a Chief Sanitary Inspector, who has in his office the necessary clerical force and three assistants. One of these assistants is especially competent in the life-history and habits of the mosquito, another in knowledge of ditching, tile-draining, etc., and the third in knowledge of general executive work. Each of the seventeen districts has a District Sanitary Inspector in charge. Each District Inspector has a sufficient force of labourers (forty to fifty) to do the necessary ditching and draining. A force of carpenters is required to keep the screening in repair, and there are one or two quinine dispensers who are kept constantly going around giving doses of quinine to those who desire it. It is not attempted to enforce prophylactic quinine. The Chief Sanitary Assistants are kept constantly going over the work advising and instructing the District Inspectors. The District Physician sends in daily to the central office a report of the number of cases of malaria and the number of the employees from which these cases come. This report is made up weekly in the central office. A copy is sent to each District Inspector, and he is held responsible for any excess of malaria in his district. If the admission rate for malaria during the week rises above 1} per cent. something is considered wrong, and the assistants of the Chief Sanitary Inspector are sent to look over the 133 ground to try to discover the cause. The District Inspector, for the purpose of doing away with the breeding places of larvae, puts down tile drains wherever they would be suitable. This is considered the most effective and economical form of drainage. After it is laid down it requires no more attention. There is no breeding place left for mosquitos, as no water whatever is exposed at the surface. A horse mower or scythe can be used for cutting the grass over it. Where tile-drainage cannot be used, open concrete ditches are put down. The first cost of this is nearly as great as that of tiling, and a certain amount of labour is necessary to keep the ditches clear. They must be swept out once a week. If the ground cannot be drained in either of the above ways, open ditches are used. This is the least effective and most expensive form of drainage. In Panama they fill up rapidly with grass, and have to be cleaned out about once in two weeks. They are always breeding places for mosquitos, and have to be constantly treated with oil or larvicide. For the purpose of doing away with places which will harbour the adult mosquito, the inspector clears the ground of brush and grass for a hundred yards around the place to be protected. Where the locality is to be occupied for a year or more, it is more economical to grade the ground and plant grass, which can then be kept down with a horse mower or scythe. A limited amount of shrubbery and a few trees about a dwelling are not objectionable. The inspector keeps the wire screening in repair by constantly going over it with a force of carpenters. Good wire should last on the Isthmus at least three years. The inspector uses crude petroleum or larvicide in such places as cannot be drained, and in temporary pools caused by construction or at temporary camps where it would not be economical to drain. Usually when a new location is occupied the malaria rate is high, frequently as high as 25 per cent. a week, but always in the course of a month or two, when the ground is drained and the brush cut, this drops to a rate somewhere about 1 per cent. The above methods could be applied to a considerable extent to military organisations. Where troops remained at one camp for a week or longer, it would be practicable to clear and drain the ground. It is most important that the Sanitary Inspector should attend to the work himself. The men 134 who do the ditching and brush-cutting, etc., should be immediately under his control, and he should be held responsible for the proper performance of the work, or in other words, the work must be constantly supervised by a man who has a considerable knowledge of these anti-malarial measures. Prophylactic quinine is looked upon as an important measure, and is offered in three grain doses to all employees who will take it. It is placed on the table in all the messes in liquid and pill form, and one to three dispensers are employed in each district who go around the various villages. Fuller details may be had from articles by the following authors :—W. C. Gorgas (1904, 1906, 1907, 1908, 1909, 1910, 1912), Attia (Orenstein, (19Ul, 4d012)).Jie5 A- pile 4Pmnecs, Goes), sand S. T. Darling (1910, 1912); also from the Reports of the Department of Sanitation of the Isthmian Canal Commission by W. C. Gorgas, and from the Canal Record. The anti-malarial work, therefore, on the Canal Zone consists simply in carrying out thoroughly and in a straightforward manner the anti-malarial principles laid down by Ross soon after his discovery of the transmission of malaria by the Anopheles mosquito. Yet simple though these principles seem, the methods and organisation of Colonel Gorgas deserve the most careful study. His system has produced most remarkable results in a country which was exceedingly unhealthy and in which the difficulties were great and many, and although the construction of the Canal brought unlimited labour and material to his aid, yet it also greatly increased his difficulties, because it is well known that malaria tends to increase greatly where land excavation is going on, on account of the innumerable new breeding pools which are produced by these excavations. This alone necessitates the constant inspection and tackling of ever-changing conditions. The working population is constantly changing its location, and this also involves many difficult sanitary problems and increase of sanitary work. The working population on the Canal moves about so much that these methods are even suitable, as Colonel Gorgas has said, for military organisations in the tropics. It seems to me that the keynote of the success 1s thoroughness in every detail. With regard to mosquito- proof buildings, even the signal-boxes on the railway are mosquito- proof, and special arrangements have been made so that mosquitos OO 135 cannot gain an entry where the wires run into these buildings to the operating levers. The quarters in which the European labourers live are mosquito-proof, but it is found that they are careless in leaving the doors open at times. To safeguard them, mosquito catchers are sent into these dwellings every morning to catch the mosquitos which have gained entry. Most of the insects are filled with blood, having fed during the night. Many mosquitos which might have become infected are therefore destroyed. Dr. Orenstein (1912) has found that this procedure reduces the malaria rate. Oil and larvicide are used so freely that it is very difficult to find any trace of larvae in pools not yet drained or filled in or in ditches not yet concreted. In order to emphasise further the thoroughness of the sanitation on the Canal Zone, I give the following list of the personnel under Colonel Gorgas. The personnel of the Division of Zone Season is as follows :— 1 Chief Sanitary Inspector. 1 Chief Assistant Sanitary Inspector. 2 Division Inspectors. 20 Inspectors. 1 Inspector—Entomologist. 18 Foremen and 226 labourers. The following is a summary of the routine work accomplished by this personnel during the month of July, 1912 :— Anopheles work : lnimear feetyofiditelies, cleaned ijsccscctraobens des cxeaslistw: 129,150 Dineambect of new. Gitches ues es Tamperatu | Se ! PANE AACA ARBOR BER UE JG GE00 TECHNIQUE As stated, 10 c.c. of blood were drawn, with aseptic precautions, from the median basilic vein on two occasions, at the time when the temperature was at its height, on the 11th February and again on the 13th February (see chart). 155 The blood on both occasions was transferred to a large sterile test tube and defibrinated; it was then distributed into several smaller test tubes and incubated at 39°C. The corpuscles settled gradually, leaving about one half-inch of clear serum above them. The amount of dextrose solution added was slightly more than the amount recommended by Bass (1912). EXAMINATION OF CULTURES Experiment I. A blood smear, taken at the time of the with- drawal of the first 10 c.c. of blood on the 11th February, showed 85 per cent. of young ring parasites and 15 per cent. of three-quarter grown forms, also a few gametocytes. After nineteen hours’ incubation there were only 3 per cent. of young rings, 17 per cent. were one-quarter grown, while 70 per cent. were one-half to three- quarters grown, and 10 per cent. showed signs of presegmentation. After twenty-three hours the majority of the parasites were three- quarters grown. No further examination was made till the forty-fifth hour of incubation, and it was then found that 80 per cent. were small rings, and 20 per cent. were one-half to three-quarters grown. Sporulation had, therefore, occurred between the twenty-third and the forty-fifth hours of incubation. Experiment II. Blood smears taken at the time of the second withdrawal of 10 c.c. of blood, on 13th February, showed 96 per cent. of young rings, the remaining 4 per cent. of the parasites being about one-half grown (Pl. XIV, figs. 1-3). After eight hours’ incubation, 4 per cent. were young rings, 80 per cent. were one- quarter grown, and the remaining 16 per cent. were three-quarters to full-grown (figs. 4-10). After twenty hours, 65 per cent. were one-half to three-quarters grown, with only 2 per cent. of young rings, and a few sporulating forms (figs. 11-17). After twenty-nine hours, 89 per cent. were young rings (fig. 18), 8 per cent. were three-quarters grown, and again a few sporulating forms were found. It is, therefore, evident that in this experiment the maximum sporulation, which we again unfortunately missed, occurred between the twentieth and twenty-ninth hours of incubation. Although the blood was drawn during the height of the temperature, that is, when the majority of the parasites would have 156 been expected to be very young, yet they attained their full development and sporulated in little more than half the time which would have been thought requisite for this development. This, however, is in accord with the results obtained in the cultivation of P. falciparum by J. G. Thomson and McLellan (1912). They found that sporulation occurred after twenty-six hours of incubation. We do not think that the 96 per cent. of young rings shown at the time the blood was drawn could have been twenty hours old, so that it must be concluded that growth took place more rapidly in the tube than it would have done in the circulating blood of the patient. MORPHOLOGY OF THE PARASITES IN CULTURES The following remarks refer to Experiment II, and are illustrated by the accompanying plate (Pl. XIV). The morphology of the benign tertian parasite (Plasmodium vivax), as it occurs in the peripheral circulation, is well known. Unlike the malignant parasite, all stages may be found in the peripheral blood. In malignant tertian, as a rule, only ring forms are found, as the further development of the parasite takes place in the capillaries of the internal organs. In the benign tertian several stages of the development are usually found in a single smear of peripheral blood. Even the full-grown parasites of benign tertian, which, by the way, are much larger than the full-grown malignant Plasmodia, are not arrested in the fine capillaries of the internal organs, but are apparently able to circulate freely. A pure case of benign tertian malaria, in which the parasites are all in the same stage of development, is very rarely obtained, yet, as a rule, the majority of the Plasmodia are found to be of the same age. In the cultivation of Plasmodium vivax, therefore, if a full-grown parasite, or even a sporulating form, is found, it must not be concluded that development has taken place in the culture tube. In order to avoid being deceived by this, we have made careful differential counts in the smears taken at different periods of cultivation. In our cultures the blood was drawn shortly after sporulation of most of the parasites, so that the majority were young rings. Figs. 1 and 2 show corpuscles with young rings, and already the corpuscles contain many Schiiffner’s dots, and are somewhat 157 enlarged, having a diameter of Qu and 10m respectively. In fig. 3 the corpuscle is 11°5“ in diameter, and contains a parasite rather more than one-quarter grown. After eight hours’ incubation the blood was again examined, and it was found that the parasites had increased in size in a very marked degree; the majority were now one-quarter grown, and many were three-quarters to full-grown. Fig. 5 shows a parasite about one- quarter grown. Figs. 7 and 8 show half-grown parasites with scattered pigment and the chromatin greatly increased in quantity. Figs. 9 and Io represent full-grown parasites, and show the collection of pigment into a loose mass and marked increase of chromatin. In twenty hours the majority of the parasites were half, three- quarters, or full-grown, and several were sporulating (figs. 11-17). Fig. 15 shows the parasite with chromatin divided into seven particles, or daughter portions. Figs. 16 and 17 show typical sporulation, with production of 14 or 15 merozoites. In culture it appears that the number of spores produced in benign tertian is less than that produced in the malignant form of the parasite (Thomson and McLellan, 1912). The maximum number of spores in Plasmodium vivax averages about sixteen, but in some cases a few more may be produced. The culture was not again examined until the twenty-ninth hour, and it was found that the majority of the parasites were then young rings (fig. 18). There was, therefore, evidence that sporulation had occurred and the young merozoites produced had entered new corpuscles. No further asexual development took place. DISCUSSION OF RESULTS In the paper by J. G. Thomson and McLellan (1912) it was noted that the parasites, after an incubation of twelve hours, showed a definite increase in size, the pigment being collected together into a circular compact mass. In twenty-four hours the malignant tertian parasite was found to undergo segmentation. The maximum number of merozoites counted was thirty, and it is quite probable that thirty-two is the greatest number of merozoites produced by Plasmodium falciparum after complete segmentation. In their remarks, J. G. Thomson and McLellan pointed out the 158 great difference of opinion among various observers regarding the number of spores produced by the segmentation of the malignant tertian parasites, and reasons were given for this. In some slides of smears of the spleen and bone marrow made during autopsies of several cases of comatose malaria (P. faldci- parum), sent by Dr. James from Panama, very numerous sporulating parasites were found. The number of spores in these preparations was counted by Sir Ronald Ross and one of us (D. T.). The following table gives the number of spores found in one hundred fully-developed malignant tertian parasites (P. falczparum) from a spleen smear of a case of untreated comatose malaria, Panama :-— 13 per cent. contained 32 spores. 2 ” ” ” 31 ” ner eee » BOs ae oa 7 ” ” ” 29 38) 25 ” ” ” 28 ” 3 ” ” ” 27 ” 17 »9 ” ” 26 ” 2 55 ” ” 25 yy 10 49 9 ” 24 ” 3 ” ” ” 23 ” 5 >” >” 9 22 ”’ 2 D9, ”’ ”’ 20 LB) UGS “13 5, 37 ,, from 20 to 32 spores. These numbers lead us to conclude that the maximum number of spores, after complete segmentation, may be as high as thirty- two. This is entirely in agreement with the findings in culture by J. G. Thomson and McLellan, and is a higher number than has, up till recently, been given. Some observers have put the number as low as eight to ten (Stephens and Christophers, 1908). Again, in a placenta smear from a case of comatose malaria, P. falciparum, Panama, sporulating parasites were found which did not fill the entire corpuscle. The average number of spores found in these was only eight. The patient, however, had received 60 grains of quinine during the twenty-four hours preceding death, so that the sporulation was either atypical due to the quinine, or else the parasites had not reached their maximum development. Dr. James, of Panama, also believes that those forms containing few spores are not completely developed, or are atypical sporulating forms due to the action of quinine. 159 It may now be stated with confidence that the malignant tertian parasite is capable of producing, under favourable conditions, a maximum of thirty-two spores, as proved both by culture and by examination of autopsy smears of cases of untreated comatose malaria. Last year Sir Ronald Ross taught his students that the maximum number of spores produced by the malignant tertian parasite was 2°, while that of the benign tertian parasite was 2+. Another point of great importance, noted in the cultivation of P. falctparum by Thomson and McLellan (1912), was that there was a great tendency to clumping of the parasites, both before and after segmentation. This point is of great interest since we have found no such tendency in the case of cultures of benign tertian. This phenomenon explains why the malignant tertian parasites accumulate in the capillaries of the internal organs after attaining a certain size, only the corpuscles containing the young forms being able to circulate in the peripheral blood. It would also explain the blocking of the capillaries in the brain in the comatose form of the disease. In our cultivation of benign tertian, as already stated, no such tendency to clumping of the parasites has been noted, and they always remain well distributed throughout the smears. This observation is in accordance with what we should expect, since in the benign tertian there is little tendency to blocking of the capillaries, with the resulting coma and other pernicious symptoms. The corpuscles containing all stages in the development of this parasite are able to circulate in the peripheral blood. Another marked feature to be made out in cultivation is the distribution of the pigment. In the figures given by J.°G. Thomson and McLellan for malignant tertian, it is clearly shown that the pigment collects into a circular compact mass before segmentation occurs, and remains so until the complete formation of merozoites. This has also been seen in smears from the internal organs. In the cultivation of P. vzvax the pigment does not collect so early in the growth of the parasite, and at no time does it appear to form so dense or compact a mass as found in cultures of P. falczparum. There was no evidence of haemolysis in the culture tubes. 160 POSSIBLE FORMATION OF GAMETOCYTES IN CULTURES In a paper by one of us (D. Thomson, 1911), it was concluded that crescents develop from the youngest forms of the asexual parasite when a certain amount of immunity or resistance towards the latter had developed in the human blood, and that the time required for their development into an adult gametocyte was about ten days. Reverting now to a consideration of the cultural development of P. vivax, it was found that only one generation of asexual parasites developed in the culture tubes. After the first sporulation the parasites remained small, and did not pass through another asexual phase. On the 5th day, however, it was noticed in our cultures (P. vzvar) that the parasites had changed much in appearance, since all of them had become heavily pigmented and compact (figs. 19-22), even the very youngest forms contaimed pigment (fig. 19). There were no amoeboid straggling forms, all were round and compact, and varied in size from very small circular pigmented bodies to forms as large as 4m to 5m in diameter (fig. 22). The pigment was scattered more or less evenly throughout, and the chromatin consisted of a single mass (fig. 21). The protoplasm had a greyish-blue colour, quite unlike that of the asexual phase. On the eighth day there was a very marked increase in the number of large forms, varying from 44 to 6p in diameter (figs. 23, 24). All were circular, compact, and very heavily pigmented. On the tenth day the cultures had dried up too much to permit of a further examination. We are doubtful as to the true explanation of these bodies. They suggest gametocyte formation from their appearance, being circular, compact, and heavily pigmented, and there were many more large forms on the eighth day (figs. 23, 24) than on the fifth day, which would appear to indicate that these bodies had been growing in size. On the other hand, we must consider the possibility that these are degenerating parasites, though it is difficult to understand why degenerate parasites should develop such a large amount of pigment, or why they should become compact and show an apparent slow growth. We were unable to obtain any flagellation in these bodies, and the corpuscles containing them appeared to be shrunken. e ——_- 161 SUMMARY 1. The benign tertian malarial parasite is capable of being cultivated up to the stage of sporulation, for at least one generation. 2. In our cultures the growth of the parasite from young rings to sporulating forms took place more rapidly than in the blood of the patient. 3. The cultures of benign tertian differed from those of malignant tertian in that there was no tendency to clumping of the parasites in the former, either before or during sporulation. 4. This difference appears to us to explain in a satisfactory manner why only young forms of malignant tertian are found in the peripheral blood, as the clumping tendency of the larger forms causes them to be arrested in the finer capillaries of the internal organs. It also explains the tendency to pernicious symptoms, such as coma, in malignant tertian malaria. All stages of the benign tertian parasite are found in the peripheral blood, and there are seldom pernicious symptoms, because there is no tendency to clumping. 5. Heavily pigmented, compact parasites were found in our cultures of benign tertian on the fifth day. On the eighth day these had grown larger, and their appearance suggested the development of gametocytes. 6. The malignant tertian parasite (P. falczparum) is capable of producing, in maximum segmentation, thirty-two spores. On the other hand, benign tertian (P. vzvax) produces, as a rule, during maximum segmentation, sixteen spores; sometimes more may be produced, but the number is never thirty-two. 7. The pigment in P. falciparum collects into a definite, circular, and very compact mass early in the growth of the parasite. On the other hand, during the growth of P. vivax the pigment remains scattered in definite granules throughout the body of the parasite, till just before segmentation, when it collects into a loose mass of granules in the centre of the full-grown Plasmodium. REFERENCES Bass, ©. C. & Jouns, f. M. (1912). The Cultivation of Malarial Plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. Journ, Exp. Med., XVI, pp. 567-579. Sreruens, J. W. W. & Cunisrornens, S. R. (190%). The Practical Study of Malaria, p. 44. Tnomson, D. (1911). A Research into the Production, Life, and Death of Crescents in Malignant ‘Tertian Malaria, in Treated and Untreated Cases, by an Enumerative Method, Ann. Trop. Med, and Parasitol., V, pp. 57-82. 6 charts. ‘T'nomson, J. G. & McLean, S. W. (1912), The Cultivation of one Generation of Malarial Parasites (Plasmodium falciparum) in vitro, by Bass's method. Ann. Trop. Med, and Parasitol,, VJ, pp 449-462. 2 plates. 3 CEM aren 3o°-porEmOUR w eboeh ee eii Bdidit pie tie OREO Arvest AT rpbyds sunersanh: in thoreict 1 “tie « Bik } Welows > aed 4 Wdergiq wf! Aero tee tay} ant) pieenanat A ott Jeeins zw * in megiqois ae 3) eteds Soe. (isieroirciag 4. TE Ripe ttt a Sie it ere? whe avers eaTiziige ‘wos ’ 2% af By* fd sures) Sqr Woe Tt oF > rat mi Sethe; DidTys “pnp mes ' it Osa : J ad Maw + : i Srateh sate ieee) 21 ts) pisetgey Qed 4 - ges tt ; ¢ See é 2 FE SQUZRIEG Leute eee) tt ‘irksgsg2 ' co"! 7% 7 ok. >it ‘SI IauE OG ules wv&D > P . : 3 . 4 ts ane Asoo sid) apifine 8 ’ » - ‘ | boast ae elteet 9 5 4 ‘ M ~ stele oma fee bal (bic niaan bP USEERLet verb eet ag Volume VII June, 1913 No. 2. ANNALS OF TROPICAL MEDICINE AND PARASITOLOGY ISSUED BY THE LIVERPOOL'SCHOOL OF ‘TROPICAL MEDICINE Editor Prorrssor Sir RONALD ROSS, K.C.B., F.R.S., Mayor I.M.S. (REt.), NED? DP... Fates. Dieses bbsb. In Collaboration with Prorrssor J. W. W. STEPHENS, M.D. Cantab., D.P.H. Peoressor kk: NWEWSPEAD, M.Sc. J-P.,, F.R:S.,. ALS. EF.E.S., Hon. F.R.H.S. Prarmsor J. L. TODD, B.A. M.D‘, -C.M. McGill, “D.Sc: Liv H. WOLFERSTAN THOMAS, M.D., C.M. McGill. ANTON BREINL, M.D. feeb. PANTHAM, 1).Sc..Lond.,. -B:Ac Cantab, A-R-C.S,. F:Z:5. Editorial Secretary Dr. H. B. FANTHAM, School of Tropical Medicine, The University, Liverpool. C. Tinling & Co., Ltd. Printers to the University Press of Liverpool 53 Victoria Street * = * ct , ee 7 A aid soreseGay — - ; seine ~ tr ) é Pea «| orasionT . s PS ® | f a aq A t4 oth ig - { a d = - - % rT a A — > ~ oa - ~~ - 2 nes 3 =~ >. 4 LIVERPOOL THE INCORPORATED SCHOOL oi, ERoriCcalL, MEDICINE FouUNDED By SiR ALFERD LEWIS JONES, K.C.M.G. Hon. President: Chairman Vice-Chairman : Hon. Vice-Presidents : MrT i |: EAD, Vice-Chancellor Si Sir W. B. BOWRING, Bart. Professor R. CATON ) Professor W. HERDMAN, F.R.S. Professor C. SHERRINGTON, F.R.S. ! Dr. C. J. MACALISTER Mr. G. P. NEWBOLT j Mr. C. BOOTH (Jun.) Mr. T. F. HARRISON Mr. A. R. MARSHALL Mr. W. ROBERTS Mr. J. W. ALsop (Affiliated with the University of Liverpooi) H.R.H. Princess CHRISTIAN OF SCHLESWIG-HOLSTEIN. : Mr. F. C. DANSON. Professor R. CATON. THE DuKE oF NORTHUMBERLAND, K.G. THE EARL. UB CEREY,. (1.G-V.0. EARL CROMER, G.C.B. ViIscouNT MILNER, G.C.B. Lorp PIRRIE, K.P. SrR OWEN PHI LIpps, K.C.M.G. Sirk Epwin DurRNING LAWRENCE, Bart. SIR WILLIAM H. LEVER, BART. Mr. O. HARRISON WILLIAMS COMMITTEE C.M.G. Colonial Office RALFREDDALE University of Liverpool Council of University of Liverpool Senate of University of Liverpool | Royal Southern Hospital j Steamship Owners’ Association Shipowners’ Association ) | | } Mr. G. BROCKLEHURST Mr. C. LIVINGSTON Mr. J. A. TINNE Mr. J. W. W. DANSON Professor E. W. HOPE Mr. R. RANKIN Professor E. GLYNN Mr. A. L. REA, Hon. Treasurer Mr. A. H. MILNE, C-M:..Ge; Secretary. B 10 Exchange Buildings, Liverpool Staff, 1913 1. At the University of Liverpool Professors - - JOHN WILLIAM WATSON STEPHENS, M.D., Cantab., D.P.H. Sir Alfred Fones Professor of Tropical Medicine ROBERT NEW SPEAD, BUR GS. Wisc. lees. F.E.S., Dutton Memorial Professor of Entomology MAjJOR “Sik RONALD] ROSS, KGB, BRS CE De ER €)S., DEH. MoD: Dzse sNobel Laureate 1902. (Indian Medical Service, retired). Professor of Tropical Sanitation Vacant. Walter Myers Professor in Parasitology Assistant Lecturers - HAROLD BENJAMIN FANTHAM, D.Sc., B.A.., Assistant Lecturer in Parasitology HENRY FRANCIS CARTER, S.E.A.C. Dipt., F.E.S., Assistant Lecturer in Entomology HAROLD WOLFERSTAN THOMAS, M.D., C.M. Flonorary Lecturers - J.O. WAKELIN BARRATT, M.D., D.Sc., (Lond.) Mayor JOSEPH FITZGERALD BLOOD, M.D., M.Ch. (Indian Medical Service, retired) PRoF. ERNEST EDWARD GLYNN, M.A., M.D., (Cantab.), M.R.C.P., MIR.C:S: Pron. EW. HORE Maps. DSc. WILLIAM THOMAS PROUT, M.B., C.M.G. Honorary Statistician WALTER STOTT Yellow Fever Bureau HARALD SEIDELIN, M.D. (Scientific Secretary). Assistant in Cryotherapy JOHN GORDON THOMSON, M.A., M.B., Ch.B. Bibliographer - - WALTER DRAWZ 2. At the Runcorn Research Laboratories Director - - - WARRINGTON YORKE, M.D. Assistant - = - B. BLACKLOCK, M.D., D. PH. 3. At the Royal Southern Hospital, Liverpool Physicians - - CHARLES JOHN MACALISTER, M.D., FUR (CxRe JOHN LLOYD ROBERTS, M.D., M.R.C.P. Surgeons - = - D. DOUGLAS CRAWFORD, F.R.GE:S. ROBERT JONES, F.R.C.S., Ch.M. GEORGE PALMERSTON NEWBOLT, M.B., BRE .S: Tropical Pathologist - JOHN WILLIAM WATSON STEPHENS, M.D., Cantab. Clinical Pathological Assistant - DAVID THOMSON, M.B., Ch.B., D.P.H. 4. On Expedition HAROLD WOLFERSTAN THOMAS, M.D., C.M. Laboratory JOHNSTON LABORATORY, UNIVERSITY OF LIVERPOOL Research Laboratory : RUNCORN Hospital : ROYAL SOUTHERN HOSPITAL, LIVERPOOL Secretary's Office: B 10, EXCHANGE BUILDINGS, LIVERPOOL NOTICE The following courses of instruction will be given by the Liverpool School of Tropical Medicine during 1913 :— Full Course begins 6 January. Advanced Course begins 2 June. Diploma Examination, 7 April. Certificate Examination, 30 June. Full Course begins 15 September. Diploma Examination, 15 December. The full Course of Instruction is open to all qualified medical men, and the Examination to all students who have taken out this full course. Fee for the full Course of Instruction—Thirteen Guineas. Fee for the Diploma Examination—Five Guineas, Fee for the Short Course of Instruction—Four Guineas. Fee for the use of a School microscope during one term—Ten shillings and sixpence. For prospectus and further information, application should be made to the Dean of the Medical Faculty, University of Liverpool. The following have obtained the Diploma in Tropical Medicine of the University of Liverpool :— Diploma in Tropical Medicine Date of Date of Diploma Diploma 1904 Augustine, Henry Joshua 1905 Macfarlane, Robert Maxwell 1904 Bennett, Arthur King 1905 Maddock, Edward Cecil Gordon 1904 Bruce, William James 1905 Moore, James Jackson 1904 Byrne, John Scott 1905 Nightingale, Samuel Shore 1904 Clayton, Thomas Morrison 1905 Radcliffe, Percy Alexander Hurst 1904 Dalziel, John McEwen 1g05 Young, John Cameron 1904 Dee, Peter 1906 Adie, Joseph Rosamond 1904 Greenidge, Oliver Campbell nee ce Pedi Laie 1904 Hehir, Patrick 1906 Bate, John Brabant 1904 Khan, Saiduzzafor 1906 Bennetts, Harold Graves 1904 Laurie, Robert : 1906 Carter, Robert Markham 1904 Maclurkin, Alfred Robert 1906 Chisholm, James Alexander 1904 McConnell, Robert Ernest 1906 Clements, Robert William 1904 Nicholson, James Edward 1906 Dundas, James 1904 Philipson, Nicholas | 1906 Faichnie, Norman 1904 Sharman, Eric Harding 1906 Jeffreys, Herbert Castelman 1904 Thomson, Frank Wyville 1906 Mackenzie, Donald Francis 1904 Walker, George Francis Clegg 1906 Pailthorpe, Mary Elizabeth 1g05 Anderson, Catherine Elmslie 1906 Palmer, Harold Thornbury 1905 Brown, Alexander 1906 Pearse, Albert * 1905 Caldwell, Thomas Cathcart 1906 Sampey, Alexander William 1905 Critien, Attilio 1906 Smithson, Arthur Ernest 1905 Hooton, Alfred 1906 Taylor, Joseph van Someron 1905 Hudson, Charles Tilson 1906 Taylor, William Irwin tgo5 Illington, Edmund Moritz 1906 Tynan, Edward Joseph Date of Diploma 1g0b 1906 1906 1907 1907 1907 1907 1907 1907 1907 1907 1907 1907 1907 ey N87) L387) E007, mOe7 2927, Ley 5907, nOe7, SET) Watson, Cecil Francis Willcocks, Roger Durant Williamson, George Alexander Allan, Alexander Smith Allwood, James Aldred Bond, Ashton Branch, Stanley Collinson, Walter Julius Davey, John Bernard Donaldson, Anson Scott Fell, Matthew Henry Gregson Gann, Thomas William Francis Graham, James Drummond Hiscock, Robert Carroll Keane, Joseph Gerald Kennan, Richard Henry Kenrick, William Hamilton Le Fanu, George Ernest Hugh Mackey, Charles Maddox, Ralph Henry McCarthy, John McDonald Raikes, Cuthbert Taunton Ryan, Joseph Charles Vallance, Hugh Caverhill, Austin Mack Crawford, Gilbert Stewart Dalal, Kaikhusroo Rustomji Dansey-Browning, George Davidson, James Dickson, John Rhodes Dowdall, Arthur Melville Glover, Henry Joseph Greaves, Francis Wood Goodbody, Cecil Maurice Harrison, James Herbert Hugh Joshi, Lemuel Lucas Le Fanu, Cecil Vivian Luethgen, Carl Wilhelm Ludwig Mama, Jamshed Byramji McCay, Frederick William McLellan, Samuel Wilson Pearce, Charles Ross Schoorel, Alexander Frederik Smith, John Macgregor Stewart, George Edward Tate, Gerald William Whyte, Robert Abercrombie, Rudolph George Allin, John Richard Percy Armstrong, Edward Randolph Barrow, Harold Percy Waller Beatty, Guy Carr-White, Percy Chevallier, Claude Lionel Clark, William Scott Cope, Ricardo Fleming, William Hanschell, Hother McCormick Hayward, William Davey Henry, Sydney Alexander Innes, Francis Alexander Date of Diploma 1909 1909 1909 1909 zcy 1g09 1909 aoe 1909 zgc9 1909 IgIo 1910 IgIO IgIO 1910 IgIO 1910 IgIO IgIO 1910 IgIO 1910 I9IO IgIO Igio I9IO IgIo 1910 IgIO 1910 IgIO 1910 I9IO IgIO 1910 IgIO 1910 IgIO IQII IgII IglI IQII IQII IgII IgII IQII IQII I9II IQII IgII IQII IQII IgII Igii IgII IQII IgII IgII IQII Jackson, Arthur Frame Kaka, Sorabji Manekji McCabe-Dallas, Alfred Alexander Donald Meldrum, William Percy Murphy, John Cullinan Samuel, Mysore Gnananandaraju Shroff, Kawasjee Byramjee Thornely, Michael Harris Turkhud, Violet Ackroyd Webb, William Spinks Yen, Fu-Chun Brabazon, Edward Castellino, Louis Caulcrick, James Akilade Dowden, Richard Haigh, William Edwin Hamilton, Henry Fleming Hefferman, William St. Michael Hipweil, Abraham Homer, Jonathan Houston, William Mitchell James, William Robert Wallace Johnstone, David Patrick Korke, Vishnu Tatyaji Macdonald, Angus Graham Macfie, John Wm. Scott Manuk, Mack Walter Murison, Cecil Charles Nanavati, Kishavlal Balabhai Nauss, Ralph Welty Oakley, Philip Douglas Pratt, Ishmael Charles Sabastian, Thiruchelvam Shaw, Hugh Thomas Sieger, Edward Louis Sousa, Pascal John de Souza, Antonio Bernardo de Waterhouse, John Howard White, Maurice Forbes Blacklock, Breadalbane Brown, Frederick Forrest Chand, Diwan Jai Holmes, John Morgan Tevers, Charles Langley Iles, Charles Cochrane Ingram, Alexander Kirkwood, Thomas Knowles, Benjamin Liddle, George Marcus Berkeley Lomas, Emanuel Kenworthy Mackarell, William Wright MacKnight, Dundas Simpson Mascarenhas, Joseph Victor Murray, Ronald Roderick Oluwole, Akidiya Ladapo Rao, Koka Ahobala Sinton, John Alexander Tarapurvalla, Byramji Shavakshah Taylor, John Archibald Woods, William Medlicott Date of Date of Diploma Diploma Igt2 Aeria, Joseph Reginald 1912 Smalley, James 1912 Anderson, Edmund Litchfield {g12 Strickland, Percy Charles 1912 Borle, James Hutchison 1912 Bowie, John Tait 1912 Watson, William Russell 1912 Brassey, Laurence Percival Ig12_ Christie, David 1912 Dillon, Henry de Courcy 1913 Becker, Johann Gerhardus 1912 Dunn, Lillie Eleanor 1913 Forsyth, Charles 1912 Hardwicke, Charles 1913 Grahame, Malcolm Claude Russell 1912 Jagose, Jamshed Rustonji 1913 Grieve, Kelburne King 1912 Kochhar, Mela Ram 1913 Hargreaves, Alfred Ridley 1912 McGusty, Victor William Tighe 1913 Hiranand, Pandit 1912 Milne, Arthur James 1913 Jackson, Oswald Egbert 1912 Mitra, Manmatha Nath 1913 MacKelvie, Maxwell 1912 Myles, Charles Duncan 1913 MacKinnon, John MacPhail 1912 Pelly, Huntly Nevins 1913 Macmillan, Robert James Alan 1912 Prasad, Bindeshwari 1913 Mouat-Biggs, Charles Edward 1912 Prentice, George Forbes 1912 Ross, Frank 1913 Olubomi-Beckley, Emanuel 1912 Russell, Alexander James 1913 Puttanna, Dodballapur Sivappa Hutchison 1913 Reford, John Hope 1912 Ruthven, Morton Wood 1913 Walker, Frederick Dearden 1912 Sandilands, John 1913 Yin, Ulg Ba Ig12 Seddon, Harold 1913 Young, William Alexander EDITORIAL NOTICE By order of the Committee of the Incorporated Liverpool School of Tropical Medicine, the series of the Reports of the School, which had been issued since 1899, were followed, from January 1, 1907, by the Annals of Tropical Medicine and Parasitology, of which this is the second number of the seventh volume. Altogether twenty-one Memoirs, besides other works, were published by the School since 1899, and of these ten, containing 519 quarto or octavo pages and 95 plates and figures, were published during the two years 1904 and 1905. The Annais are issued by the Committee of the School, and will contain all such matter as was formerly printed in the Reports—that is to say, accounts of the various expeditions cf the School and of the scientific work done in its laboratories at the University of Liverpool and at Runcorn. In addition, however, to School work, original articles from outside on any subject connected with Tropical Medicine and Hygiene, and Parasitology may be published if found suitable (see notice on back of cover); so that, in all probability, not less than four numbers of the Annals will be issued annually. Each number will be brought out when material sufficient for it has been accumulated. Du plems * a a age Sigg: fo a Shane “ On eae Bo skit a0 hava aeRO: suai £101 ; Let, Saath = Tis a oeee , : tots) nt edt: assed figt - segpmt) het roi thizeo 4 Seek cateald M uae” E108 ‘sth Ht Sty! belt eae porsin) $rat a fA Gants, Seem: E16! AMAT, ots yteta ener 1% de Bien A) coeAs eo ENE : fomees (or ain afi SAHA £i0Y TESS ee hilt folie att e101 with Sime PS sox Fe Pas me WEI ees as! th ne hi yirt TRG £10 ‘. a Ct ae : i al Ad-tahauley © 3 rot Bitte PAUSE. , sanggied » p10} Atte? Brot: vi fight = sy Mrdbnt a: aM: p1e% ioe = ew ay gg! agit Se welinay sth tneiileV Quay. Er ~~ a Wat “oebh ft j ba wy bee —* te a — ae 1g tty ome ates st a cod Daas - ee ys ty 8 + ones - 1 T Ou Rr tetapsisicy ili er 1 = eo — eat: P3 ood Wags syts Is30 sk inttl witli to, 2950s Testy (owe ie diy b soins “eure botdiec a te) eBied: ra5L. oy to bal 39% aut aesthM T aig ae 4¢ \OGE oe be of MAE, mot ,psw ollot JisW 608 Sonus, purer “oad b ay = Dwele ot. Ritsu te “gem swegiaties s1 bas om pibeM Total ie “stpue A add oniuloy dingyoe on td dina broged ont BRONTE HA “goqigdens, euem ve ed Able Pe Johre ae { Ale Matai siios tes Bese niorf brs: 0082 same loodse-ote ed bodail ie sthariciicy San, 29a ne bis Zoda! in 20° bis, SS98q. OF 8190. 10; ou 20g! bith ‘pogt eisoy owas Terps - iwi TA 4 fiw. bar “i att i gottivamto.) Et es boned’ ioe ot feed Gor it ar botabiey vhs oimtaok emy26 io1 ain ie. liso dt Ad has lopdee, ail enoiithaqns, bpotey SAT 1 Bifitooos 2: or sa } RY . HM 38 oO I joodisvi_lote nase nid om is esis pameretek 2 “weiter? bined E,., te ieYeticee! cap Of fret fence" a fon sa He Sig ity oirtei aie bi bar a) tai ive Us sins a i! arb tid aga s AW Gees yi toed: stoke “hey AE 4 ‘i a4) Dis - 2 22 i f rw « a mt -_- c ee _— < ye _ - ° =~ = ci) &- ‘ a od =a _ = a a .- Fe 183 PINAL REPORT OF LEE LUANGWA SLEEPING SICKNESS COMMISSION OF THE BRITISH SOUTH AFRICA COMPANY LGLL-fGE2 BY ALLAN KINGHORN, WARRINGTON YORKE AND BLEWELLYN 7 LLOMD, ENTOMOLOGIST TO THE COMMISSION (Received for publication 7 February, 1913) PLATES XV-XXVI CONTENTS PAGE INTRODUCTION ... oc Abe Es ae a So sins ae oc soo aye! Section I. Tue Human TrypanosomMe— (a) Distribution of the Disease ... ne win te sa ee Be WEE (6) Clinical Features = : es 5 XD ae AoC wa) S186 (c) Identity of Parasite with The Chidesene sone ae oe ae eae BLOT, (d) Transmission of the Trypanosome ... 187 (e) Influence of Meteorological Conditions on Dey elopment a the Try pano- some in Glossina morsitans ... = ate x. be Sot guar (f) Reservoir of the Trypanosome xe gat p2A! (g) Occurrence of the Try panosome in Glossina morsitans in Nature eo BG (4) Identity of ‘Game’ and ‘Fly’ Strains with the ‘Human’ Strain of T. rhodesiense ... sie as ois wan dee se SH AG) (4) Summary " cx ie he Eas 6¢ sor S06 blac) Section IJ. Trypanosomes or Game aND Domestic StocK— (a) Methods se 227 (6) Examination of Game at Naw alia (itera Vi alley) and iNeon (Congo- Zambesi Watershed) * 228 (c) Examination of Domestic Stock... ase “ie Gh 360 S235 (d) Summary 237 Section III. Trypanosomes FouND IN WILD Glossina morsitans— (a) At Nawalia... ate be At ee Fe spi Se <2 OG (6) At Ngoa as was A ws oa ae see x6 sayy BEL (c) Summary mie nee sis ae oz? ae ae oc sau 24G Section [V. Description oF TRYPANOSOMES— (a) Trypanosoma rhodestense 245 (6) Trypanosoma vivax 245 (c) Trypanosoma nanum 248 (d) Trypanosoma pecorum 251 (e) Trypanosoma multiforme, sp. nov. 254 (f) Trypanosoma montgomery1 ... “oe a soe es B6¢ op 2 (g) Trypanosoma ignotum, sp. nov. 486 soc ste sae 263 (b) Trypanosoma tragelaphi, sp. nov. ... a Hee 50 Se .. 269 Section V. Devetopment oF Trypanosoma rhodesiense in Glossina morsitans— 273 Summary as 35 ae Sac 58 Oc 00 as nae a2 Section VI. Report or ENromMoLoGist— (a) Glossina morsitans in the Laboratory mas ac 506 so sereee'S (b) Breeding Places of Glossina morsitans fc 406 508 eee 250 (c) Bloodsucking Insects collected at Nawalia and Ngoa sc x, 5 45rt 293 APPENDIX A. An Experiment to ascertain whether Tabanids transmit Segal in Nature - 299 B. An Attempt to Transmit "Trypanosoma rhodesiense “by means of Ornithodoros moubata ... oi Soe 5 oc 506 fs 301 184 INTRODUCTION During the years 1909 and 1910, the diagnosis of several cases of human trypanosomiasis amongst Europeans, who had never. been in contact with Glossina palpalis, drew attention to the occurrence of the infection in portions of Rhodesia and Nyasaland in which this particular insect was not known to exist. As the result of prolonged and careful search, it was definitely proved that Glossina palpalis did not occur in these areas, and accordingly, in the beginning of 1911, this Commission was instituted by the Chartered Company to ascertain the transmitting agent. Nawalia, in the Luangwa Valley, was chosen as the site of the laboratory (Pl. XV, figs. 1-2). Work was commenced at the end of June, 1911, and was continued until April, 1912. Our investiga- tions quickly placed it beyond doubt that Gloss¢na morsitans was the vector of the human trypanosome, and further revealed the fact that a considerable percentage of game and of ‘wild’ Glossina morsitans were infected with the same parasite. In April, 1912, the headquarters of the Commission were removed to Ngoa, on the Congo-Zambesi watershed, in order that experiments might be undertaken to ascertain what influence, if any, was exerted by climatic conditions on the transmission of the trypanosome. Work at Ngoa (Pl. XVI, figs. I-2) was continued until the end of August, 1912, when the Commission left for England. By this time it had been definitely determined that the relatively low temperatures experienced during the cold season on the plateau were inhibitory to the completion of the developmental cycle of the human trypanosome in Glossina morsitans. Records of the greater portion of the work embodied in this report have been published from time to time in the form of separate papers. As, however, these preliminary communications were necessarily somewhat incomplete, it was considered desirable to collect and correlate all the results in the final report. We desire to acknowledge our indebtedness to Dr. Aylmer May, Principal Medical Officer, Northern Rhodesia; to Dr. A. F. Wallace, _M-O2;""RL""Ritodestay "and “to E, “ATA. [pnes, su Assistant Magistrate, Mpika, for the great assistance they rendered the Commission. The report was completed at the Runcorn Research Laboratories of the Liverpool School of Tropical Medicine. 185 SECTION I THE HUMAN: PRYPANOSOME BY ALLAN KINGHORN AND WARRINGTON YORKE (a) OCCURRENCE OF THE DISEASE The distribution of human trypanosomiasis, due to infection with 7. rhodesiense, is comparatively wide. When the parasite was first described,* the Luangwa Valley was the only region known to be implicated, but it has since become apparent that the disease is much more widely disseminated. In the territory formerly known as North-Eastern Rhodesia, cases have been diagnosed not only in the Luangwa Valley, but also in the districts south of Fort Jameson and to the west of Serenje. It may be mentioned further that this parasite was isolated from wild game in the vicinity of Ngoa, and from a native dog living in a village on the Nyasaland boundary. It is possible, therefore, that sporadic cases of the disease may exist in these localities also, but on this point no definite information can be given. In North-Western Rhodesia, it would appear that 7. rodesiense exists in at least one locality, namely, between Broken Hill and the Anglo-Belgian boundary. The details of a case, due to this organism, and contracted in the district named, have recently been described by Ellacombe.t It is, of course, occasionally very difficult to ascertain the exact locality in which the infection was acquired, more particularly in native cases, but there are good grounds for believing that those from the districts mentioned were autochthonous. *Stephens and Fantham, (1g1o). Roy. Soc. Proc., B, Vol. 83, p. 28. Annals of Trop. Med. and Parasit., IV, p. 343. t Ellacombe, G. W. Sleeping Sickness Bulletin, 1912, Vol. IV, p. 185. 186 Beyond the confines of Northern Rhodesia, an instance of infection by 7. rhodesiense in Nyasaland has been recorded by Stannus and Yorke,t an observation which has been confirmed more recently by the Royal Society’s Commission.{ The parasite has been isolated from a case of sleeping sickness originating in Portuguese East Africa,§, and it is accordingly probable that cases reported from the neighbouring portions of German East Africa are due to the same trypanosome. Finally, indigenous cases of sleeping sickness have recently been diagnosed in Southern Rhodesia, though we are not in a position to state the identity of the organism. It will be seen, therefore, that Trypanosoma rhodesiense 1s widely spread over South Central Africa, and its distribution is in close association with that of Glossina morsitans, which has been shown to be the vector. (b) CLINICAL FEATURES OF THE INFECTION There is no essential difference between the clinical manifesta- tions of the disease caused by 7. vhodesiense and that due to T. gambiense, except possibly the greater virulence of the former. In the earlier stages, enlargement of the lymphatic glands is commonly seen, and more or less irregular fever, with the accompanying symptoms of malaise, anorexia, headache, pains in the limbs, &c., is the rule. During the attacks of fever trypano- somes can readily be demonstrated in the peripheral blood, but as a rule are rather scanty. As the disease progresses oedemata of various parts of the body, more particularly the face, are common features, together with emaciation, loss of muscular power, ataxic gait, tremors, difficult speech, loss of memory, and other signs of nervous derangement. These symptoms gradually increase in intensity until finally the patient sinks into a condition of coma, and dies. No exact data exist as to the duration of the disease in natives, but it would appear to be short. Many of the cases complained of + Stannus and Yorke. Proc. Roy. Soc., B 84, 1911, p. 156. + Reyal Society Commission. Roy. Soc. Proc., B 85, 1912, p. 423. § Sleeping Sickness Diary. Nyasaland Protectorate. 187 no subjective symptoms of the disease when diagnosed, and presented very few objective signs, but in general they lived only a few months. Occasionally, however, a patient is more resistant, and one native definitely proved to be infected with 7. rhodesiense is still alive and in a state of apparent good health a year later. (c) IDENTITY OF TRYPANOSOME WITH T. RHODESIENSE The essential characteristic of 7. rvhodesiense, Stephens and Fantham, is the occurrence of ‘posterior nuclei,’ i.e., amongst the short forms of the parasite, organisms in which the macronucleus is markedly displaced from the usual central to a decidedly posterior position are commonly seen in certain laboratory animals, more especially rats. This displacement may proceed to such a degree that the macronucleus may actually be situated behind the micronucleus at the aflagellar end of the trypanosome. As this peculiarity has not been observed in 7. gambzense, the two strains of human trypanosomes can readily be distinguished. A further difference is noticed in the pathogenicity of the parasites, T. rhodestense being much more virulent for all species of animals than T. gambiense. At Nawalia and Ngoa, rats were subinoculated from sixteen cases of sleeping sickness, and in every instance the posterior nuclei were observed in stained preparations. The pathogenicity of these strains agreed closely with that of the Armstrong strain,* from which 7. rhodesizense was originally described. It will be understood, therefore, that in all our transmission experiments the strains of human trypanosomes utilised answered in all respects to the description of 7. rhodesiense. (d) TRANSMISSION OF THE TRYPANOSOME Experiments to transmit the human parasite by Glossina morsitans were made at Nawalia in the Luangwa Valley, and at Ngoa on the Congo-Zambesi watershed. Nawalia, the site of an old station, is situated on the right bank of the Nyamadzi river, * Bevan. Journal of Comparative Path. and Therapeutics, 1910, p- 160. Yorke. Annals of Tropical Medicine and Parasitology, 1910, p. 351. 188 a tributary of the Luangwa, at an altitude of about 2,100 feet above the sea. Its position is approximately 12° 25’ S. and 32° 2’ E. Ngoa, 11° 40’ S. and 31° 30’ E. lies some 30 miles North of the station of Mpika, at a height of 4,400 feet above sea level. The meteorological observations at Nawalia and Ngoa are synopsised in Tables 1 and 2. Certain general conditions which attach to all the experiments may be mentioned. The identity of the flies has been controlled both by direct examination of the external characters and by the preparation of the male genitalia, as recommended by Newstead,* so that we can state with some degree of confidence that we have been dealing only with Glossina morsitans, Westw. All the experimental animals have been kept in fly-proof cages, the fronts of which were protected by a double layer of wire gauze, the inner composed of coarse, and the outer of mosquito meshing. The two layers were separated by a space of one inch in order to obviate the possibility of an animal being bitten while pressing its body against the front of the cage. The feeding of the flies, and the changing into fresh bottles daily, was supervised personally, while the flies were kept in such a manner that they had no opportunity of obtaining food from other than the animals used in the actual experiments. EXPERIMENTS AT NAWALIA A. WITH LABORATORY-BRED Glossina morsitans Experiment 1 Commenced August 20th, IQI1. It is somewhat difficult to tabulate this experiment, owing to the fact that it was not started on a definite date with a definite number of flies. Between August 20th and September 29th twenty-six flies had hatched out, and each, as it did so, was given its first meal on an animal showing numerous parasites in the peripheral blood, so that on any given date the periods which had elapsed since the infecting feeds of the flies varied considerably. In Table 3 the main facts in connection with the flies are given. * Newstead, R. Bull. Entomol. Research, Vol. II, Part 1, May, 1grt. Annals Trop. Med. & Parasitol., Vol. VII J2IL ATO, DG *talee + Kk ri Fic. 2. Laboratory at Nawalia, Luangwa Valley. To face p. 188 C. Tinling & Co., Ltd., Imp. 189 69 $o,9f | I 10,0 ie Srh "6.15" | hebg wien Roding 2 6.16 6.62 (496 09) judy 9 o1.$ S.z9 S.Z9 8-98 mor 74 0-19 a _ 9-26 0.62 ee UORETAT tak 22 waa ed g.tZ 0.12 6.48 , 1.22 1-99 | o,06) 7-62 pa Areniqay 91 £6,+1 L.L£L 3.22 1.28 $.gZ oLg wen cb fe) 9-08 IS AEA oz $$. 1.69 or 4 $.16 9.62 | 8-49 2.101 £.zg " Jaquiadaqq g 19-1 1.It $.#2 | 0.96 9-8 | 6.29 'g.Zo1 1.28 “+ JaquUIaAONy 3 gz-0 | ge1f High £.66 S.tg £.79 g-Lor 1.98 son 88 T9qOIIO) -— ) S.1€ $-19 | $:26 on 0.4 €.£01 SLL “ aquuaqydag — ° BSE g.2S §.S¢ zl Ze1S 8-96 £.€2 eee Faris ty: — ° L.S+ g.1S Z-ZQ +.Zg g-tb 0.16 Z.g9 oO fd Ajnt -— ° 9-gt _— | — —- £.bP 0.68 zg Ma Che aunt a wnat =| winurxea | Uap umunu | umunxew | uy | [fy ures sayout % ainposqy = aa NOSq aqnjosqy aanjosqy yoryn esareyy Ayprany | |_|} —_ A J | | zt 161 uo sec] PALLY SAMALVAICNAT, AMOLVAORV'T SAMALVAIANA YT, ACVAG IWNAALXG *(Ajayeunrxoidde) qaay oor'z apnynayy =“ Zz zt GS Sz ZI SeIsopoyy “N ‘ereMeEN Je sUOTILAIASGO [edTFO[OIOD}aI—"1 TAV,T, 190 [ey Utes yorym uo skeq 10-0 10.0 sayout Treyereed o/ /O Ayrpramy DATILPIY, | uuInuirurur g-94 UINUIITXelW aynposqyy ayn[osqy ueboIy SAUNLVAIAWAT, AUOLVAORV’T zSb €.z4 £.v€ 1.S¥ 1-4 uanurturur aynjosqy SHUALVAIGWAY, AAVHG IVNUALXY +.98 L.L9 0.£g $.79 S.$g | 9-19 ¢.06 | 0.69 1.68 | ved | | I— ee UINUITXP UL | se) AN aqnjosqy (yaok -(Ajageuntxosdde) 3aay Sth apnanpy “gq of QIf “gs oF 11 ‘eIsapoyy “N ‘voSyy ye suOTeArasqo [eorsofosOajay—'t AAV], 19! Taste 3.—Giving date of infecting meal, date of death, and duration of life after infecting meal. No. of fly Date of infecting meal | Date on which fly died | Duration of life from date of infection A I 20.8.11 22.9.11 | 33 days AY 2 21.8.11 20.10.11 fo 1 25.8.11 | 6.9.11 12 AL le | 26.8.11 | 23.10.11 Chee Aas | 27.8.11 | 28.10.11 | 624 *., A 6 27.8.11 | 17.10.11 | GR op IAG) 28.8.11 II.10.11 Ake A 8 29.8.11 | IT.11.11 oih ee A 9 30.8.11 12.g.11 Tigi A Io | 31.8.11 20.10.11 KONe, All 31.8.11 12.9.11 [2a ag A 12 | 3.9.11 | 27.9.11 | 2 Aaron Ay | 3.9.11 5.9.11 2a A 14 5-9-11 | 28.9.11 273) ins A 15 6.9.11 25.10.11 AQuNs, A 16 7-9.11 4.11.11 Gls ING) 8.9.11 3.10.11 | Zen A 18 8.9.11 14.10.11 | HO op A 19 9.9.11 19.10.1T 45,55 A 20 9-9.11 13.9.11 Ages. AS2Y 13.9.11 23.10.11 4Ope5 A 22 | 16.9.11 14.11.11 Ga) es A 23 | 17.9.1 | 27.10.11 40.733 A 24 | 25.9.11 | 3.1N 11 Ewe A 25 | 26.9.1 29.9.11 3253 A 26 26.9.11 | 29.10.11 33 192 Twenty-four hours after each fly had fed on an infected animal, it was afforded an opportunity of feeding on a clean monkey (No. 41), after forty-eight hours on a second (No. 42), and from the third day onwards on a third (No. 52). The schedule of feedings is given in Table 4. Taste 4.—Showing transmission of human trypanosomes by laboratory-bred Glossina morsitans, Date | Animal | No. flies Result Remarks | fed Aug. 21—Sept. 18...) Monkey 41 Gd Negative | Flies fed 24 hours after infecting | | feed. ; mal 2 Jr 28-0 ‘ 42 14* af | Flies fed 48 hours after infecting feed. ' ye 2 oy 2 Ons A, 52 23 Infection | Flies fed 72 hours and onwards after infecting feed. ( 55 68 | 5 Negative | (a) Infecting feed over 30 days | | | before. Bept..27andeasi. caine bss 69 | 6 + (b) Infecting feed between 20 to 30 | | | | days before. 5 FOE | 5 Infection | (c) Infecting feed less than 20 | | | days before. Sept. 29—Oct. 3... - sea 16 Ee _ All the flies fed. | | Oct/> Ay te ...| White rat 77 16 5 All the flies fed. Pan lle tcc ...| Monkey 68 14 Negative | Infected fly did not feed. 33) OO re =| 3 58 16 Infection | All the flies fed. PR lh bob 5a £ 61 I sete Infected fly, only, fed. $e aoe x 68 14 “4 Infected fly fed on 13th and 14th as well. 3) 13—16 see - 69 13 Negative | Infected fly did not feed. 5) 16—19 a0 b 83 12 Infection | Infected fly commenced feeding on 16th, others on 17th. 3) 20—28 aa 5 69 10 Negative | Infected fly did not feed. 9» 29—Nov. 11... 3 56 4 | 3 | Infected fly did not feed. } * The remaining flies refused to feed. From this table it will be seen that neither of the first two monkeys on which the flies were allowed to feed became infected, whereas No. 52 did so on the 27th September. A reference to Table 3 will show that up to, and including the 26th September, twenty-three flies had fed on an_ infected animal more than three days previously, and had, accordingly, ene + 193 been fed on Monkey No. 52. The three flies, A 24, 25 and 26, had never fed on this animal, and therefore had not to be considered in the attempt to isolate the infected fly. Moreover, six of the flies, A 1, 3, 9, II, 13 and 20, had died prior to the 26th September, and of these, three proved to be negative throughout on examination. The other three, namely, A 3, 11 and 20, were found to show a heavy intestinal infection of trypano- somes. Fly A 3 died on September 6th, A 11 on September 12th, and A 20 on September 13th, while the monkey did not become infected until September 27th, much too long an incubation period for one of these flies to have been the infecting one. We have additional proof for the conclusion in that the abdominal contents (gut+salivary glands) of flies A 3 and A 20, on inoculation into monkeys, did not determine an infection. On the 26th September there were, then, twenty flies with which to deal, amongst which was at least one infective fly. As stated above, three flies, A 24, 25 and 26, had never fed on Monkey No. 52, so that the inquiry was limited to seventeen, and this was further reduced to 15 by the death of flies A 12 and 14 on September 27th and 28th—both flies negative on examination. These were accordingly split up into three groups, based on the length of time which had elapsed since the date of the infecting feed, and each group was allowed to feed for two days on a clean monkey, Nos. 68, 69 and 70. Group (a) Infecting meal over thirty days previously. ppait:! (6) Ns 5, between twenty and thirty days previously. Rew) FF 5, less than twenty days previously. Of the three monkeys, No. 70 was the only one to become infected, and the transmitting fly was thus located in Group (c), consisting of A/10;:2%;22:and 23. While waiting to ascertain which of the three monkeys would become infected, all the flies were fed from September 29th to October 3rd on Monkey No. 72, and on October 4th on White Rat No. 77. Both of these animals became infected in due course. On October 5th all the flies, with the exception of A 19, were re-fed on Monkey No. 68, and from the 6th to the oth all were fed on Monkey No. 58, except on October 9th when fly A 1g alone, 194 was fed on Monkey No. 61. Of these animals, No. 68 did not become infected, while Nos. 58 and 61 did. The fly A 19 (¢) was thus proved to be the infecting one. No particular interest attaches to the further experiments. From the table it will be seen that both those animals on which fly A 19 fed became infected (Nos. 68 and 83), whereas those on which it did not feed remained quite healthy (Nos. 69 and 56). When it had been definitely ascertained which was the infecting fly, it was possible to determine fairly accurately the duration of the cycle of the trypanosomes in the insect. Fly A 1g had its infecting meal on September oth, and Monkey No. 52, the first to become infected, showed trypanosomes in the peripheral blood: on September 27th. On the 26th, therefore, the last day on which the flies were fed on this animal, a period of eighteen days had elapsed since A 1g fed on the infected animal. The average , Incubation period of the local strain of human trypanosomes in monkeys is four to five days, and subtracting this from the eighteen days, it is evident that the fly must have become infective in thirteen days. This fly, A 19, lived for forty days from the date of the infecting meal, and between the time of becoming capable of transmitting the parasite and the date of death had fed on eight animals, all of which became infected. The other flies were fed continuously from the date of the possible infecting meal to that of death, which occurred at varying periods from two to seventy-four days, but none of them became infective. Experiment 2 Commenced November 14th, 1911, with sixteen laboratory-bred Glossina morsitans. sc has In this experiment the flies were infected directly on a case of human trypanosomiasis, each fly being allowed to feed on one occasion only. Ten fed on the 14th of November, when the patient showed three trypanosomes per field in the blood, and the remaining six on the 15th, when there was, on an average, one trypanosome to seven fields (Zeiss Oc. 4, Obj. D.D.). The subsequent meals were as shown in Table 5. 195 ‘Taste 5.—-Showing transmission of human trypanosome by laboratory-bred Glossina morsttans, Days after No. of infecting feed | Animal flies fed Result Remarks a SSS SS SS TitOa5 White rat 116 15 | Negative 6 to 10 > 118 15 | It to 15 5 124 15 Infection 16 to 20 + 116 13 a | CAKE) 22 5 118 12 | 45 23 to 27 Monkey 137 6 Negative | ) Flies divided into two groups to separate the infective one. 23 to 27 $ 138 a Infection ) 28 to 42 3 148 | Varied Negative * One fly of this group (6) refused to feed, and died on the 26th day of the experiment. Rat No. 124 became infected on December 4th, five days after the flies had fed last, and as the incubation period of the trypano- some in these animals, on an average, is five days, it seems probable that the fifteenth day was the one on which the infecting fly became capable of transmitting the parasite. On the 7th of December (23rd day after infecting meal) the twelve flies then alive were divided into two groups in order to effect an isolation of the infective one, and were fed on Monkey Nos. 137 and 138, as indicated in the table. On December 12th, the fly numbered B 23 (¢) died, and on examination proved to be heavily infected throughout the alimentary canal, and in the salivary glands. No infection of the proboscis, however, was observed. The other flies were fed on a clean monkey until the 42nd day, but without result. Experiment 3. Commenced December 29th, 1911, with twenty laboratory-bred flies. These flies were infected directly from a patient in whose peripheral blood three trypanosomes per field (Zeiss Oc. 4, 196 Obj. D.D.) were seen. They were afterwards fed daily for sixty- five days on a series of healthy monkeys, none of which became infected. From the 67th—7oth day of the experiment, the seven flies then alive were fed on a guinea-pig heavily infected with the human trypanosome, and were then fed for a further period of thirty days on a clean monkey. This did not become infected. Lx periment 4 Commenced January 12th, 1912, with twenty-three laboratory- bred flies. These were fed for four days on a patient showing, on an average, one trypanosome to three fields in the peripheral blood, and afterwards on healthy monkeys, as indicated in Table 6. Tasce 6.—Showing transmission of human trypanosome by laboratory-bred Glossina morsitans. Day Animal | No. of Result | Remarks | flies fed | | ae | | oto.3. |. Patient | 23 | = | | | | pee 4 oo —_ -— | Flies starved. sto 8 Monkey 237 | 22 | Negative | g to 12 | 3 238 | 22 a | | 13 to 16 : 240 | 22 6 | 17 to 20 || PF: aoa | 20 Infection | 21 to 23 «OI 3 207 18 , Died on 24th day. 24 a4 240 | 17 Infection | | | | 25 to 29 260 | 9 | Negative 1) ia - Flies divided into two groups. 261 | 9 Infection | | | | | - . | 3 30 to 60s} 5 272, | 16—o | Negative | Infected fly did not feed. | | On February 20th, the 29th day of the experiment, the fly numbered D 18 died, and on dissection was found to show a massive intestinal infection of trypanosomes. Unfortunately, the fly had been dead for some hours before it was examined, and it was found impossible to dissect out the salivary glands. The whole abdominal contents, therefore (gut and glands) were crushed 7 up in normal saline solution and inoculated into a healthy monkey, which became infected five days later. The disease ran a typical course. None of the other flies—dissected as they died—were found to harbour trypanosomes in the proboscis, gut, or salivary glands. In this instance the time which elapsed from the date of the first infective meal until the date on which the fly became capable of transmitting the trypanosome (allowing five days for the incubation period in the monkey) was nineteen days. B. By ‘WILD’ Glossina morsitans Experiment 5 Commenced on November 14th, 1911, with ninety-eight ‘wild’ flies. Prior to infecting these flies with the human trypanosome, they were fed for three days, November 14th-16th, on a_ healthy monkey (No. 95), and for the next four days on a native fowl. The monkey never became infected. From the 2Ist to the 24th of November the insects then alive, fifty-seven in number, were fed on an infected monkey showing twenty to thirty trypanosomes per field in the peripheral blood, and were afterwards fed on healthy animals, as in Table 7. Taste 7.—Result of feeding ‘ wild’ Glossina morsitans on clean monkeys, after a preliminary meal on an animal infected with the human trypanosome. Days after first Animal No. of Result | Remarks infecting feed | flies fed gto 6 | Monkey 125 48 — | Monkey died on 7th day. 7 to g 4 127 41 Negative © to 13 130 34. Infection 14 to 16 119 31 aS - 141 10 Infection | Flies divided into two groups to isolate infected one. | | 17 to 18 5 140 7 Negative | Monkey died on rgth day. 198 This experiment was finished after the flies had fed on the 25th day, the flies being then killed and embedded. The duration of the cycle of the parasites in the flies, in this instance, would appear to be slightly over eleven days. The first infecting meal was taken on November 21, and Monkey No. 130 showed parasites in the peripheral blood on December 7, a difference oi sixteen days. As stated already, the incubation period of this trypanosome in monkeys is about five days, and by subtracting this from the sixteen days, we obtain eleven for the duration of the development cycle. Experiment 0. Commenced January 12, 1912, with forty-two freshly-caught flies. After being fed for one day on a monkey infected with the human trypanosome, and showing numerous parasites in the peripheral blood, the flies were fed on a clean monkey for nine days. They were then starved for one day, and subsequently allowed to feed on clean monkeys and rats from the 11th to the 33rd day. None of these animals became infected. The flies were dissected as they died, and while trypanosomes were found in the gut and proboscis of several, in no instance was an infection of the salivary glands observed. Experiment 7 Commenced January 12, 1912, with forty-two freshly-caught flies. The details of this experiment are exactly similar to those of Experiment 6, with the exception that from the Ist to the oth day the flies were fed on a native fowl instead of on a monkey. They were starved on the 1oth day, as before, and afterwards fed on clean monkeys and rats from the 11th to the 38th day. None of these animals became infected. Trypanosomes were found in the proboscis and gut of several of the flies when dissected, but in no case were the salivary glands implicated. Experiment 8 Commenced February 14th, 1912, with 104 freshly-caught flies. On the 13th of February, the flies were fed on a healthy monkey 199 which did not become infected, thus excluding the possibility that they were already infected with the trypanosome. On the four succeeding days they were fed on a guinea-pig infected with the and showing numerous parasites in the human trypanosome, peripheral blood, and afterwards on clean monkeys as indicated in Table 8. Tasie 8.—Showing transmission of the human trypanosome by freshly-caught Glossina morsitans. Day 13 to 27 13 to 29 28 to 29 30 30 to 38 30 to 4o 30 to 4o 31 to 33 31 to 37 31 to 34 34 to 39 35 to 52 39 to 52 41 to 52 Animal Monkey 269 269 280 286 No. of flies fed 98 Result Negative > >b] Infection Negative Infection Negative Infection Negative Remarks Flies starved. Flies starved. Died on 28th day. Flies divided into two groups, A and B. Group A, only, fed. Group B, only, fed. At Flies of group A divided Az into 3 sub-groups, A 1, A 2, A 3. A 3 Br Flies in group B divided B2 into three sub-groups, B 1, B 2, and B 3. B3 Sub-group B 1 fed. Sub-group B 3 fed. Sub-group A 1 and B 2 fed. Sub-group A 2, A 3, and B 1 fed. The insects were dissected as they died, but only in one, the infective fly, was an infection of the salivary glands observed, though in a considerable number an infection of the proboscis and gut was found. 200 The duration of the development cycle of the trypanosomes in the fly would appear to be twenty-five days in this experiment. The flies were fed for the fivst time on the infected guinea-pig on February 14th, and the first monkey became infected on March 15th, thirty days later. The average incubation period of the disease in monkeys is five days, so that the cycle took twenty-five days to complete. DISCUSSION OF RESULTS In these transmission experiments, there are at least three sources of error which must be considered, (1) accidental infection of the experimental animals by other than the experimental flies, (2) hereditary transmission of trypanosomes from infected female flies to their progeny, and (3) natural infection in the experimental animals. (1) With regard to the first of these, the conditions under which the experimental flies and animals were kept have been mentioned already, and it seems more than improbable that accidental infection would account for the unfailing regularity with which the animals became infected after the infective flies had fed on them. More- over, in all our experiments, 644, such an occurrence as the unexpected infection of an animal has not been observed. (2) The number of bred flies which we have been able to obtain has been too small to permit us to examine many of them prior to use in the experiments, but such as were, have been found uniformly free of infection. Stuhlmann,* Kleine,t and Brucet with his colleagues have examined large numbers of bred flies belonging to the species Glossina brevipalpis, Glossina morsitans, and Glossina palpalts, and are unanimous in the opinion that hereditary trans- mission of trypanosomes does not occur amongst the tsetse flies. (3) With reference to the third point, we have used in the course some 256 monkeys, and have never seen a naturally-occurring trypanosome infection in any of them. Plasmodium kochi and microfilaria have been observed, but beyond these, nothing. * Stuhlmann. Arbeit aus d. Kaiser. Gesundheitsamte, Band XXVI, Heft 3, p. 374. + Kleine. Deutsche med. Wochenschrift, No. 45, 1909. t Bruce, Hamerton, Bateman and Mackie. Reports of $.S. Commission of the Royal Society, No. 11, pp. 122-125. 201 Experiment 2 was specially devised to obviate the possibility of error through the use of local monkeys (Cercopithecus pygerythrus). The flies, as they were obtained, were fed on healthy, imported rabbits which showed no signs of infection throughout; they were infected directly from the human host; and they were then fed on white rats. With the exception of Experiment 1, all the bred flies used in the transmission work were infected directly on the human host. The trypanosomes transmitted by these flies were identical with the human one, both morphologically and in their animal reactions. There are certain points in connection with the experiments which appear to be worthy of emphasis. The number of bred flies which has been used in each is strikingly small, very much more so than in any other similar work, of which the records are available. In the four experiments a total of eighty-five was employed, and of these three only became infective. A percentage of 3°5. The time occupied by the trypanosomes in completing their cycle in the flies is also strikingly short, approximately two weeks (thirteen, fifteen, eleven, nineteen and twenty-five days). It may be pointed out, however, that all our estimations of the latent periods of the trypanosomes in the flies represent the probable durations only. Although the average incubation period in monkeys is five days, this has been found to vary from three to eight days, and it is possible, therefore, that the cycle may have been slightly shorter, or longer, in any one instance. Moreover, a further source of error is introduced in those experiments in which the flies were fed on an infected animal for more than a single day. It has yet to be determined whether only a definite percentage of flies are inherently capable of transmitting the disease, or whether azy fly will do so provided that it has an opportunity of feeding on an infected animal at some particular time during its existence. If the latter alternative be correct, the peculiar factors governing their infectability have still to be ascertained. Assuming the first view to be correct, then the latent period of the trypanosomes in the flies must date from the fs¢ occasion on which the insects were fed on the infected animal, while, if the second be correct, the latent period may date from any of the meals on the infected animal. 202 So far as our results go, we have seen no indication of late infection in any of our flies, although some of them have lived as long as seventy-four days after the potentially infecting meal.* All our results go to show that mechanical transmission of the trypanosomes does not occur, that is, 1f a period of twenty-four hours has elapsed since the infecting meal. We have not made any experiments to ascertain whether infection could be accomplished by interrupted feeding. This has been proved with various insects, but practically, would account for very few, if any, cases of the disease. The infective flies have been found to retain the power of transmitting the parasites during life, and do not require to feed more than a single time on an animal in order to infect it, neither do they require, prior to becoming infected, to feed more than once on an animal suffering from trypanosomiasis. With regard to Experiment No. 5, although only two flies were definitely proved to transmit the trypanosome, infection of the salivary glands was found, on dissection, in four others. As will be seen in Section V, this is strong presumptive evidence that these four were also infective. EXPERIMENTS AT NGOA A. WITH LABORATORY-BRED Glossina morsitans Experiment 9 Commenced on June 23rd, 1912, with nineteen laboratory-bred flies. They were fed for five days on a heavily-infected guinea-pig, and afterwards for fifty-five days on a healthy monkey which did not become infected. B. By ‘WILD’ Glossina morsitans Experiment 10 Commenced on May 18th, 1912, with 116 ‘wild’ Glosszna morsitans, which had previously been shown to be non-infective by being fed on a healthy monkey. The flies were fed for four days on a heavily-infected guinea-pig and afterwards for sixty-seven days on three healthy monkeys, none of which became infected. * See, however, page 209. Annals Trop. Med. & Parasitol., Vol. VI1 PLARLRE XTi Fic. 1. Camp at Ngoa, Congo-Zambesi Watershed. Fic. 2. Laboratory at Ngoa, Congo-Zambesi Watershed. To face p. 202 C. Tinling & Co., Ltd., Imp- 203 Experiment U1 Commenced on June 13th, 1912, with ninety ‘wild’ flies previously proved not to be transmitting the human trypanosome. They were allowed to feed for three days on a heavily-infected guinea-pig, and were afterwards allowed to feed on a healthy monkey until the 4oth day of the experiment. This monkey did not become infected. On the 41st day of the experiment the flies then alive, forty-two in number, were placed in an incubator, kept at a temperature of 85° F. Three flies were found to be infective eight days later. Experiment 12 Commenced on June 14th, 1912, with 119 ‘wild’ Glossina morsitans, proved to be non-infective. The flies were fed from the Ist to the 3rd day of the experiment on a heavily-infected guinea-pig, and afterwards to the 6oth day on a healthy monkey, which did not become infected. On the 61st day the thirty-eight remaining flies were placed in the incubator kept at 83° F., and were fed from the OIst-75th day onaclean monkey. The monkey died on the 76th day without becoming infected. However, on dissecting the flies, one was found to harbour trypanosomes in the gut and salivary glands, and the contents of these structures inoculated into healthy monkeys caused the animals to become infected with 7. rhodesiense. Experiment 13 Commenced on July 11th, 1912, with 176 ‘wild’ Glossina morsitans, previously shown not to harbour the human trypanosome. These flies were fed for three days on a heavily-infected guinea-pig, and afterwards from the 5th—5ist day on healthy monkeys, none of which became infected. Experiment 14. Commenced on July 24th, 1912, with 160 ‘wild’ Glossina morsitans, previously proved to be non-infective. From the Ist—4th day they were fed on a heavily-infected guinea-pig, and from the 5th—36th on a healthy monkey, which did not become infected. 204 (e) INFLUENCE OF METEOROLOGICAL CONDITIONS ON THE DEVELOPMENT OF THE TRYPANOSOME IN GLOSSINA MORSITANS At Nawalia, eight transmission experiments were made, four with laboratory-bred, and four with ‘wild’ Glossina morsitans. At Ngoa, six experiments were carried out, five with ‘wild’ and one with bred flies. It is unfortunate that the bred flies in the latter series were not more numerous, but owing to the low temperature the majority of the flies did not emerge from the puparia, and many of those which did were malformed, and quickly died. In all the experiments in which ‘wild’ flies were used, however, the possibility that they were already infected with the trypanosome was excluded by first feeding them on healthy monkeys. Synopses of the two series of experiments are given in Tables 9 and io. It will be seen from these tables that whereas, in the Luangwa Valley, T7ypanosoma rhodesiense was successfully transmitted by Glossina morsitans, all efforts in this direction on the Congo- Zambesi watershed have been in vain. Of 330 flies used in the valley experiments, six, and probably ten, became infective. The larger figure is based on the number of salivary gland infections found in the flies. Our experience indicates that the implication of these structures is intimately connected with the ability of Glossina morsitans to transmit JT7ypanosoma rhodesiense, and ‘that until they are invaded by the organisms _ the flies are non-infective. Salivary gland infections have been found in all the flies which were capable of transmitting the parasite. In Experiment No. 5 (Table 7) six flies were found to harbour trypanosomes in the glands, but of these only two were actually proved to transmit T7yfanosoma rhodesiense. As in all other instances, it was shown conclusively that those flies in which trypanosomes were found in the salivary glands were infective, it may be concluded that the remaining four flies in this experiment were also capable of transmitting the parasite. Invasion of the salivary glands has not been observed except in those flies which were known to transmit 77ypanosoma rhodesiense. In the six plateau experiments, 680 Glossina morsitans were employed without a single fly becoming infective. Pa 7 205 Dee So eee o.LP o.1$ 1.9 L.+9 — es af og! e zuLhz 9 0.0 1.6+ o.7£ 0-29 — oe PIEM. gLI ae zuL11 S 0.28 o.zh g.1Z Z-09 _ ob pog 61 «“ z1'9'0% $ 0.2$ o.th $.2Z 1.6 — se ‘ 611 oe z1'9'FI ¢ 0.2 o.zh $272 1.6$ — OG t 06 “ z1g'f1 z 0.2$ $.o$ $.bZ 4.29 _ dATIESON, PIEM git A1q ziS°gi I % apoAo ayoso ayoAo jequaurdojaaap | jequeurdoyaaap | jeyuewdoyasap aqpoAo peqieys Ajiprumny surinp sulinp Surimp [equeurdoyaaap qNsoxy pasn sary pasn sary woseas yoryas ‘ON DATILPOY umurruru wINUWTxeur ainqeiadura} jo uorjeing jo Aare A jo ‘ON uo 21%q aqnjosqy aqnjosqy uray “poysiaqeM Isaquivz-o8u0y ‘eos ye Jno patsivd ‘suppsssom DuULSso]Z) PUL asuaisapogs “J, YIM syuouttiodxa uorsstursuvsy jo sisdoukg—or atavy, ‘dquo skep of sip ay} 10} pazepNoyeo usaq aavy AIprumy eAT}e[aI oy pur ainqeiaduia} uvout 94} ‘syuaurtiadxe pnjssaoonsun ay} JO sasvd dy} UT y | $.99 0.1 0-98 €.L2 Sz ¥ PIEAA tol " Zeek g $.SZ gz £.$8 6.22 61 uoTqaJUyT pag tz CA ani £ z-bL g-zZ L.$8 0.gZ a < is a7 a ZUI'ZI 9 Zbl gz £4.58 o.gZ = f PIM zb Aurey ZZ S SZ gz 1.28 Z-QL -— 9ATIEDIN pug oz Aurey I1'Z1°6z + gt S.bZ $.£6 g-£8 11 2 PIEM LS ) IV1Ub1 € suivi Jo Juour g-th S.bZ $.£6 S.£g $1 st MY gI -sououmod +) | riribr Zz o.S€ $.09 0.06 1.$2 £1 uoT}IOjUT pag gz 41g I1"g*07Z I % ayoso ayoAo aposo skep [equourdoyaaap | jeyuowdoyaaap | jeyuaurdoyaaap ; peqie3s ,Auprumy Surinp Sutinp Sutinp apodo yNsoy pasn sary posn sory wosvas yory a ‘ON IATILPY wumnurTuTUT uINUWITxe Ur ainqesoduray | [equourdoyaAap jo Ajarse A, jo ‘ON uo 23eq aynjosqy aynjosqy uvayy jo wuorqring i es eee D0 206 The explanation of these apparently contradictory results is at first sight not very obvious, more particularly in view of the fact that even on the plateau ‘wild’ Glossina morsitans capable of infecting healthy monkeys with Trypanosoma rhodesiense were occasionally encountered. If the climatic conditions under which the valley experiments were carried out be compared with those obtaining during the plateau experiments, it will be seen at once that the most striking difference is one of temperature. As a rule, the temperature during the former series of experiments was roughly from 15-20° F. higher than during the latter series. With a view to ascertaining the influence, if any, exerted by temperature on the developmental cycle of T7ypanosoma rhodesiense in the tsetse fly, a further series of experiments were performed on the plateau, in which, by means of an incubator, the flies were kept at a temperature approximating to that of the valley at the most favourable season. In the first two experiments ‘wild’ flies were used. No water was placed in the incubator, and the warm dry air was found to have a very deleterious effect on the insects. Within the first seven days, twenty-five of the sixty-one flies with which Experiment No. 1 was commenced, and fifty-three of the seventy- two in Experiment No. 2 had died. Notwithstanding the small number alive at the end of the second week, two infective flies were obtained in the first experiment and one in the second. In the third experiment laboratory-bred flies were employed instead of ‘ wild’ ones. Attention was drawn to the fact that the low temperatures obtaining at Ngoa in the cold season were very unfavourable to the pupation of Glossina morsitans; in fact, so slow was the process that in spite of the large number of pupae at our disposal, we were unable to procure sufficient flies for experimental purposes. The difficulty was all the greater as many of those which did emerge were malformed, and quickly died. In order to obtain a sufficient number of bred flies for this experiment we resorted to the expedient of placing the pupae, some of which had been deposited over two months previously, in the incubator (85° F.). Within three or four days a large number of flies were procured. The experiment was commenced on August 8th with thirty bred 207 flies, to which were added twelve on the oth, eleven on the 1oth, and three on the 11th. These groups were fed for four, three, two and one days, respectively, on a heavily-infected guinea-pig and after- wards on a healthy monkey. Parasites were found in the blood of the animal on which the flies of the fourth group were fed on the 18th after the insects fed on the guinea-pig, so that, allowing five days for the incubation of the disease in the monkey, the fly was infective on the 13th day. The monkey on which the flies in Group 1 were fed became infected on the 26th day of the experi- ment, so that the duration of the developmental cycle of the parasite in the fly would be twenty-one days. As we cannot be certain that the infective fly fed on the infected guinea-pig on each of the four days, the latent period in the insect may be anything from seventeen to twenty-one days. The animals on which Groups 2 and 3 were fed did not become infected. It will be seen from Table 11 that the total number of flies used in the incubator experiments was 189—133 ‘wild’ and 56 ‘bred.’ Of the ‘wild’ flies, three became infective, and of the ‘bred’ two, i.e., 26% of the total number used. This figure is, however, hardly a fair estimate, as of the 133 ‘wild’ flies only seventy-eight were alive at the end of the first seven days of the experiment. This heavy mortality was probably due to the sudden change from the cold external air to the warm, dry atmosphere of the incubator. As a general rule, in our transmission experiments it was found that about 10 % of the flies died during the first week. This was approximately the case in the incubator experiment in which bred flies were used, as, owing to the fact that the insects were hatched out in the incubator they were not subjected to any sudden change of atmospheric conditions. We consider, therefore, that had the mortality of the flies in these two experiments been the customary 10% instead of over 40 %, the proportion of infective flies would be three of ninety instead of three of 133, or 3°3% instead of 22%. This figure, 3°3, approximates closely to that obtained in the incubator experiment in which bred flies were used, namely, 3°5, and also to that obtained in the valley experi- ments, 3°5. The results of these three series of experiments, viz., those carried out at Nawalia at laboratory temperatures 75°-84° F., 208 O/ /O Aqrprumy DATILPOY oLL z-6g 9:78 Iz puv {1 ge S.b£ 3-28 9-08 €1 et S.bZ 9-28 9-08 b UOT}OOFUT aposo aposo apodo skep [equourdoyaaap | [eyuourdoyaaop | jeyuowdooaop suimnp surinp surinp apo qyNsoyy wmnuwrurur wInUITxeUl ainqeiadura} | Jeyuoudoyaaap aynjosqy aynjosqy uvayl jo worjeing pag ia posn sary jo Ajore A zL 19 pasn sory JO “ON “ ZTI'Q'g £ ue Zl Ler Zz Ag ZI"g'0f I peqieys wosvas yory ‘ON uo 93eq *10geqnour ur ydoy suy7jtsiom vurssozH Jo suvour Aq ‘payssaqeAA Isequie7z-o8uo0g ‘voSN ye asuatsapogs “7, ywuusuesy 07 syusurtiedxe jo stsdouAg—11 alavy, 209 those made in the laboratory at Ngoa at from 59°-65° F., and the incubator experiments at Ngoa 80'6°-82°6° F., show in a most conclusive manner that comparatively high temperatures, 75°-85° F., are necessary for the completion of the developmental cycle of Trypanosoma rhodesiense in Glossina morsitans. In addition to temperature, there is another factor in the climatic conditions which might possibly influence the develop- mental cycle of the trypanosome in Glossina morsitans. We refer to the relative humidity of the atmosphere. At the most favourable season of the year in the Luangwa Valley for transmission experi- ments, and also in the case of the first two carried out in the incubator, the relative humidity was extremely low. In order to decide the point, the relative humidity of the atmosphere in the incubator in the bred fly experiment described above was kept at from 70-72'5%. As the incubation period of the parasite in the flies (twelve and seventeen to twenty-one days respectively) and also the percentage of infective flies obtained (3°5 %) were approximately the same as those in other experiments in which the relative humidity was very low, we can only conclude that this factor does not exert any appreciable influence on the developmental cycle of T. rhodestense in Glossina morsitans. The following experiments were devised with a view to ascertaining more definitely the influence of temperature on the development of the parasite. Experiment 15 Two batches of ‘wild’ Glossina morsitans (Batch A consisting of 95 and Batch B of 119) in which the possibility of the presence of an infective fly had previously been excluded by feeding the insects on clean monkeys, were fed for three consecutive days on a guinea-pig infected with 7. rhodesiense. After being starved for a day, each batch was fed on a healthy monkey until the goth day after the first feed on the infected animal. Neither of the monkeys became infected. Batch A, in which there were forty-two flies still alive, was placed in the incubator, whilst Batch B, in which there were now fifty-eight flies, was kept at laboratory temperature. The sudden change from the laboratory to the warm, dry air of the incubator proved very fatal to the flies 210 in Batch A, and on the 43rd day only six were alive. From the AIst to the 47th day the flies in this batch were fed on a monkey, and from the 48th day on a rat. The rat became infected on the 53rd day, so that, allowing five days for the incubation of the disease in the animal, Batch A contained an infective fly on the 48th day after the first feed on the infected guinea-pig, and eight days after being placed in the incubator. As the monkey died on the 47th day, we are unable to state whether the fly became infec- tive before the 48th day. The four flies still alive on the 53rd day were fed on four clean rats, and three of these became infected. The monkey on which Batch B was fed was still negative at the end of sixty days, when there were thirty-eight flies alive. Experiment 16 This is really a continuation of the former experiment. The thirty-eight flies in Batch B were placed in the incubator on the 61st day after the first feed on the infected guinea-pig, and were fed from the OIst-75th day on a healthy monkey (No. 443). Unfortunately the animal died on the 76th day, so that we were unable to determine with certainty whether any of the flies became infective. All the flies were dissected as they died, and one was found to harbour trypanosomes in the gut and salivary glands. Animals inoculated with the contents of these structures became infected with T. rhodeszense. As in our experience all flies in which salivary infection was observed were capable of infecting animals with the human trypano- some, we may assume that had the monkey (No. 443) lived a few days longer it would have been found to be infected. In Experiment 15 the relative humidity of the air in the incubator was very low (36%), while in Experiment 16 the relative humidity was comparatively high (72 %). In addition to confirming the view that a relatively high temperature is essential to the completion of the developmental cycle of 7. vhodeszense in Glossina morsitans, and that the relative humidity of the atmosphere is not an important factor, these experiments afford more definite information. It is apparent that the earlier stages of the develop- ment of the parasite in the fly can occur at comparatively low temperatures (60° F.), and that trypanosomes can persist in this “dep puzZ oy qnoge 9aatqoazut (Aep 4s19) aueseq 9A aug | oz o.LZ£ 0.16 0.£g gt ZI'Q 1 o-7$ o.zh t.2Z 1.6$ aw 611 s zi'gtt | gt ep WIgh uo satjoozurT (4ep ystt) aumedeq sat saiyy, | 2-9 oLL $.06 0.$g ca zrL-vz 0.78 o.¢F bezL 1-6$ PIEM. 06 Aig ZIOse weal ne 1ojeqnout _ ur qnd Ys " ‘uIny “UTUT *xeur | -dura3 qs JoJeqnout “Uru xeut | ‘duaq PU ‘Josqy | ‘fosqy | uvayy | orm fay} ayy ‘uny | ‘josqy | ‘josqy | uray pasn aut} ur qnd PY sof yo | sore | uosvag | paqiezs | ‘on SyIVUIDY ——__'———__|-___|______| oy qe_s | arom sary ——_——_|______!_______| Ayatie, | Jo on a1eq pale UP es SNOILIGNOD, AOLVANONT sary uo 93eq SNOILIGNOD ANOLVAOAV'T FORON eee ee ee eee eee eee eee Ee eee “SUDISLOUML DUISSOI) Ul asuatsapogs “J, Jo yuoudojasep ay Uo ainqvioduray ay} Surster fo Joyo Zurmoys syusuradxa jo stsdoudg—'zI alavy, 212 stage for at least sixty days. It is obvious that the developmental cycle of the parasite is not complete, since the flies are non- infective, and inoculation of the gut contents into susceptible animals is followed by negative results. For the completion of the cycle it is necessary for the temperature to which the flies are subjected to be raised to a considerable extent (75°-85° F.). It is interesting to note that the flies in Batch A (Experiment 15) became infective eight days, possibly less, after being placed in the incubator. This is three days less than the shortest incubation period observed in any of our successful transmission experiments, a fact which supports the view that the developmental cycle of the parasite in the fly had proceeded to a certain point at laboratory temperature (60° F.) before the insects were subjected to the higher temperature (80° F.) of the incubator. The fact that an occasional infective ‘wild’ fly was encountered on the plateau during a period (May, June and July) when attempts to transmit in the laboratory were invariably unsuccessful requires some explanation. A possible solution may be that the flies in question were infected during the warmer season of the year and had survived into the cold season. If the results obtained by feeding freshly-caught flies on healthy monkeys in the valley are compared with those from flies caught on the plateau, a marked difference in the number of infections resulting is apparent. In the Luangwa Valley, 3,202 flies were fed in twenty-nine batches, and 77ypanosoma rhodesiense was isolated in six of the experiments, giving a ratio of 1 infective fly to 534, whereas on the Congo-Zambesi watershed, 5,250 freshly-caught Glossina morsitans were fed in groups on forty-one monkeys, with four positive results—1 infective fly to 1,312. As tsetse flies and game are about equally numerous at Nawalia and Ngoa, and as the disease was presumably introduced into the two localities, which are less than seventy miles apart, about the same time, it appears to us that the only essential difference which can account for the fact that the percentage of infective ‘wild’ flies at Nawalia is two and a half times as great as at Ngoa is the difference in the climatic conditions. It will be seen from Tables 1 and 2 that the temperatures experienced on the Congo-Zambesi watershed during May, June and July, are very much lower than those at Nawalia Annals Trop. Med. & Parasitol., Vol. VII PEATE XVit Fic. 1. Feeding Glossina morsitans on experimental animals. Fic. 2. Feeding Glossina morsitans on experimental animals. To face p. 213 C. Tinling & Co., Ltd., Imp. 213 from September to March. It was during the months named that our experiments were carried out at the two places. Finally, it might be mentioned that the percentage of infective ‘wild’ flies caught in the valley was greater in the hot than in the cold season. This point is illustrated in Table 13. TaBLe 13.—Percentage of Glossina morsitans found infected with Trypanosoma rhodesiense at Nawalia at different seasons of the year No. infective | Ratio of infective IQII-IgI2 Mean external No. of flies fed with to non-infective shade temp. | I. rbodesiense flies syatysi nt ot stn) she) [23 POT June ... set 67-2 18 | fo) uly se: Soo 68-7 385 | ° re : 790 August GEG 193 ° September... 77°5 : 194 | fo) | October ae 86-1 = | — November... 87-1 270 | I | December... 82°3 205 ° I : 338 January ope 80-6 538 2 | February 500 79°2 , 104 | ° | March “oc 79:0 823 | 2 + 1: 466 April (to 9th) 79°5 472 | I | The facts brought to light in this investigation afford a satisfactory explanation of some phenomena which have hitherto appeared to be contradictory. Kleine* was unable to transmit T. gambiense by Glossina morsitans on the Victoria Nyanza (altitude 3,700 feet), whereas Tautet was successful on Lake Tanganyika at a lower altitude (2,680 feet). Although we were unable to transmit 7. rhodesiense experimentally on the Congo- Zambesi watershed in the cold season, nevertheless we found that a certain percentage of ‘wild’ flies was infective. This apparent * Kleine. Deutsche med. Wochenschrift, No. 45, 1909. + Taute. Reviewed in Bull. S.S. Bureau, No. 31, Nov., rgrt. 214 discrepancy may be explained on the assumption that the flies became infective during the warm season and that a certain number survived into the colder season of the year. (f) THE RESERVOIR OF THE TRYPANOSOME The possibility that game might act as a reservoir of infection of sleeping sickness areas has been recognised almost since the inception of work on the disease, but up to the present it would appear that the trypanosomes have never been demonstrated in such animals under natural conditions. In Uganda, Bruce, Hamerton and Bateman* have proved that certain species of buck, notably waterbuck, bushbuck, and reedbuck, can readily be infected with T7ypanosoma gambiense by allowing infected Glossina palpalis to feed on them, and that healthy flies, in turn, may be infected from game harbouring parasites in their blood. They were unable, however, to examine a sufficiently large number of head to ascertain whether a natural infection was present. The importance of the question is obvious, and the results of our investigations on the point afford a striking commentary on the potential danger involved in the infection of the game. The Luangwa Valley is particularly rich in a widely-varied fauna, and owing to the fact that in the dry season the great bulk of the game tends to collect in the vicinity of the few permanent streams, it has been comparatively simple to shoot buck for the purposes of experimentation. At Ngoa, on the Congo-Zambesi watershed, game is plentiful at certain seasons of the year. Trypanosomes indistinguishable from TJ. rhodesiense were isolated from buck in both these localities, and, in addition, from one native dog living in a village some fifty miles to the East of Nawalia. In the Luangwa Valley 127 head of game were examined, and sub-inoculations into healthy monkeys and rats were made from 56. In this manner the human parasite was recovered from the following animals : — 4 Waterbuck, 1 Hartebeest, 2 Mpala, 1 Bushbuck, 1 Warthog. * Bruce, Hamerton and Bateman. Proc. Roy. Soc., B, Vol. 83, 1911. 215 On the plateau 124 head were examined, and sub-inoculations were made from sixty. The trypanosome was isolated from two waterbuck only. There is thus a marked difference in the percentage of game infected with 7. rhodesiense in the two districts, the parasite being five times as frequent in the valley as in the plateau game. As the game was shot without discrimination, the figures are probably a fair indication of the proportion of the total game infected in the two areas. TaBLe 14.—Percentage of game infected with T. rhodestense. NAwWALIA NGoa (Luangwa Valley) (Congo-Zambesi Watershed) No. inoculations No. infected we No. inoculations | No. infected Woe made made 56 9 16 60 | 2 353 As stated above, the trypanosome was isolated from one native dog only of thirty-five domestic animals examined—cattle, goats and dogs. Our experience indicates that big game is much the most important reservoir of the infection. During our sojourn in the country some 256 monkeys, 142 wild rats, and 15 wild mice were examined, with negative results. In all, therefore, 698 animals were examined. Apart from the bigger species of game, most of the smaller wild animals are nocturnal in their habits, and it seems unlikely that many of them would be exposed to infection. (g) OCCURRENCE OF THE TRYPANOSOME IN GLOSSINA MORSITANS IN NATURE Series of experiments were carried out in the Luangwa Valley and on the plateau to determine the species of trypanosome transmitted, in nature, by Glossina morsitans. A number of fly-boys were sent out from time to time to capture and bring into the laboratory ‘ wild’ tsetse flies, which were allowed to feed on healthy monkeys. Full details of these experiments will be found in a later section of this report. At Nawalia (Luangwa Valley) the human trypanosome was 216 isolated in six of twenty-nine experiments, in which 3,202 tsetse flies were used. Possibly it was present in a seventh, but as the monkey died on the day after becoming infected, no definite state- ment can be made other than that the incubation period of the disease was the same as that in known 7. rhodesiense infections. At Ngoa (Congo-Zambesi watershed) 5,250 freshly-caught Glossina morsitans were fed in forty-two batches on _ healthy monkeys, and the trypanosome was isolated on four occasions. The ratio of infective to non-infective flies in the two localities, assuming that only one was capable of transmitting the virus in each instance, is, therefore, At Nawalia, At Ngoa, ia Ion Sie: No definite comparison can be made between these figures, as the experiments were not carried out under identical conditions. Those at Nawalia were made during both the dry and wet seasons, while those at Ngoa were carried out during the height of the winter at a time when it was impossible to transmit the human trypanosome in the laboratory. | However, the difference is so marked that it may safely be concluded that the plateau flies are infective, in nature, to a much smaller extent, than those in the Luangwa Valley. In one of these experiments the actual infective fly was isolated. Experiment 17 Commenced October 30th, 1911, with sixty freshly-caught flies, to which were added twenty-two additional ones on the next day. The flies were fed as indicated in Table 15. On November 13th, the flies still alive, thirteen in number, were killed and embedded. In the sections, numerous parasites were found in the gut and salivary gland of only one of them. Tasie 15.—Showing the transmission of the human trypanosome by naturally-infected Glossina morsitans. No. of Date Animal | flies fed Result Remarks Oct. 30—Nov. 4 ...| Monkey 96 60-22 Infection Nov. 6 20 Bae -p 105 29 a: Nov. 7—10 53 =f 108 19 “ Nov. 11—12 sa e 113 7 Negative Flies divided into two Nov. 11—12 | oi II4 6 Infection groups to isolate the infected fly. 217 (4) IDENTITY OF ‘GAME’ AND ‘FLY’ STRAINS_WITH THE ‘HUMAN’ STRAIN OF T. RHODESIENSE The conclusion that one of the trypanosomes isolated from game and from naturally-infected tsetse flies is identical with T. rhodesiense has been based on a careful study of the morphology and pathogenicity of the three strains in question. 1. Morphology (Pl. XVIII) In fresh preparations, all three strains show the same mixture of short, slowly-moving, and long, active forms, the relative numbers of which vary in the peripheral blood of any animal from day to day. In stained preparations, it is sufficient to say that it is impossible to distinguish any one of the three strains from the others. Short forms in which the macronucleus lies actually posterior to the blepharoplast have been observed in each of the three strains. The measurements of the three strains also show an extremely close agreement. Eleven hundred individuals of each have been measured, and the results are given in Tables 16, 17 and 18. The total number of parasites drawn from each variety of laboratory animal is the same in the case of each strain, and only twenty-five have been measured from any one preparation, as it has been found that the average length varies within wide limits, from day to day, in any given animal. We are of the opinion that the measurement of twenty-five individuals from one preparation gives a fair estimate of the average length of the trypanosome present in the particular blood film, and that the error is less if twenty-five parasites are measured on each of forty-four different days than if, for example, 100 are measured on each of eleven different days. In measuring the parasites the following technique was adopted:—Thin blood smears, dried in the air, were fixed in absolute alcohol and stained with Giemsa’s solution. The trypano- somes were then outlined at a magnification of 2,000 diameters with the aid of an Abbé camera lucida, and the length along the middle line of the body measured by the tangent method described by Stephens and Fantham.* * Roy. Soc. Proc., B, Vol. 85, p. 223 (1912). 218 Taste 16.—Giving details of measurement of 1,100 individuals of the ‘ human’ strain, Animal Monkey Day of disease Number measured 1100 | Average 21°03 1 19°5 19°41 26°3 228 19°97 21°28 19°57 24°2 20°59 22°81 22°41 19°95 22:26 20°16 272, 19°7 23°5 18-11 19°52 21°91 21°09 22°03 22°21 20°66 18-2 18-4 20°03 21-08 22°98 22°44 22°12 19°64 20°17 20°59 19°32 19°69 23°33 20°88 18-66 20°95 19:94. 23°94, 28-65 2125 Length in microns Maximum 27°75 26°19 28-0 315 30°3 26°25 28°25 29°75 28°75 29°75 315 29°25 27°0 29°25 24°5 31°25 22°5 30°5 24°75 39:25 29°0 30°25 31°75 33°25 27°25 26°5 28-0 25°5 28°25 33°25 30°75 31°25 31-0 27-5 30°0 23°25 24°5 31-0 32°5 22°5 26°75 230 27°25 33°9 3970 Minimum 15°5 13°27 13°5 19°67 17-2 13°25 18-0 15°25 16°75 15°29 18-25 18:5 170 18-75 E75 18-25 17°25 14°5 Lee) 14°5 16°75 La5 16:0 14°5 15°75 14:0 13°75 15°75 16°75 15°70 18°75 18-0 14°25 17°0 16°75 16°75 17°0 18°75 15°5 13°75 18-5 17°25 18-5 21°5 13°25 219 TasLe 17.—Giving details of measurement of 1100 individuals of ‘ game’ strain, Day of | Number Length in microns Animal disease | measured |———————_____»________ Average Maximum Minimum Monkey 71 7 25 24°79 32°9 17°0 » 71 9 25 19°84 23°8 15°3 » 99 - 38 25 26°36 34°25 1g'0 5 120 8 25 20°02 23°5 18-0 “6 120 It 25 21°9 29°25 17°25 > 120 13 25 17°4 20°0 15°0 » 130 8 25 25°97 35°5 19°0 + 130 II 25 22°05 30.5 16°25 ” ughy) 5 25 ee, 25°75 15°75 » 199 7 25 23°6 32°25 16°75 » 201 iD 25 23°4 31-0 17-75 “5 201 8 25 21°62 25°5 17°5 %9 201 9 25 19°58 21°75 17°25 Dog; native: ...) <2. es: ? 25 1g'l 26:0 1575 ~ PAS coc oor 5 25 21°69 25°75 18-5 of ZODE Sone ae 7 25 19°13 23°5 13°5 > AS Gee hee II 25 18-34 2295 16°25 Rabbit 7h: ocomeacee II 25 20°02 29:0 15°2 ~ ZAQY face eae 9 25 16°18 19°5 13°75 5 ZAQi toes) Bees 13 25 22°29 32°0 15°25 ” ZA ON eened Mee 13 25 20°91 28°5 15°75 Guinea-pig 251 ... ... fe) 25 20°87 33°25 15°25 “6 Aisi Reo bas II 25 22°87 34°5 15°75 5 ZISf Wicoo. “ba 13 25 23°11 33°75 15°0 oe ZED voce) aes 15 25 23°5 32°25 14°75 of PIS eich) e509 17 25 24°09 34°25 13°75 5 ART cas Wooe 21 25 21°67 29°75 14°5 Rat Bie bag sas 14 25 2105 Br5 16-0 55 L289) soot Meee 20 25 20°25 21°75 17°5 - HAMS | G05 cos 22 25 20°3 23°75 16:0 oe WAKO), Goo cos ? 25 20°9 28-0 16°25 5 WEG? | song on. 21 25 25°65 30°5 14°5 + 7) | oct son 42 25 19°27 21°5 16°75 *~ TET ess. San 49 25 22°8 32°5 16°25 “5 KOS esse 26 25 21°38 3570 16°5 3 EOGG ies) tot 36 25 19°0 24°5 17'0 59 DLR ws) Met 17 25 17°38 19°0 14°5 op PUY 65) bos 26 25 22°31 34°25 17°5 a PPB cog ek 7 25 18-91 23:0 16°5 oD ZG eco bos 14 25 21°91 35°5 11°75 Mouse 170 ee eee 9 25 20°13 26°5 17°5 a WGlD ) ooo oa 14 25 20°99 26:5 16°75 33 72} Loco cs 6 25 22°89 29°5 17.25 ) 178 7 25 21°6 27°70 16°5 1100 21°38 35°5 11°75 220 Taste 18.—Giving details of measurement of r1oo individuals of ‘ fly’ strain. Day of | Number Length in microns Animal disease | measured. | —————__.—_ — Average Maximum Minimum Monkey 96 7 25 Hiei 32:0 16:0 ” 96 8 25 24:8 33°5 16°5 = 96 9 25 25°6 36°25 16:0 » 96 10 25 23°3 30°75 15°75 -. 96 II 25 22°6 31-0 15°25 ” 96 14 25 20°3 23°5 16°5 ze 114 27 25 22°0 28-0 16°25 “6 114 32 25 20°9 25°25 18-0 “5 114 41 25 20°8 30°75 15°25 =a 210 8 25 24°66 30°56 1725 “ 210 fe) 25 20°29 23°25 18-25 on 217 9 25 26:03 30°75 22°0 » 316 9 25 24°69 52725 17°5 Dog 235 5 25 26°7 33°0 19°0 p 235 Gi 25 21-4 28-0 19:0 A 235 9 25 20°0 28-0 18-25 6 235 13 25 20°0 21°25 18°5 Rabbit 245 7 25 Bae 29°5 16°5 ~ 245 8 25 20°0 28-0 14°5 ” 245 9 25 18-75 27°75 16°25 a 245 13 25 22°84 30°0 17:0 Guinea-pig 246 13 25 19°87 23°5 16°7 BS 246 15 25 20°88 26:0 16°25 a 246 18 25 17°63 21°5 130 x 246 19 29 19°0 27°25 16°5 * 246 20 25 18-95 25°25 14°25 + 246 21 25 21°0 27°5 15°25 Rat 103 4 25 24°1 30°0 17°0 os 103 5 25 20°3 30°0 16°5 ” 103 8 25 18-8 30°75 14°5 %» 218 6 25 19°47 24°75 16°5 3 218 9 25 19°3 29°0 14°5 “5 218 14 25 20°1 2295 17°0 os 218 16 25 19°41 26°75 16°0 as 218 18 25 22°0 30°5 18-0 » 229 6 25 24°55 29°5 UGS 9 229 8 25 21°09 29°25 17:0 ” 229 9 25 19°5 21°75 17°5 ” 229 13 25 223 29°5 18°5 5 229 15 25 20°31 22°75 17:0 Mouse 247 4 25 23°1 29°5 19°5 247 6 25 23°66 29°5 19°25 » 247 9 25 22°6 34/0 18-75 5 247 14 25 20°91 25°25 17°25 221 TasBLe 19.—Comparison of the measurements of the ‘human,’ ‘ game,’ and ’ ’ Length in microns Strain | - Average Maximum SEbomanit tes. “30 a 21°25 39°0 Gamer) | i. dei oo 21°38 35°5 Bly ab a: a 21°67 36°25 ‘fly’ strains. Minimum The similarity in the measurements is, perhaps, best appreciated by a glance at the curves obtained by plotting out the distribution of the various lengths of the parasites, expressed in percentages of the total numbers measured (see Chart I). A comparison of the percentages of ‘short and stumpy,’ ‘ inter- mediate’ and ‘long’ forms is also of interest. TasLe 20.—Comparison of percentages of ‘ short and stumpy,’ ‘ intermediate,’ and the ‘human,’ ‘ game,’ and ‘ fly’ strains. ‘long’ forms of Short and stumpy Intermediate forms Strain forms 11-21 22-24 SHumian!~ pace ae ae 64:78 15°98 = Gamer ye oe oo 300 62°37 15°34 ‘Fly’ ae be re 58-68 18-31 Long forms 25-39" 19°14 21°56 22°41 2. Pathogenicity The pathogenicity of the three strains is synopsised in Table 21, p- 223. A glance at this table will show how remarkably the three strains agree in this respect. V2 ce alone Sede As ee ‘ Seeceeeeessesee ‘ , ourey), ——__—____ Ujeyjs , Ueunyy , ‘ uv Oe (CU CE Fe Ot (6 1 8) een kon Ge acs *4e wige Se te CE tel ce DetGr 4c xZe. oF, Pel lh Eee ile os ya ies az a fea! ig ea Pa ee = a E Be it ia pees a 2 & SPOVINIAIAFA [OEE Ce ee eee f ‘(uIvijs yous Jo Corr) posnsvour sxoquinu [e307 oy} JO sa8vzusored ur passoidxa ‘saqisesed ayy JO syjBua sora oy} JO UOIyNqIIsIP oy} Jno BuNj0[d Aq sures , dg, pue ‘sues, ‘uewMY, ay} JO saAino jo uosVduIOD “[ LUVHD 223 26 PE a8e1aay gs—6 (sdep $$ sa9ye oatpe 1) tL asvi0ay Lz1—Lh 6z a8eioay 6£—61 gz 981 bz a8eisay og—Z skep uorjeing + + a8vi0ay L—z Fr a8vioay Z4I—I1 $ a8vioay L—+ $ 9 oseI0Ay 11—+ skep uorjeqnout a NIVYLS , ATT, ol pesn ‘ON Erb asviay 1S—eh S€ a8eiaay Zg—11 eZ a8viaay oor—£S¢ gz aSeioay zf—t1 Sz O1z 21 o8viaay f9—Z skep uorjeing fb a8eioay s—r+ $ a8eiay g—t Il asvisay Zi—?+ 9 asei0ay g—t $ Z 9 o8eI0ay 11—+ ————_—_________ sdep uorjeqnouy NIVYLS ,INVD, gf cf aseioay €g—S1 $Lz o8vi0Ay fg—Z gf a8visay 611—gh If a8eisay zg—S1 gz 60z 81 asevioay zb—g skep uorjeing t $ a8eioay g—z f11 asei0ay 61—9 $ a8eisay g—b $ vi $ o8ei0ay 6—£ skep uorjeqnouy NIVULG , NVWOP,, ‘asuatsapogs *7, JO suresys , Ay, pure , awed , deere 3 Number of positive inoculations in which no parasites were found in buck’s blood 7 5 Number of negative inoculations in which parasites were found in buck’s blood ... Capos: 6 Votal-mumber: found infected. ....<....¢-05-0. Di tm TA These figures show that at least 37°5% (Nawalia) and 233% (Ngoa) of the local fauna were infected with trypanosomes. Both 7. vivax and T. nanum have been found in game, and to both these species monkeys and rats are refractory, so that no conclusions can be drawn regarding the presence or absence of these trypanosomes in animals in which parasites were not found in the blood smears. Had sheep and goats been available for inocula- tion, it is probable that many more buck would have been shown to harbour the two organisms in question. As a conservative estimate, the percentage of game actually infected with trypanosomes in the vicinity of Nawalia might be placed at 50, and at Ngoa 35. A further point which is brought out in the tables is that different species of buck appear to vary widely in their susceptibility. Amongst the commoner varieties, trypanosomes were never found either by direct examination, or by inoculation in zebra, buffalo, wildebeest and bushpig, and only rarely in roan, hartebeest, puku, mpala and warthog. Waterbuck, eland, bush- buck and kudu were the species found to be most heavily infected. To a certain extent, perhaps, these differences may be accounted for by the habitats affected by the various species of game. Kudu and bushbuck, and waterbuck to a lesser extent, are usually found in thick cover from which they seldom emerge, and where they are more constantly exposed to the bites of tsetse flies. Mpala, puku and wildebeest are usually found in open country, frequently remaining for the greater part of the day on wide, bare plains, and here the flies are less noticeable than in the bush. Specific 232 TaBLe 24.—Percentages of various species of game found infected with trypanosomes at Nawalia. Percentage Animal Number examined harbouring trypanosomes Bushbuck ae sins ae ins se a 9 66-6 Waterbuck 586 aa st = See we 28 60°7 Kudu =. oS a soe = “ef. tre 7 57°71 Hartebeest es $6 moe Rec io an 6 16°6 Roan ... ave ae ack adc a8 “e 8 12°5 Warthog si sn pee So sn wie Q Itt Puku... “oe eds $3 S56 at: Bee 10 10° Mpala ... bet oa ae = a f 29 6-9 Tale 25.—Percentages of various species of game found infected with trypanosomes at Ngoa. | Percentage Animal Number examined harbouring trypanosomes Sitatunga 3 Ex et a3 he ae 2 5° Waterbuck ... she ee soe sos 536 27 44°4 Eland ... oe och ae see aa ae 15 26:6 Duiker ... so sp BE 3 oe 20 9 2252 RGan aes ae oor oe eos aes nee 5 20 Pukuji3 te he ext me abe i 8 12°5 differences in the amount of immunity enjoyed by buck are probably, however, of much greater importance. In Tables 26 and 27 are given the species of trypanosomes occurring in each animal in which parasites were found. In compiling the tables, information obtained from the result of inoculations, where these were made, has been utilised. This enables a _ differentiation to be made between such parasites as JZ. pecorum and T. nanum, which are morphologically indistinguishable. Z. vzvax has a characteristic morphology, and can thus be identified in blood smears without difficulty. Animal Bushbuck ” ” ” ”? »” Waterbuck ” 9 ” ” ” ” ol ”» ” ” sf 99 ” rb} ” ” Kudu ” ” 3%) Roan Warthog Puku = Mpala bP) Hartebeest I | fo) II 12 _ 2 I T iE: vivax Ti a TasLe 26.—Trypanosomes found in game at Nawalia. 233 Trypanosomes found in peripheral blood Negative T. pecorum or T. nanum T. multiforme, sp. nov. T. pecorum or T. nanum T. pecorum or T. nanum Negative T. pecorum or T. nanum T. pecorum or T. nanum T. pecorum or T. nanum and T. vivax fF. pecorum or T. nanum Negative T. vivax T. vivax . rhodestense and J. vivax TE, T. pecorum or T. nanum T. pecorum or T. nanum (?) I. rbodestense . rhodesiense . rhodestense and T. vivax . vivax (?) I. rhodesiense and T. vivax (?) I. rbodestense Negative T. pecorum or T. nanum T. pecorum or T. nanum T. pecorum or T. nanum T. pecorum or T. nanum Negative T. vivax Negative T. pecorum or T. nanum Negative Trypanosomes isolated by inoculation into monkeys and rats T. pecorum Negative T. multiforme, sp. nov. No inoculation T. rhodestense T. pecorum T. pecorum and T. rhodesiense Negative PP) T. pecorum Negative ” T. rbodestense Negative No inoculation Animal died day after inoculation T. rhodesiense and T. pecorum No inoculation T. rhodestense No inoculation ” ” T. pecorum No inoculation T. pecorum No inoculation pb] T. rhodesiense No inoculation T. rhodesiense T. pecorum or T. rhodesiense T. rhodesiense N XQ Ny Ny . . . : . . . . . She ee ae ee oie ee ei Ny Nyy rn Ny vu —= ed wv = Ww yy Se ge te Ae a ee Diagnosis pecorum nanum multiforme, sp. nov. pecorum or T. nanum pecorum or T. nanum rhodestense pecorum pecorum and T. rhodestense nanum and T. vivax nanum , pecorum vivax vivax rhodestense nanum and T. vivax pecorum or T. nanum T. rhodesiense rhodestense and T. pecorum vivax rhodesiense and T. vivax vivax T. rhodesiense and T. vivax T. rhodestense pecorum pecorum or T. nanum pecorum pecorum or T. nanum pecorum or T. nanum rhodesiense V1Vax rhodesiense pecorum and T. rhodesiense rhodesiense —— er — — — — — ——— ————SSSSSSSSSSSSSSSSSSSSSSSSSSSMsssssssssesese Animal Waterbuck 1 ” 2 ” 3 ” + ” 5 » 6 ” 7 ” 8 ” 9 ” 10 =p II Bs 1 Eland I ” 2 33 3 ” 4 Roan I Puku I Sitatunga 1 Duiker I tN 39 Trypanosomes found in peripheral blood 234 TasLe 27.—T'rypanosomes found in game at Ngoa. ie de YN NN NON . Uivax vivax . vivax UtVax - U1Vax vivax vivax . UIVax . vivax . vivax vivax vivax Negative Ti T. tragelaphi, sp. nov. 3”) ” vivax T. vivax Te pecorum or T. nanum Trypanosomes isolated by inoculations into monkeys and rats No inoculation rp) Negative ” T. rhodestense T. rhodesiense Negative T. pecorum Negative No inoculation ” T. pecorum T. pecorum T. pecorum T. pecorum T. pecorum Negative No inoculation Negative No inoculation NM oN ON Be SSE Se AES NYMR NN NNN NN KH AN Diagnosis vivax . U1lVvax . VIVax ViVaXx vivax . vivax and T. rhodesiense . vivax and T. rhodestense . vivax . vivax and T. pecorum . vivax vivax . vivax . pecorum . pecorum « pecorum - pecorum + pecorum . vivax . tragelaphi, sp. nov. . vivax . pecorum or T. nanum As would be expected, double infections in game are not uncommon, tables. and several instances of this are recorded in the No data exist as to the ultimate effect of infection on game. All the animals which were shot appeared to be in_ perfect condition, and presented no objective signs of disease. Whether 235 or not buck succumb to trypanosomiasis it is impossible to say, but as they have increased steadily since rinderpest swept through the country, it may be assumed that their tolerance to trypanosomes is very great. (c) EXAMINATION OF DOMESTIC STOCK The domestic animals examined, and the species of trypano- somes found in them, are given in Tables 28 and 29. Taste 28.—Examination of domestic stock for trypanosomes at Nawalia. Animal Cow ” Goat 39 ” 94 Trypanosomes found in peripheral blood . pecorum or T. nanum . pecorum or T. nanum . VIVaX 4 oN YON . vivax and T. nanum or T. pecorum 9) +e 202 | T. pecorum or T. nanum ” 258 | T. vivax Dog T. rbodesiense 55 T. pecorum 35 T. pecorum a T. pecorum ) T. sp. (montgomeryi ?) TABLE Animal Trypanosomes found in peripheral blood Goat 369 «| T. nanum or T. pecorum +5 375 | T. vivax and T. nanum or T. pecorum » 378 | I. vivax and T. nanum or T. pecorum Trypanosomes isolated by inoculation into monkeys and rats No inoculation ” Negative Negative by) T. rhodesiense T. pecorum No inoculation ” Negative Trypanosomes isolated by inoculation into monkeys and rats No inoculation T. pecorum Diagnosis T. pecorum or T. nanum T. pecorum or T. nanum 29.—Examination of domestic stock for trypanosomes S Sh Gie sh a. aa Se . vIVax . vivax and T. nanum - nanum . V1VaXx rhodestense pecorum pecorum pecorum sp. (montgomeryi ?) at Ngoa. Diagnosis T. nanum or T. pecorum T. vivax and T. nanum or T. pecorum . vivax and T. pecorum 236 The only native village in which cattle were found was Kambwiri’s, some forty miles south-west of Nawalia. At present there are only three head, all that are left of a big herd which existed there some four or five years ago. Two of the three appeared to be in good condition when seen, but the headman of the village fully expected to lose them within a few months. The third beast was obviously ill. The cow in which trypanosomes were found at Fort Jameson was bred on the Government Farm, and had never been beyond the limits of the township. Tsetse have never been seen within some miles of the place, but Stomoxys is abundant in the kraals, and at certain seasons of the year various species of Zabanidae are common. . In several of the villages on the main road from Nawalia to Fort Jameson, a number of goats were found at the end of August, IQII, and again at the beginning of April, 1912, but at the end of that month not a single animal was alive. Glossina morsitans was found around all these villages. The four goats mentioned in Table 28 were under observation at Nawalia for a considerable length of time. During this period, parasites were found in the peripheral blood only at rare intervals. Two were rather thin, but not markedly so, and, apart from this, there were no signs of disease. Goat No. 258 was examined at frequent intervals for two: months before parasites were first found, while in the others, trypanosomes were seen on the first occasion. Nos. 39 and 258, after having been under observation for nine and four months respectively, died on the road when the Commission left Nawalia, most probably from being over-driven. The other two are still alive, seven and four months after the diagnosis was made. The dog in which 7. rhodesiense was found came from a village just on the Nyasaland border. The natives said that it had not been out of the village for over a year previously. As the disease runs an extremely acute course in these animals, there can be no doubt that the dog was infected locally. 237 (d) EXAMINATION OF SMALL VERMIN It has been suggested that the small vermin might also act as reservoirs of trypanosomiasis. It must be remembered, however, that many of the small vermin of Tropical Africa are nocturnal, and are, therefore, not subjected to the same extent as are the big game to the bites of G/. morsitans. At Nawalia and at Ngoa we examined in all 142 wild rats, 15 wild mice, 1 wild rabbit, 1 giant rat, I squirrel, 1 galago, and 2 genet; the results were uniformly negative. Furthermore, it might be remarked that there is no evidence to show that the small vermin are tolerant of the human trypanosome as are the big game. In those which we infected experimentally the disease ran an acute course, and the animals died. If this be the case with the majority of the small vermin they cannot have the same significance as reservoirs of the virus as have the big game, which can probably harbour the parasite for long periods without exhibiting signs of disease. Not a single case of infection with trypanosomes was found in the 256 monkeys (Cercopithecus pygerythus) examined by us, although infection with filaria and Plasmodium kochi was common. The probable explanation of this is that the monkeys during the daytime catch the tsetse fly before the insects have time to feed on them, whereas, on the other hand, they are frequently bitten by mosquitos whilst they are asleep at night. Moreover, it must be remembered that in these animals infection with the human trypano- some runs an acute course, and those animals which contract the disease quickly succumb. SUMMARY Trypanosomes are of frequent occurrence in game and domestic stock in North Eastern Rhodesia. As a conservative estimate the percentage of big game infected with trypanosomes pathogenic to man and domestic stock may at Nawalia (Luangwa Valley) be placed at 50, and at Ngoa (Congo-Zambesi watershed) at 35. At Nawalia six species of trypanosomes were isolated from game 238 and domestic stock, viz., 7. rhodesiense, T. vivax, T. nanum, L. pecorum, T. montgomeryi, and T. multiforme; whilst at Ngoa five species were found, viz., 7. rhodesiense, T. vivax, T. nanum, T. pecorum, and T. tragelaphi. The results of examination of over 400 monkeys, wild rats and mice were invariably negative. 239 SECTION. .iI TRYPANOSOMES FOUND IN WILD GLOSSINA MORSITANS BY ALLAN KINGHORN AND WARRINGTON YORKE During the sojourn of the Commission at Nawalia and at Ngoa, experiments were undertaken with the object of ascertaining the species of trypanosomes transmitted, in nature, by Glossina morsitans, \Westw. The flies, as they were brought to the laboratory, were fed on clean monkeys, which were the only animals available for the purpose. Unfortunately, owing to the lack of healthy goats and sheep, no definite conclusions can be drawn as to whether the fly was infected with such species as Trypanosoma vivax and Trypanosoma nanum, both of which are of common occurrence in game and domestic stock. At Nawalia, in the Luangwa Valley, freshly-caught Glossina morsitans were fed on healthy monkeys from day to day. In all, 3,410 flies were fed in batches, as they were brought to the laboratory, on thirty-three monkeys, but as five of the latter died within two or three days, inferences can only be drawn as to the infectivity of the 3,202 flies fed on the remaining twenty-nine animals. Details of the experiments are given in Table 30. It will be seen from the table that three species of trypanosomes were isolated, namely, 77yfanosoma rhodesiense, Trypanosoma pecorum, and a third, hitherto undescribed parasite, for which we propose the name 77ypanosoma ignotum. 240 Tare 30.—Result of feeding freshly-caught Glossina morsitans on healthy monkeys at Nawalia. Number of Experiment 30.10.11 30.10.11 1411.11 4.12.11 6.12.11 Number of flies fed Result _ Trypanosom?s isolated 160 | fele) 194 Negative Infection Negative Infection ” Negative Infection Negative bb) Infection Negative ” Infection ” Negative Infection ” Negative Infection ” Negative Infection Negative Infection T. ignotum, sp. nov. T. 1gnotum, sp. nov. T. ignotum. sp. nov. 1. rbodestense ?>T. rhodestense, animal died day after becoming infected T. ignatum, sp. nov. T. rhodesiense T. rhodesiense T. rhodesiense T. pecorum T. ignotum, sp. nov. T. ignotum, sp. nov. T. rhodesiense T. ignotum, sp. nov. T. ignotum, sp. nov. T. rhodesiense and T. 1gnotum, sp. nov. T. pecorum and T. ignotum, sp. nov. Goat ; subinoculated monkeys and rats did not become infected 241 An analysis shows that of the 3,202 flies used, at least nineteen were capable of infecting monkeys. This figure is based on the assumption that, with the exception of Experiments Nos. 343 and 353, each batch contained but a single infective fly. In each of these experiments it is highly probable that at least two infective flies were present, as in the former both 77yfanosoma rhodesiense and Trypanosoma ignotum, sp. nov., were found in the monkey’s blood, and in the latter both Trypanosoma pecorum and Trypanosoma ignotum, sp. nov. The percentage of flies infected with each of the three trypanosomes is given in tabular form. Tasre 31.—Proportion of wild Glossina morsttans infected with T. rhodestense, T. pecorum and T. ignotum, sp. nov. ) | Number Number Ratio of infected Species of of to non-infected flies fed infections flies T. ignotum ... ses ae “sae ees 3,008 10 1 2300 T. rhodestense Soc oe 50 ane BA202 (Se I 2 534 T. pecorum ... 300 50 ase S00 3,202 2 I : 1600 fn our second interim report* one experiment in which 7. rhodestense was obtained was inadvertently omitted, so that this parasite was isolated in six, instead of five instances. In addition, Monkey No. 100 became infected four days after the flies had been fed on it. As the animal died the same day, we were unable to decide the species of trypanosome present, but from the short inoculation it is.. highly probable that the parasite was T. rhodestense. On this assumption the ratio of infected to non-infected flies would be 1 to 455. At Ngoa, on the Congo-Zambesi watershed, 5,250 freshly- caught Glossina morsitans were fed in batches on forty monkeys and two goats. Details are given in Table 32. *Kinghorn, A., and Yorke, W. A Further Report on the Transmission of Human Trypanosomes by Glossina morsitans, Annals of Tropical Medicine and Parasitology, 1912, Vol. VI, p. 269. 242 Taste 32.—Results of feeding freshly-caught Glossina morsitans on healthy monkeys at Ngoa. Number of Experiment 2 goats 366 367 368 377, 379 395 404 Number of flies fed 248 223 212 211 130 121 124 146 109 151 118 | Result Negative Infection ” ” Negative Infection Negative Infection ” Negative Infection re Negative Infection Negative ” PP] Infection Negative ” Trypanosomes isolated Subinoculated monkeys did not become Te infected with T. rhodestense or T. pecorum tgnotum T. ignotum my a Te T . ignotum . ignotum . 1gnotum . rhodestense and T. ignotum . Ignotum . 1gnotum . ignotum rhodestense .agnotum TABLE 32,—continued. Number of 481 485 487 Experiment Date Baa 6.7.12 8.7.12 11.712 12.7.12 13.7-12 15-7.12 16.7.12 17.7312 19.7.12 19.7-12 20.7002, 2.8.12 3.8.12 Number of flies fed 80 160 243 Result Negative ” Infection oP) Negative 9) Infection Negative Infection Negative ‘Trypanosomes isolated T. rhodestense MN NON . tgnotum and T. pecorum . ignotum . tgnotum . ignotum . ignotum . rhodestense and T. ignotum It will be seen from Table 33 that 7. rhodestense, T. pecorum and 7. zgnotum were isolated from the plateau flies in the proportion of 1 in 312, I in 1312, and I in 5250 respectively. TaBLeE 33.—Proportion of wild Glossina morsitans infected with T. ignotum, T. rhodesiense and T. pecorum. Species T. ignotum ... T. rhodestense T. pecorum ... Number of flies fed Number Ratio of infected of to non-infected infections flies 16 eg Quid 4 Deyn I Tes 250 244 SUMMARY T. rhodesiense, T. ignotum and T. pecorum are transmitted by Glossina morsitans in nature, and were obtained by feeding wild freshly-caught Gloss¢na morsitans on healthy monkeys. 245 SECTION:..1TV DESCRIPTION OF TRYPANOSOMES FOUND BY ALLAN KINGHORN AND WARRINGTON YORKE 1 LKYPANOSOMA ‘RHODESIENSE \(P1. XVID This parasite has been fully dealt with in a previous section, and requires, therefore, no further description. It was isolated from all the cases of Sleeping Sickness, sixteen in number, observed by the Commission. At Nawalia it was found in four waterbuck, two mpala, one hartebeest, one bushbuck and one warthog— 10% of the game from which inoculations were made—and from one native dog. Parasites resembling 7. rhodestense were found in blood films made from three other waterbuck, from which no sub-inoculations were made. At least six, and possibly seven, of 3,202 freshly-caught Glossina morsitans were found capable of transmitting this organism. At Ngoa the trypanosome was isolated from two waterbuck, and from four of 5,250 freshly-caught tsetse flies. 22 LRYPANOSOMA: VIVAX (PL (XIX, figs. | 1-8) At Nawalia this organism was found in eight waterbuck, one puku and three goats, and at Ngoa in twelve waterbuck, one puku, one duiker and two goats. MORPHOLOGY (z) In fresh preparations it appears as a club-shaped organism, characterised chiefly by the extraordinary rapidity with which it moves across the field. (6) In stained preparations it is seen to be more or less club- shaped, with a long free flagellum. The greatest width is posterior to the nucleus, which is situated about the middle of the body. 246 TaBLe 34.—Measurements of T. vivax. Length in microns Animal | Day of Number = |————————_|—— — disease measured Average Maximum Minimum Goatle.. a0 ? 25 24°35 26 22°5 39 t 25 23°67 26-25 21°25 rte ho One ? 25 22°43 25°5 19°75 » - 258 ? 25 22°74 25°5 21°25 Geo BES ? 25 23°48 28°25 20°25 39 ses 448 17 25 24°69 28°25 18-75 Sees sas 18 25 24°5 28°75 20°25 het 445 19 26 23°22 265 19°25 200 23°63 28-75 18:75 Microns AQ ABIVh (25 7LE 127 Be Die PERCENTAGES Cuart 2.—Giving the curve representing the distribution, by percentages, in respect of length, of Trypanosoma vivax. 247 The blepharoplast is large and rounded, and lies close to the posterior extremity of the parasite. The undulating membrane 1s, as a rule, very feebly developed, or absent. The mean length of 200 trypanosomes was 236, the maximum 28:75, and the minimum 18°75 (see Table 34). The greatest width varied between 2 and 4'25m, average 32h. PATHOGENICITY Inoculations were made into the following animals : — 8 monkeys... = .... all remained negative. 2 rabbits 42 ae : oat | OES 3 Sratsecs: uae Be 4 alk e 4 TRANSMISSION Owing to the fact that we were unable to obtain a number of clean goats, we could not ascertain definitely that Glosszna morsitans transmitted this trypanosome in nature. Nevertheless, that this fly can transmit 7. vivax is shown by the following two experiments. The wild Glossina morsitans used for breeding purposes were fed regularly for over two months on goats which were naturally infected with 7. vzvax and 7. pecorum. ‘These goats were obtained from Ngoa, in the vicinity of which tsetse flies were abundant. Experiment 1 On July 3rd, forty-eight of the breeding flies, which had previously been starved for five days, were fed on a young, healthy goat. The goat’s blood had been examined regularly for ten days before the commencement of the experiment, and no parasites were found. Ten days later the animal became infected with Trypanosoma vivax. Experiment 2 Three other flies were fed on a healthy kid on June 24th, and twelve days later 7. vzvax appeared in the peripheral circulation. The insects were dissected on the day after they had fed on the goat, and trypanosomes were found in one only. The infection 248 was confined to the proboscis, in which the parasites were extremely numerous and disposed in large rosettes. Both these goats were brought, in mosquito-proof cages, from a fly-free area. 3. LRYPANOSOMA NANUM (PI. XIX, figs. 9-16) Found in the following animals at Nawalia:—One bushbuck, three waterbuck and two goats. Possibly it was also present in two other bushbuck, one other waterbuck, two kudu, a roan and two cattle, but as no sub-inoculations were made, it was impossible to differentiate it from 77ypanosoma pecorum. At Ngoa the parasite may have been present in a duiker and two goats, but in the absence of sub-inoculations it could not be distinguished from 7. pecorum. MORPHOLOGY (a) In fresh preparations it appears as a short, sluggish organism. As a rule, it does not progress. (6) In stained preparations it is found to be short, with a more or less rounded posterior extremity from which the body tapers forwards to the acute anterior end; the nucleus is placed at the centre of the body. The blepharoplast is small, and is situated near the posterior extremity. There is no free flagellum, and the undulating membrane is absent, or, at most, very slightly developed. The protoplasm is free of vacuoles and granules, in general. The mean length of 200 individuals was 14°3”, the maximum I9#, and the minimum 10m (see Table 35). The breadth, at the level of the nucleus, varied from 1 to 2'25m, the average being I°5ph. PATHOGENICITY The following animals were inoculated :— 3 monkeys... 7 ... all remained negative. 1 rabbit ot. = is did oy 9 i 3 Eatsr:. « Star ec sal By AL 50 249 ‘Tasre 35.—Measurements of T. nanum. | Length in microns Animal Day of Number = disease measured Average Maximum § Minimum | | r | Goat ... 202 Naturally 25 15°2 19:0 | 12°25 infected | Set 2O2 “6 25 14.77 18°5 115 | ! no) WeaeneO2k sl *) 25 14.0 170 TI'5 : “he Croeese? “y | 25 14°61 | 18'o 115 = | P : . ” 202 ” | 25 13°99 170 115 “5 on Ber 5 25 13°51 18-0 | 10°5 a. wey 202 f 25 14°43 | 18°75 10°0 99 tee 202 ” 25 14°39 | 17° 12'0 | | { | 200 14°36 19:0 10:0 | Microns PERCENTAGES Cuart 3.—Giving the curve representing the distribution, by percentages, in respect of length, of Trypanosoma nanum. 250 DESCRIPTION OF PLATE XIX Trypanosoma vivax and Trypanosoma nanum. Films fixed in alcohol and stained with Giemsa. The figures were drawn with the aid of a camera lucida at a magnification of 2,000 diameters. Figs. 1-8. TJ. vivax. Figs. 9-16. ZT. nanum. 251 TRANSMISSION The vector of 7. xanum has not been determined with certainty. 4. TRYPANOSOMA PECORUM (P). XX, figs. 1-8) At Nawalia this parasite was found in one bushbuck, one mpala, four waterbuck, two kudu, three dogs and one wild rat. It was possibly present in two other bushbuck, a fifth waterbuck, two other kudu, a roan and two cattle, but as mentioned before, it could not be distinguished from 7. xanum in the absence of sub-inoculations. It was also isolated from 2 of 3,202 freshly- caught Glossina morsitans. At Ngoa it was obtained from one waterbuck, four eland, one roan and one goat. It was possibly present in one duiker and two other goats. It was also found in one wild tsetse fly. MORPHOLOGY (a) In fresh preparations, this parasite resembles 7. xanum very closely. It is a short, sluggish organism, showing no degree of translatory power. (6) In stained preparations it appears as a short organism, with an obtuse or rounded posterior extremity from which it tapers to the attenuated anterior end. The nucleus is oval or rounded, and situated near the middle of the body. The blepharoplast is small and rounded, and lies close to the posterior extremity. The undulating membrane, if present, is very feebly marked, and there is no free flagellum. The general protoplasm stains uniformly, and 1s devoid of granules and vacuoles. The mean length of 500 individuals was 13°64, the maximum 19H, and the minimum Q’5y (see Table 36). The breadth, at the level of the nucleus, varied from 1 to 2’5m, the average being 1'5p. PATHOGENICITY A synopsis of the pathogenicity is given in Table 37, p. 253. 252 TaBLe 36.—Measurements of T. pecorum. Length in microns Animal Day of Number = |————————— disease measured Average Maximum Minimum Ox 393 II 25 13°73 18-25 10°25 ” 393 14 25 WE 15°5 9°75 Monkey 2 21 25 13°27 16°25 10°0 E 9 9 25 13:0 16°38 10°3 » 37 29 25 1447 16°75 11-0 Tog 27 25 13°37 16°75 10°5 9 109 36 25 13°09 15°75 10°75 - 123 28 25 15°26 19:0 12:0 ” 317 43 25 14:28 17°5 Ito a 317 47 25 13°54 17°5 Ilo %» 339 14 25 15°61 17°75 11°75 Dog 274 | (?) Naturally 25 14:09 16:75 11-0 infected Guinea-pig 31 21 25 12°33 15°5 9°5 Rat 49 9 25 rgs07 16-0 98 “s 163 20 25 13°26 15°25 10°75 x 163 23 25 13°13 16°25 10°75 0 282 5 25 12°38 14°75 9°5 _ 282 II 25 13°75 16:0 10°25 Mouse II 12 25 | 13°4 16:0 10°6 “ 12 5 25 14-1 16:8 9°8 500 13°6 19:0 9°5 253 Microns 13 /4- PERCENTAGES ae Ae a aoa ed CS a 7 Cuart 4.—Giving the curve representing the distribution, by percentages, in respect of length, of Trypanosoma pecorum. TaBLe 37.—Pathogenicity of Trypanosoma pecorum. Animal No. inoculated Incubation period Duration days days Monkey ne abe 17 8-21, average 12} 23-225 (still alive) Rabbit se Ae 3 8-11, os 10 66-215 Guinea-pig... Ses I 5 39 RACE st BSc 14 3-14, average 64 6-32 Mouse 2 5 Dgpasfs Goat 3 11-18, average 15 53-58 (still alive) (Chee saat ae 508 |= I 9 254 TRANSMISSION T. pecorum was obtained in two of twenty-eight experiments in which freshly-caught Glossina morsitans were fed on clean monkeys at Nawalia, and in one of forty experiments at Ngoa. In all, therefore, 3 of 8,452 wild flies were found to be naturally infected. At Ngoa batches of breeding flies which had fed for two to three months on goats, naturally-infected with 7. pecorum, were allowed to feed on three healthy goats. That on which the first batch, consisting of thirty flies, were fed became infected with the parasite on the 11th day of the experiment. Trypanosomes were found in the blood of the second goat, on which eight flies were fed, on the 12th day. The third goat, on which three flies were fed, became infected after an incubation period of eighteen days. The flies in the last two groups were dissected the day after they had fed. Trypanosomes were found in the gut and proboscis of a number, but in no instance was an infection of the salivary glands observed. These experiments afford satisfactory proof that Glossina morsitans transmits the parasite in nature. 5. TRYPANOSOMA MULTIFORME, sp. nov. (Pl. XX, figs. 9-16) This parasite was isolated from a bushbuck at Nawalia. MORPHOLOGY (2) In fresh preparations, the trypanosome is seen to be markedly polymorphic, resembling in this particular 77ypanosoma rhodesiense, Trypanosoma gambiense and Trypanosoma pecaudt. Short sluggishly-moving forms are seen, as well as long free- flagellated, active ones. As a rule, the short varieties are more or less stationary, while the long ones progress fairly rapidly. The proportions of each of the forms varies widely, from day to day, in any one animal. (2) In stained preparations, every gradation between extremely short, aflagellar forms, indistinguishable from 7. fecorum, and long, slender, free-flagellated parasites are seen. In general, the 55 trypanosome morphologically closely resembles 7. gambzense or T. brucei, except for the presence of occasional pecorum-like forms. The mean length of 1,000 individuals was 21°18, the maximum 33°5#, and the minimum 10’5m@. The curve (Chart 5, p. 259) representing the distribution of the various lengths of the trypano- somes, expressed in percentages of the total number measured (1,000) differs from that of 7. gambzense in that the apex occurs at 17h. Tasie 38.—Measurements of T. multiforme, sp. nov. | Den einbeaiierare Animal Day of | Number — = disease | measured | Average Maximum Minimum Monkey 166 47 25 18-26 26:0 | 14°25 ; 166 62 25 16-2 18°5 | 13°75 Be 166 78 25 15°34 18-0 | 12-0 ” 312 12 25 17°93 24°75 | 10°75 5 312 13 25 16°84 23°0 13°75 » 312 19 25 18-09 221/45 15°75 a 312 42 25 19°65 28-0 17°25 ” 312 23 25 24°73 30:25 13°0 ” 312 44 25 17°48 28:0 13:0 ” 360 9 25 23°73 30°75 15°0 55 360 14 25 18-88 24°5 14°75 ~ 360 19 25 20°22 27°75 15°5 Rabbit 370 7 25 18-36 28:25 11°75 370 15 25 24°1 29°75 16°25 p 370 16 25 20°55 29°0 13°25 ” 379 81 25 23°49 28°75 19°5 ” 414 5 25 25°82 33°5 14:0 » 44 12 25 25°76 31-0 15°5 ” 414 13 25 23°42 30°0 12:0 op 414 16 25 21'1 29°25 10+§ Rabbit Animal 219 219 Day of disease 256 Tasre. 38.—T. multiforme, continued. - - Number measured 1,000 Length in Microns Average Maximum 21°49 28-5 26°03. 32°25 26-41 315 25°34 30°5 23°8 29°5 21°44 28°75 PEST 5 32525 24°79 3Z0 20°32 26:25 18-54 26:0 20:0 30:0 16-61 23°25 16°08 25°75 25°38 33°0 22°11 33:25 21°46 30°25 22597, 32°25 21°64 30:0 19:23 30°0 17°24, 1955 21718 3355 Minimum 10°5 257 TABLE 39.—Pathogenicity of Trypanosoma multiforme. Animal Incubation period Duration Remarks days days Monkey 166 8 94 Tryps. last seen on 78th day. * 312 10 74 Tryps. last seen on 55th day. 5 360 54 156 Tryps. last seen on 65th day. = 455 4 Still alive rroth | Tryps. last seen on 16th day. day Rabbit 370 6 11g Tryps. last seen on rrath day. Pa AT4 = -- Did not become infected. 5 after 2nd 69 Tryps. present at time of death. inoculation Guinea-pig 371 — — Did not become infected. Inocu- lated twice. 5 413 -- - - » 454 =F ai ” ” Rat 219 Gi 14 Tryps. last seen 44th day. “5 253 12 125 Tryps. last seen 86th day. 3 361 10 67 Tryps. last seen 61st day. Ox 391 — — Did not become infected. Inocu- lated twice. Goat Sol = = » DIAGNOSIS This parasite is at once distinguished from 7. pecaudi and I. rhodesiense by the absence of posterior nuclear forms and by its slight pathogenicity for laboratory animals. It more closely resembles 7. gambiense than any other known species, and is not easily distinguished from this parasite. A study of the biometric curves of the two trypanosomes shows certain differences, and if the percentages of short, intermediate and long forms of each be compared, as in Table 4o, the difference is more clearly brought out, the parasite in question exhibiting very few intermediate forms. 258 Taste 40.—Comparison of percentages of ‘short and stumpy,’ ‘intermediate,’ and ‘long’ forms of T. multiforme and T. gambtense. | | | Short and stumpy | Intermediate Long and slender 1O—2I gM 22—24p 25—33e | é Trypanosoma multiforme ; 55°6 12°5 319 } Trypanosoma gambiense Bie? 23°1 25°7 The extreme chronicity of the disease caused by this parasite in laboratory animals is an additional point of distinction from T. gambiense. Ne conclude, therefore, that the parasite is a new species, and in view of its marked polymorphism we propose for it the name 77ypanosoma multiforme. ON ee ze “audofezjnu vuosoungds yz, yO y{3ue] JO y09dsaq ut ‘sasequaosed 452 18 25 16:7 20°25 13°25 ” 452 20 25 15543 1935 1255 oc Soc oe 452 21 25 15°93 20°75 14:0 op 497 1 25 1688 21-0 14°0 ” 497 25) 25 16-93 19°5 13°25 —————————— Ee ee 265 Microns PERCENTAGES Cuarr 7.—Giving the curve representing the distribution, by percentages, in respect of length, of Trypanosoma ignotum, PATHOGENICITY In monkeys, the virulence of 77yp. zgnotum was found to be very great. The disease is of a fulminating character, the parasites increasing rapidly in number until the animal’s death. The trypanosome is of equal virulence in those animals infected directly by the bites of the tsetse flies and in those cases where the strain was passed from monkey to monkey. The incubation period varied from three to ten days, the average being seven, and death occurred two or three days after the parasites appeared in the peripheral blood. It is interesting to note that on reaching the plateau three monkeys failed to become infected when inoculated with the valley strain of the parasite. At Nawalia, a failure was not recorded, and 266 it is difficult to understand those mentioned, more particularly as the parasite is of frequent occurrence in the plateau flies, and monkeys infected with this strain react in the same manner as those infected by the valley strain. Three rabbits were successfully infected with the strain by sub-inoculations from a monkey. The incubation period varied from 13 to 22 days, and the duration of the disease 66 to 106 (still alive). Two guinea-pigs, twelve rats, four mice, an ox, a goat and a dog were found to be refractory. Moreover, negative results were obtained by feeding infective flies on rats. TABLE 43.—Pathogenicity of Trypanosoma ignotum, } Animal Number used Incubation Duration Remarks days days Monkey ... ... 36 3—I10 5—16 Three did not become Average 7 Average 14 infected. Rat cose 12 —_ — None became infected. Guinea-pig ... 2 = _ Did not become infected. Rabbit secs ers 3 13—22 16—106 Two alive after over 100 Average 17 days Mouse ..2) == 4 _- -— Did not become infected. (ORE aos Ge dex I — = 5 35 Godt Woon I 17 68 Tryps. only seen once. Still alive Monkey inoculated on ssth day did not become infected. Dog Sen pases I — _ Did not become infected. DIAGNOSIS Morphologically the parasite appears to be distinct from any hitherto described species. The graph (Chart 7) showing the distribu- tion of the trypanosomes in respect of length resembles very closely that of T7ypanosoma uniforme, but the parasites are at once distinguished by the fact that whereas Trypanosoma uniforme is na Se eireiepty ’ OO FE Cet LE Wye Fe 267 invariably furnished with a free flagellum,* this, as mentioned above, is of rare occurrence in 77ypfanosoma ignotum, sp. nov. Moreover, the difference between the two trypanosomes is clearly demonstrated by reaction of sub-inoculated animals. According to the Royal Society Commission, T77yfanosoma uniforme is innocuous to monkeys, an observation which has been confirmed by Fraser and Duke,+t who record that they were unable to infect these animals by sub-inoculation from game harbouring the parasite, although goats were readily infected. However, the fact that a large number of monkeys, and one rabbit, quickly succumbed to the disease indicates clearly that the two parasites are not identical. No information is at present available regarding the alternative host of the trypanosome. Although the parasite has been isolated from wild Glossina morsitans much more frequently than any other trypanosome, it has never been found in game or domestic stock. Nothing resembling it has been seen in the peripheral blood of any animal examined in the Luangwa Valley and on the Congo-Zambesi watershed. These include 250 wild animals (elephant, rhinoceros, hippopotamus, buffalo, eland, zebra, wildebeest, roan, kudu, hartebeest, true waterbuck, Crawshay’s waterbuck, puku, mpala, bushbuck, duiker, klipspringer, bushpig, warthog, lion, hunting dog, caracal, galago, squirrel, genet, giant rat and rabbit), 35 domestic animals (cattle, goats and dogs), 256 monkeys, 142 wild rats and 15 wild mice—making a total of 698. Eighty-six monkeys have been sub-inoculated from game and domestic animals, and in no instance has an infection with this trypanosome been observed. In view of the fact that we have been unable to find the vertebrate host, we propose to name the parasite Trypanosoma ignotum.t *Reports of Sleeping Sickness Commission of the Royal Society, No. XI, 1911, pp. 160-164. TFraser, Capt. A. D., and Duke, H. C. Bull. S.S. Bureau, Vol. IV, No. 36, April, 1912, pp. 11-152. t On returning to England at the conclusion of the work of the Commission, we found that the Royal Society Commission had published an account of a parasite which they called T. szmiae. This is almost certainly identical with I. ignotum. As the paper of the Royal Society Commission appeared a few days before our paper on T. ignotum, this name must be dropped in favour of T. simiae, should the two eventually prove to be identical. (W.Y.) 268 DESCRIPTION SOR ELATE =. x0 Trypanosoma ignotum and Trypanosoma montgomeryt. Films fixed in alcohol and stained with Giemsa. The figures were drawn with the aid of a camera lucida at a magnification of 2,000 diameters. Figs. 1-8. TJ. ignotum. Figs. 9-16. T. montgomeryz. 269 TRANSMISSION In one experiment the infective fly was determined to be one of a group of ten. These were then killed and dissected. Nine of the flies were found to show no trypanosomes in the gut, proboscis, salivary glands or sucking stomach, whereas in the tenth a heavy infection of the proboscis was encountered. The gut, salivary glands and sucking stomach were negative. This observation would indicate that the development of the trypanosome, 7. zgnuotum, occurs in the proboscis. 8. TRYPANOSOMA TRAGELAPAHI, sp. nov. (Pl. XXII) This parasite was found in blood films made from a sitatunga (Tragelaphus spekez) shot near Mpika. MORPHOLOGY It is at once distinguished from all known mammalian trypano- somes, with the exception of 7. zugens, to which it bears a very close general resemblance—so close, in fact, that in a previous report we referred to it by this name. It is a long, fairly broad trypanosome with a well-marked undulating membrane, sometimes terminating in a short free flagellum. The posterior extremity is effilated. The nucleus appears as a broad band lying transversely across the centre portion of the body, and stains faintly with Giemsa. The blepharoplast is small but distinct, and is situated slightly posterior to the nucleus. The length of the five specimens seen in the single film examined were respectively 52°5, 53, 66, 70 and 72°5 microns, whilst the breadth at the level of the nucleus was 6, 8°5, 5, 6 and 7 microns. DIAGNOSIS Whether or not this parasite is 7. zzgens, we are, from the small amount of material available, unable to state with certainty. The general resemblance of the two trypanosomes is striking, but if the camera lucida drawings of this parasite be compared with those of I. ingens published in the report of the Royal Society Commission, 270 it will be seen that the trypanosome in question is shorter and more slender than 7. zmgens, and that the undulating membrane 1s broader and more pronounced. In the original description of T. ingens the length of five specimens are given as 72, 77, 82, 88 and 122 microns. The breadth is stated to be from 7 to 10 microns. It will be thus seen that 7. zzgens is on an average considerably longer than the parasite in question. The average length of the specimens of 7. zwgens measures 88 microns, whereas that of the specimens of this parasite is only 63 microns. We propose T. tragelaphi as a name for this parasite. TRANSMISSION Unknown. This infected animal was one of a herd which lived in a large swamp formed by the Luitikila river, about four miles from Mpika. The sitatunga only emerge from the swamp in the late evening and very early morning for an hour or two in order to feed, and even then do not wander more than a few hundred yards from the water. Glossina morsitans have never been found within a radius of fifteen miles, and it is therefore exceedingly improbable that they are the vector. Leeches are found in enormous numbers in the swamp, and feed voraciously on human beings. It seems possible that these animals are the vectors, more particularly as the parasite bears a very close resemblance to amphibian trypanosomes. Mosquitos are also extremely plentiful in this swamp. Filaria were seen in the blood films of the same animal. 272 DESCRIPTION “OF SPEATE x Trypanosoma tragelaphi. Films fixed in alcohol and stained with Giemsa. The figures were drawn with the aid of the camera lucida at a magnification of 2,000 diameters. an3 SECTION V ON THE DEVELOPMENT OF T. RHODESIENSE IN GLOSSINA MORSITANS BY ALLAN KINGHORN, WARRINGTON YORKE AND LEE WEELYN ELOYD In the course of our investigations, we endeavoured to accumulate information regarding the development of the human trypanosome in G. morsitans. Reference has already been made to this subject in a previous paper,* and it is here intended to correlate the facts at our disposal. It may be remarked at once that owing to the comparatively small number of laboratory-bred G. morsitans available, the information we have collected is by no means so definite as could have been desired. Up to the present, comparatively little work has been done on this subject, and the records are more or less contradictory. Kleine, +t who was the first investigator to write on the development of T. gambiense in G. palpalis, is of the opinion that the complete cycle takes place in, and is limited to, the intestine, whereas the Royal Society Commissioners} in Uganda consider that involve- ment of the salivary glands is essential. They state that without invasion of the salivary glands there is no infectivity of the fly. TECHNIQUE The method of dissection of the flies used by us was that described by one of us in a previous paper. Briefly, it consists in splitting the dorsum of the thorax longitudinally, and, after separating the muscles and loosening the tissues with needles, “Kinghorn and Yorke. Annals of Tropical Med. and Parasitology, 1912, Vol. VI. t Kleine. ‘Trypanosomenstudien.’ Arb. aus d. kaiserl. Gesundheitsamte, Bd. XXXI, Heft 2. 1 Bruce, etc. Reports of the Roy. Soc. Commission in Uganda, 1911. 274 drawing out the salivary glands attached to the pharynx through the waist. This method has obvious advantages over that described by the Royal Society Commission, in which after snipping off the terminal segment of the abdomen the whole contents were expressed on toa glass slide, and the salivary glands subsequently separated from the mass of intestines and other structures. We claim for the technique adopted by us that the process is quicker, more certain, and that the danger of contamination from the intestines is reduced to a minimum. In fact, the only lesion in the alimentary canal accompanying the operation occurs in the anterior portion of the oesophagus. To a certain point the information obtained from our dissections is exceedingly definite. | We found that in every fly capable of infecting animals with the human _ trypanosome (IT. rhodestense) the salivary glands were invaded. Of the 160 laboratory-bred Glossina morsitans utilised in various experiments to transmit 7. rhodesiense, 132 were dissected as they died. The remaining twenty-eight were too dry when discovered to allow of dissection. ‘Twenty-seven of those dissected were found to be infected with trypanosomes. The day of the experiment on which the flies died and the results of dissection are given in Table 44. A glance at the table shows that, of these 132 flies, five became capable of infecting animals with the human trypanosome. In each of these there was an enormous invasion of the salivary glands by trypanosomes. In the 127 flies which remained incapable of transmitting the parasite, the salivary glands were not involved, although trypanosomes were found in the intestines of twenty-two. A precisely comparable state of affairs was observed on dissection of ‘ wild’ Glossina morsitans which had become infective after feeding on infected animals. The salivary glands were found to be infected only in those insects which were capable of transmitting the human trypanosome. In all, go6 ‘wild’ Glosszna morsitans were used in these experiments, and of this number 620 were dissected. The remainder were for various reasons too dry to admit of dissection. Of these 620 flies the salivary glands of fourteen were invaded by trypanosomes. All except four of these were definitely proved to transmit the human trypanosome. In the case of the other four, the animal upon which the flies had been 275 Taste 44.—Results of dissection of laboratory-bred Gi. morsitans which were found to contain parasites after being fed on infected animals, See eee ee ee ene No. no wm N Date of infective feed 9-9-11 10.8.12 22.1.12 9.8.12 10.8.12 23.6.12 8.8.12 9.8.12 8.8.12 23.6.12 10.8.12 8.8.12 10.8.12 8.8.12 25.8.11 31.8.11 8.8.12 5.3502) 11.8.12 10.8.12 9.8.12 9.8.12 8.8.12 8.8.12 14.11.11 22.1.12 9-9-11 Day of dissection after infective feed mS Ss “N ao mn Resutts or DissECcTION Proboscis % uOueeeie, © © teflon Ore. ©. Cho goro. Ge © + + + 2 tryps. seen oO I tryp. seen a O oO oO Intestine +++ + + ++ ++ - +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Salivary gland @ © OS =O2 On CO. Gk GUO MOR Ce oO: CO ait @ © © An el ch vo 2 at ° +++ +++ +++ +++ Remarks Infective on 12th day Infective on 17th to 21st day On 15th On 19th On 13th N.B.—O = Negative, + = Scanty, ++ = Considerable numbers, +++ = Swarming. 276 allowed to feed died before a diagnosis could be made. None of the 606 flies in which the salivary glands were not involved were able to infect animals with the human trypanosome. Again, the infectivity of Glossina morsitans in nature was examined, as mentioned in a previous section, by feeding batches of freshly- caught flies on healthy monkeys. Certain of the groups infected monkeys, and from one of these infective groups the actual infective fly was isolated. This, on dissection, was found to have the salivary glands swarming with trypanosomes. The remaining 242 flies in this group, which had been shown to be non-infective when fed on monkeys, were dissected, and in no instance was an infection of the salivary glands observed. Tasre 45.—Results of dissection of wild Gl. morsitans found to be capable of infecting animals with T. rhodestense. Resutt or DissecTIoN Date of Day of | |) No. | infective | dissection after Salivary Remarks feed infective feed | Proboscis | Intestine glands it |) oieiinn 25 O +++ +++ | Infective on rith day. 2) || Arie 25 O ae te te +++ | Infective on 11th day. 3 1.7.12 28 O +++ +++ | Infective on 13th day. 4 PA Ep i Ep 30 + +++ +++ 5 2d 30 + +++ +++ Not proved to be infective. 6 PIG ioihit 30 O ++4 +44 7, 21.11.11 30 + +++ +++ 8 14.2.12 39 Oo +++ +++ Infective on 25th day. 9 4.10.11 40 Oo +++ +44 Infective in nature. ite) 30.6.12 42 O JE SESE Seta Infective on 14th day. II 30.6.12 47 O ah oipets +++ | Infective on 14th day. 12 13.6.12 58 O +++ +++) | Infective on 48th day after infected meal, or 8 13 13.6.12 58 O +++ sear an | days after having been placed in the incubator. 14 | 13.6.12 59 O seaea5 tis) 15 14.6.12 71 O +++ +++ Not proved to be infective, but inoculation of try- panosomes from gut and salivary glands followed by positive results. a + ; . 277 In all, twenty Glossina morsitans were found to have invasion of the salivary glands by trypanosomes, and of these, sixteen were definitely found to be capable of infecting animals with T. rhodesiense. Owing to unavoidable circumstances, we were unable to prove the point in the case of the remaining four, but there is no reason to doubt that had the animals on which these flies were fed survived beyond the necessary five or six days they would have proved to be infected. In order to anticipate the criticism that the trypanosomes were not really inside the salivary glands, but lying outside these structures, and due to contamination from the gut, our examina- tions were conducted with extreme care. In the first place, the glands were removed uninjured and attached to the pharynx, placed on a microscope slide, and gently covered with a coverslip. By careful focussing, it could easily be decided that the parasites were actually in the lumen of the tubes and not outside. Moreover, they were usually present in such enormous numbers as absolutely to exclude the possibility that they were the result of contamination from the intestine. Again, the glands of other flies were removed with care and immediately fixed, and subsequently imbedded and cut. In the sections the parasites could be seen to be inside the glands. Finally, in order to remove any possibility of doubt, sections of the whole abdomen of these infective flies were made, and the glands found to be loaded with trypanosomes. It will be seen from Table 44 that it was by no means a rare occurrence for trypanosomes to be present in the intestines in the earlier stages, especially in the case of those flies examined within a few days of the infected meal. As a general rule, however, most of the insects dissected after the first five or six days were negative. In a certain proportion multiplication of the parasites took place in the intestine. As to the reason for this multiplication in the gut of occasional flies only, and as to the manner in which it occurs, we have obtained but little information. On one occasion a fly, which died on the 12th day after having been fed on a guinea-pig infected with IT. rhodesiense, was found to have an enormous gut infection. Possibly there were also a very few trypanosomes in the salivary glands, but on this point we could not be absolutely certain, as the 278 insect had been dead for some time before the dissection was made. In the mid-gut were found a number of cysts containing swarms of trypanosomes. Some of the cysts had thin walls and were filled with a seething mass of flagellates, while others had thicker walls and the contents were quiescent. The cysts ranged in diameter from 27 to 32m. Unfortunately, we are unable to state whether the fly was infective at the time of death. It had refused to feed for two or three days previously, and the animal on which it had last fed (oth day of the experiment) did not become infected. The gut contents were inoculated into a monkey, but the animal died from some unknown cause a couple of days later. Although multiplication of the parasites occurred in the guts of a proportion of the flies, we met no instance in which a fly was infective and in which inoculation of the gut parasites into experi- mental animals gave rise to infection, unless there was an accompanying invasion of the salivary glands. On the other hand, it appears that on every occasion on which the salivary glands are involved the trypanosomes, both in these structures and also in the intestines, are virulent, i.e., the fly infects when fed on a healthy animal, and inoculation of the parasites from either the salivary glands or the intestine gives rise to infection. The results of inoculation of trypanosomes from laboratory- bred flies in different stages of infection, and also from the wild flies which were proved to be infective, are given in Table 46. Our knowledge of the manner in which the salivary glands become infected is uncertain, but there is a certain amount of evidence which would cause one to believe that it is secondary to the intestinal infection, and that it only occurs when the trypanosomes in the gut have reached a certain stage of development, and only then when the conditions of temperature are suitable for the further development of the parasites. In the first place, of 752 flies dissected at various intervals after having fed on infected animals, we never found trypanosomes in the salivary glands in the earlier stages before the flies were infective. Again, whenever trypanosomes were found in the salivary glands there were also enormous numbers present in the intestine. Moreover, it is significant that whenever trypanosomes were found in the salivary glands they were always infective, as were also those present in the gut. | “poazoofur oq 0} punof usoq aavy ppnoa yr ‘ysnouas Zuo] paar ‘pay sem Ay ay} YSIYM wo ‘eurue ayi pey yyy afqeqoad AyYsry st at ‘syvr poqpMooUrqns 07 dATIoFUT OOM OUT]sazUT OYJ UT puL spuUL{T Arearpes oy} YI0q Ut soztsesed oy] qey3 qovF DY} JO MITA UT *10JeqQnouT 943 UT paoeid Avp ys1g OY} UO puL ‘ainqesodura, AToyLIOQLT ye poof AATJOOZUT YY Jaqze Shep Og 10F ydoy sea _YoryA Satias ay} JO au0 sem Ay ay, “JOU JO DATIOOFUT seM JOOSUT OY} JOYIOYM UreZLodsv 0} a[quuN 19M 9M pay sem AY styy YOTYM UO AdyUOUT ay} Jo YIap aywuNgr0;UN oy} 07 SUIMGO y ‘aatqisod yer $sjuaquoo purjs Arvarres = ‘aAtqisod yer $s}ua}U0d ynD +++ + +--+ O rye Bil g1 SPpIVMUO Poe} IAT} *poqoagur Adyuour {sjuaqzuOd (3Ja]) purys Arvat[es -Oafut ysiy s0qze Avp yIgr *paqoayur Aoyuour £ syuaquod (34811) purys AreArpes pue sozeqnour ur Zurqynd *poqoayur yer { sjuaquo0s yng te taaets Sea O gs Jaye Aep yag wor oarytsog | Zi *poqoayur yer $syuoquU0d purys Arvar[es *poqooyur Aayuowr {s}uUo}U0d nF 10g | spzeauo *paqooyur yer $squoquos 4yn3 pulpy rer Sty oleae O 6z Aep yyfr wor aarytsog | gt “pagoayut Aayuour {szuajzUo0 purys Arwayyeg spieMuo *poqoayur Aoyuour { syuequ0s yn + Sicectaente O 6z Aep y361 woz dATIISOg SI “BUIppoqure IOF pasn spurys Arvaryes spremuo *poqoozut Aoyuour £ sytaqU0. nH ae SPS SRP He O Bz Aep yySi utosz aatytsog | +1 *paqoayur Aayuour {sjua}u09 spurys Areatyes spieMuo *paqoayur Aayuour { squaquoo yng Site ar eteiateeat ats zz Avp ystz-y3Z1 wosy aatytsog | £1 ns ee O a IE uaas ‘dA33 1 (a4 ce ZI ce ce | oO + -+ + oO Iz ce ith = *paqoajur you Asyuour { syuaquo. yng Oo bet uaas *sdA1q z dAtqeZaN | OF Nn *paqoayur Adyuour {syuaju09 purzs Areates *paqoazur yer $ syuazuo0s yng spieMuo *paqoayur Aoyuour £ s}UaqU0d stosoqorg Gr aie ar Siar eect 61 Aep y}ZI wtorz aATyIsOg | 6 “ ce Oo zs oe pes oO hee “ 8 + M O sas O TI a Z oe ce O + ++ O Il ce 9 oe 6 O fb O Ol ‘73 S “ “ O a O 6 a 14 “ “ | Oo cet O 8 ue £ “ “ O ue O ¢ Ae Zz *pazoajut you Aoyuour { s}u9}U09 yng Oo +++ O M4 aatqeson | I sje 10 skayuour spurs autqsazuy st9soqoig poey poqoasstp sear ATF ay uva]o ojur AY ayy jo suorjiod juaraytp Arearyes dAT}OOFUT oY arojaq Avp oy} [eurue wor} saurosourdds} Jo WorzE[NIOUT Jo yNsoy rare narracern | rrrernre + em = 280 Attention has already been drawn in a previous section to experiments which suggest that although the parasites can multiply and develop up to a certain stage in the intestine at comparatively low temperatures (55°-65° F.), yet the flies do not become infective until the temperature to which they are subjected is raised to at least 75°-80° F. In none of our experiments were trypanosomes found in the salivary glands of flies which had not been subjected to the higher temperatures. Probably the salivary glands become invaded by parasites which have reached a certain stage in their developmental cycle in the intestine. The remarkably short period (eight days or less) in which three flies, which had been kept forty days after the infective feed at laboratory temperature, became infected after being placed in the incubator at 85° F. can be best explained on the assumption that some portion of the cycle must have occurred in the gut during the first forty days at laboratory temperature. Whilst the evidence that invasion of the salivary glands is secondary to that of the intestine is fairly conclusive, our knowledge of the path by which the parasites reach the glands from the intestine and of the morphological characteristics of the forms which migrate is by no means so definite. It is at once apparent that there are two alternative routes by which the trypanosomes might reach the salivary glands from the gut, viz.:—(1) By way of the proboscis; (11) by penetrating the gut wall, and after crossing the coelom traversing the wall of the salivary glands. We have accumulated no evidence enabling us to decide along which of these two routes the parasites migrate. Parasites were but rarely seen in the proboscis, and then, with one exception, only in small numbers. In the case referred to, trypano- somes were present in the proboscis in considerable numbers, but as both the gut and salivary glands contained enormous numbers of flagellates, their occurrence in the proboscis could easily be explained on the assumption that they had been discharged from the salivary glands and were passing down the proboscis with the secretion of these structures. The parasites from the gut, from the salivary glands, and from the proboscis were inoculated into monkeys and rats, all of which became infected, showing that the trypanosomes in each of these structures were virulent. 281 As will be seen from Plate XXIII, the parasites present in the gut and in the salivary glands of infective flies exhibit marked differences in morphological characteristics. The forms encountered in the intestine were many and diverse, but the predominant type was undoubtedly that depicted in figs. 7-12. It may be described as a large broad flagellate, with a feebly-developed undulating membrane and little or no free flagellum. The nucleus is usually compact and situated near the centre of the body, but not infrequently it lies in a more posterior position. The blepharoplast is, as a rule, situated near the posterior extremity, but sometimes lies further forward approaching the nucleus. In addition to these forms, long thin parasites were found. Here the nucleus was, as a rule, diffuse, and occupied a central position. The parasites stained more faintly, the undulating membrane was narrow, and there was generally a distinct free flagellum. Many more or less rounded bodies were also seen. The form met with in the salivary glands of infective flies was almost invariably that represented by figs. 1-5. It approximates somewhat closely to the short form of the trypanosome in the blood of the vertebrate host, but is obviously not identical with this. The nucleus is compact, and is situated at the middle of the body; the blepharoplast is distinct, and lies near the posterior extremity. The undulating membrane is well developed; there is only occasionally a short free flagellum. The length of this form is 15-18u. In addition to this form, long attenuated flagellates were occasionally seen, but they were very few in number, and it was necessary to search for a considerable period before discovering one of them. These forms resemble the corresponding variety seen in the gut. The nucleus is diffuse, and situated near the centre of the body. The parasite stains a faint pink with Giemsa. In so far as we could determine, the predominant gut type was not met with in the salivary glands, and the predominant salivary gland type did not occur in the intestine. In spite of this, however, we are faced with the fact that the parasites in both situations proved to be virulent when inoculated into experimental animals. It appears to us that two hypotheses can be advanced to explain this; either that the salivary type is the only virulent form and that a certain number of these reach the intestine from the 282 salivary glands by way of the proboscis—this may be quite fortuitous, depending on the fact that a certain quantity of infected saliva is from time to time drawn into the intestine—and are then lost amongst the multitude of diverse gut forms, or that there is some unknown form (the one which migrates from the intestine to the salivary glands) which precedes the typical salivary gland type and which is also virulent for laboratory animals. In conclusion, we might remark that invasion of the salivary glands was only observed in the case of flies infected with the human trypanosome (7. vhodeszense) and not in the case of any of the other trypanosomes with which we had to deal either in the Luangwa Valley or on the Congo-Zambesi watershed. This was the case both with the strain of 7. rhkcedesiense derived from man and also with that found in ‘wild’ Glossina morsitans which had been infected in nature. It is of interest to note that of 310 ‘wild’ Glossina morsitans which were dissected as they were brought into the laboratory recognisable mammalian red corpuscles were found in the intestine of seventy, whilst nucleated red corpuscles were only found on twelve occasions. SUMMARY 1. The salivary glands of all Glossina morsitans capable of transmitting T. rhodesiense are infected, and conversely without invasion of the salivary glands there is no infectivity of the fly. 2. Invasion of the salivary glands is secondary to that of the intestine. 3. The first portion of the developmental cycle of the trypano- some takes place in the gut. In order for its completion and for invasion of the salivary glands to occur, a relatively high mean temperature, 75°-85° F., is necessary. 4. Invasion of the salivary glands was only found in flies infected with the human trypanosome, 7. rhodesiense. 5. The predominant type of the trypanosome in the intestine of infected Glossina morsitans—a large broad form—is quite 283 different from that which predominates in the salivary glands, where the parasite resembles somewhat the short form seen in the blood of the vertebrate host. 6. Both the intestinal forms and also those from the salivary glands of infective Glossina morsitans are virulent when inoculated into healthy animals. 284 DESCRIPTION “OF PEATE Xx Trypanosoma rhodesiense in Glossina morsitans. Films fixed in alcohol and stained with Giemsa. The figures were drawn with the aid of a camera lucida at a magnification of 2,000 diameters. Figs. 1-5. Typical forms seen in the salivary glands. Figs. 6-14. Intestinal forms. 285 SECTION VI REPORT’ OF THE ENTOMOLOGIST; LLEWELLYN, LLOYD (a) GLOSSINA MORSITANS IN THE LABORATORY The breeding of these flies was carried out continuously from the middle of June, Ig911, to the end of May, 1Ig!2. Kinghorn* has already outlined the method adopted, and has described the larva and pupa. The flies were fed on native fowls at Nawalia, except for a short period when goats were used. At Ngoa the flies were fed at first on fowls, but afterwards entirely upon goats, which died quickly under the repeated feedings, doubtless owing to the trypanosomes with which the flies were naturally infected. owls would appear to be unsuitable food, in spite of the fact that the flies feed much more readily upon them than upon mammals. Fowl blood in the ‘sucking’ stomachs of the flies forms large firm clots, and apparently blood in this condition cannot be utilised by the flies, as the clots have been found to persist for several weeks, monkeys having been used as blood donors in the interim. A fly with so large a clot in the body cannot retain a full-grown larva, and consequently many abort. The phenomenon is rare with mammalian blood, and it has not been observed in nature. The bottles used for the stock flies were glass cylinders, five inches deep and two inches in diameter. At first only three flies, two females and a male, were kept in each tube, but it was found later that the number could be increased to six without damaging the vitality of the insects. In damp weather many flies die through fouling themselves with the copious semi-fluid faecal deposits. This may be obviated by inverting the tubes over sheets of blotting paper, which absorb the excreta, but precautions must then be taken to avoid the escape of the larvae. This was done by placing the tubes in dishes and paper-lined boxes. When flies are kept under these conditions the daily changing of the tubes becomes unnecessary—a great saving of labour when many flies are kept. *Kinghorn, A. Bulletin Entomological Research, 1912. 286 It is sometimes difficult to obtain a sufficient supply of female flies, but natives learn to distinguish between the sexes and to select and bring in females. In a collection made without discrimination, the proportion of females is often as low as 2%. During the twelve months the number of females in stock has varied considerably. From July to February, with the exception of a period at the end of August when all were destroyed by an accident, the number varied between 200 and 300. During April and May the number of females was between 500 and 600. The flies breed most readily at the beginning and at the end of the rains. This is confirmed by observations on the number of flies in nature at different periods of the year. Males and females emerged from the pupae in approximately equal proportions for the twelve months. From June to December the males in each month were somewhat in excess of the females, while from January to April the reverse was the case. The pupation period of a male is on the average about a day longer than that of a female. The variation in the duration of the pupation period is given in Table 47, p. 288. It shows more fully the influence of the climatic conditions on the pupal life; the pupae have been divided into groups which were deposited in periods of half months. For each of these periods the mean temperature in the laboratory and the relative humidity of the air are given. A fuller account of the meteorological conditions is given in a previous section of this report. The approximate mean temperatures to which the various groups of pupae were exposed have been calculated by averaging the mean temperatures for the periods in which the groups were deposited, and for those periods following which could have influenced the duration of the pupation period. Chart 8 indicates how readily the pupae respond to changes of temperature. The humidity of the air has little or no influence on the pupal life, as will be seen by comparing the data of June and July with those of the second half of March. While the relative humidity of the latter portion was 10 % higher than in the former, the temperature and the duration of the pupal life were approximately the same in each case. It should be mentioned that the observations from June to the middle of March were made at Nawalia (altitude 2,100 feet), 287 (AU SEYAN THONG, IER IEOIO Tee JO) 1\ SES & faye 2 ” 3 Le S ‘wn ra) lo BY ‘the influence of temperature on the pupation period of Glossina morsttans. Curt 8,—Showing JYNLYYJIWIL NVIW -}- pede | Seeeraeerad el eb pas The pupation periods are represented by the continuous line, the temperatures by the broken line. 288 Taste 47.—Showing the influence of Temperature on the Pupation Period of Glossina morsitans. = | ie : Be Soe sees cle. Fr [CeoBbae id | eh ecg Ja8 eee) Pee | SEP] 82 bel ieee | oe Bape ce er A a eo i oes Ba E-28 Es= | he] 8 ez Sf | S85 e| SEs aE Zee | 2am |/ese 2S |

9207) vanbia RS were fc dst opis yoy ainattion eoludidt bealayncrstit Vo wiladiap 7 bus = Huan e‘nsnrwol to siloliqn ainilsd? ni peqoks ' tm bevinctio ols, 2] syne ytic} ella Hiraetioly <3 7 5 ; bsdzam zen i FEL ; 2 ' | : Sion zecrley fo redone qite) stisial feat: ai Rerewets HA eq esiseld te eden] sgiiilermann att Ver a bailing : ~ a @ : iey.2)- Sone eoflt “deel ube “alt al taludw | ~ 7 Sorvade elisa leitsiliqn adi to. sine ino ,beolism sini 7 sion $n Soin - > 474 PLATE: XOXO Technique as indicated for Plate XXX (x 360). Fig. Fig. Fig. ie N Suprarenal capsule (Case 55) cortex. A considerable amount of fat is present in nearly all epithelial cells. Kidney (Case 62). Moderate fatty change, both convo- luted tubules and glomeruli cells being affected, the latter less than the former, but in the case of both only minute fat droplets are observed in the cells. Kidney (Case 47). Intense fatty metamorphosis, the epithelia of the convoluted tubules contains very large fat drops; in the lining epithelia of Bowman’s capsules and the glomeruli cells fatty change is also observed, but much less marked. Kidney (Case 47). Intense fatty metamorphosis of the epithelia of the ascending limbs of MHenle’s loops, whilst in the descending limbs the change is very little marked, only some of the epithelial cells showing minute fat droplets. DNAS ans Ataanocmoviuguiats, | pittetinle eet a, een ae al meieqoes brs cousiitiigs biqanryt al robe, nig anti oT isons ‘ , Ra] x 7 : shi tersoruich avitBiely hag eboaly een BH TO eidinendl ~ *izoten “lamdreque ots, fom. stetad ecrt Jano eutt Jug casse ete Se2colsn 20s .xbaely batirshilwig aild vd = Bao pith erwin aaiweile tae? nowy) sil rt git Ie cee oihsboig Dine eportipais | NOSE % 5 rota ctl: semtih elie seat boworle svi ii eehacs waite (Cee ent 2) eternal e INE He Fig. Fig. Fig. Fig. NO 1oS) 476 PLATE XXXII MICROPHOTOGRAPHS Mucosa of stomach (Case 55), showing hyperaemia, diffuse lymphoid infiltration, and superficial necrosis and haemorrhages. To the right, part of a lymphoid follicle issseen.. X O5. Mucosa of duodenum (Case 34), showing hyperplasia of Brunner’s glands and diffuse microcellular infiltration; also superficial necrosis. Remnants of the muscularis mucosae are seen, but this coat has been entirely broken up by the proliferated glands. x 95. Liver (Case 55), showing intense diffuse fatty meta- morphosis and moderate necrobiotic changes, likewise of a diffuse character (x 370). Other parts of the same liver showed intense capillary hyperaemia. Pancreas (Case 43), showing marked necrobiotic changes. Kot | | “aug © ¢ BATARIOTORIOD IY 2 ~ ad We : 1 ba nba siginwd Fo suit iH. alubnit igand i onigquil | 1 { i ? - Q " ad ‘ 1a enragyllennaeige tea TY ‘otto pe : ary etfs at : 478 PLATE XXXII MICROPHOTOGRAPHS | Hyperaemia of lymph nodule. Spleen: Hyperaemia. x 140. Kidney: Necrobiotic changes. Kidney: Intense fatty change. x 140. x 370. x 370. Volume VII December, 1913 No. 4 ANNALS OF TROPICAL MEDICINE AND PARASITOLOGY ISSUED BY THe LIVERPOOL SCHOOL OF TROPICAL MEDICINE Edited by Proressor J. W. W. STEPHENS, M.D. Cantab., D.P.H. Pronessor sR. INEWoTEAD, MSc... J-P) Roo, A128; FES. Hon. F.R-H.S. AND Proressor Sir RONALD ROSS, K.C.B., F.R.S., M.D., F.R.CS., Mayor I.M.S. (Ret.) Editorial Secretary Dr. H. B. FANTHAM, School of Tropical Medicine, The University, Liverpool. C. rane & Co., Lid. x, a ee Printers to the University Press of Liverpool - 53 Victoria Street - on - La. s - : AF . +4 - . = : i ~? ’ ‘ « | 1.) * are . i pe = ; : a ¢ ‘ . 13 < i = A r 4 ~ cy’ J / iy ast bees ~*~ at } » z } > OFF Gg i 7 fT AS oe ay =< - gel sada de , ea, ® e steele ge eS & y ~ : = r J ha 7 : == 7 _ aun « » oa : : i ~ ve — ° ° "ian ae ee b | P rt » i , PR Ree BLA A CILLA KO8 at 7.5 Tt Fi fais «& sin GOLA BY: fa ’ . : ; > 7 =5 . Pd > + re le = = ‘4 et = ~ 4 é - 7 a ~— * -— £ THE INCORPORATED PiVvER POOL, SCHOGESeOr~PROPIC ATL. MEDICINE FoUNDED BY Sir ALFRED LEWIS JONES, K.C.M.G. (Affiliated with the University of Liverpool) Hon. President: H.R.H. PRINCESS CHRISTIAN OF SCHLESWIG-HOLSTEIN. Chairman: MR. F. C. DANSON. Vice-Chairman: Professor R. CATON. Hon. Vice-Presidents : THE DUKE oF NORTHUMBERLAND, K.G. THE EARL oF DERBY, G.C.V.O. EARL CROMER, G.C.B. VISCOUNT MILNER, G.C.B. LorpD PIRRIE, K.P. SIR OWEN PHILIPPS, K.C.M.G. SIR EDWIN DURNING LAWRENCE, Bart. SIR WILLIAM H. LEVER, BART. Mr. O. HARRISON WILLIAMS COMMITTEE Mi Ei READ, 'C.M.G. Colonial Office Vice-Chancellor SiR ALFREDDALE = University of Liverpool oS * . peta Sp ane Bore | Council of University of Liverpool Professor W. HERDMAN, F.R.S. ) Professor J. M. BEATTIE Drege ee a ene ts | Royal Southern Hospital Mr. C. BOOTH (Jun.) oe Mr. T. F. HARRISON j Steamship Owners’ Association Senate of University of Liverpool Pit ee Ree _ Shipowners’ Association Mr. W. ROBERTS j F Mr. J. W. ALsop Mr. G. BROCKLEHURST Mr. C. LIVINGSTON Mr. J. W. W, DANSON Professor E. W. HOPE Mr. R. RANKIN Professor E. GLYNN Mr. E. G. BUCKLEY Mr. A. L. REA, Hon. Treasurer Mr. A. H. MILNE, C.M.G., Secretary. B 10 Exchange Buildings, Liverpool Staff, 1913 1. At the University of Liverpool Professors - - JOHN WILLIAM WATSON STEPHENS, M.D., Cantab., D.P.H. Sir Alfred Fones Professor of Tropical Medicine ROBERT NEWSTEAD, *FIR°Ss, M:Sc., -A-L.S., F.E.S., Dutton Memorial Professor of Entomology Major SiR RONALD ROSS, K.C.B:, F.R.S., vss EORC:S:, OSes, Nts 1) Scam Nobel Laureate 1902. (Indian Medical Service, retired). Professor of Tropical Sanitation Vacant. Walter Myers Professor in Parasitology Lecturers - - HAROLD BENJAMIN FANTHAM, D.Sc., B.A., Lecturer in Parasitology HENRY FRANCIS CARTER, S.E.A.C. Dipr., F.E.S., Lecturer in Entomology WILLIAM THOMAS PROUT, M.B., C.M.G., Lecturer on Tropical Sanitation HAROLD WOLFERSTAN THOMAS, M.D., C.M. PROK. E,W, HOPE, MoD) Disc: Honorary Lecturers - MAJOR JOSEPH FITZGERALD BLOOD, M.D., M.Ch. (Indian Medical Service, retired) PRoF. ERNEST EDWARD GLYNN, M.A., M.D., (Cantab.), M.R.C.P., M.R.C.S. Honorary Statistician WALTER STOTT Yellow Fever Bureau HARALD SEIDELIN, M.D. (Scientific Secretary). Bibliographer - - WALTER DRAWZ 2. At the Runcorn Research Laboratories Director - = - WARRINGTON YORKE, M.D. Assistant - - = Be bUACKEOCK Meb ber ars 3. At the Royal Southern Hospital, Liverpool Physicians = - CHARLES JOHN MACALISTER, M.Dz BARGER: JOHN LLOYD ROBERTS, M.D., M-R.C.P. Surgeons - : - D. DOUGLAS CRAWFORD, F-R.C.S: ROBER® JONES, F.R.C-S., Ch.M. GEORGE PALMERSTON NEWBOLT, M.B., BR-GS, Tropical Pathologist - JOHN WILLIAM WATSON STEPHENS, M.D. Cantab. Clinical Pathological Assistant - JOHN GORDON THOMSON, M.A., M.B., Ch.B. 4. On Expedition HAROLD WOLFERSTAN THOMAS, M.D. es SEIDELIN, M.D. Laboratory JOHNSTON LABORATORY, UNIVERSITY OF LIVERPOOL Research Laboratory : RUNCORN Hospital : ROYAL SOUTHERN HOSPITAL, LIVERPOOL Secretary's Office : B 10, EXCHANGE BUILDINGS, LIVERPOOL NOTICE The following courses of instruction will be given by the Liverpool School of Tropical Medicine during 1g14 :— Full Course begins 6 January. Advanced Course begins 1 June. Diploma Examination, 6 April. Certificate Examination, 30 June. Full Course begins 15 September. Diploma Examination, 14 December. These dates are subject to revision. The full Course of Instruction is open to all qualified medical men, and the Examination to all students who have taken out this full course. Fee for the full Course of Instruction—Thirteen Guineas. Fee for the Diploma Examination—Five Guineas. Fee for the Short Course of Instruction—Four Guineas. Fee for the use of a School microscope during one term—Ten shillings and sixpence. For prospectus and further information, application should be made to the Dean of the Medical Faculty, University of Liverpool. The following have obtained the Diploma in Tropical Medicine of the University of Liverpool : — Diploma in Tropical Medicine Date of Date of Diploma Diploma 1904 Augustine, Henry Joshua 1905 Macfarlane, Kobert Maxwell 1904 Bennett, Arthur King 1905 Maddock, Edward Cecil Gordon 1904 Bruce, William James 1905 Moore, James Jackson 1904 Byrne, John Scott 1905 Nightingale, Samuel Shore 1g04 Clayton, Thomas Morrison 1g05_ Radcliffe, Percy Alexander Hurst 1904 Dalziel, John McEwen 1905 Young, John Cameron 1904 Dee, Peter : 1906 Adie, Joseph Rosamond 1904 Greenidge, Oliver Campbell ers fase, Brink Arthur 1904 Hehir, Patrick 1906 6Bate, John Brabant 1904 Khan, Saiduzzafor 1906 Bennetts, Harold Graves 7994 Laurie, Robert 1906 Carter, Robert Markham 1904 Maclurkin, Alfred Robert 1906 Chisholm, James Alexander 1904 McConnell, Robert Ernest 1906 Clements, Robert William 1904 Nicholson, James Edward Tagen Dina a : go undas, James 1904 Philipson, Nicholas _ 1906 Faichnie, Norman #904 Sharman, Eric arate 1906 Jeffreys, Herbert Castelman t904 Thomson, Frank Wyville 1906 Mackenzie, Donald Francis 1904 Walker, George Francis Clegg 1906 Pailthorpe, Mary Elizabeth 1g05 Anderson, Catherine Elmslie 1900 Palmer, Harold Thornbury tg05 Brown, Alexander 1906 Pearse, Albert fas 1905 Caldwell, Thomas Cathcart 1906 Sampey, Alexander William 1905 Critien, Attilio 1906 Smithson, Arthur Ernest 1g05 Hooton, Alfred 1006 Taylor, Joseph van Someron 1905 Hudson, Charles Tilson 1906 Taylor, William Irwin tgos_ Illington, Edmund Moritz 1906 Tynan, Edward Joseph Date of Daze of Diploma Diploma 1g00 Watson, Cecil Francis 1909 Jackson, Arthur Frame 1906 Willcocks, Roger Durant 1909 Kaka, Sorabji Manelkji tg06 Williamson, George Alexander 1909 McCabe-Dallas, Alfred Alexander Donald 1907 Allan, Alexander Smith 1909 Meldrum, William Percy 1907 Allwood, James Aldred 1gog Murphy, John Cullinan 1907. Bond, Ashton 1909 Samuel, Mysore Gnananandaraju 1907 Branch, Stanley 1909 Shroff, Kawasjee Byramijee 1907 Collinson, Walter Julius 1g09 Thornely, Michael Harris 1907. Davey, John Bernard 1909 Turkhud, Violet Ackroyd 1907 Donaldson, Anson Scott 1909 Webb, William Spinks 1907. Fell, Matthew Henry Gregson 1909 Yen, Fu-Chun 1907. Gann, Thomas William Francis 1907. Graham, James Drummond 1907. Hiscock, Robert Carroll 1910 Brabazon, Edward 1907 Keane, Joseph Gerald 1910 Castellino, Louis 1907 . Kennan, Richard Henry 1910 ©6Caulcrick, James Akilade 1907. Kenrick, William Hamilton 1910 Dowden, Richard 1907 Le Fanu, George Ernest Hugh 1910 Haigh, William Edwin 1907. Mackey, Charles 1910 Hamilton, Henry Fleming 1907 Maddox, Ralph Henry 1910 Hefferman, William St. Michael 1907 McCarthy, John McDonald 1910 Hipwell, Abraham 1907 Raikes, Cuthbert Taunton 1910 Homer, Jonathan 1907. Ryan, Joseph Charles 1910 Houston, William Mitchell 1907. Vallance, Hugh . Ig10 James, William Robert Wallace 1910 Johnstone, David Patrick : F 1910 Korke, Vishnu Tatyaji 1g08 Caverhill, Austin Mack 1910 Macdonald, Angus Graham 1908 Crawford, Gilbert Stewart : : 1908 Dalal, Kaikhusroo Rustomji oa ee ee 1908 Dansey-Browning, George 1908 Davidson, James 1g08 Dickson, John Rhodes 1908 Dowdall, Arthur Melville 1908 Glover, Henry Joseph 1908 Greaves, Francis Wood 1908 Goodbody, Cecil Maurice 1908 Harrison, James Herbert Hugh 1908 Joshi, Lemuel Lucas 1908 Le Fanu, Cecil Vivian 1908 Luethgen, Carl Wilhelm Ludwig 1908 Mama, Jamshed Byramji 1908 McCay, Frederick William 1908 McLellan, Samuel Wilson 1910 Murison, Cecil Charles 1910 Nanavati, Kishavlal Balabhai 1910 Nauss, Ralph Welty 1910 Oakley, Philip Douglas 1910 Pratt, Ishmael Charles 1910 Sabastian, Thiruchelvam 1910 Shaw, Hugh Thomas 1910 Sieger, Edward Louis 1910 Sousa, Pascal John de 1910 Souza, Antonio Bernardo de 1910 Waterhouse, John Howard 1910 White, Maurice Forbes 1908 Pearce, Charles Ross 1911 Blacklock, Breadalbane 1908 Schoorel, Alexander Frederik IQII Brown, Frederick Forrest 1908 Smith, John Macgregor Ig1I Chand, Diwan Jai 1908 Stewart, George Edward 19trt Holmes, John Morgan 1908 Tate, Gerald William 1g11 levers, Charles Langley 1908 Whyte, Robert 1911 Iles, Charles Cochrane t9gtt Ingram, Alexander 1911 Kirkwood, Thomas 1909 Abercrombie, Rudolph George ceeds: Peake 1909 Allin, John Richard Percy AOS ES a 1909 Armstrong, Edward Randolph 1911 Liddle, George Marcus Berkeley 1909 Barrow, Harold Percy Waller 1911 Lomas, Emanuel Kenworthy 1909 Beatty, Guy IgII Mackarell, William Wright 7909 Carr-White, Percy 1911 MacKnight, Dundas Simpson 1909 Chevallier, Claude Lionel 1911 Mascarenhas, Joseph Victor 1909 Clark, William Scott tgt1 Murray, Ronald Roderick 1911 Oluwole, Akidiya Ladapo aes aere Igtrt Rao; Koka Ahobala sis EA eeenael McCormick ges into, Jone aexanley shor i, Hawa itech DAs AMS rr a. 7, Sy 7 xa ‘ Be! : ae Be dig Ca oe, ro1n Woods, Willigmaedlicctt 1909 Cope, Ricardo Date of Date of Diploma Diploma Igt2 Aeria, Joseph Reginald 1913 Becker, Johann Gerhardus 1912 Anderson, Edmund Litchfield 1913 Carrasco, Milton 1912 Borle, James 1913 Clark, James McKillican 1912 Bowie, John Tait 1913 Forsyth, Charles 1912 Brassey, Laurence Percival 1913 Grahame, Malcolm Claude Russell Ig!2 Christie, David 1913 Grieve, Kelburne King 1912 Dillon, Henry de Courcy 1913 Hargreaves, Alfred Ridley Igt2 Dunn, Lillie Eleanor 1913 Hepper, Evelyn Charles 1912 Hardwicke, Charles 1913 Hiranand, Pandit 1912 Jagose, Jamshed Rustomji 1913 Jackson, Oswald Egbert 1g12 Kocbhar, Mela Ram 1913 Khaw, Ignatius Oo Kek 1912 McGusty, Victor William Tighe 1913 MackKelvie, Maxwell 1912 Milne, Arthur James 1913 MacKinnon, John MacPhail 1912 Mitra, Manmatha Nath 1913 Macmillan, Robert James Alan 1912 Myles, Charles Duncan 1913 Mouat-Biggs, Charles Edward 1912 Pelly, Huntly Nevins Forbes 1912 Prasad, Bindeshwari 1913 Noronha, Jobn Carmel Jg12 Prentice, George 1913 O’Connor, Edward 1912 Koss, Frank 1913 Olubomi-Beckley, Emanuel 1912 Russell, Alexander James 1913 Pestonji, Ardeshir Behramshah Hutchison 1913 Puttanna, Dodballapur Sivappa 1912 Ruthven, Morton Wood 1913 Reford, John Hope 1912 Sandilands, John 1913 Smith, Edward Arthur 1912 Seddon, Harold 1913 Stewart, Samuel Dudley 1912 Smalley, James 1913 Walker, Frederick Dearden {912 Strickland, Perey Charles 1013 Wilbe, Ernest Edward Hutchison 1913 Wilson, Hubert Francis 1912 Watson, William Russel 1913 Yin, Ulg Ba 1913 Young, William Alexander 1913 Austin, Charles Miller 1913 Banker, Shiavux Sorabji PDITORTAL NOTICE By order of the Committee of the Incorporated Liverpool School of Tropical Medicine, the series of the Reports of the School, which had been issued since 1899, were followed, from January 1, 1907, by the Annals of Tropical Medicine and Parasitology, of which this is the fourth number of the seventh volume. Altogether twenty-one Memoirs, besides other works, were published by the School since 1899, and of these ten, containing 519 quarto cr octavo pages and gs plates and figures, were published during the two years 1904 and 1905. The Annais are issued by the Committee of the School, and will contain all such matter as was formerly printed in the Reports—that is to say, accounts of the various expeditions cf the School and of the scientific work done in its laboratories at the University of Liverpool and at Runcorn. In. addition, however, to School work, original articles from outside on any subject connected with Tropical Medicine and Hygiene, and Parasitology may be published if found suitable (see notice on back of cover); so that, in all probability, not less than four numbers of the Annals will be issued annually. Each number will be brought out when material sufficient for it has been accumulated. ting x ii ites 5 Vive b, * te. per Me F 3 Z 2 Ht ., ‘ nth ent: : Latent s meee by ped Ts FAA ER Salle " Ks} ‘ 1S: ‘ u - Aas 3 i 4 reac, } y 7) ‘5 , , ' : =a) , - a & p : " ; Lae ma it ? es yt , wide iG Hen ; 4) lt ‘ 2 : ; far SES oe 7 i tee! tte J si on ’ eT 7 h peas Mh é “ i os ee Sart : ’ 9 oar # es Ly e san} 4 J é fe 4 > na f a? ‘3 uF ih een hs ‘ : 7 - * - = : aU ae | i ~~! sect \ erp Uplate 2 a 5 hy ries oe x ee a i ee eee is ar ’ mia Tanti ~ » i 4 aN 4 : dpgtrds salah! s* tia ew fen “ : abe .* : i } yo Seite J - nig tt y : tn ; Ja?p Binet Raat WL e- Mfaadoaghe aie | - ; rte * es Seah FS" i : ° i a ik “fs “The 4 Sut! “¥ hi A ne 4 & A. J kat or = v1 . , v} : a. / ergy oT : | , 6 Lng! Roar prs » i 4 t ; : -o OMSL Oe ae nl econ m a ir ye, Poaienr WoenD sf / ' a Te a at : . sent Beagles ie cts i i Oe Chie 5-3 ¥ ‘ log ity , von | an j i} ROD Welsh pate = = s i ! ’ . a's ; : ‘ 1 | / i 4 vi } py OP ame “ee ‘ ' i : Ss . 2 ' Ay x | ii tt ~ ; / y { ‘ 7 ‘ n @ ex} Sie ass »f.a } my +m, x — ‘) Jue s >See + P ped P tik +3) bis - i i i 7 es * ; a } ae date rent ee or) ie odes a es x x j Artal iat gael ree. 2a ae 5) many . . rf iy sre - fs : r ( aan 1} f rut. a ’ r 5 a yer } f ' ¥ : : Ar “ 4 ; ‘ AY ' , . ‘i ‘ it Hy: 9 if wi, oat Denice ‘ 1 pathabew it vy ws : = ‘ j - ’ + « ti ied ot 2! ! ltohety f aor - x - ” = ‘ ¥ rye j ue ( ee gs an, me was ¥ { oe i » - ye ¥ 4 / $ = - \ a ‘~ - + 479 STUDIES IN BLACKWATER. FEVER (1) STATISTICAL BY J. W. W. STEPHENS, M.D. (CANTAB.) In this paper I propose to consider some of the facts bearing on the malarial origin of blackwater fever which I have been able to collect from an examination of recent literature. Arguments of a general nature, though there are many, I shall not consider here, but shall examine the facts so far as I have been able to ascertain them under the following headings :— (1) Malaria parasites. (2) Pigmented leucocytes. (3) Post-mortem examinations. (4) Influence of malaria. (5) Relationship to species of malaria parasite. (6) Effect of period of residence. (7) Seasonal prevalence. (8) Correlation between malaria and blackwater statistics. (9) Second attacks. In 1901, Christophers and myself (1901), as a result of our microscopical studies of blackwater fever, came definitely to the conclusion that blackwater is malarial in origin but is not simply an attack of malaria, but occurs only in those who are in a condition induced by repeated malarial infection lasting over a certain time. MALARIA PARASITES One of the most important facts, in my opinion, bearing on the aetiology of blackwater fever is the presence of malaria parasites in the blood of blackwater cases. In the literature I have been able to collect 390 cases. I divide them into three categories, those on the day before, those on the day of, and those on the day after the onset of blackwater, as in the accompanying Table I. My reason for doing this is that in my experience, and also in that of others, parasites when found rapidly disappear. 480 “punoy s}Uodse19 pure pasiyednyt4squd. pooyq “wos o1 Jazze Aep, 10 , Jo Aep , LOYIOYM AvITD ION ‘yjeap ‘dep yas “PF “E uo ‘Sou ‘dep puz ‘1 uo ‘sod “1g -Avp yag pure ‘2 “p ‘1 uo quosaid sozisvard +15 ‘souuef 2 syooq{ a8 sasvo Joyjo roy $€1 pur OL sased (doe Avp, 10 _ Jo dep , doyqoYM Avayo OU ST 4] SYAV UOT Z ca ol 61 - -~ aatqtsod ‘on VINANISOTOONIH JO LASNO WALAV AVC] Sasvo JO “ONT aattsod *oN Z gt Ol ZI 9 Or 808V9 JO “ON VINANIAO TOONS 410 LASNO AO AVCT ] 21av 1, ——— aatytsod ‘ony VINVANIEPOTOONIH 40 LASNO FNOTIM AVC Sosvo jo ‘ON oO) (1061) ourpy ae (1161) sjosunf one (4061) ueqqeig (01-6061) vorrpy yseq URWIOD (1161) gia8nojng yw noqsey re *** (0161) d1017 (1161) sowef x syooq ** — (Zo61) yotsopeoq BO (g061) eys09 ep (go61) Adpueg wy ssoydoqsiayD un “~ (4061) tsonpaeg o0 ** (1161) styeuepirD a “+ (0161) tssoasodeg : oh *t* (go61) waig see wee *** (£061) qneag as (6061) oyi0X Y yQeameg oy Ny fg1 06£ LE 191 dATIISOg Sased [20], Il to aarzisod ‘ony syIRWAY SOSED jo “ON VINONIGOTSONTH 40 LASNO WALA AVG O° aatqtsod ony | saseo yo aatqtsod ‘ony VINONIDOTSOWIN 40 LASNO dO AVC] % EL a4 £z Sased JO "ON VIYONIFOTSOWIH 4O LISNO AXO4da AVC LS (6061) afddiypy (061) suaydays “* (zo61) a8ny (c161) uosdung zy uoswoyy, ‘ssoy (1161) saw0Y (3681) Temod *** (zo61) 0719 an (061) aw *** (z161) anySou0g,o aoe (z161) vary ep (9061) urwaajseyy (0161) yeysaryy eee see (0161) alias! (g061) zjny (to61) ssneiy Joyiny i en eee panutjuor—y aiavy, 482 This means that in 100 cases of blackwater examined on the day before the onset, parasites will be found in 73 cases, in 100 cases examined on the day of onset in 47°5* and in 100 cases examined on the day after in 23.T An important deduction that I think may safely be made from these figures is that parasites disappear, and indeed rapidly, during the course of the disease, as we had observed in our original studies. This disappearance of parasites is in all probability associated with the acute haemotysis that is taking place in blackwater fever, and should be borne in mind in all comparisons between the parasite rates in blackwater fever and malaria. Further, the fact that on the day before the onset of the black- water parasites are found in 73% of cases seems to point to the initial attack being closely associated with a malarial attack. That relapses do occur during the course of the disease (which relapses in some cases at least can be brought on by quinine) without the occurrence of parasites in the blood, seems to be certain, though I have not collected the data on the subject. And even if parasites were rarely found in blackwater, it would not necessarily exclude its malarial origin, because it might be that it was just in the chronic cases of malaria where parasites are absent or scanty that blackwater occurred, but I will not pursue the argument further, as I wish as far as possible in this paper to ascertain facts and avoid discussion. The following table, compiled from Panse’s (1902) cases, 35 in number, enables one to show in more detail how time affects the result of the examination for parasites in blackwater : — TABLE JA Time of blood examination Cases Positive | Percentage Day before onset ... oe nc Sct Ach 9 8 88-9 Day of onset (a) before onset... =e See II 8 72:7 a (b) after onset ~ yy 6 54°5 Within 12 hours after onset... sce ave 12 6 50°0 More than 12 hours after onset 208 os 31 9 290 * A correction is probably necessary for those cases occurring on the day of the blackwater for some of these will probably have been examined defore the onset of the blackwater and some (probably the majority) after, so that on the assumption that the parasite rate before the onset and that of the day before is the same, this figure should be reduced, but it is impossible to say by how much. + Or put in a different way of 100 fosztive cases, 51 will occur on the day before, 32°8 on the day of, and 16 on the day after the blackwater. 483 The next tables, Table II and III, I give separately, as the data were compiled in a different way to that in Table I. Taste I].—Parasires 1N BrackwaTter Fever. Compiled from Deeks and James (1911) 75 cases examined 4—1 days before onset ... Pos. 48 =" 64-0.% ys oa os 1 day before ,, nee re 14 = so:0% na x5 day of onset ... a 12 = 333% 62 5; + 1 — 4 days after onset ae a TZ) == TOG 40,928 ,, of Malaria examined on admission ... Se SALON — 57-2. 260105 Amoebic dysentery ... wae see +, ASS oI viel 5s Liver abscess 22 e ae 33 LOLs Ban Typhoid Ss Ae —? ,, Tuberculosis 53 is seh About 12:°0% —? ,, Pneumonia ... ak $3 de Less than 12:0% Taste III Maperra~Mamore Rairway Co., Porro Veruo, Brazit. Compiled from Lovelace (1912) Total blood-examinations Positive Rate (26 months) Malaria... om ad 16,434 93155 BO, Blackwater* 333 178 465% The tables are also of value because we are able to compare the percentages in blackwater with those made in malaria under the same conditions of observer, staining, and probably of time devoted to each examination. In the next table, Table IIIA, the data of the first three tables are summarized. From it we see that parasites are found in blackwater fever in 42°9% of cases (taking no account of the day of the disease on which the examination is made), while in malaria, parasites are found in 56°8% of cases. If we accept the conclusion drawn from Table I, viz., that parasites disappear during the course of the *In the majority of cases only one blood examination was made, and it was made during the period of haemoglobinuria. 484 disease, the lower percentage in blackwater is readily accounted for. The figure for malaria is probably low, but in the Panama figures of Deeks and James it should be remembered that only a single examination was made on admission to hospital. Tasre II]la.—Parasite Rate in Blackwater Fever and Malaria BLACKWATER FEVER Cases | Positive oe | Observer | ieee 390 163 41°8 | Various observers (Table I) 230 | 89 38°7 Deeks & James (1911) 383 178 46°5 Lovelace (1913) 1003 430 42°9" | Matraria 40,928 23,410 57:2 Deeks & James (1911) 16,434 | 95155 EGY! Lovelace (1913) 57362 32,565 56:8 The objection has been brought against figures of this kind that in a malarial country parasites would be found in the same or about the same percentage in people not suffering from blackwater fever, but such objections have ot been supported by actual data. Christophers and myself (1901), moreover, examined 44 Euro- peans at Lagos, taken at random. They were especially from communities suffering much from malaria. We found parasites in 2, pigmented leucocytes in 2, 1.e, 4 infected out of 44, about 9%. Christophers and Bentley (1908) examined 66 babus in the Duars, Bengal, and found parasites or pigment in 14, 1.e., 21:2%. In 10 cases of blackwater on the day of onset they found parasites or pigment in 8, or 80%. Further we have the control figures of Table II. * This figure represents the positive parasite rate irrespective of the day of the disease and should be compared with the figures of Tables I and II, 485 If, then, these data are correct, they imply that in the general population and those suffering from diseases other than malaria or blackwater fever the parasite rate is not comparable with that in blackwater fever. Moreover, I believe we are justified in meeting the above objection more forcibly still. We have in blackwater a disease in which immediately before the onset of the disease malaria parasites are found in 73% of cases, taking the figure of Table I. If this does not signify that the disease is malarial, then the finding of parasites in 73% of cases of ‘malaria’ has also no significance, and ‘malaria’ is also due to some unknown cause. PIGMENTED LEUCOCYTES In blackwater fever it is the general experience that parasites when found rapidly disappear. We still, however, have a means of detecting a malarial infection if present, viz., by means of pigmented leucocytes. In the literature most frequently no state- ment is made with regard to pigmented leucocytes. The following figures of Christophers and Bentley are small and only suggestive, and more observations are required. Taste IV.*—Pigmented Leucocytes in Blackwater Fever (Christophers & Bentley, 1908). | First Day SEconp Day FourtH-Sixtu Day No. of No. No. of No. No. of No. cases positive cases positive cases | positive | | r = - | i Parasites... 60 aoe fe) 6 7 fe) fe) I Pigment... — as 10 8 7 | 6 iio) 4 POST-MORTEM EXAMINATIONS In 31 cases where definite statements positive or negative have been made malaria pigment was found P.M. in 26, i.e., 83°9%. The evidence of malaria then in 5 cases is negative. If the absence of pigment P.M., supposing the facts correct, definitely excludes malaria then some blackwater cases must be due to other causes, which one must admit is not impossible, although I believe the facts prove that malaria is the dominant factor. * In this table, the data are obtained from the same 10 cases. 486 Taste V.—Post-mortem Records of those cases only where definite statement made. Parasites Cases or pigment Negative Authority Positive 3 fe) 3 Barratt & Yorke (1909). But in one of these scanty pigmented leucocytes, found during life. 6 6 fe) H. Werner (1907). 13 13 fo) G. H. Whipple (1999). 2 2 ° Christophers & Bentley (1908). 5 5 fr) Stephens & Christophers (1go1). (B.W. cases IX-XVI, p. 24.) 2 ) 2 Brem (1906). In one case only a smear from a rib examined. | 31 26 5 83-9 % 161% INFLUENCE OF MALARIA The influence of malaria in determining blackwater is shown in a different way by the following table compiled from Deeks and James. Taste VI.—Influence of Malaria. Ancon Hospital, July, 1904—Sept., 1910. Among 40,928 cases diagnosed as Malaria, 102 cases of Blackwater developed subsequent to admission. Among 42,000 cases diagnosed as Typhoid, Pneumonia, Amoebic dysentery, and Tuber- culosis, etc. (medical and surgical). 4 cases of Blackwater developed subsequent to admission.* RELATIONSHIP TO SPECIES OF MALARIA PARASITE The statistics on this point are often fallacious. What we require to know is not the percentage of malignant tertian or other species of parasite in blackwater cases, but rather what number of blackwater cases there is in 100 cases of malignant tertian, simple tertian and quartan respectively. The following figures show this. * In the surgical wards: 3 after well defined malarial paroxysms, 1 after quinine ina patient with a history of much malaria. 487 Taste VII.—Compiled from Deeks & James (1911) Of 23,410 Of 89 positive Ratio of positive blackwater blackwater % Ratio of malaria cases to malaria % | M.T. to S.T. cases Malignant Tertian TALK, 76:4% | 103'2% 100 Simple Tertian ... 26% 236% go8 % 88 i.e., if x cases of malignant tertian malaria give 100 cases of blackwater. then x ” ” » 88 ” ” Compiled from Lovelace (1913) Of 9,155 Of 178 Ratio of Ratio of positive positive blackwater % | M.T. to S.T. malaria cases blackwater to malaria % and to cases M.T. & S.T. respectively Malignant tertian ..[5988 =.65:4% | 87 = 48:9% 74:8 % 100 Simple tertian ..|2760 = 301% | 74 = 416% 138:2 % 185 MND. Se. Le cA ees Se COR Ie eh = CHB 223°3% 299 Quartan ... 16 —= "0-2 45 o= 0-0 00 fo) i.e., if x cases of malignant tertian malaria give 100 cases of blackwater. then ,, simple ” ” » 185 ” ” anu ces M.T. + S.T. " 299 ss a ebayel 63 4s quartan + A ° rr ae The discrepancy between these two results I cannot at present explain, assuming they are both correct, but it should be noted that Lovelace (1913) in his paper states that the simple tertian parasite ‘is here an extremely persistent infection. More time and more quinine are required for its eradication than for that of the aestivo- autumnal parasite.’ And again he speaks of ‘the rapid blood destruction in, the great prostration incident to, and the diabolical persistence of tertian infections.’ It would seem as if here we had an example of increased virulence of the simple tertian parasite. One’s own clinical experience of the malignant tertian parasite in the tropics and in temperate zones, seems certainly to point to differences in virulence. 488 INFLUENCE OF PERIOD OF RESIDENCE There is a general impression that blackwater is most prevalent in the second or third year of residence, but the statistics on this point are nearly all subject to the objection that no account has been taken of the number of people in each residential period, first, second, third, etc., years, and this may vary much from year to year. Here again, what we require to know in the first place is not out of one hundred cases of blackwater how many occur in the first, second, etc., years, but how many cases of blackwater occur in each one hundred people or similar sample of the different periods of residence, one, two, three, etc., years. Knowing this, we can then calculate how many out of one hundred cases of black- water occur in the first, second, third, etc., years. Plehn (1901!) 1s the only observer who supplies us with what we require to know, though, unfortunately, his figures extend only to two years. The figures in the second row of Table VIII indicate the number of people in their first, second, third, etc., month of residence under observation (for Hgb. estimations). Thus in the second month of residence there were seventy-five, therefore eight of these were not under observation in their first month, but whether the remaining sixty-seven were the same people as the sixty-seven in their first month, the table does not tell us; probably they were, and at the end of twenty-four months they were reduced to five, but it is not a material point. Tasre VIII.—Blackwater Fever: Effect of Residence (Plehn, rgor). First YEAR | | Month... bs a3 te el eee | Bbw Al BS 6 | 7 iler8 he ol Hebe beatae | | | | Residents no “Ae cee -sh OIRO SL 75 hegn 67 | 62 | 61 | 57 | 55 | 5° 46 | 47 Malaria cases ... 28 | 50 | 6o | 42 | 41 | 43 | 38 | 44 | 37 | 33 | 26 | 24 | } B.W. cases 489 Seconp YEAR | | Month... ee 358 ee sa Sle Pests lO lz | Se ne |-20;]:20 || 22} 23 |24 > oie St |S re me | tp als Residents... Le Meee eaaet a9). 35° |. 33 | es | ar jorge} ie tot Gg | 5 Malaria cases ... aa Bee none” neo Salle) | 18 Oita. (meer eae conl aha seh ep) | | | B.W. cases... nes ane Sod) YY) EE eA ey a Te | eOn eee Ciel| Om by xOuly 12 | | | Tasre VIII. B. (compiled from previous table.) Time of Residence ... es ae see ...| First 6 months | First year Second year Number of residents 200 508 — 69°5 61-1 24 Cases of blackwater ane aes ws wes 3 22 : 12 B.W. rate per roo residents nb ene wes 4°32 36:0 50°0 Of 100 cases of B.W. there occur ort 3c 502 41°86 58-14 i.e., Among a population of 100 4°32 cases will occur in 1st 6 months residents, 36-0 ae 3 ist year “ 50°0 * 2nd year “f And of too cases of B.W. occurring during the 2 years’ period : 502 will occur in the 1st 6 months. 41°86 5 = Ist year. 58-14 - a 2nd year. Malaria cases 500 505 505 aia oo 264 466 127 B.W. cases ... aa est bes 56 mise 3 22 12 B.W. rate per roo malaria cases ... ae oe Iel4 4°72 9°45 i.e., For 100 cases of malaria in 1st 6 months’ residents there will be 1-14 blackwater. on ay 5 Ist year’s _ 6 4°72 3 ” ” ” 2nd ,, ” yy) 9°45 ” And of 100 cases of B.W. in 2 years there will be 8-05 among x malarial cases in the rst 6 months. 33°31 “ a5 - Ist year. 66-69 = 5S Fe 2nd year. The next table gives the distribution of 1,050 cases of which I have found records. The data for the first six months and first year, and possibly second year, are probably fairly correct, as they have been taken from large and probably approximately equal popula- tions. But in the third and subsequent years we do not know what proportion the population bears to that of the first year, and as I have already pointed out, this is essential before a comparison can be made. I should note, however, that Daniels in his figures has made this correction, and that the difference from the uncorrected figures was only slight. 490 (6681) yoo ‘savad bz 19qye youyqe qsuyq, “(2061) paremoxy -(1161) samef 7 sy0q *(1061) sjaturq *(g061) v4sod ep *(g061) Aspuag 2p ssoydoystay5 ‘(zo61) syrups *(1061) ypuodadiq 7 jnoyusdure) ‘savod + uvyy ra8u0T, *(46g1) pueisay xy joing ‘(g061) uapoig ‘(1161) wag ‘(pZgr) pnesigg 1a3dueI9g (6061) axI0X IWwIIKg dIUIIOJOY ise) I9qv'] Ivad WHA ZI gt al Lz z e S ZI é q € I oz t+ u 9 I —_ (Sox) | (% zt) ES 6L I = iva Ieak LO PaTGL ‘QuUapIsoy JO Joy :IOAI_ JayeMYyI[G— KX] ITaV], CT Ivok puosas 1Z IZ (% +-S) ol reak SIT an. no syquour 9 SII] 491 ee % 1-4 AU % 4-6 % 6.2 % 0.€€ % £-1z % eb *saseo oSO‘r ‘syquow g ysiy Sutmnp gf ssa] gzit SE ad Zor 1g9z ott bez gf (S061) Aospqoyy Aq porto ‘Apaa + Zz S gz £ $ Q *(66g1) sppeudoy 1 _— — — iz Zz 1 (66g1) aJoog é 2 d d d “1 d (3681) ‘a “uyetd = = I 2 8 6 t ‘reoh puz ay} uryy sajey, ‘(£061) *y ‘uystg — — — ai It 44 ZI *(zo6r) osurg € — — t t oD + *syqUOUL gI qsea] qv ROyy ul useq pry [Vy “(66g1) ossuayy — oo — -- ol — — ‘sread ys pure ‘yyh ‘pif Sutinp sased xtg, ($061) Aosyqoy 6f @ AE eke io z d | ad UaTOJOY | 19e'] Ivak ivak iva arad Irak syquow WaT WEEN PAL [pdeselakeks) TIAL Oneal “ponurqu0I—aouapIsay JO e2YA : IOAI{ JaywMyov[g—X[ AAV L 492 I believe that we may deduce from these figures the fact that blackwater is not a disease of the first six months or first year, but mainly of the second year. I believe this fact is capable of a simple explanation, viz., that blackwater fever only shows itself in the majority of cases when the patient has been subjected to ‘repeated malarial infection lasting over a certain time.’ THE SEASONAL PREVALENCE In Tables XA, XB the distribution of 226 European cases of blackwater fever in Northern Nigeria (1912) month by month is given. The figures have not been corrected for monthly variations in the European population as no figures are available for this purpose. From 1900-1911 the total European population has increased from about 175 to 775, but there is no reason to suppose that the increase has not been a uniform one, spread over the whole of each year, or that if in any year any disproportionate increase occurred in any particular month it would not be equalized in other months in other years. Tarte Xa.—N. Nigeria. Showing the distribution by month of blackwater cases from 1899-1911. Jan. | Feb. | Mar. | Apr. | May | June | July | Aug. | Sept. | Oct. | Nov. | Dec. | Total 1899 10 2 I ae oe aa So 2 2 On I 2 20 1900 2 2 3 I I I I I 12 1901 I 2 I I 2 3 2 12 1902 4 I I 2 I 3 I B 2 2 20 1903 I I 2 2 3 3 2 3 17 1904 3 3 I 4 2 4 6 5 I 6 35 1905 I 2 I I I 3 I 3 3 4 20 1906 3 I 2 5 2 4 3 2 4 26 1907 3 2 I I B 2 12 1908 1 I I 2 I 3 2 3 2 16 Cpls, I 2 I 2 I I 2 I 2 I 14 Igto 2 I I 2 2 I I 10 1911 2 I 2 I 2 2 I I 12 Total} 30 14 14 9 8 9 18 27 26 23 20 28 | 226 493 Tasie Xs.—Northern Nigeria | Corrected for a Departure + or — Months Actuals month of 30-4368 from average 18-8 days 30 29°5 | + 10°7 2 14 | 15:2 — 3:6 3 14 13°7 = opr 4 9 | gr yy 5 8 | 79 — 10°9 6 9 | git = OF 7 18 17°7 see 8 27 26°5 a FG) 9 26 26-4 + 7-6 Co) 23 226 Bac Il 20 20°3 + 1-5 12 28 27°5 + 87 226 225°5 Column I represents the months. Column 2 the actuals, taken from the previous table. Column 3 the corrected figures for a month of 30°4368 days. Column 4 the departure + or — from the average figure 18°8. We observe in this table that the cases below the average come together, and Mr. Stott has calculated for me that it is 131 to I against the distribution being simply a random one. Further, he has calculated that if we take into account the amount of departure, that it is 1,000 to 1 in favour of the effect being due to season. 494 The next Tables XIA, XIB show similar figures for Southern Nigeria, Graham (1912). Taste XIa.—Southern Nigeria. Showing the distribution by month of blackwater cases from 1899-191. Jan. | Feb. | Mar. | Apr. | May | June | July | Aug. | Sept. | Oct. | Nov. | Dec. | Total 1899 I I 2 I 5 1900 I I I 3 1gOI 2 2 I I I 2 5 I g 2 20 1902 5 3 3 I I I I 5 3 23 1903 I I 2 2 I 2 I 2 I I 2 I 18 1g04 2 2. 2 2 4 3 I I 17 I I 2 I 2 3 2 I I 14 I 3 I 2 I 2 2 3 3 3 4 2 5 I I I I 5 3 3 I 4 x ars 3 2 4 4 I I 3 I I I 21 I 2 2 I 2 I g 2 2 16 I I 3 2 I 3 3 5 6 5 30 B 2 I I 4 2 I 4 3 I 22 jp ff 495 Tasie XIz.—Southern Nigeria. © ene een Corrected figures for | Departure + or — Months Actuals a month of 30-4368 from the average days 20°1 I 20 19°6 — 6 2 20 21-7 + 1:6 3 20 19°6 — 5 + 19 19°3 san8 5 14 13°7 — 64 6 14 14:2 = 459 7 28 274 a waa 8 3° 29°5 + 9-4 9 13 13-2 — 649 10 22 21:6 SEP ol Ir 25 2554 ar bys 12 16 15°7 — 44 241 240°9 Column 1 represents the months. Column 2 the actuals, taken from the previous table corrected for a month of 30°4308 days. Column 3 the departure + or — from the average figure 20°1. In this case Mr. Stott remarks ‘ some other factor seems to be at work and the probability is reduced to 7 to I.’ CuHart A fe lake ROWER fever EAS Ones « | weIDENCE APR MAY AU SEP ocT | wov | eee SS ea Northern Nigeria QRS Cacsie’ 5; 1S96-1971 Sovlhern Nigeria 240 cases 1839 s94H1 Lay | SS sae eae cree ae a ES ae eS Hee aun all 7 Me eres) ne eee ee \ Zh oo (aed 2 2 a | ee) SZ (ot cara (ee en a es ee EY ae =| —_ — f+ __} f _-__ eae PE ee | a ae Ce eee \ha [ he SS Sse cae ee Stee oe ee AST Mae A A ams | eda Spee al : Se ee ae ae: Pe | See Ppa Se il fe ee ae Eee |e her erty 496 The next table represents similar figures for the European cases of blackwater admitted into Ancon Hospital. Tasie XII.—Ancon Hospital, Panama. Showing the distribution by month of blackwater cases among Europeans from 1go8-1912. | Departure Oe 1908 1g09 IgIo IQIt 1912 | Totals from the average 20 January ... ... 3 | 17 I 5 2 28 + 8 February Bee I 9 4 6 3 23 + 3 March Ys. es 3 10 3 4 | 3 23 + 3 April ae see I 6 3 8 I 1g — I May seo SEE fo} 7 3 4 I 15 — 5 June Soo) bc ° 3 4 7 I 5 — 5 July Beet eee I fo) 2 4 5 13 —7 Ausust” ees 9 e- I 4 3 8 4 20 ° September... ° I 2 10 6 19 — I October=-.) << 8 3 4 5 3 23 3 November .... II 7 2 6 2 29 +9 December... 5 | I 7 ° I 14 — 6 241 In Table XIII, I have given the uncorrected totals of Table XII and the totals corrected for monthly variations in the European population to show that the general conclusion is not materially affected. 497 Taste XIII.—Europeans at Ancon Hospital. CORRECTED FOR POPULATION AND FOR LENGTH OF MONTHS UNCORRECTED Average European Population, | Blackwater 1908-1912 cases Deviations | Blackwater | Calculated | Deviations admitted from rate to make 241 from 1908-1912 average per 10,000 average it, nba pee 6,061 28 amt) 46°18 27°8 + 79-9 2 6,112 23 + 3 37°63 2571 + 5:0 3 6,090 23 a as 37°77 22°8 F257 4 6,436 19 = i 29 18-4 — 17 5 6,324 15 = 25 3°7 14°3 — 53 6 6,143 15 15 24°42 15°2 =" 49 7 6,138 13 = 9 21°18 12°8 — 73 8 5,982 20 fe) 33°43 20°1 — 00 9 55953 19 td 31°92 198 = 6°3 fe) 6,241 23 + 3 36-85 22°2 ap Sth 11 6,071 29 +9 47°77 29°7 + 96 12 6,290 14 — 6 22:26 134 = YY 73,841 241 392°65 241°6 Average ... 6,153 20 20°! Accordingly, in Panama also, as Mr. Stott observes, we have six cases below the average coming together, making a probability of 131 to 1 in favour of a seasonal incidence, although in this case our confidence is reduced by one-third owing to the average being only over 5 years, instead of 13. A similar conclusion can also be drawn from the figures for blackwater for all employees admitted to the Commission hospitals (Table XIV) showing that the Ancon figures are a fair sample of the conditions existing over the whole zone. For comparative purposes the monthly distribution of malaria 1s also given, in which the seasonal malarial rise is clearly shown. It should be noted that there are slight discrepancies between the returns (Table XII) from Ancon Hospital (Kuropeans), privately communicated by Dr. James, and those given in the monthly official reports. I believe that the Ancon figures are reliable, as they are the hospital records, whereas it is easily intelligible that errors may occur in the official figures for the whole zone. 498 Taste XIV.—Showing the monthly distribution of malaria and blackwater among all employees admitted to the Isthmian Canal Commission Hospitals, 1907-1912. Macaria | Departure from ad 1908 1909 IgIo fgII 1gi2 Total | average (5,226) January ... ...| 1,822 | 641 1,245 479 331 496 5,014 — 212 February ene} BLAOO7, 570 832 354 375 502 4,240 — 986 March ~ =<. ~==.|| 15570 §25 657 528 402 343 4,025 —1,201 April sc. aelh 022 402 524 436 319 235 25738 —2,488 May Pee ccr|| ey, 393 576 613 920 257 3,366 —1,860 June pooe ccd. Et 629 737 1,261 1,736 424 5,715 + 489 July i] 7 ' 4 x . 4 2 * | Ps 2 f eS ié- pee | ~- \ 1 as ; i a t = 4 f c a 7 ; 2 2 ‘ee i i all axes 1 2s a a ‘ r %% : ; od 7 ~ al 5 = - 7 Hi Se. 5: - i “ Pe 7 3 View “or oy = ; i. J io, E = ~ 2 , oo li a id - 7 = , 3 7 . re - “elt e# 2 = st 7 J ei , , ’ Xs i, ; A oe = -s i. we ae 509 THE GROWTH AND SPORULATION OF THE BENIGN AND MALIGNANT TERTIAN MALARIAL PARASITES IN THE “CULTURE (EUBEYAND® IN THE HUMAN HOST BY JOHN GORDON THOMSON, M.A., M.B., CH.B. (Edin.) SIR EDWIN DURNING-LAWRENCE RESEARCH ASSISTANT, AND PATHOLOGIST, ROYAL SOUTHERN HOSPITAL AND DAVID THOMSON, M.B., CH.B. (Edin.), D.P.H. (Cantab.) GROCERS’ RESEARCH SCHOLAR, LATELY CLINICAL AND PATHOLOGICAL RESEARCH ASSISTANT, SCHOOL OF TROPICAL MEDICINE, LIVERPOOL (RKecewved for publication 14 Settember, 1913) PLATES XXXIV, XXXV CONTENTS PAGE Prefatory note by Sir RonatpD Ross ace ae 5 509 Introduction Be ais oe ade ae os a3 510 Cultivation Technique ... sas est a eas ote 510 Further observations on cultivation ... oe = ons 511 Optimum temperature, Haemolysis, Clumping ... Jats 512 Resistant forms... ido bet as Br Se Sa 513 Morphology of P. falciparum in the culture tube ... oar 513 Ar an = “6 human host ... Bhs 516 3 », P. vivax in the culture tube ae me 518 Bf . ; » human host ie Sos 519 Summary ... 00 306 ee sec 50 ele cee 520 References ... are bac o0C 00 550 20 one 521 Explanation of Plates... BS oct de ae 300 522 PREFATORY NOTE BY SIR RONALD ROSS Researches on the cultivation of the parasites of malaria in Liverpool were commenced some time ago at my suggestion by Dr. Sinton, and then, with better success, by Drs. J. G. Thomson and McLellan, and by Dr. D. Thomson. We are greatly obliged to Sir Edwin Durning-Lawrence, Bart., for giving us the services of Dr. J. G. Thomson for this important enquiry. 510 INTRODUCTION The successful cultivation of malarial parasites was first announced by Bass and Johns (1912). Since then several workers, Thomson and McLellan (1912), J. G. Thomson and D. Thomson (1913) and Ziemann (1913) have successfully repeated these cultivation experiments. This achievement has led the way to new discoveries regarding the malarial parasite, and suggests that it may be possible to cultivate zz vitro any protozoal parasite however specialised it may be. So far, only the asexual generation of the malarial parasite has been grown zz vitro. The next step is to cultivate the sexual generation as it occurs in the human host and in the mosquito. We have attempted both, but so far without definite success. We have no doubt, however, that this will also be accomplished sooner or later. In 1911, J. G. Thomson and Sinton successfully cultivated the human trypanosome, and the development they obtained was apparently that which takes place in the stomach of the tsetse fly. Joukoff (1913) states that he has cultivated the mosquito cycle of the malarial parasite, though his results have not yet been confirmed. There is no reason to doubt that the phases of development of protozoa in insects may be produced in the culture tube. An abridged account of these researches, illustrated by Plate XXXIV, was published in the Proceedings of the Royal Society, B, Vol. 87 (1913), pp. 77-87. . We have much pleasure in returning thanks to the Royal Society for permission to reprint Plate XXXIV. We wish to thank Dr. H. B. Fantham for his very valuable help during our work on this subject. CULTIVATION TECHNIQUE Our method of cultivation is almost the same as that of Bass and Johns, except that it is less complicated. Ten c.c. of blood is drawn from a vein and transferred to a sterile test tube containing a thick wire leading to the bottom of the tube from the cotton wool plug. 1/10 c.c. of a 50 per cent. aqueous solution of glucose is added to this tube, preferably before adding the blood. "ee S11 The blood is defibrinated by gently stirring with the thick wire. Defibrination should be complete in about 5 minutes. The wire with the clot is then removed and the blood is poured into several smaller sterile tubes (about one inch column of blood in each). A rubber cap is placed over the cotton plugs to prevent evaporation and the tubes are then transferred (standing upright) to an incubator at a temperature of 37° to 41°C. The corpuscles settle in a short time, leaving about one half-inch of clear serum at the top. It is apparently unnecessary to remove the leucocytes by centrifugalisation. FURTHER OBSERVATIONS ON CULTIVATION We have grown four complete generations of parasites in one tube by the above method, and we do not see why their growth should not continue indefinitely, provided fresh serum and corpuscles be added. It is not the presence of leucocytes which prevents further development. This is due to degenerative changes taking place in the corpuscles and serum. If the serum and corpuscles be kept in a sterile condition in an ice chest they remain unchanged for a long time; but at the temperature required for the growth of the parasites visible changes take place in a few days. The corpuscles become fragmented and form a brownish débris, and the serum becomes dark brown in colour. When this occurs the parasites are unable to continue their developmental cycle. Bass and Johns in their original paper (1912) stated that the parasites grow only on the surface layer of corpuscles, and that no growth took place in the deeper layers. They also stated that the serum destroyed the parasites when they escaped from the corpuscles, so that when the spores escape they must enter immediately into a contiguous corpuscle in order to survive. The blood also, in their opinion, required to be heated to a temperature of 40° C. to destroy the complement in the serum; furthermore, in order to cultivate several generations, the leucocytes had to be removed. We do not believe that they have sufficient evidence for these deductions. We find that the parasites develop even in the very deepest layer of the column of corpuscles. Also, it seems rather a contradiction that the parasites should grow best on the surface layer of corpuscles 512 next to the serum and the leucocytes which are supposed to destroy them. No doubt the leucocytes ingest some of the parasites, but they are never able to ingest all of them. Again, in one of our most successful cultures the temperature of the blood never exceeded 38° C., and we have found that they are able to grow at a tem- perature as low as 36°C. This is rather against the theory of complement destruction. In an able paper by Mary Rowley Lawson (1913), considerable evidence is brought forward to show that the parasites are extra-corpuscular during their entire development. If this is true, then the parasites would be constantly in contact with the serum which is supposed to destroy them. It appears to us that the only conditions necessary for the successful cultivation of the parasites are fresh corpuscles, fresh serum, a temperature of 37° to 41°C. and the requisite amount of glucose. The presence of leucocytes and complement is apparently immaterial. THE OPTIMUM TEMPERATURE In our opinion the optimum temperature for cultivation 1s about 38°C. On two occasions we incubated identical culture tubes from the same patient, some at 37° C. and some at 41°C. On both the occasions the parasites developed much more successfully in the tubes incubated at 37°C. HAEMOLYSIS IN THE CULTURE TUBES This is a rare occurrence, having been seen only once out of fifteen cases. A kind of haemolysis occurs after the blood has remained in the incubator for several days. The corpuscles degenerate into a brownish débris, and the serum shows a brown discoloration. CLUMPING OF THE MALIGNANT TERTIAN PARASITES This phenomenon has been observed in all our malignant tertian cultures (twelve cases). It occurs even when the parasites are scarce. It is best observed in wet films, as smearing of the blood tends to break up the clumps. No tendency to clumping occurred in our benign tertian cultures. 553 RESISTANT FORMS OF PARASITES Sometimes it will be found that the parasites do not grow well in vitro. They may grow only partially, stopping short of segmentation. This is liable to occur if quinine has been given to the patient before the blood is drawn. In other cases, only a few of the parasites reach maturity, these apparently being able to resist successfully adverse conditions in the culture tube. THE MORPHOLOGY OF PLASMODIUM FALCIPARUM IN CULTURE The blood examined before incubation always showed the typical small ring parasites of malignant tertian malaria; these varied in size, with a maximum diameter of about 3“. There was no enlargement of the red blood corpuscles, and no Schiiffner’s dots. Plate XXXIV, fig. 1, shows a small ring parasite from the peripheral blood before incubation. After many experiments in Liverpool it has been found that there is great variation in the rate of growth of P. falctparum in artificial media, and many suggestions can be offered to explain this phenomenon. J. G. Thomson and S. W. McLellan (1912) found in one case that maximum sporulation of P. falczparum occurred in twenty-five hours. This culture was made from a case with very heavy infection of parasites, and the patient had taken no quinine. The temperature of the incubator was only 38° C., but the glucose added was slightly in excess of that recommended by Bass (1912). The rapid segmentation of the plasmodia in this case, therefore, may have been due to two causes: (1) the age of the parasites when introduced into the culture tube, since it is to be noted that they were fairly large rings, about 3m in diameter, and (2) the quantity of glucose may in some way have hastened the growth. In other experiments, however, where the glucose was slightly in excess there was no such rapid growth, so that in all probability the real cause of the rapid segmentation of parasites zz vztvo is due to the age of the plasmodia when drawn for incubation purposes. Another factor which seems to influence the rate of growth is the previous administration of quinine, and, in fact, this may inhibit the growth entirely. On several occasions we have made unsuccessful attempts 514 to cultivate the malignant tertain parasite, and these have usually been from patients to whom quinine had been administered. On two occasions complete sporulation did not take place till after fifty hours’ incubation at 41°C. On one of these occasions the parasites completed several generations, as illustrated by the accom- panying coloured plate (Plate XXXIV, figs. 1-20). We found that maximum segmentation took place in fifty-two hours. Division of the chromatin into daughter nuclei began in about thirty-six hours (figs. 4 and 5). In forty-seven hours the number of spores had increased (figs. 6 and 7), and more or less complete segmenta- tion took place in fifty-two hours. Fig. 11 shows a parasite which has produced thirty-two daughter cells, and these have broken loose from the corpuscle. It is to be noticed that in the peripheral blood the small ring parasites show no pigment. After incubation the rings gradually grow in size (fig. 3), and at a certain stage, immediately previous to the splitting of the chromatin a round compact mass of pigment appears, usually situated near one margin of the parasite, which now measures about 5 or 6p in longest diameter. In the culture under discussion this round mass of pigment was seen in thirty-six hours (figs. 4 and 5). J. G. Thomson and S. W. McLellan (1912) found that the pigment had collected in twelve hours. The parasites immediately previous to segmenta- tion measure about 5, to 6u in their longest diameter, and all show the circular mass of compact pigment. We may call this stage the pre-segmenting stage. The chromatin now begins to split into two, and if segmentation is complete thirty-two daughter cells may be formed (fig. 11). All stages of segmentation can be found, from two spores up to thirty-two spores as a maximum. During segmentation the circular mass of pigment takes a central position, and the spores form in a circular arrangement around these (figs. 7 to 11). In this culture segmenting forms were found at different stages up to fifty-six hours, this being, no doubt, due to the fact that the parasites were not all of the same age when introduced into the culture tubes. In seventy-five hours all segmenting forms had disappeared and only very young parasites were found, about 1’5m in diameter and containing no vacuole (fig. 13). This represents the beginning of a second generation. It is to be concluded, therefore, that many B15 of the young merozoites escape ingestion by the leucocytes and enter a new red cell. The same culture examined in three days again showed segmenting forms (fig. 14); but in this case the spores were never so numerous as 32, and this can easily be explained by the adverse conditions which have now developed in the culture tube. These conditions did not, however, prevent the young parasites attempting to segment. In four and a half days (fig. 15) we again only found young rings, which represent the beginning of a third generation; and in six days sporulating forms were again found (fig. 16). Here, only eight spores have formed, and it was evident that the power of the parasite to undergo full segmentation was getting gradually less. On the seventh day, again, only very young plasmodia were found, which represented a fourth genera- tion, and these again attempted to sporulate (figs. 18 and 19) on the eighth and ninth days respectively. On the tenth day only young parasites again were found, which represented a fifth genera- tion. No further development, however, occurred, as the conditions in the culture were gradually becoming more and more adverse. Thus we have evidence that the parasites will, under very suitable conditions, actually proceed in the original culture tube through four complete generations without the medium being in any way renewed. We now wish to draw particular attention to several points of great interest which occur in the cultures of P. falciparum. In these there is a definite tendency for the parasites to clump together into masses immediately the circular mass of pigment appears, and even before segmentation begins. A mass of seven parasites is seen in the microphotograph, Plate XXXV, fig. 2. This photograph was taken after twelve hours’ incubation at 38°C. In all of these note the dark circular mass of pigment, the parasite having a diameter of about 6. At this stage the chromatin is dispersed into the proto- plasm of the plasmodium, and as yet shows no division into daughter cells. This tendency to clump becomes more marked when sporulation actually begins, and fig. 3, Plate XXXV, shows a large mass of sporulating parasites, the photograph being taken after twenty-five hours’ cultivation. This picture shows all stages of sporulation and illustrates in a very striking manner the tendency of the parasites to agglomerate into masses. This clumping in 516 malignant tertian malaria was seen by Dr. Cropper (1908) in the peripheral blood of a patient in Jerusalem, and with his kind permission we have been able to reproduce a microphotograph (Plate XXXV, fig. 5) of his plate in the ‘Lancet,’ which shows a mass of these pre-segmenting parasites, each with a circular mass of pigment. This must have been a most unusual case, as these forms are seldom seen in the peripheral blood in malignant tertian malaria; but it is of extreme interest, since it proves the tendency of the parasites to clump together during the pre-segmenting stage of development. When we compare this figure of Dr. Cropper’s, photographed from the peripheral blood of a case of malignant tertian malaria (fig. 5), with our own microphotograph, obtained from a culture twelve hours old (fig. 2), we are at once impressed with this phenomenon; and it is to be noted that this tendency to clump remains until full sporulation. Fig. 3 shows a whole field of a microscopic preparation filled with segmenting forms. In our opinion this phenomenon explains why the sporulation of P. fal- ciparum occurs, as a rule, only in the capillaries of the internal organs. All the large parasites tend to get caught in the fine capillaries of the internal organs, and thus only young rings, whose containing corpuscles have not yet acquired the clumping tendency, are seen in the peripheral circulation. This fact explains many phenomena of pernicious malaria, and shows how comatose malaria may occur in infections with Plasmodzum falciparum, im which condition the brain capillaries are filled with sporulating parasites. Why Dr. Cropper’s case showed pre-segmenting forms and also segmenting forms in the peripheral blood is uncertain, but it could well be suggested that these were the overflow from fine capillaries of the internal organs which were already packed with parasites, and could not hold more. It has been observed by us that segmenting forms of P. falctparum are only found in the peripheral blood of patients suffering from extremely heavy infections. THE MORPHOLOGY OF PLASMODIUM FALCIPARUM IN THE HUMAN HOST This is identical in every respect with that obtained zz vztvo, so that it 1s hardly necessary to describe it in detail. As already stated, only the young ring forms of this parasite are found, as a 517 rule, in the peripheral blood. This is due to the fact that when these parasites have grown larger than the ring stage, the containing ‘corpuscles stick together, forming clumps which are unable to circulate, and thus are arrested in the fine capillaries of the inner organs. The study of the further stages of this parasite 1s obtained by examining smears of the inner organs, such as the spleen, liver, brain, etc., of deceased patients. The autopsy smears of over one hundred cases have recently been examined by one Gf us (D203) im conjunction- with, Dr. Ws M.-_ James, in Panama (the results of this work will be published later). In such smears the parasite is found in different stages of development. In some, pre-segmenters only are found; some show only young rings, and others only sporulating forms. This depends entirely on the stage of development of the parasite at the time of the patients’ death. In some cases, of course, several stages of development are found, since one patient may contain several broods of parasites which are in different stages of development. In only one case did we find the parasites in the stage of maximum sporulation. All of them contained over 20 spores, and 13 per cent. had 32 spores. One of these is represented in the microphoto- graph (Plate XXXV, fig. 4). We think that there can be little doubt that we have proved conclusively, both from observations in the culture tube and in the human host, that P. falczparum is capable of producing a maximum of 32 spores under favourable circumstances. This is a much larger number than has been pre- viously given in the writings of competent observers. Ross (1912) quotes the figures of Welch (1897) at 6 to 20 or more spores. Later, however, from observations made by himself and one of us, he taught that the maximum number was 2° or 32. Marchiafava, Bignami and Mannaberg (1894) state that the numbers vary from 8 to 15, while the following authors give the numbers as follows. Stephens and Christophers (1908) 8 to 10, Deaderick (1910) 5 to 25 and even 30, Gulland and Goodall (1912) 8 to 15. It is probable that these discrepancies are due to two reasons: (a) observations of autopsy smears in which the sporulation had not reached its full maturity, and (4) observations on autopsy smears of patients to whom quinine had been given before death. In such cases incomplete and atypical sporulation is seen. We do not claim, 518 of course, that 32 spores are always produced, even under favour- able conditions, without quinine administration. It is likely that the numbers produced vary considerably, just as in the case of benign tertian, which produces numbers varying from 16 to 26. The clumping phenomenon in the human host is represented in Plate XXXV, fig. 5. This clump was, as already stated, found by Dr. Cropper in a peripheral blood smear. A similar phenomenon is seen in the brain capillaries of cases of comatose malaria. THE MORPHOLOGY OF PLASMODIUM VIVAX IN CULTURE Here we are studying a parasite in which all stages, from small rings up to full sporulation, may be seen in the peripheral blood. Sporulation does not necessarily occur in the internal organs, and it is quite usual to find these forms in ordinary peripheral blood smears. When cultivating this parasite, therefore, it is important to draw the blood from the patient when the young forms pre- dominate, so that it is then certain whether or not we are obtaining further development in the culture tubes. In the culture illustrated in Plate XXXIV, figs. 21 to 30, we obtained the blood when young rings (fig. 21) predominated, and no segmenting forms were found. After eight hours’ incubation at a temperature of 39° C., a marked increase in size of the parasites was noted, figs. 22, 23 and 24, and pigment was now evident, being scattered throughout the proto- plasm in fine granules. This arrangement of the pigment in P. vivax is in marked contrast to what occurs in P. falciparum, where the pigment always becomes arranged in a dense circular mass from the commencement. After 20 to 29 hours’ incubation (figs. 25-30), sporulation was seen at different stages, the pigment being collected into a loose mass of granules in the centre of the parasite. Fig. 29 shows a parasite with 15 daughter cells, and we have found on several occasions sixteen spores, which we think is the usual maximum of daughter cells found in P. vivax, although again we find all stages, from two spores up to sixteen, according to the stage of development at which we examine the cultures. We are quite certain that the spores are never so numerous as in the malignant tertian parasite. In these cultures clumping has not been found, and this explains why the parasites do not tend to be 519 arrested in the internal organs during sporulation, although when full grown they are much larger than the malignant tertian parasite. The absence of clumping in the case of the benign tertian parasite explains satisfactorily the lack of pernicious symptoms in this infection, and, hence, the absence of comatose malaria, and it also explains why all stages of this parasite are found in the peripheral blood, even up to sporulation. THE MORPHOLOGY OF THE BENIGN TERTIAN PARASITE IN THE HUMAN HOST This is identical with that which we have just described under morphology in the culture tubes (Plate XXXIV, figs. 21-30). The chief features in which this parasite differs from P. falciparum, are the large size of the containing corpuscle with the presence of Schiiffner’s dots, the straggling form of the medium-sized parasites, the scattered pigment in the pre-segmenting stages and the smaller number of spores. These spores are larger than the spores of the malignant tertian parasite. The pigment in the sporulating forms is collected into a loose mass. With regard to the number of spores produced by this parasite, we have found as many as twenty-four, but the most usual number is 16 to 18. In culture the largest number we obtained on three occasions was 18. The following are the numbers of spores produced by P. vivax in the human host, according to several observers. Ross (1910) quotes the figures of Grassi and Feletti, 15 to 20 spores. Marchiafava, Bignami and Mannaberg (1894), 16 spores. Golgi quotes 14 to 19 spores. Stephens and Christophers (1908), 15 or more spores. Deaderick (1910) gives 12 to 26 spores, most often 16 spores. In our opinion Deaderick’s figures are most correct. All stages of this parasite are found in the peripheral blood, and the phenomenon of clumping has never been observed. Ross has taught recently that there are four splits with this parasite—that is, sixteen spores; three splits with the quartan parasites—that is, eight spores; and five splits, or thirty-two spores, with the malignant parasite. 520 SUMMARY (1) The malignant tertian parasite has been successfully cultivated, after the method of Bass and Johns, on twelve occasions, and the benign tertian parasite on three occasions by the present writers. (2) It is unnecessary to remove the leucocytes from the blood before incubation. The optimum temperature would appear to be about 38°C., and the parasites may grow successfully at a temperature of 36°C. or 37°C. (3) The time required for the full development of the parasite zn vitro varies, but this variation is partly due to the age of the parasite at the time of incubation. (4) The cultures of benign tertian differed from those of malignant tertian in that there was no tendency to clumping of the parasites in the former, either before or during sporulation. (5) This difference appears to us to explain in a satisfactory manner why only young forms of malignant tertian are found in the peripheral blood, as the clumping tendency of the larger forms causes them to be arrested in the finer capillaries of the internal organs. It also explains the tendency to pernicious symptoms, such as coma, in malignant tertian malaria. All stages of the benign tertian parasite are found in the peripheral blood, and there are seldom pernicious symptoms, because there is no tendency to clumping. (6) The malignant tertian parasite (P. falczparum) is capable of producing, in maximum segmentation, thirty-two spores. On the other hand, benign tertian (P. vevaxr) produces, as a rule, during maximum segmentation, sixteen spores; sometimes more may be produced, but the number is never thirty-two. (7) The pigment in P. falciparum collects into a definite, circular, and very compact mass early in the growth of the parasite. On the other hand, during the growth of P. vivax the pigment remains scattered in definite granules throughout the body of the parasite, till just before segmentation, when it collects into a loose mass of granules in the centre of the full-grown Plasmodium. (8) The morphology of P. falczparum and P. vivax in the human host is identical with the morphology of these parasites, as obtained in the culture tube. | 521 REFERENCES Bass and Jouns (1912). The Cultivation of Malarial Plasmodia (Plasmodium vivax and Plasmodium falctparum) in vitro. Journ. Exper. Med., XVI, pp. 567-579. Cropper, J. (1908). Phenomenal Abundance of Parasites in a Fatal Case of Pernicious Malaria. Lancet, July 4th, 1908. Deaperick (1910). Malaria. Gutianp, G. L., and Goopatt, A. (1912). The Blood: a guide to its Examination and to the Diagnosis and Treatment of its Diseases. (William Green & Sons, Edinburgh and London.) Jouxorr, N. M. (1913). Culture du Parasite de la Malaria. Comp. Rend. Soc. Biol., Vol. LXXIV, No. 3, pp. 136-138. Marcuiarava, E., BigNami, A., and MannaBerG (1894). "Two Monographs on Malaria and the Parasites of Malarial Fever. (1) By Marchiafava and Bignami; (2) by Mannaberg. Ross, Sir Ronarp (1910). The Prevention of Malaria, p. 89. Rowrey-Lawson, Mary (1913). The Extracellular Relation of the Malarial Parasite to the Red Corpuscles and its Method of Securing Attachment to the External Surface of the Red Corpuscle. Journ. Exper. Med., Vol. XVII, No. 3, pp. 324-335. With 6 plates. STEPHENS, J. W. W., and Curisropuers, S. R. (1908). The Practical Study of Malaria, p. 34. Tuomson, J. G., and McLexran, S. W. (1912). The Cultivation of one generation of Malarial Parasites (Plasmodium falciparum) in vitro, by Bass’s method. Ann. Trop. Med. and Parasitol., VI, pp. 449-462. Two plates. Tuomson, J. G., and THomson, D. (1913). The Cultivation of one generation of Benign Tertian Malarial Parasites (Plasmodium vivax) in vitro, by Bass’s method. With 1 plate. Ann. Trop. Med. and Parasitol., Vol. VII, No. 1, March, pp. 153-165. Tuomson, J. G., and Sinton, A. (1912). The Morphology of Trypanosoma gambiense and Trypanosoma rhodestense in Cultures : and a Comparison with the Developmental Forms. described in Glossina palpalis. Ann. Trop. Med. and Parasitol., Vol. VI, pp. 331-356 Three Plates. : 522 EXPLANATION \OF PLATES PLATE XXXIV All the figures in this plate represent the growth of the parasites in the culture tube. The figures were drawn with an Abbé camera lucida from stained preparations. Magnification 1,600 diameters. Piet Pigs Biss 3 Figs. 4 Figs. 6 Figs. 8, Pig, 11 Fig. 12 Pion ai Fig. 14 Fig. 15 is a malignant tertian parasite at the time of inoculation of the culture tube. Corpuscle is shrunken. No pigment is seen and no stipulation of the corpuscle. represents 12 hours’ growth at 36°C. No pigment is yet visible. represents 23 hours’ growth at 41°C. Pigment is not yet evident, but the parasite has increased in size. and 5 represent 30 hours’ growth; note the appearance of a compact mass of pigment. Fig. 5 is a double parasite. and 7 show commencing segmentation after 47 hours’ incubation. Fig. 6 shows five daughter cells, and fig. 7 twenty spores. g and 10 show sporulating forms after 51 hours’ incubation. shows complete sporulation (32 spores) after 52 hours’ incubation. |The containing corpuscle has burst and liberated the spores. was obtained after 56 hours’ incubation. represents a young merozoite of the second generation which has entered a new corpuscle after 75 hours’ incubation. Note that there is no vacuole. The parasite is only 1°5m in its longest diameter. represents sporulation of the second generation after 3 days’ incubation. shows a young merozoite of the third generation after 44 days’ incubation. PS eee 523 Fig. 16 represents sporulation in the third generation after 6 days’ incubation. Only eight spores have formed. Fig. 17 is a young merozoite of the fourth generation after 7 days’ incubation. Fig. 18. Eight days’ incubation, showing commencing segmenta- tion in the fourth generation. Fig. 19. Nine days’ incubation, showing sporulation in the fourth generation. Fig. 20 shows a young merozoite of the fifth generation after 10 days’ incubation. All of the above figures were obtained from one culture tube, without the removal of leucocytes and without the addition of fresh serum or corpuscles. Fig. 21 shows a young benign tertian parasite at the time of inoculation of the culture tube. The culture was made at the period when these young rings predominated. Note the large size of the corpuscle and the Schiiffner’s dots. Figs. 22, 23 and 24 represent 8 hours’ growth in culture tube at 39° C. The parasites have now distinctly increased in size, and fine granules of pigment are now seen scattered in the protoplasm. Figs. 25-30 show parasites obtained from the culture tube after 20 to 29 hours’ incubation. Fig. 25 shows four masses of chromatin. Fig. 30 is a female gamete (undivided chromatin and _ scattered pigment) found in the culture tube; similar gametes were found in the blood at the time of inoculation of cultures, so it is probable that this gamete was one of these and that it had not developed in the culture. Note the large size of the corpuscles in the benign tertian as compared with those in the malignant tertian; also the Schiiffner’s dots and the scattered pigment. The spores also are larger and fewer in number in the case of the malignant tertian parasites. 524 PLATE XXXV Microphotographs of malignant tertian parasite. Figs. 1 to 3 are from the same culture of Plasmodium falciparum, and are all photographed at the same magnification, namely, 1,500 diameters. A photograph of the malignant tertian parasite as seen in the peripheral blood before incubation. No pigment is seen. The small dark mass is the chromatin. The malignant tertian parasite after 12 hours’ incubation at a temperature of 37°C. to 38°C. The parasites have grown to at least twice the size. A circular mass of compact pigment is now evident. This photograph illustrates the clumping tendency and seven parasites are seen grouped together. No division into daughter cells has yet taken place. The dark circular area is pigment, and the darker area, towards the margins, is due to the chromatin. The malignant tertian parasite after 25 hours’ incubation at a temparature of 37°C. to 38°C. This photograph shows well the tendency to clump and the parasites are now seen at all stages of sporulation. Shows two parasites from a smear of the spleen showing segmentation. Thirty-two spores can be counted in the one in the centre of the field. This is a photograph taken from a coloured plate drawn by Dr. Cropper and shows the clumping of the red cells containing pre-segmenting forms of the malignant tertian parasites. This drawing was made from a smear of the peripheral blood (vzde ‘ Lancet,’ July 4th, 1908, and coloured plate fig. 1). (Magnification about 1,000 diameters). THE TRICHONOCARDIASES BY ALBERT: J CHALMERS M.D. Me R.C,S. PH. DIRECTOR, WELLCOME TROPICAL RESEARCH LABORATORIES, AND CAPTAIN W. R. O’PFARRELL, R.A.M.C., PROTOZOOLOGIST, WELLCOME TROPICAL RESEARCH LABORATORIES, KHARTOUM (Recewved for publication 27 September, 1913) PLATES XXXVI—XXXVII INTRODUCTORY The literature relating to the Trichonocardiases being of such recent date and of such small bulk, together with the fact that this is the first record of their occurrence in the Anglo-Egyptian Sudan, and, as far as we know, in Africa, has induced us to bring forward the following observations. HISTORICAL A number of affections of hairs have been described which are characterized by the presence of variously coloured nodes, nodosities and more or less elongated unilateral, bilateral or annular thickenings on hairs in various parts of the body. The nodes are confined to the shaft, and do not penetrate into the hair follicle. These affections may be classified as the Trichosporoses, Lepothrix and the Trichonocardiases. Trichosporosis was first described as a rare disease of the hair of the head in native women living in the valleys of the province of Cauca on the slopes of the Eastern Cordilleras of Colombia. It has been observed in Rio de Janeiro and Germany. It also occurs on the hair of the beard in men. Osorio gave it the name ‘ Piedra,’ because of the stony hardness of the nodules, but the more correct name is Trichosporosis tropica, as its causal organism is T77icho- sporum giganteum, Behrend, 1890. A similar condition has been 526 briefly described by Castellani as occurring in India and Ceylon, but so far, he has not named the fungus, which appears to differ from 7. gzganteum. The remaining Trichosporoses belong to temperate climates ; they are :— Biegel’s Trichosporosis, or Tinea nodosa of Cheadle and Morris, due to Trichosporum beigeli (Rabenhorst, 1867). It was first found by Lindemann in 1865, and is often called the Chignon fungus. It occurs on the hair of the head. Unna’s Trichosporosis, or Piedra nostras, found on the hairs of the beard and moustache, and due to 7. ovale (Unna, 1896). Behrend’s Trichosporosis, or Nodular trichomycosis, found on the hairs of the beard, and due to 7. ovoides (Behrend, 1890). Du Bois’ Trichosporosis, found on the hairs of the pubic region, and due to 7. glycophile (Du Bois, 1910). Another nodular hair disease is Lepothrix, or scaly hair, which was discovered by Paxton in 1869, was named by Wilson, and described by Eisner and Crocker. It is characterized by variously coloured grey, yellow, or brown nodules, which surround the hairs of persons who perspire profusely. The hairs most usually attacked are those of the axillae, but the disease may also be found on those of the chest, pubes, and inside of the thighs. It is common in Germany, and is believed to be due to a diplococcus forming zoogloea masses. Allied to Lepothrix is the affection called Trichomycosis capillitii by Winternitz in 1903, which is said to be due to a straight or slightly bent bacillus. The descriptions of these conditions suggest that there may be some confusion, and that in reality they may be Trichonocardiases in which either only the diplococcus has been observed or in which only the bacilliform fungus has been seen. In any case it would seem that the disease Lepothrix requires further investigation, especially as Crocker’s excellent description closely agrees with Trichonocardiasis. The Trichonocardiases are allied in appearance to Lepothrix, but differ therefrom in the fact that they are all due to a fungus Nocardia tenuis (Castellani, 1911), either alone as in Trichono- cardiasis flava (Pl. XXXVI, figs. 1 and 2; Pl. XXXVII, fig. 13), or in association with a Micrococcus as in Trichonocardiasis nigra (Pl. XXXVI, figs. 4 and 5; Pl. XXXVII, figs. 14, 15 and 20), and §27 T. rubra (Pl. XXXVI, figs. 6 and 7; Pl. XXXVII, figs. 16, 19 and 22). They were first described by Castellani in IQII in a paper published in the British Journal of Dermatology, and this description was extended by the same author in 1912 in an article published in the Transactions of the Royal Society of Medicine, and still later, in 1913, in the second edition of the Manual of Tropical Medicine published by himself and one of us. He named the disease Trichomycosis axillaris, and sub-divided it into three varieties, flava, rubra and nigra; but as it occurs on the hairs of the pubes, in some cases without any axillary infection, we are of the opinion that Trichonocardiasis is a better term. Attention has, very briefly, been drawn to Castellani’s work by Jackson and McMurty in their book on ‘Diseases of the Hair,’ published in 1913, where they devote a small section to the subject under the title ‘A Disease resembling Lepothrix.’ So far as we know, the above comprise the total literature on the subject of the Trichonocardiases. GEOGRAPHICAL DISTRIBUTION We have found the three varieties of Trichonocardiasis in the Anglo-Egyptian Sudan, and one of us has observed a condition resembling Trichonocardiasis flava in Europeans on the Gold Coast, West Africa, as long ago as 1808. As the three varieties may be found on one and the same person, and, indeed, in the same axilla, we do not propose to give separate descriptions of each variety, but to describe the disease Trichono- cardiasis as a whole, mentioning the difference between the three varieties when necessary. At present the Trichonocardiases are only known to exist in the Tropics, 1.e., in Ceylon, the Anglo-Egyptian Sudan, and on the Gold Coast. These regions being so widely separate one from the other, it is reasonable to suppose that the disease will be found in other tropical regions when looked for. Castellani has drawn special attention to the fact that he found his cases in the hot, damp districts of Ceylon, and adds that it will probably be found occurring in every low-lying tropical region. While our Gold Coast cases support Castellani’s views, the cases we 528 have found in Khartoum demonstrate that the disease may also occur in a hot and dry climate and at a certain elevation. We found our first cases here in the month of May of this year, when the highest shade temperature was 112°2°F. (44°5°C.), and the mean shade temperature was 105°9°F. (41°C.). During this time the least percentage humidity of the air was 4% and the average 21%, while the evaporation from Piche’s tube rose as high as 30°1 milli- metres. As the elevation of Khartoum is 390 metres above sea-level at Alexandria, it is not low-lying. We, however, agree with Castellani that the disease will probably be found in many tropical regions, low-lying or elevated, if looked for. RACIAL DISTRIBUTION Most of our cases have occurred in Europeans, but Castellani has seen it among the Sinhalese and we have met with it in a negro. Possibly its occurrence was noticed long ago by native doctors, and may be one reason why so many native races shave the axillae. BODY DISTRIBUTION So far we have only seen the infection on the hairs of the axillae, pubic regions, and folds of the groin and in this we are entirely in agreement with Castellani. The reason for the distribution appears to us to be the nutrition of the causal fungus Nocardia tenuis, as it would appear to require a mixture of human proteid material such as is found in blood serum and normal saline before it will sprout. As we shall point out later, it derives its nutriment from the lymph of the cortex of the hair while at the same time it is bathed by the saline sweat of the axillae, pubic regions, and groins, and this appears to be the reason of its affinity for these particular parts of the body. AETIOLOGY The causal agent 1s Nocardia tenuis (Castellani, 1911) (PI. XXXVI, fig.g; Pl. XXXVII, fig. 17), either alone as in the variety Trichonocardiasis flava, or associated with Mzcrococcus nigrescens (Castellani, 1911) as in Trichonocardiasis nigra, or with Micrococcus castellanu (Chalmers and O'Farrell, 1913) (Pl. XXXVII, fig. 18) as in Trichonocardiasis rubra. We will now consider these aetiological factors seriatim. 529 NOCARDIA TENUIS (Castellani, 1911). This organism can be readily seen in cleared specimens of the infected hairs mounted in gelatin or Canada balsam as minute rod-like or bacillary forms often lying more or less parallel to one another and embedded in an amorphous ground substance (Pl. XXXVII, fig. 22), probably excreted by the fungus. This ground substance, as wiJl be explained later, is fixed to the cortex of the hair, and lies under the cuticle and some of the superficial layers of the cortex. When individual hyphae are examined after coloration by any of the ordinary stains, they are seen to be narrow, elongated, unbranched, non-septate rods resembling bacilli (Pl. XXXVI, fig.g; Pl. XXXVII, fig. 17). They may be short or long, straight or slightly curved, but they are always fairly and usually very narrow. A series of measurements gives the average length as varying from about 2°0 to over 7’0m, and the average breadth from 014 to O34. Each hypha is enclosed in a cell wall, inside which lies the cytoplasm, which may stain very lightly with the colouring agent used, but may also stain fairly darkly, especially in certain areas. No nucleus can be demonstrated and the cytoplasm appears to be homogeneous except at certain places where well-defined deeply staining rounded granules may be seen (Pl. XXXVI, fig. 9). These granules, which measure about 0°14 to 0°3m in transverse diameter, may be situated at one end of the hypha, as in Pl. XXXVI, figs. 9, 10, 12, and Pl. XXXVII, fig. 21, or this may be placed at regular intervals through the cytoplasm (Pl. XXXVI, fig. g), or one may be at one end while others may be close together near the opposite pole. We have not met with any branched forms, except in cultivation, but we have seen forms with one end rather bulbous, resembling in miniature the club forms of some other Nocardias. We have not succeeded in cultivating this fungus permanently, in bulk, or pure cultivation, but we have succeeded in inducing growth by placing scrapings from the hairs in equal parts of human blood serum and 0°85% normal saline solution in hanging drop prepara- tions. These were first kept for twenty-four hours at the room temperature, which varied from go° F. (32°2°C.) at night to about 105° F. (40°6° C.) in the day, and were then placed in an incubator at ro," Fy (40°C): 530 Under these conditions the fungus quickly grew out of its amorphous bed (Pl. XXXVI, fig. 10) into the surrounding liquid, and proceeded to develop in the manner depicted in Pl. XXXVI, fig “fey"and” Pi RKAVAL fig. 21, the latter of which isa microphotograph. It will be noticed that the branching is monopodial, and that the hyphae are non-septate. With regard to its position in the vegetable kingdom it is obvious that, as it possesses neither chlorophyll nor chromatophores, it must belong to the class Fungi, and that it belongs to the fungal order Deuteromycetes or Fungi imperfecti, because its life-cycle is unknown. Further, it can be placed in the sub-order Hyphomycetes because perithecia, asci and immersed stroma are also unknown. Of the families of the Hyphomycetes it belongs to that of the Mucedineae because its hyphae are pallid and do not cohere to form a compound stem-like structure. Lastly, it may be grouped with other forms in the genus Nocardza (Toni and Trevisan, 1899), which is defined as consisting of mycelial filaments of various sizes, but generally very thin (one micron or less in diameter), often branching, non-septate, and without differentiated nuclei. Nocardia tenuzs (Castellani, 1911) may therefore be defined as follows :— Nocardia composed of thin, bacillary-like hyphae, varying from about 2 to over 7# in length, and from o'14 to o°3 in breadth, embedded in an amorphous ground substance attached to the cortex of the axillary and pubic hairs of man in tropical countries. MICROCOCCUS NIGRESCENS (Castellani, 1911). This coccus has been grown in pure culture by Castellani, who has described its biological characters on page 1521, and has illustrated its cultural appearances in fig. 8 on Plate V of the second edition of the Manual of Tropical Medicine mentioned above. It is there- fore unnecessary for us to go into details with regard to this organism, except to say that Trichonocardiasis nigra appears to be much rarer here than the flava or rubra, while the pigmentation on the hairs and in the growths we have obtained is relatively scanty, and we have found it difficult to isolate and grow Micrococcus castellani (Chalmers and O'Farrell, 1913). Castellani in Ceylon found that it was more difficult to isolate and grow this coccus than was the case with Micrococcus nigrescens. On the other hand, we, 531 in Khartoum, have found quite the reverse: we have found it exceedingly difficult to obtain the black coccus with anything beyond a trace of black pigment, while the red coccus grows easily and well. It is very readily separated out by sowing scrapings from the hair, or pieces of the hair on sloped agar, and picking out either the red colonies which require some time to develop the reddish pigment, or the yellow colonies which always appear first and in which the red pigment subsequently shows itself. MICROCOCCUS CASTELLANII is a round or oval coccus, measuring from about 0°3 to about 07m in diameter (P]. XXXVI, fig. 18). It is divided medianly by a clear central line into two half-moon shaped segments, thus producing the well-known diplococcal appearance. The cocci, which may occur singly or in groups, are colourless and non-motile, but excrete an amorphous non-granular lemon chrome-coloured pigment (classified according to Ridgway’s Colour Standards), which lies between the individual coccl. In old cultures another pigment of dark brownish-red colour appears (according to Ridgway’s Standards this colour is madder- brown), but its relationship to the earlier yellow pigment can easily be proved by removing some of it and suspending in a sufficiency of distilled water, when the fluid at once resembles a similar suspension of the yellow pigment. If, however, this is done in a very small quantity of water, a faint reddish tinge can be observed. The best medium for showing the striking yellow pigment is the potato, when in twenty-four hours the growth assumes the appearances depicted on Pl. XXXVI, fig. 11. In order to demonstrate the red pigment it is necessary to cultivate the microccocus for some time. The medium on which this pigment. shows best is the ordinary agar slope, which, when old, exhibits the dark-red pigment in the centre and the yellow pigment at the sides (Pl. XXXVI, fig. 8). The yellow pigment is seen both in aérobic and anaérobic growths. In broth and peptone water or liquid blood serum, whether grown aérobically or anaérobically, the pigment is not visible until a very abundant growth appears, when the yellow colour can be seen. The coccus colours with all the ordinary staining re-agents, and as a rule is decidedly Gram positive. It must, however, be admitted that even in preparations showing most of the cocci well coloured 532 when treated by Gram’s methods, a few may be seen quite decolourised, and we have also seen cocci with one demi-lunar segment well stained while the other was completely decolourised. We have failed to demonstrate a capsule around the coccus. The organism grows aérobically and also anaérobically. The optimum temperature appears to be 37°C., but it can also be cultivated at the room temperature, which in Khartoum at this season of the year is seldom less than 32°2°C. It also grows at 20° C. on agar slopes, but not as abundantly as at 37°C., and 1s therefore less pigmented. Its rate of growth depends somewhat upon the medium, as it grows quickest and best on potato, and next best on ordinary or glycerin agar. On solid media it gives rise to a yellow growth at first; but on most agar media, if kept long enough, some red coloration will subsequently be found. The best medium for the red coloration appears to be the ordinary agar slope. With regard to the other agar media, it grows well on glucose and maltose agar. Like Micrococcus nigrescens, it produces neither acid nor gas in the following media : — Peptone-sugars: Glucose, Laevulose, Galactose, Arabinose, Maltose, Lactose, Saccharose, and Raffinose. Peptone-carbohydrates: Dextrin and Inulin. Peptone-glucostide: Amygdalin. Peptone-alcohols: Erythrite, Adonite, Dulcite, Isodulcite, Mannite, Sorbite, and Inosite. It grows slowly on blood serum, which it does not liquefy, and well in broth and peptone water, in which it forms a general turbidity. It does not produce indol. In agar stabs the growth is confined to the line of puncture and to the formation of a small yellow knob on the surface, in other words, the growth is filiform. With regard to its systematic position in the family Coccaceae (Zopf, emended Migula), it must certainly be grouped with the genus Mzcrococcus (Hallier, 1866, emended Cohn, 1872). In this genus it certainly belongs to those forms which grow well on agar media and are Gram positive, and in this division it belongs to the sub-division which produces colours. The cocci of this sub-division which possess red coloration are 533 only three in number, Mzcrococcus rvoseus (Bumm, 1885), Mzcrococcus ruber (Trommsdorff, 1904), and Micrococcus rubzdus (Hefferan). Under the term M/. voseus (Bumm, emended Lehmann and Neumann) are gathered a large number of rose coloured diplococci which are not known to be parasitic, and which produce growths on potato which are limited to the streaks. These growths are faint rose red in colour within an oily lustre, and are often surrounded by a whitish glistening zone, thus giving rise to a very different appearance from that produced by M. castellanw. Micrococcus ruber (Tromsdorff, 1904) or, as it is sometimes named, M. chro- midrogenus ruber, which was isolated from axillary hairs, 1s characterised by the fact that it does not grow on potato, its colouring matter 1s not soluble in water, and when treated by sulphuric acid the red colour turns blue-green, while M/. castellanz does grow on potato and its colouring matter is unaffected by 25 per cent. sulphuric acid. Moreover, /. vuber is associated with chromidrosis, while M@. castellaniz is not. With regard to Mzcro- coccus rubidus, it is impossible to obtain any information as to its characters in Khartoum, but it appears to be closely related to M. roseus var carneus, and to be non-parasitic. We, therefore, are of the opinion that the organism which we are describing is not the same as any species of Micrococcus so far named, and therefore give it the appellation Micrococcus castellaniz, Chalmers and O’Farrell, 1913. METHOD OF INFECTION There is no doubt in our minds that the usual method of infection is from man to man, and we base this opinion upon the following observation : — A young European, known to be quite uninfected with these parasites, became greatly interested in our work; he examined the cases which came to the laboratory, and handled and examined the fresh hairs removed from the axillae. When he visited the laboratory he was clad in tennis attire, and as he moved about, his shirt rucked up from below repeatedly, and whenever this happened he adjusted it by pulling forward his leather belt with one hand while he pushed the soft shirt downwards inside his trousers with the other. Two weeks after examining the 534 cases he first noticed a reddish appearance on the hairs of the pubes. On examination it was found that the hairs were infected with the red variety of Trichonocardiasis, that is to say, with the variety which he saw and handled in abundance. It grew rapidly on the pubic hairs, but did not extend to the axillae. Moreover, it only grew in the fold of the left groin, where sweat is apt to accumulate. It did not spread to the right groin. INCUBATION PERIOD It would therefore appear as though two weeks were required to elapse before an infection produces sufficient growth on the hairs to attract attention, even when it is being looked for. PATHOLOGY The pathology of the infection appears to be quite simple. The fungus arriving upon the hair grows and pushes its way at first under a cuticular scale and then quickly works its passage into the cortex, raising, in so doing, its superficial fibres, which, together with the cuticular scale, form a covering or pro- tection for the fungus (Pl. XX XVII, fig. 19), which probably finds the nutriment it requires in the fluids of the cortex of the hair. Once established in position the fungus does not penetrate deeper into the cortex, but, on the contrary, grows outwards, forming the nodosities, rings and sheaths to the hair which have already been mentioned. It is owing to this method of growth that the hair is so little affected, as the deeper layers of the cortex, the medulla and the root of the hair are not involved in our cases. MICROSCOPICAL ANATOMY In an early stage of the infection the cuticle and some fibres of the cortex (Pl. XX XVII, fig. 13, shows this stage at three places between the large clumps of fungus) may be seen to be raised in ridges, which run in wavy lines across the shaft of the hair transversely to its long axis. Under these ridges masses of the fungus may be seen growing. A parasitic mass 1s composed of a firm homogeneous ground substance, in which the rod-like hyphae of the Nocardia may be seen 535 (Pl. XXXVII, fig. 22). In this the cocci are also found which are responsible for the coloration of the black and red Trichono- cardiases. SYMPTOMATOLOGY As already stated the shortest incubation period with which we are acquainted is two weeks. The infection causes no general symptoms and is only discovered by accident by the patient, who then seeks advice in order to know what is taking place. Very often the infection is first met with while the patient is being examined by his medical attendant for some other disease. In regiments it is usually found during a general medical inspection. The regions of the body which are affected are the axillae and the pubes, most commonly the former alone or in conjunction with the latter, and less commonly only the latter. The hairs of these regions may be affected with one or more of the three varieties already mentioned. On examining with the naked eye an axilla affected by the yellow variety, it will be noticed that the hairs are covered with a thick or thin yellow deposit (Pl. XXXVI, fig. 1), as though the natural grease had accumulated, and, indeed, this is the reason why some persons ask about the complaint, as they consider that it reflects upon their habits. If the black variety is present it will appear as black or very dark patches, short or long, thick or thin, on one side of or ensheathing the hair (Pl. XXXVI, fig. 5), and the same description holds good for the red variety (Pl. XXXVI, fig. 7), with the exception of the difference in colour. These observations are easily confirmed by microscopical examination, when the masses may be seen encircling the hair, lying on both sides or merely forming excrescences on one side of the shaft. The cuticle and fibres of the cortex may be seen covering the small masses or embedded in the large ones. As already stated, the disease is confined to the shaft of the hair, the health of which it does not affect. The infection of the hair does not appear in any way associated with excessive sweating, though, of course, the amount of this excretion produced by the axillae is considerably greater in the tropics than in the temperate zone, and, therefore, in warm climates the axillae are generally 536 damp and the pubic regions are also damper than usual. Probably it is the dampness of these areas of the body which determines the distribution of the infection, as it has so far never been seen on the hairs of the head, face, arms and legs. The infection dies away on return to a temperate climate, especially in winter. DIAGNOSIS The appearance of minute nodules, rings or sheaths of a yellow, black or red colour on the hairs of the axillae or pubes indicates the presence of either Lepothrix or Trichonocardiasis. The latter may be differentiated from the former by demon- strating the presence of Nocardia tenuis, Castellani, 1911, which is absent in Lepothrix. From Trichosporosis tropica or Piedra it may be distinguished by the fact that it does not attack the hair of the head or face, and also by the difference in the causal parasite. From the other Trichosporoses it may be separated by its distribution and by the presence of Nocardia tenuis, as well as by the absence of a Trichosporum. PROGNOSIS It appears to have a marked tendency to remain in the region or regions which it first invaded, and does not, as far as our observations go, tend to spread over the body. TREATMENT We have found the treatment recommended by Castellani to be quite useful, viz., a lotion of one drachm of formalin to one ounce of water applied twice daily to the affected hairs, while a 2% sulphur ointment is used at night. PROPHYLAXIS The infection appears to be only mildly contagious; still it does tend to spread in communities. Cases which are likely to be sources of infection should have their clothes disinfected before being washed. During the infection the underclothing should be dusted with some antiseptic powder, e.g., dermatol, one drachm in one ounce of venetian talc, or with a boracic acid dusting powder. 537 ACKNOWLEDGMENTS We wish to acknowledge with many thanks the kindness received from Major Forrest, R.A.M.C., during the preparation of this paper, and the skill with which our artist, Mr. A. Marshall, Senior Laboratory Assistant in these laboratories, has prepared the illustrations. KHARTOUM, September 15th, 1913. REFERENCES (Arranged in chronological sequence.) CasTELLANI (1911). British Journal of Dermatology, XXIil, p. 341. CasTELLANI (1912). Transactions of the Royal Society of Medicine, VI, No. 2, Dec., p. 23. CasTELLANI, and Cuatmers (1913). Manual of Tropical Medicine, 2nd ed., pp. 820 and 1521, Plate V, fig. 8. Jackson and McMurry (1913). Diseases of the Hair, p. 294. 538 EXPLANATION OF PLATES. PLATE XXXVI Fig. 1. Trichonocardiasis flava. Natural size. Fig. Trichonocardiasis flava. ~ 60. NO Fig. 3. Trichonocardiasis flava. x 40. Fig. 4. Trichonocardiasis nigra. x 60. Fig. 5. Trichonocardiasis nigra. Natural size. Fig. 6. Trichonocardiasis rubra. x 20. Fig. 7. Trichonocardiasis rubra. Natural size. Fig. 8. Micrococcus castellanu,;, old culture on an agar slope. Natural size. Fig. 9. Nocardia tenuis; separate hyphae. x 1,200. Fig. 10. Nocardia tenuis sprouting out from the ground-substance ; hanging drop preparation. x 1,500. Fig. 11. Micrococcus castellaniz; 24 hours’ growth on potato. Natural size. Fig. 12. Nocardia tenuis; growing in hanging drop preparation. x 1,500. yg? ‘ “onan p Velo) eryrte Fo come 4 = - oa) ou mboanedain 1 soil te, a ie aired ; Ts wy 4, 7 who ig my coe. ayeh di#bibasonodsinT =i = vo ‘100: ay gift eee iDeetonaitacrt a ry - a sa as : ® - ia pit Giibieonotois j : 7 ‘° “- Sadi skbmssrodanTe } ‘ ¢ (ar 70) [ite ae Canes sain A “a LT Vis by Avy: ei oa Gy ys Mi _ e10l Misa PO bik Aainilen) O08, | a Ar ae be 10 s in ’ hi - b. a 7 , Lt Stoitys ati wieysia al Hwo ny leona set pelwotle tH 2 ee od! doles a istoiisgae Dalles | m4! 2TGUT peut ecbarasn! ie mcmik 3 mK] peat, cars at ys wore YRS. A 50 Vi bias ae Mas hi prt sala: OLS i pve te ieee Ol a" ond piss diy Cat sis #46) colerigagq feo, pit is & aedqy:! gnisibey ot) guiwore srl sleuily iy as nt onliqual 547 Vo oie leony sly das iatiqnag A, ee . Je; r : (OG5 @ — .oTtH52 7 ms. our ae ts) Poe t — " Batak, Megs wit a" Wee ae - waebjtte, 3 apis, mind rue Fig. Fig. Fig. Fig. PANG 22. 540 PLATE XXXVII Microphotographs of the Trichonocardiases. Trichonocardiasis flava. x 60. Trichonocardiasis nigra. x 60. Trichonocardiasis nigra. x 60. Trichonocardiasis rubra. x 60. Nocardia tenuis, Castellani, 1911. x 1,200. Micrococcus castellantzz, Chalmers and O’Farrell, 1913. x 1,500. Hair showing the fungal growth elevating the cuticle and the superficial fibres of the cortex. x 500. Trichonocardiasis nigra. x 60. Nocardia tenuis growing in a hanging drop preparation. x 1,200; Fresh preparation of a hair affected with Trichono- cardiasis rubra, showing the radiating hyphae at the periphery and the optical section of the hyphae in the centre. x 700. 541 EPIDEMIC TRICHONOCARDIASIS BY ALLBEI. “|. “CHALME Ret. MeD 2, Tek-© S49, D:P> Hi: DIRECTOR, THE WELLCOME TROPICAL RESEARCH LABORATORIES AND CaPTAIN A. D. STIRLING, R.A.M.C. SENIOR MEDICAL OFFICER, BRITISH TROOPS, KHARTOUM (Recezved for publication 25 November, 1913) INTRODUCTORY The literature connected with Trichonocardiasis being new and of small extent, it may be of interest to record an epidemic which recently took place in the Welsh Regiment in Khartoum. Before commencing the account of the epidemic we may mention that we have heard of a case of Trichonocardiasis rubra in a European while in Aden, but whether it originated there or was merely imported we are not in a position to state. It would, however, appear that the Trichonocardiases are widely distributed throughout the tropics and, further, that they may occur in epidemics. Full accounts of the history, aetiology, etc., having been already given during this year by Castellani, O’Farrell and one of us, we restrict our remarks entirely to the present epidemic, its probable origin and its suppression. THE EPIDEMIC About the middle of September, 1913, a Private in the Welsh Regiment, which had been stationed in Khartoum for some months, applied for medical treatment on account of irritation of the skin in both axillae. On investigation it was found that the hairs of these regions were heavily infected with Trichonocardiasis rubra. Figure I is a photograph of one of his axillae and shows the remarkably heavy infection of the 542 hairs which were matted together. The skin surrounding this mass of hairs was red, congested, and very irritable. The individual hairs, separated out and photographed, are depicted, natural size, in Figure II, which is improved if examined by a lens. As, however, this affection of the hair has been recently described by Castellani and by one of us in conjunction with Captain O’Farrell, R.A.M.C., no further remarks will be made as to the clinical appearances or the microscopical findings, except to state that they entirely agree with those already reported. | ai) Fic. i.— Photograph of an axilla, showing Fic. II.—Photograph of hairs from the axilla infection with Trichonocardiasis rubra. depicted in Fig. I, natural size. This Note the thickenings and the matting figure is improved if examined by means of of the hairs. a lens. The occurrence of this case led to a systematic investigation of the regiment, when no less than 42 cases, in all, were discovered. Some of the men complained of slight irritation in the axillae, and these men invariably had an area of erythema surrounding the hairs of the axilla, but the majority of men had felt no symptoms and did not appear to have noticed the altered colour of the hair. None of the cases showed any affection of the hairs on the pubis or any other part of the body. The most common variety was Trichonocardiasis flava, while a 543 number of cases showed Trichonocardiasis rubra, but no Trichono- cardiasis nigra could be found. The erythema noted was only seen in men suffering from Trichonocardiasis rubra. A thorough investigation was made as to the possible source of infection, and very early suspicion was aroused that the washer- men might possibly be the spreaders. There were, in all, five washermen and four washerwomen for the cleansing of the regimental clothing. All these, save one, worked in the central washhouse and these were found to be free from Trichonocardiasis. The remaining washerman, who was occasionally assisted by one of the other washermen, went from bungalow to bungalow in the barracks for the purpose of doing extra washing for the men, as the regulation washing was insufficient to keep the clothing clean in a hot dusty country like the Anglo-Egyptian Sudan. This man washed, in particular, the shirts and the khaki trousers for the men. He was found to be infected with Trichonocardiasis flava and rubra, and he was strongly suspected to be the spreader of the infection, as it was not confined to the men of any one company but scattered through all the companies of the regiment. This washerman and the infected men were duly treated, with the result that during the last inspection, made a few days ago, no new cases were found. TREATMENT As so many men were affected it afforded ample opportunity for observing the results of various forms of anti-parasitic treatment. Most of the cases were treated by means of a lotion of formalin 40 per cent., one drachm to six ounces of rectified spirit. This lotion was carefully applied to the hair twice daily. It was advisable to restrict this application entirely to the hairs as it was often found to irritate the skin if it was carelessly rubbed into the axilla. At might a 2 per cent. sulphur ointment was applied, thus completing the routine recommended by Castellani. The result was excellent, but it required time, and, on an average, a cure was not effected under three to four weeks. 544 If the cases did not improve under this treatment, application of tincture of iodine effected a cure in three to four days. Tincture of iodine alone was also applied, but with doubtful advantage. Perchloride of mercury was also tried, but it required three to four weeks to bring about a cure. The best remedy in our hands appeared to be a combination of the formalin-sulphur method for about two days, followed by tincture of iodine, when a cure was effected in several cases in less than a week. ; With regard to the erythematous areas mentioned above, these were readily healed by the application of Calamine lotion, associated with the anti-parasitic treatment to the hairs, as already described. In addition, all the underclothing was dusted regularly with a boric acid powder of the strength of one drachm to the ounce of starch powder. This is not the best powder for the purpose, but it is the cheapest and is readily available. The use of dermatol, europhen or of xeroform with Venetian talc would be better, but the boric acid served its purpose quite well. KHARTOUM, November 12th, 1913. REFERENCES CasreLLani and Cuarmers (1913)" Manual of Tropical Medicine, second edition, p. 1525. Cuatmers and O’Farrect (1913). The Trichonocardiases. Annals of Tropical Medicine and Parasitology, Vol. VII, pp. 525-540. 545 BERBOLCA RN sec PION. WIT EL SPEGIAL)\REFRRENCE (fOr TLS OCCURRENCE: IN™ AYALOMMA MEG) RLLOM AEE CEE DW. 11 Et CRITHIDIA HYALOMMAE BY Cxrprarn W. RL O FARRELL, BR-AM-C: PROTOZOOLOGIST TO THE WELLCOME TROPICAL RESEARCH LABORATORIES, KHARTOUM (Recezved for publication 8 October, 1913) PLATES XXXVIII—XL INTRODUCTION Our knowledge of the réle played by the blood-sucking parasites as transmitters of human and animal diseases has consider- ably advanced during recent years. Consequently, the study of these vehicles of transmission has increased, and many cases of hyper-parasitism have been discovered. During the search for the © pathogenic varieties others have been encountered, and these, though their pathogenicity be confined only to their insect hosts, must not be overlooked. In three ways, a comprehensive study of their hfe cycles is of value: Firstly, in order to prevent their stages from being confused with the stages in the life histories of more important parasites. Secondly, to act as a guide to the possible behaviour of the disease- producing varieties, and thirdly, as a weapon of offence against their parasitic hosts. Probably the most important stage in the life cycle of all hyper- parasites is the hereditary infection stage. Hereditary infection does not deal with individuals but with generations, and _ this method of infection 1s a higher grade of organisation and of evolu- tion than simpler methods of infection. The complete life cycles of but few hereditary infections are known, though a considerable number of hereditary infection-transmissions are known to exist. 546 Consequently any addition to our knowledge of such infections is of value. Moreover, any example that can be studied with any degree of facility is worthy of note. The hereditary infection of Hyalomma aegyplium by Crithidia hyalommae is one of these, and will be described in the following pages. HISTORICAL The occurrence of hereditary infection has, been suggested, observed and worked out in the insect flagellates already, and among the authors who advanced this line of research I have primarily to mention Miss A. Porter, whose work on Crethidza melophagia, Flu, 1908, has proved of valuable assistance. As early as 1903 Dr. L. W. Sambon put forward the hypothesis of the possibility of the occurrence of hereditary infection of the tsetse fly by Trypanosoma gambiense. P. C. Flu (1908) published an account of a flagellate parasitic in the ovaries and small larval forms of the sheep-ked, Melophagus ovinus, Linnaeus. L. D. Swingle (1909) working in Nebraska, described rounded and plasmodial stages of the same parasite, occurring in the egg of Melophagus ovinus. A. Porter (1910) described the intestinal contaminative method of infection and the full cycle and mode of hereditary infection of the same parasite in the host, egg, developing embryo, and fully hatched Melophagus. In a preliminary note published in August, 1913, I have given a brief description of the flagellate stage and discussed the move- ments of Crzthzdia hyalommae, O'Farrell, 1913. The movements will not therefore be further described, except the special movement connected with the entry of the flagellate into the ovarian system of the tick. The life cycle passed in the larva and nymph will form the subject of a future communication. THEORETICAL APPLICATIONS Apart from the practical proofs of the hereditary transmission of Crethidia hyalommae in Hyalomma aegyptium, certain theo- retical conclusions formed the basis of the search for such proofs. These conclusions were :— N Oo 547 The infection was not inoculative, i.e the vertebrate host, because :— ., derived from (az) All the ticks collected from one given cow were not infected. (6) Ticks other than Hyalomma aegyptium, although among batches of infected ticks, never showed infection. (c) The cows from which the infected ticks were collected were invariably healthy. The infection was not contaminative, because :— (a) Ticks, unlike other blood-sucking parasites gener- ally, do not move about much on their hosts (except the males), but settle in one place and slowly secure an attach- ment, where they remain till replete. (6) The natural secretions and excretions dry very rapidly, and are often voided on the earth after the tick detaches itself from its host. The infection was hereditary, because :— (a) The height of crithidial infection occurred about the time of oviposition. (6) The occurrence of the infection in batches of ticks all of which probably belong to the same generation. The practical application of these lines of thought worked out as follows :— lie NO The intestinal diverticula of the ticks were never found to harbour parasites. The haemocoelic fluid was the habitat of the early pre- flagellate forms. These early pre-flagellate forms developed into full grown Crithidia in the haemocoelic fluid. When these were present in large numbers they were found to migrate to the ovarian system. The hereditary infecting forms were found entering the ova. Plasmodial forms were found in the deposited eggs. MATERIAL AND METHODS Many specimens of Hyalomma aegyptium were examined from different localities, but always from cows, which are their common hosts. Some batches of ticks never showed infection, while in others infection was the rule. 548 To study the living organism, living specimens were examined with ordinary ight and with dark ground illumination, but with the latter the Crithidia were found to degenerate and die rapidly because of the strong light and heat produced by the source of illumination used in working with a paraboloid condenser. Vital staining was also employed. To obtain the haemocoelic fluid a portion of any of the legs is removed with a fine pair of scissors. For stained specimens of tissues all ticks were carefully dissected under a dissecting microscope, and the organs after carefully washing in saline solution, as recommended by Sir William Leishman, were smeared out on slides. Osmic acid and alcohol were employed as fixatives, and also special methods of wet fixation were used, such as Schaudinn’s fluid (corrosive-acetic-alcohol), and Bouin’s fluid (picro-formol- acetic-alcohol). Various stains were used—Leishman’s and Giemsa’s solutions gave good results. Bohmer’s haematoxylin (which in this climate is rather superior to Delafield’s haematoxylin) and Heidenhain’s iron haematoxylin and its modifications were employed. Cultivation of the flagellate has proved negative up to the present, together with attempts to inoculate animals. Frequent examinations and cultivation of the blood of the cows have given negative results. DISTRIBUTION OF THE PARASITE IN THE HOST The majority of insect flagellates are found parasitic in the alimentary tracts of their hosts, but in this respect the Crithidia of Hyalomma aegyptium is an exception. The haemocoelic fluid is the seat of election for the development and subsequent distribution of the parasite in the adult tick. Shortly after removal of the ticks from their hosts (cattle in this instance) the early pre-flagellate forms may be found in the haemocoelic fluid (Pl. XXXVIII, figs. 1-5), which is a dark amber colour, and is of about the consistency of human blood serum. Microscopically it is seen to contain among other elements the large vacuolated haemocoelic corpuscles. The nuclear and protoplasmic portions of these cells, when coloured with Leishman’s or Giemsa’s 549 solutions, show a tendency to stain, as do the nuclei, blepharoplasts and protoplasm of human and animal parasites. This point is of importance, as fragments of these cells might easily be mistaken at first sight for parasites. A few days after the ticks have been collected, the early pre- flagellate forms show evidences of division and increase in number (figs. 5, 6, 8). After about four to ten days, that is, a little time previous to oviposition, early flagellate forms make their appearance (figs. 11-18). Rapid multiplication of the young flagellates now takes place, with the formation of division rosettes (figs. 18 and 19), and the growth to adult Crithidia (Pl. XXXVIII, figs. 20-29; Pl. XXXIX, figs. 31-33) soon follows :— The fully-grown Crithidia multiply by longitudinal division (Pl. XXXIX, figs. 30, 34-39) till the smallest drop of haemocoelic fluid presents a felted mass of crithidial bodies and waving flagella. Owing to the great numbers of parasites present, the haemocoelic fluid is a milk-white colour. Towards the end of oviposition, and just previous to the death of the tick, post-flagellate forms may be found (figs. 40-44) in the haemocoelic fluid, although in some cases all forms disappear from it. The post-flagellate forms also occur in the genital tract, and are perhaps better termed ovarian forms: they will be dealt with under the description of hereditary infection. The intestinal diverticula and malpighian tubes have not been found to harbour any of the parasites in the adult tick. Sometimes early division forms (Pl. XL, fig. 62) are found in the salivary glands, but this seems to be the exception rather than the rule. The parasites were found more commonly in female than in male ticks. MORPHOLOGY The life cycle of Crithidia hyalommae in the adult tick may, for the sake of description, be divided into four periods which, however, merge into each other. These are the pre-flagellate, the flagellate, the post-flagellate, and ovarian stages. The morphology of the ovarian stage will be dealt with under a subsequent heading. The morphology of the pre-flagellate, the flagellate and _post- flagellate stages may be described as follows :— 550 THE PRE-FLAGELLATE STAGE The early pre-flagellate stages of C. hyalommae found in the haemocoelic fluid are rounded or oval Leishmania-like bodies (Pl. XXXVIII, figs. 1-5, 7, 8), which vary from 4 to 12y in length and from 4 to Irm in breadth. The protoplasm of these forms is of a finely reticulate nature, containing distinct vacuoles (fig. 4) and a rounded nucleus which is situate near the periphery of the parasite (figs. 1, 2). The blepharoplast is rounded or bow shaped - (figs. 1, 2), and acquires the characteristic dark tint of such structures when coloured by the various stains. It may be placed at the side of, or opposite to, the nucleus (figs. 3, 4). These early forms soon show evidences of division (fig. 5), two daughter parasites resulting, smaller in size and with more granular protoplasm than their precursors. Plasmodial forms (fig. 6) may result from rapid division. Increase in the size of the flagellate bodies follows and the flagellum makes its appearance, arising in the neighbourhood of the blepharoplast. The flagellum in its outward growth carries with it a portion of the periplast which forms the undulating membrane. After the formation of the flagellum rapid division takes place, rosettes being formed (figs. 18, 19), indeed nearly all stages from rounded flagellates to nearly adult Crithidia may be seen in the same specimen and in the same rosette (figs. 18, 19, 27). It may also be noted that individual members of a division rosette grow at unequal rates. The aflagellar end now grows outwards, at first wide and blunted (figs. 25, 26, 29), but it soon narrows down and tapers off into a fine point (figs. 27, 28). THE FLAGELLATE STAGE The fully-grown flagellates present the crithidial type of body, but are unusually long and slender (Pl. XXXIX, figs. 28a-36). The length of the body and flagellum varies from 26p to 50m, the length of the flagellum alone being 12°5¢. The average breadth of the nucleus varies from 2m to 2°5 pm. The aflagellar extremity tapers gradually to a fine point, while the flagellate end of the body is continued for some distance along the flagellum. 552 The flagellum, taking its origin from the vicinity of the blepharoplast, runs along the free border of the undulating membrane until the extremity of the body is reached. Here it escapes from the periplast and projects freely for a considerable distance. The flagellum is never connected with the blepharoplast. A basal granule has been noted (fig. 31), but its presence is not always evident, probably because of its close relation to the blepharoplast. The undulating membrane is well marked, and in living specimens its movements may be observed. In stained preparations it may be seen thrown into folds. The nucleus is roundish (figs. 28a-36) or oval, and generally lies about the middle of the body, the whole breadth of which it nearly occupies (figs. 28a-36). The structure of the nucleus varies. At times the nucleus appears somewhat vesicular with a marked karyosome (figs. 31-33); at other times the chromatin of the nucleus is distributed in granules within the nuclear membrane (fig. 30). When division is about to take place, the nuclear material is generally more granular (fig. 30). The blepharoplast of C. hyalommae is well defined in all stained specimens, and colours deeply. Its position varies within what might be termed the limits of crithidial definition (Patton, 1908). It may be closely applied to or a little distance from the flagellar or aflagellar side of the nucleus (figs. 28a-33). In all cases the flagellum arises in the close vicinity of the blepharoplast, in whatever position the latter may be situated. Two blepharoplasts may often be observed (figs. 30 and 35-38), but this is really an early division phase. The blepharoplast is usually rod-shaped (fig. 28a), and sometimes shows a diplosome appear- ance (fig. 34), probably indicative of approaching division. Chromatoid granules may be present in the protoplasm. In fresh specimens the protoplasm of the flagellar extremity of the body is markedly granular. THE POST-FLAGELLATE STAGE The post-flagellate forms seen in the haemocoelic fluid are merely rounding-up parasites (Pl. XX XIX, figs. 40-44). They are similar to the ovarian forms which undergo the same process in 552 the walls of the female generative tract, and which will be described when dealing with the hereditary infection. DIVISION In the pre-flagellate stages division is initiated by the blepharo- plast, which divides into two nearly equal masses. These masses now move one on either side of the nucleus (Pl. XX XVIII, fig. 5), or form the base of a triangle with the nucleus at the apex (fig. 8). The nucleus now divides into two, and when the binucleate system is completed the protoplasm splits, two daughter forms resulting. Large plasmodial forms may be produced in the haemocoelic fluid before the flagella are formed (fig. 6). In the early flagellate stages, the same phenomena are observed; the blepharoplast is often bow-shaped before splitting (fig. 11). The flagellum divides, commencing at its blepharoplastic end (fig. 11) with the division of the blepharoplast and basal granule. The blepharoplast and flagellum may have divided, and protoplasmic cleavage may have commenced before the nucleus has completed its division (fig. 15). The lashing movements of the flagella at this stage may assist in this early protoplasmic division. Fig. 15 shows this condition, with details of nuclear division: the nucleus still shows the mantle fibres surrounding the centrodesmose which connects the equatorial plates. Division in the early flagellates is rapid, and division rosettes are formed (figs. 18, 19). Two or more phases in division may be observed in the same rosette (figs. 18, 19). In the full-grown Crithidia longitudinal division takes place. Before division the blepharoplast presents a diplosome appearance (Pl. XXXIX, fig. 34), and divides first, with the flagellum (fig. 35). One portion of the blepharoplast and its future flagellum migrates to the opposite side of the nucleus. The blepharoplasts may be connected together by a thin chromatin line (fig. 36). The nucleus, which is generally granular at this stage, divides into two. The two flagellar ends separate, and the flagella, moving rapidly, separate the remainder of the organism into two similar adult Crithidia. Division of the post-flagellate stages may occur in the haemo- 553 coelic fluid, but takes place to a marked extent in the ovarian tissues (Pl. XXXIX, figs. 51, 52), ovarian cells (Pl. XL, figs. 55-57), and young ova (Pl. XL, figs. 60, 61). Their division is similar to the division of the pre-flagellate stages, and requires no special description. Aggregation rosettes are formed (Pl. XXXIX, fig. 50). Multiple division takes place in the deposited ova as in the early stages in the haemocoelic fluid. These forms, the result of multiple division, are multi-nucleated masses of protoplasm, and have been termed ‘plasmodial forms’ (Pl. XL, figs. 60, 61). THE HEREDITARY INFECTION The proofs of the occurrence of hereditary infection may be studied by the examination of living specimens and _ stained preparations. The process of events in the ovarian system is as follows: About the time of oviposition, and during this event, the adult Crithidia pass by means of the haemocoelic fluid to the ovary and oviducts. A large number of the crithidial forms here begin the post- flagellate ovarian stage of their existence in the walls (Pl. XXXIX, figs. 45-54) and cells (Pl. XL, figs. 55-57) of these organs. This post-flagellate or ovarian stage begins with the absorption of the flagella and the gradual rounding off of the parasites (Pl. XXXIX, figs. 45, 46) to Leishmania-like forms (figs. 47-49). A very large number of the parasites perform this evolution (fig. 50). A considerable number of the adult Crithidia, however, pierce the walls of the oviducts, preferably high up where the oval capsules are soft. Here they proceed to pierce the walls of the ova (Pl. XL, fig. 58), more than one parasite often entering the same ovum (Pl. XL, fig. 59). The aflagellar end is the fore- most end in entering. A boring motion may now be observed, the flagellum describing a propeliler-like movement, the whole organism being screwed forward, the undulating membrane forming the thread of the screw. Another motion has also been observed in the ovarian forms. After rounding of the protoplasm of the flagellar end, a circular motion has been noted which occurs previous to the disappearance of the flagellum and formation of the resting 554 bodies. Once within the egg the rounding-up process described above takes place, and the Leishmania-like stage results. Division of these small forms may be found in the ova (Pl. XL, figs. 60, 61), which ultimately break up into small resting bodies. A considerable number of the ova of infected ticks dry up and die. Generally the later deposited ova contain the resting forms; the ova deposited early in oviposition are usually non-infected. SUMMARY 1. Crithidia hyalommae is a flagellate parasite occurring in the haemocoelic fluid, salivary glands, ovary, oviducts and ova of Hyalomma aegyptium, the common cattle tick of the Anglo- Egyptian Sudan. 2. The parasite has four periods in its life-cycle: a pre- flagellate stage (Pl. XXXVIII, figs. 1-8) passed chiefly in the haemocoelic fluid, a flagellate stage in the haemocoelic fluid (Pls. XXXVIII, XXXIX, figs. 9-39) and in the ovary and oviducts (Pl. XXXIX, fig. 50 and Pl. XL, figs. 58, 59), a post-flagellate stage (Pl. XX XIX, figs. 40-44) in the haemocoelic fluid, and a post- flagellate or ovarian stage in the ovary and oviducts (PI. XX XVIII, XXXIX, figs. 45-54), ovarian cells (Pl. XL, figs. 55-57), and ova (P1..XL, figs. :60,461). 3. The pre-flagellate stage is of short duration. The parasites are small rounded or oval bodies from 4 » to 11 w broad, and from 4p to 12pm long. Their protoplasm is reticulate or granular, depending on division. They contain a nucleus and a rod-shaped blepharoplast. Division of the very earliest forms takes place (PI. XXXVIII, figs. 5-8). 4. The flagellate forms vary from 26 » to 50» in length and from 2m to 2°5 mu in breadth. The protoplasm is reticulate. The consistency of the nucleus varies with division. The rod (Pl. XXXIX, fig. 28a) or diplosomic blepharoplast (fig. 34) is usually situated on the flagellar side of the nucleus, but its position varies at times, depending on the phase of division. The flagellum arises in the neighbourhood of the blepharoplast, but not from it. 555 5. The undulating membrane and flagellum are both well marked. The undulating membrane assists considerably in motion. The flagellum forms the border of the undulating membrane, and continues free from the protoplasmic body for a considerable distance. A basal granule has been noted (Pl. XXXIX, fig. 31). 6. The post-flagellate and ovarian stages occur in the haemo- coelic fluid, ovary and oviducts. A superfluity of the ovarian forms seems to be present, a large number of them entering a resting stage in the ovarian cells and tissues. 7. The ovarian stages of C7vithidia hyalommae serve for the hereditary transmission of the parasite. The flagellates pass by means of the haemocoelic fluid to the ovaries and oviducts; here they pierce the ova, the aflagellar end of the parasite being anterior. Within the ova the flagellates round up and undergo subsequent division with the production of ‘plasmodial’ forms. 8. Multiplication of C. hkyalommae by longitudinal division takes place in all its stages. g. Infection of Hyalommae aegyptium by Crithidia hyalommae in the adult is purely a hereditary infection. 10. The flagellate is a strictly parasitic flagellate of the tick, and is non-pathogenic to the tick host. ACKNOWLEDGMENTS I wish to thank Dr. Balfour, the recent Director of these Laboratories, and Dr. Chalmers, the present Director, for their valuable assistance in both work and text; also Dr. Fantham, whose experience in this line of research is well known, for his valuable advice in the text and aid with the drawings. To Dr. Bousfield and his able staff I am indebted for a plentiful supply of working material. . KHARTOUM, September 27th, 1913. 556 REFERENCES Fru, P. C. (1908). Ueber die Flagellaten im Darm von Melophagus ovinus. Archiv fiir Protistenkunde, XII, pp. 147-153. One plate. Mackinnon, D. L. (1909). Note on two New Flagellate Parasites in Fleas—Herpetomonas ctenophthalmt, n. sp., and Crithidia hystrichopsyllae, n. sp. Parasitology, II, pp. 288-296. One plate. O’Farrett, W. R. (1913). Preliminary note on a New Flagellate, Crithidia hyalommae, sp. nov., found in the tick Hyalomma aegypttum, Linnaeus, 1758. Journal of Tropical Medicine and Hygiene, Vol. XVI, pp. 245-246. Patton, W. S. (1908). The Life-Cycle of a species of Crithidia parasitic in the Intestinal Tract of Gerris fossarum, Fabr. Archiv fiir Protistenkunde, XII, pp. 131-146. One plate. ——— (1909). The Life-Cycle of a species of Crithidia parasitic in the Intestinal Tracts of Tabanus hilarius and Tabanus, sp. Archiv fiir Protistenkunde, XV, pp. 333-362. One plate. ——— (1909). A Critical Review of our Present Knowledge of the Haemoflagellates and Allied Forms. Parasitology, I, pp. 91-143. Parton, W. S., and Srrickianp, C. (1908). A Critical Review of the Relation of Blood- sucking Invertebrates to the Life-Cycles of the Trypanosomes of the Vertebrates, etc. Parasitology, I, pp. 322-346. PreirFer, E. (1905). Ueber trypanosomenahnliche Flagellaten im Darm von Melophagus ovinus. Zeitschrift fir Hygiene und Infektionskrankheiten, I, pp. 324-29. One plate. Porter, ANNIE (1909). The Morphology and Life-History of Crithidia gerridis, as found in the British Water-Bug, Gerris paludum. Parasitology, II, pp. 348-366. One plate. (1910). The Structure and Life-History of Crithidia melophagia (Flu), an Endo- parasite of the sheep-ked, Melophagus ovinus. Quarterly Journal of Microscopical Science, Vol. LV, pp. 189-224. Two plates. Samson, L. W. (1903). Sleeping Sickness in the light of recent knowledge. Journal of Tropical Medicine, Vol. V, p. 207, Col. 1, paragraph under Fig. 4. Swincte, L. D. (1909). A Study on the Life-History of a Flagellate (Crithidia melopbagi, n. sp.) in the Alimentary Tract of the sheep-tick (Melophagus ovinus). Journal of Infectious Diseases, VI, pp. 98-121. Three plates. | eitta 1 bia R, = | Dien cles seu. ad} Aili bilan ‘ww ney WA es vidal -sipceciee ten © op hy oesoiteem i9))s . ashing yalinaiieginoy 1 oO dinw ; “3 ieee a on ds tod ; . . 3 ~Brstombib -' ST oamiested WMA) to enn Pad Re ph eeiT ae > " op» ohio silsosomasd sdf mon °° Fs bint sils0x0meul mort mat ghibivib yao. 2 ig - we Riel siteeno mond seek soot Aibonmest aytt "= a —~ ©» built Gilsesomsad int eemot gevey Ay egiT —-2 Pe ~ aeuflogeh- guiwode ns) bebhyost Ore. git | Ahiph oifsosomend ni-rotellopsh whe 64-1) ght ert oe Vinie ai enendey nbiaivi O16) gre @ enn rai wf SHeooomasit ett staliogoh fect Ni ag sbiwh 4iloooofad soem moceive™ = 67-81 grt y pibisbin) rwWwors, it} o-iaamqolaved Oh-Os cegrt- oo ; . hee = fd - 6 ” “ * 2 7 . »! : 7 =A a an a7 . oe ) a ‘9 ce; ‘fet ae ate ee @ ae _ ms a : : - —_ v : > fo H a * > +4 - = J ae e me ae _ ais awe a) S. ip, _ } p ee. _ : 7 “i LT - a ve — — EXPLANATION OF PLATES PLATE XXXVIII All figures were outlined with the Abbé-Zeiss camera lucida, after magnification with a 2 mm. apochromatic objective (Zeiss) with No. 12 compensating ocular. The magnification of the figures is approximately 2,000 diameters. Figs. 1-4. Early pre-flagellate forms of Czzthidia hyalommae from the haemocoelic fluid. Fig. 5. Young dividing form from haemocoelic fluid. Fig. 6. Plasmodial form from haemocoelic fluid. Figs. 7-8. Young forms from haemocoelic fluid. Figs. 9-10. Rounded form showing flagellum. Figs. 11-12. Early flagellates in haemocoelic fluid. Figs. 13-16. Division phases in early flagellates. Fig. 17. Early flagellate in haemocoelic fluid. Figs. 18-19. Division rosettes, haemocoelic fluid. Figs. 20-29. Development to full grown Crithidia, haemocoelic fluid. yee —— =~ = EE =a Sead tal abiaind: Reig, Pict vee ait ealqouedgold, beqnde-bor~ ar te fi sitrooomand mort aietiogs®. iwong Gili sot yi _ seul te shiz astlogeh ao realqorusdgold . lari YW enubizog Jownoiib aMiwoule ft-He vegi’l eelquatiqald 30 nuteivily’ yoiatroaeno. ORE a7: es ght Jariqorsigstd smoedlgib eworle 7 an ae bite onerdiom isslion yaiwote | a \ | | ry | | _ Binh silsoroswsd ai eoyete sthtloneh-teot 2 bop ai =i be GVO sad im ao MGTtRVO 46 srallogetttead ' Ob-2h agit i _ isubive : eae br agtigyo to Stiszo1 nullggeTgyn ewoile a ; a , , . . Aoubivo remit saibcincndeis.! to argete novetvi .£¢-12 4g a ei 560 PLATE XXXIX Fig. 28a. Fully grown Crithidia from haemocoelic fluid showing rod-shaped blepharoplast. Fig. 29a. Fully grown flagellate from haemocoelic fluid showing blepharoplast on flagellar side of nucleus. Figs. 31-33. Showing different positions of blepharoplast. Figs. 34-36. Commencing division of blepharoplast. Fig. 34 shows diplosome blepharoplast. Fig. 36. Parasite showing nuclear membrane and chromatic band from haemocoelic fluid. Figs. 30, 37, 38, 39. Stages in longitudinal division from haemocoelic fluid. Figs. 40-44. Post-flagellate stages in haemocoelic fluid. Figs. 45-49. Post-flagellate or ovarian stage in the ovary and oviducts. Fig. 50. Shows aggregation rosette of ovarian stages in walls of oviduct. Figs. 51-52. Division stages of Leishmania-like forms of ovary. Lxorly fp hee ‘ti cele PE ete ; br alr beeps. Ps RSs Neri Mi eoyase WORD Die “Scitle 4 *A\CoEe o wa val att ; ' AO To lew oer pb) jlube lo vitae | Bs = 2 7 7 ‘ f J . ; al ak 7 , > » y ‘I / ; - 1 . a us ane ithe 2s 3B? 's iereit4t - a . . ; ¥ a . LY cicesie ut] vi vit a 7 a rhe vib yitbe Ay Sisay ii >= 562 PLATE XL Figs. 53-54. Division stages of ovarian forms of Cvzthidia hyalommae. Figs. 55-57. Resting and division stages in ovarian cells. Fig. 58. Entry of adult Crithidia into wall of oviduct. Fig. 59. Perforation of egg by adult Crithidia. Figs. 60-61. Plasmodial forms in deposited ova. Fig. 62. Early form (rare) in salivary gland. 563 TRYPANOSOMA VIVAX IN RABBITS BY By BLACKLEOCK AND WARRINGTON YORKE (From the Runcorn Research Laboratories of the Liverpool School of Tropical Medzcine) (Received for publication 6 October, 1913) It is generally accepted that 7. vevax (T. cazalbouz) is not pathogenic for small laboratory animals. Thus Laveran and Mesnil,* writing on 7. cazalboui, state that the dog, monkey, rabbit, guinea-pig, rat and mouse are refractory, and that this fact constitutes one of the chief characters of the trypanosome. It 1s true that Ziemannt records infection with 7. vzvax in dogs and rats, but as short aflagellar trypanosomes were found by Ziemann in his sheep and goats, it 1s highly probable that the animals from which the dogs and rats were inoculated had a double infection of T. vivax and T. congolense, and that the sub-inoculated animals became infected with the latter parasite only. We might remark in this connection that of late years it has been shown that double infection of domestic animals and game with these trypanosomes is by no means uncommon. In previous communications} we have recorded an instance of such a double infection in a horse naturally infected in the Gambia, and described the manner in which the two parasites were isolated one from the other. This horse was the source of the strain of 7. vzvax with which the present paper deals. As mentioned in a former paper,§ the vivax strain was separated fortuitously from the short aflagellar parasite by inoculation of goats from the horse at different dates; one of the sub-inoculated goats developed a pure culture of ** Laveran & Mesnil. Trypanosomes et Trypanosomiases. Paris, 1912, p. 542. t Ziemann. Beitrag zur Trypanosomen Frage. Centralb. f. Bakt. Orig., 1905, Bd. XXXVIII, p. 307, 429-662. { Yorke & Blacklock. The trypanosomes found in two horses naturally infected in the Gambia. Annals of Trop. Med. & Parasit., Vol. V, No. 3, p. 413. § Blacklock. The trypanosomes found in a horse naturally infected in the Gambia. A double infection, Annals of Trop. Med. & Parasitology, 1912, Vol. VI, No. rs, p. 107. 564 T. vivax. From this time (June 14, 1911) up to the present the strain has been preserved by passage through a series of thirty-nine goats. It is of interest to note that in spite of direct inoculation of the strain from goat to goat during a period of nearly two and a half years, no appreciable increase of virulence has been observed. Thus the average duration of the disease in the first fourteen animals used was thirty-one days, whilst that in the last fourteen was twenty-nine days. The periods of incubation and duration of the disease in this series of goats are given in Table I. TaBLE I. Showing a series of thirty-nine passages of 7. vivax in goats during a period of twenty-eight months. Horse 1464 {10 \44 2 Goat 1559 164 1 Goat 1497 5 19 (16 Goat 16 : R J 3 Goat 1605 \ 47 abbit 1604 157 - | : (8 | (9 (6 | (7 Rabbit 1668 15 mths. Goat 1673 | 22 4 Goat pa | 20 Rabbit 1667 1 1 pirhel. FILE Pie erie eet PRY | | | i 6c g. Rabbit 1717 ee Pre 5 Goat 1685 ie Rabbit 16g neg Rabbit 1747 neg, | | | Goat 1735 138 6 Goat 1733 {76 2 Rabbits 1736 neg. 7 Goat 1774 1 8 Goat 1801 {13 | (43 | | Goat 1858 ee 9 Goat 1838 {eo Rabbit 1859 _ neg. 10 Goat 1895 ee 11 Goat 1933 1 | 3 | { Goat 19658 17, 12 Goat I1965A ie | {t2 13 Goat 1986 32 14 Goat 1994 134 (7 s V/ 15 Goat ae (15 Goat 2058B ee Goat z0584a ! 16 Goat 2058c be | {16 (65 565 Taste [—conlinued .abbits 2302 .abbits 2352 roat 26320 5 17 Goat 2088 # 18 Goat pits ee : 19 Goat 21654 hizo 20 Goat 2218 rs 21 Goat 2237 138 22 Goat 2237B e 23 Goat 2278 ie | neg. 24 Goat 2288 ee | 25 Goat 2326 ie | | neg. 26 Goat 2359 | 2 27 Goat es ch 28 Goat a ie 29 Goat 2448 {5 30 Goat Dek (5, *31 Goat bon i *32 Goat 2545 {5 | { *33 Goat 2581A {2 34 Goat 26025 ie *35 Goat 261 ny | *36 Goat ee iE *37 Goat 2642 » 38 Goat 1 668 lier | 39 Goat 2718 {25 roat 2674 a * Goats marked thus used for treatment. Goat 21658 Goat 2194 hee Goat 2306 x | *Goat 2581B 2 The upper figures in each bracket indicate incubation; the lower, duration. 566 Attempts to infect rabbits, made in the earlier passages of the strain through goats, either failed entirely or resulted in a temporary infection in which the parasites were exceedingly scarce and disappeared after a few days.* It was not possible to carry on the strain to a second generation in rabbits. Inoculation made from the thirty-eighth goat proved, however, to be more successful, and four rabbits, injected intraperitoneally with small amounts (0°5 to I c.c.) of the goat’s blood, all became infected after incubation periods of five to eight days. The infection in all was well marked, and parasites were fairly numerous in the peripheral blood—up to twenty to a microscope field. Two of the four died on the seventeenth and thirty-fourth days, respectively, with numerous parasites in the peripheral blood. The spleen was greatly enlarged and the animals were very emaciated; the weights had decreased from 1220 to 970 grams and from 1390 to 1000 grams. There has been some loss of weight in the other animals, but after exhibiting parasites in the peripheral blood for from three to eleven days they have since become negative, and appear to have recovered. There is, hence, evidence that the strain is, after thirty-eight passages through goats, to a certain extent virulent for rabbits. Three rabbits were inoculated intraperitoneally from the thirty-fifth goat with about 30 c.c. of defibrinated blood. Trypanosomes were found in the peripheral blood on the next day, and remained in considerable numbers—as many as fifty to a microscope field— until the time of death, which occurred on the fifth, ninth and fourteenth days, respectively. All the animals presented marked signs of infection, the spleens were greatly enlarged, and the animals had lost a considerable amount of weight. From one of these (Rabbit 2627B), two others were inoculated intraperitoneally with 1 c.c. of citrated blood (containing about 0°25 c.c. of blood). Trypanosomes were found in the peripheral blood of one of the two sub-inoculated animals on the sixth, and it died on the fifteenth day after inoculation. The strain has since heen carried on in rabbits over a period of more than three months, until the eighth generation, as shown in Table 2. * Blacklock. A note on TZ. vivax in rabbits and white rats. Annals of Trop. Med, & Parasitology, 1912, Vol. V, No. 4, p. 537. 567 ‘“ONBINp “AMO, oy} f UOFBQnOUL sayvorpur jaxoviq yore ur aindy sraddn yy G} astte waar sh pais Sanh DL) sobte naqey CT! ote uqqey 6 I} vobte uqary e mS paca ee BT eh 6 al} gclz jvor) = cq viele yiqqry a alzlz u1qqry a | / : ames ; a) ; oz q) So et ee Bow yy iy Nqqey gy yj toe! HAGE ‘Sau z} VOO4? WAGYU a) VORgz IGqry ; O26 |e ac c oF “ AOSgz WGqQrY eat WoSgz jIQQryY c mM 16992 ia. oq 7 woggz ae gs a} c * a} I > a) > o ol vebge iqqry 6 ft votgz i1qqryy oe at agboz uqqry | + fobge JIGqeryy a} Vig es $ ct ! a Lege i ei VAtge JIQQuyy Ligz yeor ‘syjUOU 9214) JO potiad v Sutunp suqqry ui xva72 +7 jo sadvssed yysIa jo sauas v SuIMOYS—']] ATAVT, 568 From a rabbit of the sixth generation a goat was inoculated subcutaneously with 1 c.c. of citrated blood, and trypanosomes appeared in its blood on the ninth day. Although, as the table indicates, the strain has not yet become invariably pathogenic for rabbits, and a considerable number appear to have recovered, nevertheless most of the animals have developed a definite infection, and in five the disease has run an exceedingly acute course, the animals dying in from five to twenty days with numerous parasites in the peripheral blood. We have examined the strain after passage through six generations of rabbits, with a view to ascertaining whether it has undergone morphological changes. In this connection we might mention that as a general rule the trypanosomes in rabbits do not exhibit that rapidity of movement which 1s characteristic of them in goat’s blood. In stained preparations the trypanosomes appeared to be identical with those seen in goat’s blood. We intend, if possible, to carry on this strain in rabbits in the hope of rendering it acutely pathogenic for other small laboratory animals. 569 THE PATHOGENICITY OF NOSEMA FEET Shy LO oe Calony, Cae, 1A RiAl HIVE: BEES BY BB. BAN T ELAM, “D.Se>(EOND:), (B.AS o(CANTAB. ) (LECTURER ON PARASITOLOGY, LIVERPOOL SCHOOL OF TROPICAL MEDICINE) AND ANNIE PORTER 4D. SC. (LOND 3, EF J.9: (QUICK LABORATORY, CAMBRIDGE) (Recezved for publication 31 October, 1913) CONTENTS PAGE I. InTRoDUCTION an ae Scr Sa 56 es ae 0 00 569 Il. ExperIMENTAL INFECTION oF Various INSECTS ... ods 503 306 Soc 570 A. HyMENOPTERA (i) Humble Bees... ee is Sx Je ae onc 570 (ii) Mason Bees... oe ses aa Fe re vas 570 (111) Wasps ... sce sth a ae Es ae a8 571 B. LeEpimpopTera (i) Cabbage White Butterflies... eae a she ee 572 (it) Peacock Butterflies... ‘on 503 se 50 be 572 (iit) Cinnabar Moths ies as aD &s Bo a 573 (iv) Gooseberry Moths... 5o0 soe aa Bes ce 574 C. Diprera (i) Blow Flies nice 50 fae cx 200 =e “00 575 (ii) Crane Flies... BS ave se Ace bc cc 576 (iii) Sheep Keds... see 500 506 30 200 ae 576 Ill. Summary anpD CoNcLusIons ane aes mee Foc 502 300 578 REFERENCES ces ser se aes aoe sas AAG ose nee 579 I. INTRODUCTION Nosema apis is now well known as the minute, microsporidian parasite that causes a malady of hive bees, popularly known as ‘Isle of Wight’ disease, though it should be noted that several diseases of bees are often being confused under this general name. The organism, \V. afzs, is transferred from bee to bee by means of resistant spores in the dejecta of the infected host, which spores are taken up by other bees, either with food or drink, or during the varlous processes consequent on the communal life of the bees. The Nosema spores absorbed by the bee liberate amoeboid forms termed planonts, which enter the epithelial cells of the gut and 57° develop rapidly, particularly in the chyle stomach of the bee where they undergo rapid, asexual multiplication, forming meronts. The functional derangements resulting therefrom are quite sufficient to bring about the death of the host in many cases. Should the host react successfully on the parasites, spore formation ensues, and the Nosema leave the host as highly resistant spores that are well adapted for extra-corporeal life, and that serve to perpetuate the species should they be able to reach a new host. Some experiments have been carried out with insects other than A pis mellifica, and tend to show that the pathogenicity of Nosema apis is far from restricted to the hive bee. Many of the experi- ments were suggested by observation of the habits of various insects present when examination of bees was in progress. Although the numbers of the insects used were not as large as we could have wished, the results are of interest and importance. II. EXPERIMENTAL INFECTION OF VARIOUS INSECTS Experimental infections of members of the Hymenoptera, Lepidoptera and Diptera have been made. AS HYMENOPTERA: The experimental insects used were humble bees, mason bees and wasps. (1) HUMBLE BEES (Bombus terrestris, B. lapidarius, B. hortorum, B. venustus, B. latreillelus). It has been shown previously by Dr. Graham Smith and ourselves that a Nosema is a natural parasite of humble bees (Bombus spp.). It seems to be a different species of Nosema from that so destructive to hive bees, and chiefly parasitises the malpighian tubules of the humble bees. But humble bees belonging to apparently clean stocks, when kept in captivity and provided with food contaminated with excre- ment of hive bees containing Nosema apis spores, both can, and do, become infected with the parasite, and die from the effects thereof. Both meronts and spores can be found in the walls of the gut, while the malpighian tubules, as is usual with infections of Nosema apis, remain uninfected. Humble bees used as controls and supplied with pure food showed no Nosema. (i) MASON BEES. The colonies of mason bees used were a7 brought from abroad and were lodged in a piece of old wall. Some of the bees were isolated to act as controls. The rest were kept in a wall, screened off to prevent exit, and were supplied with honey and pollen contaminated with spores of Nosema apis from dead bees. The abundant food supply proved attractive and the bees fed somewhat greedily. On the fourth day after the first supply of contaminated food, it was noted that fewer bees flew out, and a few dead ones were found. On dissection, the alimentary tract of these bees was shown to be parasitised with meronts of NV. apzs. The dwindling of the bees continued, and some were found unable to fly. MW. aps in the form of meronts and young spores was demonstrable on examination. Ultimately the two colonies supplied with NV. afzs died out, and the brood also was found dead. A few larvae showed Nosema spores, but the brood had been too long dead when recovered for the determina- tion of young stages of the parasite. When both colonies were extinct, the controls that had been supplied with pure honey were examined. Seven-eighths of them had survived, and when these were dissected no parasites were found. The controls that died were also dissected, but as their food canals were empty, and their fat bodies greatly reduced, their deaths were ascribed to voluntary starvation. (a) WASPS (Vespa germanica). Two years ago we reported that a colony of wasps had been exterminated by introducing bees dead of Nosema into their nest. Since then, the experiment has been repeated, with similar results, and examination of recently dead wasps has shown that multiplicative stages (meronts) of Nosema apts were present in numbers in the alimentary tracts of the wasps. Again, wasps collect dead and dying bees and carry them away as food for their larvae. Several cases are known to us in which the wasp broods have died out entirely as the result of a liberal diet of bees dead of Nosema. As before, the parasite underwent development in the body of its host, and in the majority of the wasps, adults and larvae alike, the host was killed before the life-cycle of the parasite was completed by spore formation. The hypothesis that the newer a parasite is to its host, the greater is its virulence, thus receives further support. if 2 B: (LEPIDOPTERA: Certain common Lepidoptera were used for experimental purposes, controls being kept in each case. (i) CABBAGE WHITE BUTTERFLIES (Pzerzs brassicae). Both larvae and imagines were used for experiment, as both could possibly acquire Nosema apis naturally with their food. (a) An adult, g, was fed on the day it emerged from the pupa on syrup made with castor sugar and contaminated with spores from the gut of a bee. The butterfly was fed again three days later, and died on the fifth day after the first feed. Nosema spores and young stages of the parasite were found in the gut. The control died on the sixth day, and no parasites occurred in it. (6) An adult, 2, was fed on the second day after escape from the pupa on sugar syrup contaminated with spores. It refused to feed on the first day. It lived four days after emergence. A few Nosema meronts were found in the walls of the gut after death. A control lived six days. (c) Larvae. Cabbage plants near a badly diseased hive were found spattered with bee excrement. Dead larvae of cabbage white butterflies also were found. Several experiments were then made, of which the one cited was typical. Eight larvae were collected from clean cabbage plants. Four were fed on a cabbage leaf smeared with honey in which an infected bee’s gut had been emulsified, the remaining four on ordinary clean cabbage leaves. One experimental larva died after two days with Nosema infection in its gut. Two, also containing Nosema, died on the following day. The remaining one pupated on the third day, but the imago never emerged. Of the four control larvae, all pupated and produced imagines in due course. (ii) PEACOCK BUTTERFLIES (Vanessa 20). On two occasions when bees were under examination for Nosema apis, peacock butterflies came into the laboratory, and after the usual aimless flutterings, settled on the viscera of dead bees and proceeded to suck honey from the contents. Both were captured and were examined after death. A second butterfly was captured on each occasion to act as control. (a) Adult, S. It lived two days after capture. © When 573 dissected, no stages of Nosema apis were found in the gut walls, and only a very few Nosema spores—probably those ingested with the food—were present. (6) Adult, 9. This was a rather large female. Soon after feeding, oviposition began, and the insect died at its termination. No Nosema was found in it. The extremely small number of experiments prevents any definite conclusion being reached as to the pathogenicity of the protozoon to the insect. All that can be said is that a negative result was obtained in the two cases investigated. (111) CINNABAR MOTHS (Callimorpha jacobeae). Larvae of the cinnabar moths were used for experiment as they were observed on several occasions feeding on groundsel contaminated with bee excrement. The subjects experimented on were obtained from a locality where bee-keeping was not practised, and where the larvae seemed remarkably healthy. Fifteen young caterpillars of the cinnabar moth were placed on a plant of groundsel growing in a flowerpot and covered by a bell jar to prevent escapes. The groundsel was watered with an emulsion of dead bees containing Nosema spores, filtered through coarse muslin that readily allowed the Nosema spores to pass through, but retarded the passage of the chitinous portions of the bees. The groundsel foliage was watered daily. The larvae fed on the groundsel in a quite normal way. Control larvae were kept under similar conditions but their groundsel was watered with ordinary tap water. These control larvae never showed any form of Nosema apis. The results of the experiment were :— Three caterpillars, fed on infected groundsel, died two days after the first infective feed. Young stages of Nosema apis were present in their mid guts. The hind gut was not affected. The parasites were in the multiplicative phase, meront formation being in progress. Seven larvae died on the fourth day. Of these, one contained young spores of Nosema apis, two had a very heavy infection of young meronts in an actively dividing condition, and the remaining four showed a few young stages of the parasite. The condition of the guts of the larvae resembled that seen in bees, and the varying degrees of infection among the larvae are parallel to what 574 we have so often found when examining a series of bees from one colony. One larva died on the fifth day after the first feed, and con- tained meronts of Nosema apis. On the same day one pupated, and ultimately a deformed male imago issued from it. This insect lived three hours only. It was dissected as soon as possible after death, but no stages of Nosema apis could be recognised with certainty within it. The remaining larvae died during the night and were too decomposed when examined to permit the detection of the planonts or meronts of Nosema apis, had they been present. No spores were found. The life cycle of Nosema apis as seen in the larvae of the cinnabar moth was the same as that found in the hive bee. The spores gave rise to planonts, which became meronts in the epithelial lining of the alimentary tract, and these, in turn, produced spores. (iv) GOOSEBERRY MOTHS (Abvaxas grossulariata). |Observa- tions on a garden in a district heavily infected with apian microsporidiosis showed that, in one case, the foliage of gooseberry bushes near infected hives was spattered with the excrement of the bees, and the dejecta on the leaves contained spores of Nosema apis. Beneath the bushes a few dead larvae of the gooseberry moth were found, and as the body of one of these contained Nosema spores, experiments with larvae obtained from an uninfected district were commenced. [Twelve larvae were divided into two sets of six. Both were fed on gooseberry twigs, the twigs being moistened daily with equal quantities of water, and of water containing Nosema spores, respectively. The results may be summarised thus : — (a) Larvae supplied with food contaminated by Nosema spores. On the fifth day after the first infective feed, one larva died. Nosema spores were found in its intestine, toegther with meronts. These spores were fed to bees and reproduced the disease. On the seventh day two more larvae were found dead. Thirty- six hours had elapsed since they were last seen alive. Decay had been rapid, and it was impossible to identify young stages of Nosema apis, and no spores were found. Thus it is uncertain whether these two larvae became infected. S75 One larva died on the eighth day. It contained both spores and meronts of Nosema apis. Two larvae pupated on the tenth day. The pupae were small compared with those from the control larvae. One pupa was dissected on the twelfth day, and a few meronts were found in the body. The second pupa did not produce an imago. Whether this was the effect of the action of the Nosema cannot be stated with certainty. (6) Larvae feeding on uncontaminated food. ‘Two of the larvae were dissected. Neither showed any trace of Nosema apis. The remaining four pupated. Two pupae were dissected and examined. Nosema apis was absent, nor was any other parasite found in them. Of the remaining two pupae, one produced a beautifully shaped and marked female, but the second did not develop. Morphologically, the Nosema present in the larvae of the gooseberry moth differed in no wise from the parasite as seen in bees, and its identity was established by feeding bees with the spores obtained from the caterpillars and thereby reproducing the disease. Cu, DIPTERA The Diptera used for experiment were blow flies, crane flies and sheep keds. ) BLow FLIES (Calliphora erythrocephala). During the examinations of bees for Nosema apis, it was noticed that blow- flies settled on the viscera of the bees and fed upon them. The flies were also often seen sucking up the sweet excrement voided on the alighting board and sides of the hive; they subsequently showed Nosema infection. A number of pupae of Calliphora erythrocephala were dug up in a garden on March 26th, 1912. The adult flies began to emerge on March 28th, but could not be induced to feed on that day. On March 29th, the flies sucked a piece of meat with some Nosema spores on it. The following day they were supplied with moistened sugar contaminated with Nosema spores. They refused again to feed, but sat on the sugar most of the time. From April 1st to April oth, the blow flies hatched out at the 576 rate of one or two a day, and were formed into experimental and control sets. Special cases are now cited :— Blow Fly 1 emerged from the pupa. It was provided with contaminated sugar and sucked it readily. It died nine days after emergence, and on dissection showed some young stages and a very few spores of Nosema apis in its gut. Infection of the Malpighian tubules also occurred. Blow Fly 2 was smaller than Fly 1, and emerged a day later than it. It also lived nine days, while its control lived ten days. At death it contained a number of Nosema spores. Blow Flies 3, 4, 5 and 6 lived from 7 to 10 days after emergence. All were fed on infected candy. Blow Fly 3 became infected, showing a few spores at death. Blow Fly 4 showed no form of Nosema apis. Blow Flies 5 and 6 contained a very few spores of N. apis. Blow Fly 7 showed no Nosema apis, but contained what is probably a new species of Nosema. Blow Fly 8 contained many young stages of Nosema apis, together with a fair number of spores. Blow Flies 9 and 10 contained no parasites. No control blow fly was found to harbour microsporidia. From the above experiments it can be inferred that a certain number of Calliphora are attacked by Nosema apis, and the latter can prove fatal to them if they are ingested by the insect with its food. (ii) CRANE FLIES (Zzpula oleracea). Two crane flies were noticed sucking the viscera of bees prepared for microscopical examination. The crane flies were captured, but refused to feed in captivity and died two days after capture. When dissected, a few young stages of Nosema apis were found. Crane flies caught in the open and used as controls showed no trace of microsporidian infection. (11) SHEEP KEDS (Melophagus ovinus). All the insects previously mentioned were able to acquire spores of Nosema apis naturally by means of their food, though the number of cases occurring in the open may not be very considerable. During the course of this investigation, a number of healthy sheep keds, Melophagus ovinus, from a prize Southdown flock, were sent to us. Though it was not very probable that these insects could obtain Nosema spores in nature, it was considered that it might be of 547. interest to see what effect, if any, was produced on them by Nosema apis. In order to ensure the ingestion of Nosema spores by the keds, spores from the guts of bees were smeared on to a limited area of the forearm of one of the writers and six keds were fed on this small area. When wounds were made by the bites, additional small drops of infected excrement were placed on them, and through the layer of spore-containing material the keds had to force their proboscides to get blood. Some of the keds after sampling the sweet excrement slowly sucked it up before taking much blood. All six fed well, the process taking nearly an hour in every case. Six control keds were fed on a carefully disinfected area of the other arm of the experimenter. The next day the treated keds were alive but not active, as it was a very cold day. They were fed again in the same way, all feeding well, but three being particularly greedy. A third feed was given on the next day, when the three weaker feeders of the previous day also fed slightly. Examination of the excrement of these three showed that they contained a fungus, as reported by one of us in 1910.* The other three showed no fungus in their faeces. At noon on the fourth day after the first feed, the three keds containing fungus were dead. Microscopical investigation showed that they contained large quantities of fungus in their Malpighian tubules and young stages of Nosema apis in their mid guts. Soon after mid-day, the remaining keds were noticed to be much more feeble, and between 3 p-m. and g p.m. that day all three died. Examination of fresh preparations of them showed the presence of meronts and empty sporocysts of Nosema apis in the gut contents. Stained prepara- tions were also made and were confirmatory of the fresh preparations; they also contained young spores. When the control keds were dissected, no form of Nosema apis was observed in any of them. From this experiment, it is shown that Hippoboscid flies, Melophagus ovinus, became infected when supplied with spores of Nosema apis, which underwent developmental changes in their bodies. The Glossinae or tsetse flies resemble the Hippoboscidae in their * Porter, A. (1g1o). The Structure and Life-history of Crithidia melophagia (Flu), an Endoparasite of the Sheep Ked, Melophagus ovinus. Quart. Journ. Microsc. Sci., LV, pp. 189- 224. Two plates. 578 mode of reproduction and blood-sucking habit. As is well known, the Glossinae are the flies that transmit sleeping sickness or trypanosomiasis of men and animals. We would suggest that a search should be made in the various Glossinae by competent observers, who are well versed by practical experience in the structure and life history of Microsporidia, for parasites allied to the Nosema so destructive to bees, and pathogenic also to mason bees, wasps, and the various Lepidoptera and Diptera cited above. Should such a pathogenic Microsporidian be found as a hyper- parasite in Glossinae, it would be a forward step in the solving of the problem of sleeping sickness. III. SUMMARY AND CONCLUSIONS 1. Nosema apis has been proved pathogenic to Hymenoptera other than bees. It can multiply in the food canals of humble bees, mason bees and wasps, and can bring about the deaths of the hosts. 2. Contamination of plants with infected excrement occurs in the neighbourhood of badly infected hives. Such contaminated food is pathogenic to the larvae of cabbage white butterflies, cinnabar moths and gooseberry moths, in which Nosema apis produces destruction of the tissue of the food canal in the same way as in bees. Both imagines and larvae of these insects became infected with microsporidiosis when supplied with food con- taminated with Nosema spores. 3. Calliphora erythrocephala. The blow fly becomes infected naturally by ingesting Nosema spores contained in the sweet excrement of bees. This infection has been repeated experimentally. Crane flies may also become infected. 4. A member of the Hippoboscidae, Melophagus ovinus, has been infected successfully with Nosema apis, which is pathogenic to the sheep ked. It is suggested that research be made by competent observers among the Glossinae for Microsporidian parasites allied to the Nosema of bees, and, possibly, equally pathogenic to the tse-tse flies that may harbour them. ayo REFERENCES Fanruam, H. B., and Porter, A. (1912). Mlicrosporidiosis, a protozoal disease of bees due to Nosema apis, and popularly known as ‘ Isle of Wight’ disease. Annals Trop. Med. and Parasitol., VI, pp. 145-161. (1912). The Morphology and Life-history of Nosema apts, and the significance of its various stages in the so-called ‘Isle of Wight’ disease in bees (Microsporidiosis). Annals Trop. Med. and Parasitol., VI, pp. 163-195. Three plates. —— —— (1912). The Dissemination of Nosema apis. Annals Trop. Med. and Parasitol., VI, pp. 197-214. Report on the ‘Isle of Wight’ Bee Disease (Microsporidiosis). Supplement No. 8, Journ. Bd. Agric., May, 1912. Further Report on the ‘Isle of Wight’ Bee Disease (Microsporidiosis). Supplement No. 10, Journ. Bd. Agric., July, 1913. WEsaRae FO 7 c i, ir - * * * =" — i i il ' "; Mi ts van : ‘ ~ -', i te te : stet aay ar | 7 : t. ~ s 7 -# } Pat i] r r ’ 5 } hid Cidsilenint Bee yauléatgroly a ‘ ‘ ee SyacTU ts Jah F . > wy , fn S7 = : lyis thar ; : eet | , ity? £4 ei th atl” halite di pies aoe shone’ Oh t q 3 7 - Ben ft Pe Son ee xT re, a sang niet Geass yan LV lait ase Ae eEegn a 4 4 Let j ] tage , ; ty + sTOrMiIn Lote f ot} t ‘ * x ee | 7 j . : - ' Ey \ ‘ ta a mel * if Vy ‘ c é ‘ ; f i TET 216 i 4 ; Fie “a i ; 7 ; : 3 le dea’ ne ran a gediw-rauahd Dente oz uh tivsitehpaset | Wei rise) war stich Sam * Mere SRT ard od o . , ; \ i ber \ = . 5 ieEtQ. lel tga " ae ; = 4 - bo > _ > ‘ ; S ‘ Es — A - 4 ' a 1 fi rh \ ia — - eh 581 ON" CERTAIN. MOSOUIT OS... OF, TELE GENERA BANKSINELLA, ‘Tueosarp, AND TAENIORHYNCAUS, Arriparzaca BY HENRY FPRCARTER, FES: (LECTURER IN ENTOMOLOGY, LIVERPOOL SCHOOL OF TROPICAL MEDICINE) (Recetved for publication 28 November, 1913) During the past few weeks I have made a critical examination of the male genital armatures of numerous species of African mosquitos and, in certain instances, somewhat interesting results have been obtained from their subsequent study. These studies are more especially connected with the affinities of certain obscure species and in reference also to the synonymy adopted by other students of this group of blood-sucking insects. Before dealing with the specific characters, it would perhaps not be out of place in this paper, if I draw attention to the fact that the structure of the male genitalia does not support the separation of the pale coloured African species of Chrysoconops, so defined by Theobald, from the genus Taenzorhynchus. YVhe armatures of typical examples of the latter genus compared with those of the African Chrysoconops are so essentially similar that I consider them to be congeneric. BANKSINELLA PALPALE (Newstead). Neomelanoconion palpale Newstead. Ann. Trop. Med. and ajasita, Wf. 30 (lOO7,): Banksinella luteolateralis, Edwards (nec Theob.). Bull. Ent Res., Pip: 0. (1or2). This species was originally described by Newstead (/.c.) from a single male collected by Drs. Dutton and Todd at Boma, Congo Free State. Unfortunately, however, the specimen was somewhat rubbed and therefore certain of the more important characters rendered obscure. Last year the type was submitted to 582 Mr. Edwards, of the British Museum, for examination, who subsequently placed the species as a synonym of B. luteolateralis, Theob. Some time ago a perfect male example* of an apparently new species was received from Broomassie, Ashanti. The armature of this specimen was prepared for microscopical examination, and, on comparison with a similar preparation of the type of N. palpale, was at once seen to be the same. Newstead’s species is therefore a valid one, and must be raised to specific rank. Male genitalia (fig. 1).T Fic. rs. Inner lateral NSFC view of right clasp sane filament (c.f.) of Fic. 1a. Male genitalia of Banksinella palpale, Newst. x 160; B. palpale, Newst. x s = lanceolate spines 2.40 Side pieces relatively large, broad posteriorly, narrow and rod- like distally, the internal edges being somewhat strongly curved, as shown in the figure. The latter bear numerous fine hairs in the central region, extending from each side towards the middle line, * Mr. Edwards has kindly examined this specimen for me. t+ In the designation of the parts I have followed Messrs. Howard, Dyar and Knab and the main lettering in all the figures is as follows :— s.p. = side piece; c.f. = clasp filament; u = unci; h = harpe;) bl: )=) basal lobe of side piece. 583 and several long, markedly curved ones on the apical lobe-like projection; between these groups of hairs are situated six or seven lanceolate spines (fig. 1 s), but the number of these appears to vary, since in the specimen from Ashanti only three are visible. Clasp filament comparatively short and stout, expanding slightly on the inner side near the middle, tapering off towards the apex, and bearing numerous curved hairs and a large conspicuous, slightly curved tooth- like projection in this region. The inner lateral surface of this organ (fig. 1 8) is considerably broader, with a blunt and rounded apex; the hairs and tooth previously mentioned, arise from the apical portion of this surface and extend towards the middle line. Basal lobes small, each bearing two distinct spines or teeth, and a few pr VN) e Pay ee, ; Ga ee FS |: Tu led ‘ ie If RN A A,/ i, i oe Jf AY ue Vy, 7, F. Wa Khir ESN (Lo) 2 SS (7 ZF ( IS las Fic. 2a. Left side-piece and appendages Fic. 28. _Left clasp filament of Banksinella luteolateralis, Theob. x 160; enlarged ( x 240) s =lanceolate spines. delicate hairs. Harpes well developed, the apical extremity pointed, forming a single tooth. Harpagones absent. Unci of complex structure bearing three pairs of comparatively large, inwardly and ventrally directed, teeth on the apical ventral margin; these teeth increase proportionately in size, the lowermost pair being the smallest. The genital armature of B. luteolateralis (fig. 2) is very similar to that of the previous species as regards the structure of the basal parts, and general form of the side pieces. The clasp filaments 584 (fig. 2 4) provide, perhaps, the most distinctive character. These are of a somewhat different shape, and are completely devoid of hairs at the apical extremity; the tooth, also, is distinctly larger. The internal edges of the side pieces bear similar arrangements of hairs and spines as in B. palpale, but the hairs of the central region are less numerous, and the lanceolate spines are smaller and about fifteen in number. As previously mentioned, the type of Banksinella palpale was in a somewhat damaged condition when described, and I therefore thought it desirable to add a few notes on the specific characters of the male. Head: The narrow, curved scales occupy a narrower median area than in B. luteolateralis, Theob., and the pale coloured region is formed to a considerable extent by cream coloured flat scales. A B Fic. 3. (a) Male palpus of Bankstinella palpale, Newst.; (6) Male palpus of Banksinella luteolateralis, Theob.; x 36. Palfi with the second or terminal segment relatively much shorter than in B. luteolateralzs; the basal segment is nearly three and one-third times the length of the apical segment, whereas in B. luteolateralis it is approximately two and one-third times as long. Proboscis sometimes showing an ill-defined yellowish band: 585 Thorax: Clothed with golden, narrow-curved scales laterally, dark curved scales and a few scattered golden ones in the central region—similar to the ornamentation usually met with in this genus. Abdomen: First two segments dark, unbanded; third and fourth with basal lateral cream coloured spots, especially noticeable on the latter; fifth, sixth and seventh segments* with well-marked yellowish-white basal bands. In the male of B. luteolateralis the first and second segments usually possess a few pale scales on the median area, the others being adorned with basal pale bands. Wings: Very similar to those of B. luteolateralis. Legs: Brown, the femora and tibiae being pale on the ventral surface; tibiae with apical knee-spots, well marked in the hind pair of legs. TAENIORHYNCHUS MACULIPENNIS (Theobald). Chrysoconops maculipennts, Theobald. Novae Cultcidae, Part I, pe-272-Aprik ror: This species, described by Theobald from Uganda, has recently been placed as a synonym of Taenzorhynchus (Chrysoconops) annettii, Theob., by Mr. Edwards. Through the kindness of Mr. H. H. King, of the Wellcome Laboratories, a male example has been received by the School, and an examination of the genitalia at once proves the species to be a valid one. Mr. Edwards writes that he has now examined preparations of the armature and is in a position to confirm the above statement. Male genitalia (fig. 4.) Side pieces large, gradually tapering to a broadly rounded apex. Clasp filaments long and relatively narrow, each bearing two short closely appressed teeth at the tip. The apical third bears four delicate hairs, and is deeply constricted at its base, on the lower side. Basal lobes large, each with a pair of dark rod-like appendages; one of the latter, in each case, is a very conspicuous, stout, elongated structure, the other much narrower and slightly shorter. The larger rod-like process is evidently composed of three or four fused spines or modified hairs, the smaller rod of one only. * The last few segments are necessarily wanting, as they were removed for microscopical examination. 586 Harpes well marked, with five comparatively large teeth at the extremities. Harpagones absent. Unci as depicted (fig. 4 2), bearing numerous minute teeth on the apical, dorsal and ventral edges. es S.P. Ci Fic. 4 Male genitalia of Taentorbynchus maculipennis, Theob.; X 160. Genital armature of T. annettu (fig. 5). The main point of difference between this and the previous species is in the structure of the clasp filaments. These are of very peculiar form, and an idea of their shape can best be gained from the figures. The apical portion is broad and very deeply cleft on the ventral surface; it bears a few delicate hairs and three teeth, closely pressed together, at the tip. The basal parts closely resemble those of 7. maculipennis, although the harpes appear to be more elongated (vzde fig.) This, however, may be due to displacement of the parts in mounting, 587 Fic. 5a. Male genitalia of Taentorhynchus annettii, Theob.; x 160. Fic. 58. Right clasp filament of T. annettit, mounted under pressure; X 240. 588 but, owing to lack of material, no definite statement can be made. The identification of these two species by other means is a matter of no little difficulty, and, unfortunately, the material at my disposal is insufficient to enable me to give any constant and well- defined characters for distinguishing purposes. TAENIORHYNCHUS METALLICUS (Theobald). Culex metallicus, Theobald. Mon. Cul., II, p. 63 (1901). Banksinella metallicus (Theobald). Mon. Cul., V, p. 408 (1910). Taeniorhynchus violaceus, Vheobald. Third Report Wellcome Labs., p. 262 (1908). The examination of preparations of the male armature of authenticated specimens of 7. metallicus from various parts of Africa, and of 7. violaceus from the Sudan (Mr. H. H. King) show that the above synonymy, previously proposed by Mr. Edwards, 1s correct. Male genitalia (fig. 6). Fic. 6. Male genitalia of Taentorhynchus metallicus, Theob.; x 160. — 589 Side pieces sub-cylindrical, adorned with the usual stout and slender hairs, and bearing basal lobes with their appendages, which greatly resemble those of the two above-mentioned species. Clasp filament very characteristic, the apical two-thirds being expanded for part of its length, then tapering gradually to the tip, on which is situated a very short, stout tooth; the lower side of the apical half is sharply re-curved, forming a distinct ridge. Harpes apparently provided with three teeth. Harpagones absent. Unci with a series of eight or nine small teeth on the ventral basal edges, and four larger teeth at the apex. REFERENCES Epwarps, F. W. (1911). ‘The African species of Culex and allicd genera. Bull. Ent. Res., II, pp. 241-268 (1912). A synopsis of the species of African Culicidac, other than Anopheles. Bull. Ent. Res., III, pp. 1-53. (1913). Further notes on African Culicidae. Bull. Ent. Res., pp. 47-59. Howarp, L. O., Dyar, H. G., AND Knas, F. (1912). The mosquitoes of North and Central America and the West Indies, I, pp. 69-71. tine et in Bes uke 1 SR eee ts sare vagaeeen gh Ra caine $ ey + yeah eae lt nel fara tay en, sl Soll li ELA = SPS Reel a3 nf ft: asc 408). —— cae As : sas A a a a fo re 2 atdiet wn ait yivnogiat ait 1b. i et ai\lcottobit aheriniend, or) J. Yaatlaeen Ares; EHF i 4, Peis irk cf fote2; fis Frases bt): oaeeee oe ‘08 _ ial Bs th jaime one? Wins cite vas rusty ha aol *, Me * ta Fr ier a : = Y : = en > Pa oOo ‘ae euOMaR TER es ~ < rial. Shell toasty beiilire deuce iv “a We hai lsient ay, aoa | ; E i Vu | fast eae ee = > at anh a la ingen : 6 a h 2 yl? = a a : . a BENE yy cual i = yA 7 . lets iS ; bin ry! Ne Paslupars >? ¥ rt = *> e ye : = = 4 P _ +4 7 n a ‘ J we =. ~ Fe “ae 59! NEW 'CULIGIDAE FROM. .THE, SUDAN BY PRED Ve TPHEORALD TAG? Ponts. lon PURTEDS: (Received for publication 24 November, 1913) Amongst a large collection of Culicidae sent me by Mr. Harold King, from the Sudan, the following undescribed species have so far been found. A considerable number of Uvanotaenzas and others have as yet only been partially examined, and will be reported upon later. The types of these new species are in the collection of the Liverpool School of Tropical Medicine. MUCIDUS NIGERRIMUS, nov. sp. ¢. Head: Brown, covered with mealy grey scales, a narrow white border around the large dark eyes, a tuft of long grey curled scales and long, thin, straight ones projecting forwards between them. Antennae brown, with deep brown plume hairs; basal segment bright brown, with small flat white scales. Palpz deep brown, with black scales irregularly disposed and scattered white scales, especially at the junctions of the last two segments, forming distinct bands; plume hairs deep brown, apical segment slightly longer than the penultimate; longer than the proboscis by about two-thirds of the last segment. Pvodosczs with scattered dark and light scales, as in the palpi, a distinct joint about the middle, the apical half being thinner than the basal. Thorax: Brown, clothed with irregular scattered grey scales, most dense in the median area and around the space before the scutellum and across the thorax between the wings. Scztellum very densely clothed with loose grey scales; the scantier scaled areas have long, thin, narrow-curved, pale scales, not typical Muczdus ones, which also form a dense scaled median area in front. Pleurae very deep brown, with patches of flat white scales. Metanotum rich brown. 592 Abdomen: Deep brown, clothed with alternate patches of black and white scales, forming marked black and white out- standing lateral patches; hairs dense, pale golden; the segments dorsally, with flat, loose white scales at the base of the segments, in the middle, with mixed dark and creamy ones apically and at the sides more evenly disposed; the apical segments more white scaled, the last but one with two black spots. Basal lobes of genitalia, with black scales; claspers long, curved, dark. Legs: Fore femora thin, with dark and pale scales, the former predominate, apex white; fore tibiae with a dull white basal band, followed by a black area with outstanding scales and then a snow- white area with outstanding scales; tarsi thin, yellow, slightly darkened apically; mid legs with the femora with dense out- standing scales, black predominating, with three narrow white bands and white apex; tibiae with white basal and apical bands, black in middle, with a narrow median white band, very shaggy ; tarsi thin and yellow; hind legs with femora and tibiae as in the mid, but not quite so shaggy or dark; first tarsal black scaled, with narrow basal white band, yellowish at apex, very shaggy ; remaining tarsals white basally, yellow apically, scales more or less out- standing; the white predominate on the last three segments; hind ungues equal and uniserrate, fore and mid with the larger claw bidentate, the smaller unidentate (viewed in one direction the second or basal tooth of the larger claw seems to stand out laterally, looking like three claws). “a Vic. 1. Muctdus nigerrimus. n. sp. 6 Fore ungucs Wzugs: Mostly dark scales, with a few scattered white ones; first fork-cell considerably longer and narrower that the second, its base nearer the base of the wing, its stem as long as the cell, stem 593 of the second longer than the cell; third long vein very close to the second; cross-veins thick, clouded, all three close together; fringe with eight white spots; the scanty dark scales give the wings a spotted appearance. Halteres yellow, knob black at the apex. Length. 7 mm. Habitat. Wadelai, Lado District, Sudan. (H. King, 8/6/11.) Observations. Described from a perfect male taken from under- erowth around trees on a grassy slope near the Nile. It was pointed out as being a distinct species by Mr. King. It is a very dark species, easily distinguished by the leg ornamentation. CHRYSOCONOPS NOCTURNUS, nov. Sp: Head golden-yellow; proboscis and palpi golden-yellow, black at their apices. Thorax shiny black, with scattered golden scales ; pleurae mostly brown, a long yellowish area before wing roots. Abdomen entirely golden-yellow. Legs golden-yellow, femora with some scattered dark scales and narrow dark apical bands; fore and mid tibiae with scattered dark scales and a small dark apical band; hind tibiae with narrow basal and broad median and apical dark bands; fore and mid tarsi golden-yellow, unbanded, last two tarsals dark; hind legs with metatars1, first, second and third tarsals with broad apical black bands, last all dark. Wings yellowish, all the veins with dusky brown and dull yellowish scales. Male palpi with three dark rings, legs darker than female and traces of dark apical abdominal bands. 2. Head: Integument greyish, clothed with golden-yellowish narrow-curved scales and dark, thin, upright, forked scales; eyes silvery. Antennae brown, basal segment pale. Palpi and proboscis golden, black scales at their apices, with black chaetae and some scattered black scales on the apical half of the proboscis, also some black scales below, at the base (one specimen shows some scattered dusky scales over the palpi). Thorax: Shiny black, with scattered golden-yellow, thin, narrow-curved scales, and black chaetae; prothoracic lobes projecting, golden-yellow, roundish and mammillate. Scaéelluim black, shiny, with golden narrow-curved scales and dark border bristles. Metanotum black. Pleurae deep brown with some 594 patches of flat white scales and a yellow, longish area at the base of the wings; a short blunt process appears on each side of the front region of the mesonotum. (Fig. 2. B.) Fic. 2. Chrysoconops nocturnus. n. sp. A. = Prothoracic lobe. B. = Mesonotal process. Abdomen: Steely, entirely clothed with bright golden-yellow scales and yellow hairs; venter golden yellow. Legs: Golden-yellow, femora with some scattered black scales and black apices, fore and mid tibiae with scattered black scales and black apices, hind with narrow dark basal rings, a broad black median and apical rings; fore and mid tarsals unbanded, the last two dusky; hind tarsals with broad apicai black bands; chaetae dark; ungues dark, equal and simple. Wzngs: Yellowish with scattered dusky and yellow scales, appearing mainly dusky in certain lights; first fork-cell longer and narrower than the second, its base nearer the base of the wing, its stem about one-third the length of the cell; stem of the second fork-cell not quite so long as the cell; posterior cross-vein about twice its own length distant from the mid. Halteres pale yellow. Length. 5 mm. S. Palpi with the last two segments nearly equal, the apical one mostly black, apex of the next two dark; scanty hair tufts, brown. Awdexnae banded yellow and brown with rich brown plume hairs. Legs: Asin 9, but more dark scales above on the femora and tibiae. Abdomen: With some dark metallic scales on the apices of the segments, almost forming bands, apical segment dark, with dark 595 chaetae and dark claspers. Fore and mid ungues very unequal, the larger uniserrate; hind ungues rather long, curved and simple, all black. Length. 5 mm. Habitat. Bier Terab (Shambe to Wau road), Bahr-el-Ghazal Province; Lau (Shambe to Wau road); Lau to Ateiba (Shambe to Wau road) and Mayo to Melangot (Naam River), Bahr-el-Ghazal Province. GH, Kine; 30/2/10; .3/a/iit, rofa/112) Observations. Described from three 2’s and one <. The specimens were taken by Mr. King in a rest house after dark and out of doors after dark. It is a vicious blood-sucker. This species comes near C. nigrithorax, Theob., which it superficially resembles, but it is distinct, as pointed out by Mr. King. The tibiae are not all black, as in C. nzgrithorax; the proboscis has a few black scales at the base, but 1s not deep black on the apical half, as in C. nuzgri thorax. The marked prothoracic lobes also differ, and the blunt lateral processes on the mesonotum are also characteristic. REEDOMYIA SUDANENSIS, nov. sp. Head dark, a black patch and then a pale patch on each side, middle with golden scales and black upright scales; eyes silvery and black; palpi, proboscis and antennae deep brown. Thorax deep rich brown with scattered small golden scales; scutellum silvery white; pleurae blackish-brown to brown with white puncta. Abdomen deep blackish brown, unbanded, with small basal lateral white spots; venter with basal yellow scales, the segments with blackish apical borders. Legs deep brown, unbanded, hind femora and tibiae with apical white spots, seen also in fore and mid legs to some extent; chaetae golden. Q. Head: Black and shiny with scattered narrow-curved pale golden scales in the middle, with black upright forked scales, a patch of flat black scales at the sides and then flat creamy white ones. Eyes silvery around the upper border and down to the antennae. Palfz black. Proboscis deep brown. Axtennae deep brown with pale pubescence and dark hairs, basal segment and base of second paler. Thorax: Deep rich brown, with small narrow-curved scattered 596 golden scales, a small patch of broader creamy ones in front of the root of each wing; chaetae dark. Scatellum with flat silvery white scales and numerous rather irregular dark border bristles. Metanotum dark with a greyish sheen. Pleurae black, with patches of white scales. Abdomen: Black, unbanded, with small basal white lateral spots, dark border bristles with dull golden apical reflections ; venter with basal creamy yellow areas, dark apical ones. Legs: Deep brownish-black, unbanded, apices of femora and tibiae white, especially in the hind legs, chaetae golden, showing markedly against the dark legs; ungues of fore and mid legs equal and uniserrate, of hind equal and simple. Wengs: First fork-cell longer and narrower than the second, its base nearer the base of the wing than that of the second, its stem about half the length of the cell; stem of the second fork-cell not quite as long as the cell; posterior cross-vein about its own length distant from the mid. Halteres with pale stems and fuscous knobs, with some pale scales on the latter. Length. 4 to 4°5 mm. Habitat. Yidu, Lado District, two 9’s; Khor Nambiri, Lado District, one 9; Nyumbe, Lado District, one 9; Such River, one @; 12 to 14 miles from Hierallah on road to Bundle, Lado Wrstrict™ (Hs King 22) 41 0 27 Ay te 27 ariel ley tee Observations. Described from six 9Q’s. The marked head adornment is very noticeable. In one Q there are traces of the thoracic scales forming two dorsal spots, but the deep rich brown with fine golden scale dusting is most noticeable in all six specimens. It comes near R. b2punctata, Theob., but the scutellum is snowy white, not pale yellowish, and the thorax has golden, not bronzy scales; from R. bzannulata, Theob., it can be told by the apex of the abodmen not being white and from R. neobiannulata, Theob., by having no basal white abdominal bands. Mr. King’s notes are as follows :— “Khor Nambiri (Kapei to Ye1), Lado District, 2/4/11. A small khor containing pools, and shaded by trees and bushes.’ ‘“Nyumbe, Lado District, 27/5/11. Amongst undergrowth in a grove by a khor.’ 97, ‘Yidu, Lado District, 31/5/11. From undergrowth on the bank ofa typical Glossina palpalis khor.’ “72-14 miles from Hhierallah on the road to Bundle, Lado District, 22/3/11. In a deep, heavy timbered ravine.’ KINGIA MACULOABDOMINALIS, nov. sp. Head black, silvery white on middle and sides; proboscis black ; palpi black, snow white tips; antennae deep brown, black at base with white scales; eyes silvery. Thorax rich deep brown, a large patch of snowy white on each side, a small one over the wings, and lines of yellowish scales posteriorly; scutellum snowy white in some lights, grey in others; pleurae deep brown with white puncta. Abdomen deep black, unbanded, 5th to 7th segments with silvery white median spots, and with basal lateral white spots. legs dark brown, femora with traces of two white spots; first and second tarsals only with narrow basal pale bands (fore and mid legs). 9. Head: Black, clothed with flat black scales, flat white ones in the middle and a patch of white ones on each side; chaetae black. Palgz black scaled with snow white scales apically. Proboscis black. Antennae deep brown, the basal segment with flat white scales, the second segment with outstanding dark scales, hairs deep brown. Thorax: Shiny black with deep bronzy black, narrow-curved scales, two large patches of snow white flat scales about the middle of the mesonotum, a small patch of narrow-curved white ones just in front of the roots of the wings, a short line of pale creamy scales running up to the bare space before the scutellum, and a line of yellowish scales on each side parallel to it and reaching the scutellum; chaetae black. Scutellum black, the flat scales are glassy and snow white in some lights, grey to almost black in others; border bristles blackish brown, four to the mid _ lobe. Metanotum deep chestnut-brown. Pleurae deep brown with silvery white puncta. Abdomen: Black with basal snow white spots, the fifth with two median silvery white spots, the sixth with one median snow white spot, the seventh with the spot almost basal, a small one also on the apex; border bristles deep brown; venter dark brown. 598 Legs: Blackish-brown, femora with two white spots; first and second tarsals of fore and mid legs with basal creamy white bands, others dark in fore and mid legs (hind legs missing); ungues equal and uniserrate. Wings: First fork-cell longer and narrower than the second, its stem more than half the length of the cell, stem of the second fork-cell nearly as long as the cell; posterior cross-vein rather more than its own length distant from the mid; scales brown, rather large. Length. 4 ™mm. Habitat. Khor Kokbwa (Yei to Abba), Lado district. (H. King, 13/4/11.) Observations. Described from a single 2 taken by a rocky, shady stream; the hind legs missing. A very marked species readily distinguished by the thoracic and abdominal adornment. AEDIMORPHUS QUINQUEPUNCTATA, nov. sp. Head black and silvery white; palpi, proboscis and antennae deep brown. Thorax rich brown with four silvery white spots and a small fifth white patch before the bare space in front of the scutellum, which is also silvery white scaled; pleurae deep brown with silvery white puncta. Abdomen dusky black, unbanded, with basal lateral silvery white spots; venter with basal silvery white lateral spots also. Legs dark, unbanded, with venter of femora pale at the base, especially on the hind legs where most of the venter is pale, femora of hind legs, each with a snow white apex and a spot before the apex. Q. Head: Black with a small median area of dull creamy narrow-curved scales, with black upright forked scales, a patch of flat snow white scales on each side in front, then flat black scales, then white and then black ones again. Clypeus, proboscis, palpi and antennae deep brown to almost black. Thorax: Dull black with small, narrow-curved, dull brown scales and four patches of small flat snow white scales, two in front near the head, two in the middle of the mesonotum, also a fifth patch before the bare space in front of the scutellum, and a small spot below in front of each wing; chaetae black. Scutellum 599 black with snow white flat scales forming three patches, border- bristles black, four large ones to the mid lobe. Med¢anotum deep black; pleurae rich brown with six snow white puncta. Abdomen: Black with dull golden border bristles and basal ‘snow white lateral patches, the scales somewhat outstanding; venter black with basal snow white lateral spots. ° Legs: Almost black, femora and tibiae with apical white spots most prominent on the hind legs, each of the hind femora with a round snow white spot near the apex and whitish on most of the venter; ungues of fore and mid legs equal and uniserrate, hind equal and simple. Wings: With a white scaled spot at the base and with the first fork-cell slightly longer, but no narrower than the second fork- cell, its stem more than half the length of the cell; stem of the second fork-cell about two-thirds the length of the cell; posterior cross-vein less than its own length distant from the mid; sixth long vein markedly curved. AHalteres with ochreous stem and ochreous and dusky knobs. Length. 3.5 mm. Habitat. Alenga and Matalee, Lado District. (11/5/11 and ZION ie = Re Sah areas Observations. Described from two Q’s. It comes near Aedimorphus punctithorax, Theob., but differs in having five, not six, thoracic white spots. CULICELSA CENTROPUNCTATA, nov. sp. Head brown with pale scales and two dark areas on each side separated by a line of white; palpi, proboscis and antennae deep brown. Thorax with brown and silvery white scales, the latter forming two spots in front, a median and more or less pronounced posterior area; scutellum entirely white scaled. Abdomen black with median white basal patches and basal white lateral spots; venter mostly creamy scaled. Legs black, with pale chaetae, especially marked on the tibiae, femora and tibiae with apical white to yellow bands, tarsi with narrow basal white bands to all the hind segments, and all but the last two in the fore and mid. Q. Head: Clothed with almost white narrow-curved scales in the middle and dark, thick, upright forked scales, then a few flat 600 white scales, then a black patch, then white, then black again; chaetae long and deep brown, except for some golden ones between the eyes. Palpi, proboscis and clypeus almost black. Antennae black with pale internodes, basal segment dark and testaceous, the second with flat black scales; verticillate hairs black. Thorax: Deep brown with mixed brown and silvery white narrow-curved scales, the latter forming two spots in front, a border around the mesothorax near the head, denser behind, and as a line above each wing; chaetae long and black. Scu¢ellum with narrow white curved scales. Metathorax black. Pleurae deep brown with patches of flat white scales. Abdomen: Black, the segments with median basal patches of white scales, and patches of basal lateral white scales which become median apically, posterior border bristles golden; venter mainly creamy scaled. Legs: Almost black, femora and tibiae with apical pale spots, base of first and second fore and mid tarsals with white bands; in the hind legs all the tarsals have basal white bands, bristles dark and golden; fore and mid ungues equal and uniserrate, hind equal and simple. Wezngs: With short fork-cells, the first longer and narrower than the second, its base a little nearer the base of the wing, its stem nearly as long as the cell; stem of the second longer than the cell; posterior cross-vein twice its own length distant from the mid. Halteres all pale ochreous. Length. 47m. 3. Head: Pale scaled with two dark patches of flat scales surrounded by white flat scales. Palfz a little longer than the proboscis, black, with the last two segments nearly equal and with small basal white bands, a creamy band at the base of the third segment and a pale area at their base, on the last two segments are brown hair-tufts on one side, and on the apex of the penultimate is a long golden chaeta. Antennae banded brown and grey, plume hairs flaxen brown. Thorax, abdomen and legs as in the 9; fore and mid ungues, unequal, uniserrate, hind equal and simple. Length. 4°5 mm. 601 Habitat. Alcholi, Lado District and River Mewri, Mongolla Province. (25/5/11, Qi; 2s aay i, EL Rig: ) Observations. Described from a perfect ¢ and 9, although taken in different places undoubtedly the same species. It is a most marked species and is related to those so far placed in the somewhat obscure genus Cadicelsa, Felt, obscure only for the fact that we cannot fix any definite characters, yet all the species included have a very marked similar appearance. HEPTAPHLEBOMYIA KINGII, nov. sp. Head black with pallid scanty scales, proboscis ochreous black at the base and apex; antennae and palpi dark brown. Thorax rich brown, somewhat paler in the middle, two snow white spots in front near the head and two on the middle of the mesothorax ; scutellum snow white scaled; pleurae deep brown with snow white puncta. Abdomen deep brown, unbanded, with basal lateral white spots ; venter with pale basal bands. Legs unbanded, deep brown, femora and tibiae with apical white spots. Wings with seventh vein very close to the wing border. Q. Head: Black with pale creamy, narrow-curved scales in the middle, becoming white in front, creamy flat lateral scales, white at eye border near the white narrow ones; upright forked scales black. Antennae, palfi and clypeus almost black. Proboscts ochreous brown, black at the base and apex. Thorax: Black with narrow-curved bronzy brown scales, two median bare parallel lines, four round spots of snow white narrow- curved scales, two in front near the head, two somewhat larger and wider apart about the middle of the thorax; the scales become paler towards the scutellum, being pale golden in some lights; chaetae brown. = Scadellum black with dense snowy white curved scales, deep brown border bristles, six to the mid lobe; metanotum black with grey reflections. Pleurae deep brown with patches of rather long, flat, white scales. Abdomen: Steely, clothed with black scales, with small snow white basal lateral spots; border bristles brown with golden reflections apically; venter black with basal white bands. 602 Legs: Dark brown, unbanded, femora pale at the base, apices of femora and tibiae white on the hind legs, traces in the mid, scarcely perceptible in the front pair; ungues equal and simple. Wings: With brown scales; first fork-cell considerably longer and a little narrower than the second fork-cell, its base nearer the base of the wing, its stem less than one-fourth the length of the cell, stem of the second posterior not quite as long as the cell; posterior cross-vein not quite twice its own length distant from the mid; the seventh scaled vein close to the inner border of the wing. Halteres pale with fuscous knobs. Length. 4mm. Habitat. Nyumbe, Lado District; Alenga, Lado District. (27/5/11, Nyumbe; 28/5/11, Alenga.) Observations. Described from two perfect Q’s. It canbe distinguished from the allied H. argenteopunctata, Ventrillon, by the abdomen having only small basal lateral spots and not ornamented as in Ventrillon’s species from Madagascar; the antennae are also black in the 9, not yellowish, and the thoracic adornment differs. The scaled seventh vein is well marked in both 9Q’s. One was taken from a small hole, containing water, in a tree, the other from tall grass near a khor. Bs onic 603 imi. RO bAbIens st DEN LET Y OF TRYPANOSOMA CONGOLENSE (BRODEN) AND T. VANUM (LAVERAN) BY Bz BLACKLOCK AND WARRINGTON YORKE (From the Runcorn Research Laboratories of the Liverpool School of Tropical Medicine) (Recewved for publication 28 November, 1913) In our paper on the identification of the more important mammalian trypanosomes, we have regarded 7. dzmorphon (sensu Laveran and Mesnil), 7. confusum (Montgomery and Kinghorn) and T. pecorum (Bruce) as synonymous with 7. congolense, which was first described by Broden in 1904. In the same year Laveran described a similar parasite found by Balfour in the Sudan, under the name of 7. nxanum. These parasites are identical morpho- logically in that they are both short aflagellar trypanosomes measuring 8 to 19m# in length. The sole distinguishing feature is their effect on small laboratory animals, 7. congolense being described as pathogenic for monkeys, dogs, rabbits, guinea-pigs, rats and mice, while 7. zanwm is considered to be incapable of infecting these animals. The object of this paper is to examine the evidence upon which this distinction 1s based and to decide whether it is sufficient to warrant such a differentiation. In previous papers* a description has been given of two trypanosomes which were present in the blood of a naturally infected horse sent over to this country from the Gambia. One of the parasites was unquestionably 7. vzvax: concerning the identity of * Yorke and Blacklock. The trypanosomes found in two horses naturally infected in the Gambia. Annals of Tropical Medicine & Parasitology, 1911, Vol. V, p. 413. Blacklock. The trypanosomes found in a horse naturally infected in the Gambia. A double infection. Annals of Tropical Medicine & Parasitology, 1912, Vol. VI, p. 107. 604. the other there was, however, considerable doubt. Morphologically, this parasite was indistinguishable from 7. dzmorphon (Laveran and Mesnil) and 7. xanum. As we finally succeeded in infecting small animals with the trypanosome, it was eventually decided that it was 7. dimorphon (T. congolense). Both the parasites, which were separated fortuitously, one from the other as already described, have been kept in experimental animals for a period of 18 months. The results of artificial passage of the short aflagellar parasite from animal to animal are so interesting and suggestive that we have decided to describe them in some detail. The sub-inoculations made with this parasite from the time of its isolation until the 51st generation are given in genealogical form in the table. A study of this table reveals two facts, viz. :— (1) Most of the early inoculations failed to infect, whereas the later were invariably successful. (2) The course of the infection in the earlier successful cases was chronic, whereas that in the later instances was acute. Thus, if we consider the animals used in the second to the fourth generation, we find that these comprise 8 rats, of which 3 were positive and 5 negative; 4 mice, of which 2 were positive and 2 negative; 3 rabbits, 2 positive and 1 negative; 4 guinea-pigs, 2 positive and 2 negative; and 3 goats, I positive and 2 negative. Again, if the duration of the disease in the earlier rats be compared with that in the later experiments, the contrast is very striking. For example, the average length of life of the first ten rats from the fifth generation to the fourteenth was 88°6 days, whereas that of the last ten rats, comprising the 42nd to the 51st generation, was only 8°6 days. It is clear, therefore, that by passage of this parasite through laboratory animals the trypanosome has been changed from one of uncertain and chronic pathogenicity to one of great virulence. This fact seems to us to be one well worthy of remark. That artificial passage of a strain through a series of animals does some- times alter its virulence for that species is well known. The results* * Warrington Yorke. On the pathogenicity of a trypanosome from a case of Sleeping Sickness contracted in Rhodesia. Annals of Tropical Medicine & Parasitology, 1910, Vol. IV, p. 351. 605 obtained by different workers with 7. gambzense are illustrative of this point. These observations appear to us to have some significance for the nomenclature of the parasite. 7. congolense and T. nanum are identical morphologically, they are both spread by the same specics of tsetse fly and infect the insect in precisely the same manner. The usual way of deciding with which parasite cattle or antelope known to harbour a short trypanosome in their blood are infected, is by sub-inoculation of rats or some other convenient laboratory animal. As a rule, these animals are not too plentiful in the tropics and one or two must suffice for the diagnosis. That inoculation of one or two small animals may not afford any conclusive evidence as to whether or not the trypanosome is pathogenic, is at once realised from observing the results of the earlier inoculation of the parasite from our horse. Had the number of our experimental animals been limited, we should probably have designated the parasite 7. manuwm. Further experiments, however, showed that the trypanosomes could be made acutely pathogenic to rats. This opens up the question as to whether there is really any difference between 7. congolense and T. nanum. It is interesting in this connection to refer to the observations of other workers. Writing in 1911 on a short aflagellar trypanosome obtained from ponies naturally infected in Togoland, Weissenborn* states that the parasite was of inconstant virulence. It was most virulent for mice, but slightly so for rabbits, rats and monkeys. Only a small proportion of rats were susceptible, whilst guinea-pigs were absolutely refractory. Morphologically the parasite ZT. frobeniusi closely resembled 7. congolense. The Sleeping Sickness Commission of the Royal Societyt write: ‘If Z. pecorum, which is usually more or less infective in the monkey, dog and rat, lives for some time in the blood of the goat, it loses its power of infecting other animals. This has given rise to the erroneous idea that a separate species—Z. nanum exists.’ * Weissenborn, E. Beitrag zur Kenntnis der kurzgeisseligen Trypanosomen. Archiv. f. Schiffs- und Tropen-Hygiene 1911, p. 477. + Bruce, Harvey, Hamerton and Lady Bruce. The susceptibility of various animals to T. simiae. Roy. Soc. Proc., 1913, Vol. 87, p. 49. 606 The Belgian Sleeping Sickness Commission* found that aflagellar trypanosomes from naturally infected dogs will not always infect guinea-pigs and rats. It is thus evident that workers in the field have found that short aflagellar trypanosomes, morphologically identical with 7. con- golense and 7. nanum, are of uncertain pathogenicity for the smaller laboratory animals. As the result of our investigations and of those of the authors mentioned above, we can see no evidence which would justify distinguishing one from the other on the ground of pathogenicity. In the present state of our knowledge we can only conclude that I. congolense and T. nanum are the same parasite. * Rodhain, Pons, Vanden Branden, and Bequaert. Rapport sur les Travaux de la Mission Scientifique du Katanga. (Octobre 1910 a Septembre 1912.) es 607 ‘UOIVAMp IaMOT 2Y} ‘UONYQnoUT 9}¥orpul yoxovAq Yora ur sainsy aoddn oy ‘stequinu [jews Aaa ut Ajuo Avp auo uo pasrvadde saysvaeg y a Sobz = “of = a LSSz e 1S a LQez pe OES sa ES "OZ a zboz yey «0S aA S/SrA SIE? OAS a Vhizzywey “61 val 1£Sz lee ‘ob a ggtz Ne (Ste = TZO1z me "QI SI giSz 4 “gb A 1S€z y ae [ VgbIz es vn ei 60Sz wy LP a Sree wy of a 90L12 pe ‘OI I toSz ie gb 8 LEoz Wee of oa TOL cat eNqeee sic ct S6bz " aa, a £fez y “62 a ofiziey FI Sct ggbz yey br oa €zEz yey «*gz Pt | TOME IE Se £) ! | : Il | zt , . bf gibz ie th 4 z1fz eu i a gloz wy ‘zi | iLbz i ‘eb ey £6ez IY *9z st Yoo? WY I out tobe yey ‘th et o6Lzz ge Sia on LL61 yey «OI a $Sbz ise ‘ob a v6Lzz Bs “bz ye } 6161 4 6 obtz me 6£ a ozSzz " 4 re 6061 ve ey | 6Ebz wy «gt on azSzz Jey *zz oe OL Om IENT see 8! 1zbe i ff a weS7z oe Iz a £991 ee 9 4. ay nt goSgI jeyy | voSgI lee AG | | ‘dou beSr yor “Bou dggSt yey cal VOQSI ey “dau OSeST asnopy “Bou WS9S1 asnojy *BaU OQOg! WY | goot astoyy =P “Bou AQOQI Jey : | VZ09QI yy | | | | | | | (PeLPD et asesi . a os vest yey Coa sos Peitia) | ; voeSi asnoyy ‘f | es | =) no geese al ‘soug yey “sou W ew | ozst Sid‘) ‘Sou 61$1 Bid 4) aa | S6bi-sid-y «*Sauobbisid'y §=*Bou 6ghr yiqqey ye | b6br VEQqeyel ee } Lobi yiqqey *z ‘sou Lobr yor) o Sghr yvor) | | | | | | | | | | | | bobi asaoyy ‘1 a ‘a cates fehaice ie euey Paycrer- meat’ seceimpad* pe jodes gains | Ve perl - i? —# _ m@) uo je 4 sbbasxicd i, iamnite SiG ¥}L ae ote i | yy Tate ‘SAR frat Ste vatenied sleet eae cp aeiient souien Tepe. ¥ urns oe 2 - hee vin oe im Ab ED y oa Pe ee Oe orn.™ gre S or See nr eee meine sce eae 8 ; 4 ‘ oft A rit vee ‘ ‘ha baa v ua? ANT ree a f nD aa mee” agin = eal get hs 1 Mi as ab ies ° ras ~~ ioe a | <% ri os =r a rd % j < r ‘ Ww a 7 7 A eres J \ ow Ce SR PLN poet Aad) ae = h _— eee | een 2 oe ee Ee © a By oy a ee ra J _ ' C is Cig riakt its pte verge nae ee e ie ‘Gary, Oust. Dik p Le ee 7 du: Rew fetigt’ We tA ay = ‘ pie) ~-as ee . A os ‘: WP. 2 ny - : » is. : ” Po Pale - fe] = a) . wm." a z ' = a ‘| ‘ ¢ > team us = . . aa - 2 > ‘ sine te Alva: AT ¢ we Sho ieee Saree Puke oni ia ; "7 - Fak fe ~ Ph z | a 1 aka & ~ 4 i ~ vw — we +4 aa ns Al - = — a ee ee eh jt gt me SS Dive ed eG) EN Ore ot: = & . _ ie eel = = as oem - = a = = a o~ ? : > a 3 z * ‘ - ge «t * ‘ & tal ~ * ts 4 5 ine , ~ al 1" ' “« v 7° A 4 A — Fr ~ - tare v : a * ~~. “ 7 . " n ‘ +7 et rf a ” ” w a 1 ps ees as erhcg Gea adie Meena law be ee re. o _ * a : wie 7 Wo ri) s = g S = i «ile (fe OT — a - ae oe So er om, 2 4 ij — s . & i , 5 ee ae a ee wit je a on 7 1" fw .Y* ev oa al re 1 >a vr i” we cnr, _ - ld oS c ashe ven : ; — S. 4 ——— - se = - “hes ; | cs oe = me Eng i = ¥ 7 — p ees om —o ° -€ a ' 4 oo ee mf i . N ; 7 mA > ee ; > 0 . “ . re Le » BoA oe 2a SS Sole ey O ene so edit cce Ms - ~ ~ye a ee peed mah epee mah ieee $10 ad “ . Tie! 7 “ . ~ } sy - fe i” «@ _ . . . - & i ‘ ‘- Py m™ 5 E id > ’ o ‘ “—. nS amy ae i 7 o - c oo a ye ” — - 4 oe + - = * nig My ~ f a Senter. c rs : A “ cal as eal nes met _ : ek) tlie Ui i Soy ee ee a ee Sing ea ie oe ; vas F $7) cree ’ 4 - a ‘ cm ee - * ‘ “ ~ ~ 7 a ¥ re ae a4 el a - - Sa - ~ .,. _ ry as | 5 a ; , eaat 9 sod . ’ a = { me He last ase sien Goalie i f sri Se Beth Wee ae » . soe ; paras 2 on wit *1¢- shes win Kates he pe 48 olepens Bits. 4 Pe Jeoreh wey ae At yom tae 609 HERPETOMONAS STRATIOMYIAE, mar. A PLAGELLAVE.PARASITE.OF)LHE BLIES, OTRATIONTAIA. CHAMELEON. AND 8S. POTAMIDA, WITH REMARKS TN Ete blOLOGY OR Pon MOS TS BY H. B. FANTHAM, D.Sc. (Lonp.), B.A. (CANTAB.) (LECTURER ON PARASITOLOGY, LIVERPOOL SCHOOL OF. TROPICAL MEDICINE) AND ANGE oP ORTCE RSC: CLONDo)y oH. 3. (QUICK LABORATORY, CAMBRIDGE) (Recezved for publication 19 November, 1913) PLATE XLI CONTENTS PAGE I. INTRODUCTION no oot ae ae Sod ae 560 609 II. Tue Brorocy or THE Host Figs, Straiiomyia chameleon anv S. potamida ... 610 III. Marertar anp Meruops ... Fee wee ae S06 Soe 612 IV. Tue Lire-History or Herpetomonas stratiomyiae (A) Tue Herperomonas IN THE Larva or THE Fry boo 2 612 (B) Tur HerperoMonaD DURING PuPaTION ae a ae 615 (C) Tue Herprromonap IN THE Imaco soe Fes ws 616 V. Moone or InFEcTION S05 ae 506 mee ie ie 617 VI. ConcrupiInc Remarks xe 500 er sot ec as 617 VII. Summary ... 618 REFERENCES “ic ae — ee se i 566 619 EXxPLaNaTION OF PLATE 620 I. INTRODUCTION The interesting flies, S¢ratzomyia* chameleon and S. potamida, are fairly common in certain districts where the drainage of the land is poor and where boggy areas with somewhat rank vegetation occur. Both the larva, pupa and imago of the insects are sometimes parasitised by a small Protozodn, belonging to the genus * The original generic name of the host flies was Stratiomys. Many modern authorities prefer the form Stratiomyia, which has been adopted in this paper. The Siratiomyia were obtained near Cambridge, and one local name for them is Chameleon flies. 610 Herpetomonas, and presenting certain differences from other common Herpetomonads that warrant its inclusion as a new species. The chief interest of the parasite lies in its relation to the life- history of the host, and thus a brief note on the biology of the hosts is considered advisable. We do not propose to enter into great cytological detail regarding the structure of the flagellate, nor to engage in fruitless discussion as to the nomenclature of such parasites, but rather to consider the flagellate in relation to the life-history of the host. The relation of insect flagellates to their hosts’ life-cycle is a most interesting though difficult subject, on which little or nothing has been done in most cases up to the present. II. THE BIOLOGY OF THE HOST FLIES, STRATIOMYIA CHAME- LEON AND S. POTAMIDA (a) Habits. The larvae of these insects are very characteristic, worm-like, legless organisms, armed with powerful jaws by which they drag the body forwards, progression being aided by bristles attached to each segment. The larger larvae examined varied from # inch to 2} inches in length at will, as the abdomen can be retracted telescopically. Their colour varies with that of the mud and decaying vegetation among which they live, yellowish, brown and green being common hues. Twenty-five to thirty respiratory tail. filaments are present and are applied by the larva to the surface film, so that in deep water the larva is suspended vertically, head downwards. The pupae are found near to the surface in the mud fringing the pond or stream. The larval skin is retained and the much smaller pupa is enclosed within it. The period of pupation is short. The imago is somewhat bee-like and is blackish in colour with bright yellow markings. Eggs are laid on aquatic plants just above the level of the water, and the larvae either escape into the water, or are hatched beneath the surface into which the plants finally fall. (4) Food. The food of the imago seems to be sweet vegetable material. The larvae, so far as can be ascertained by direct obser- vations extending over three years, are vegetable feeders.. The contents of the alimentary tracts of those examined have never 611 contained blood or other recognisable animal matter such as muscle or chitinous tissue that could be obtained from insects or Crustacea. Small organisms such as Protozoa may be ingested, but no proof that such was the case occurred. A typical set of examinations of 50 larvae from 1o different localities, representing about one-third of the total number examined, gave the following results :— From the first locality, seven larvae varying from ? inch to 2} in. long, were obtained. Two were infected with Herpetomonas, one infected larva was 1} in. long, the other 2 in. The large vegetation of the locality comprised grass, Veronica beccabunga, Myosotis palustris, various algae, and many rotting leaves. The contents of the food canals of the larvae were portions of Chara, Cladophora, Spirogyra and Zygnema. Ten larvae from the second locality were from 1 in. to 2 in. long. None were infected. Grass and Veronica beccabunga constituted the larger vegetation. There were fewer large algae than in the first locality. The alimentary canals of these larvae contained algae, chiefly species of Clostertum, Pentium, Micrastertas and Cosmarium. Six larvae from a third place varied from 1} in. to 24 in. long. Like the previous set, they were uninfected. Their gut contents consisted almost entirely of Gonzum_ sp., and the larger vegetation on the spot comprised grass, J/ yosoézs and Common Hemlock. A small pond, whose bordering vegetation consisted of grass and J/yosotzs, and in which some fine Hottonia grew, yielded seven larvae. These contained many algae, chiefly species of Sgenedesmus and Selenastrum. No parasites were found in the larvae. Six larvae were obtained from the fifth locality. All were vigorous, and varied from 1 in. to 2} in. in length. Grass, MW/yosotzs and algae were abundant. The food passages oi the larvae contained algae, chiefly species of Mavicula, Diatoma, Synedra, Gyrosigma and Nitzschia. No herpetomonads were found. Some of the smallest larvae were obtained from the sixth locality. They varied from } in. to 2 in. in length. Seven were examined. A larva, 1 in. long, had a slight infection with Herpetomonas stratiomytac, the rest were normal. Rotting leaves occurred in the water and hemlock, Veronica beccabunga and grass constituted the larger vegetation. Algae, chiefly species of Navicula, Denticula and Nitzschia, together with fragments of leaves, were found in the alimentary canals of the larvae. Three larvae only were obtained from the seventh locality, whose vegetation -was J y s the same as that of the sixth. The gut contents of the larvae also were similar. None were infected. One specimen, 13 in. long, obtained from a small brook partly choked with Potamogeton crispus and large algae, contained no parasites. Its gut contents included species of Scenedesmus and Cosmartum, together with fragments of Chara. One larva, 2 in. long, was obtained from a ditch fringed with willowherb. Its gut contained numerous specimens of Cosmarzum and a very few Herpetomonads also were present. Two larvae from the tenth locality were uninfected. The chief plants growing in the mud were willowherb and water dropwort, while the food contents of the larvae consisted of species of Cosmarium and Navicula. The presence of infected larvae coincided to some extent with the quantity of decaying matter that was present; but sometimes larvae obtained from the foulest sources (not those detailed) were as free from infection as those from relatively uncontaminated places. Larvae were in greatest abundance where much decaying material was present. 612 Altogether, nearly 150 larvae were examined, and the _per- centage infected with Herpetomonas was only about 3. The percentage of infection in the pupae examined was somewhat higher. II]. MATERIAL AND METHODS The larvae, pupae and imagiries of Svatzomyza, comprising both bred insects and those caught at large, have been used during this investigation. The Herpetomonas has been found most abundantly in the digestive tract of the larva. The alimentary canal has been dissected wherever possible, placed in physiological salt solution and examined in serial teased portions. The pupal condition usually prevented dissection of the alimentary canal, but smears of the central part of the body were sufficient to allow of identifica- tion of the parasite. Much time has been spent in observing the living Herpetomonas. Osmic acid followed by absolute alcohol, Bouin’s fluid and Flemming’s solution have been used for fixatives, and Giemsa’s solution, iron haematoxylin and glycerin haematin were used as stains. The paraboloid condenser and stereoscopic oculars have also been used and found of much service in determining depths of granules, chromatin particles and similar structures. V. THE LIFE HISTORY OF HERPETOMONAS STRATIOMYIAE The life-cycle of the herpetomonad may be divided into two main phases: (1) a resting phase, (2) an active, multiplicative phase. The resting phase again can be sub-divided according to the period at which it is present in the host. Infection is by the contaminative method, and freshly ingested resting forms of the parasite about to develop into the active form are best described as the preflagellate stages, while the forms produced by the preparation of the full flagellate for extra-corporeal life are more accurately described as post-flagellate forms. Naturally the post-flagellate form produced in the first host becomes the pre-flagellate organism in the second host. A. THE HERPETOMONAD IN THE LARVA OF STRATIOMYIA The preflagellate stage of Herfetomonas stratiomyiae differs from that of most herpetomonads in being elongate oval in shape (Bieee wel os: -1,,--2))5 Its length varies from 575m to 8, while 613 its breadth is about 3“. The cytoplasm is clear in life or slightly granular. The nucleus shows as an oval, refractile vacuole, the blepharoplast as a bar-like rod. They both lie relatively near the surface. Sometimes the end of the body near the blepharoplast appears somewhat more refractile in life than the distal end. Such an area, when stained, proved to be the chromatophile vacuole- like area from which the flagellum originates. The nucleus presents a definite nuclear membrane, seen best in haematin-stained _pre- parations. The chromatin varies in its arrangement, sometimes appearing as a more or less central concentrated karyosome (fig. 2), at other times being scattered as granules in the nucleoplasm (fig. 1). The bar-like blepharoplast stains deeply. The formation of a flagellum occurs very rapidly, and hence it is difficult to find stages in which the chromatophile area is present unless the host is dissected just at the time preceding the flagellation of the parasites. Division occurs among the preflagellates (figs. 4, 5), and will be described later. The flagellates (figs. 10-17) vary in size, the length including the flagellum being from 26°64 to 57, while the breadth is from 2u to 36m. The flagellum itself may occasionally reach 38» long. The non-flagellate or posterior end of the flagellate is elongate but somewhat blunted; the flagellar or anterior end is somewhat rounded, with the flagellum projecting from it. As with the preflagellate forms, the nucleus 1s oval and shows cyclical develop- ment. Relatively young flagellates, or flagellates that have formed rapidly from the preflagellate forms, often possess a vesicular nucleus with a central (figs. 15, 16) or slightly excentric (fig. 12) karyosome. Older parasites, those of slow growth and forms about to divide, have their nuclear grains of chromatin evenly distributed (figs. 11, 14). The blepharoplast is curved, rod-like or oval, and, except in dividing forms, is homogeneous in structure. Chromatoid granules may be present in some flagellates, usually forming small grains in the post-nuclear part of the body (figs. 11, 13). Multzplicatzon. The increase in numbers of the parasites within the host is brought about by longitudinal division, which can take place in either the preflagellate (figs. 3-5) or flagellate (figs. 18-22) stages. The division of the preflagellates is initiated by that of the blepharoplast, which becomes dumb-bell shaped (fig. 3) ’ 614 showing two concentrations of chromatin, one at each end. The heads of the dumb-bell remain attached to one another for some time by a strand of chromatin. The constriction of the nucleus rapidly follows that of the blepharoplast. The future flagellar end then becomes cleft (fig. 4), and the cleft extends backwards. As soon as parts of the two daughter organisms are free, rapid move- ments occur, each free part diverging from the other and twisting on itself as it does so, until the two organisms come to lie in almost a straight line (fig. 5). Violent movements precede the final separation, and this is usually succeeded by relative quietness on the part of both the daughter forms, which usually do not move away until some seconds and, occasionally, minutes have elapsed. When movement occurs it is not active as with the flagellates, but consists of slow undulations of the body, which gradually propel the daughter forms forwards. The stages of division of the flagellate (figs. 18-22) are like those of the preflagellate form, but the division of the blepharoplast and flagellum takes place almost simultaneously (figs. 19, 20), while nuclear division is often delayed, and at times, cleavage of the cytoplasm has commenced (fig. 20) before the nucleus has com- pletely divided. The movements of the two parts of the dividing organism (figs. 21, 22) are very active, and the final separation is effected more quickly than that of the dividing preflagellate, the lashing of the daughter flagella being of material aid. The two newly-formed individuals swim away rapidly after separation. Examination of stained specimens has been confirmatory in all respects of the above observations made on the living organisms. The formation of a post-flagellate form (figs. 23-36) is brought about by the gradual retraction and absorption of the flagellum (figs. 23-27) and the concentration of the body until it forms an oval or rounded form, which secretes a thin, closely adherent cyst around itself. When the assumption of the post-flagellate form is com- plete, there is no trace of the flagellum remaining as such. Even its root (or rhizoplast) disappears, the chromatin of it appearing to be dissolved and concentrating, in some cases, in a small part to form a chromatoid area, as well as diffusing generally through the body substance, as shown by the staining reactions of the cyst. The post-flagellate is Leishmania-like (figs. 32-36), of a somewhat 615 elongate type. The nucleus may, or may not, show a karyosome, for as with the flagellate, the structure of the nucleus varies. If there is a karyosome in being at the time of encystment, the nucleus of the post-flagellate shows the same feature (figs. 34, 36). Should encystment follow rapidly on division, the nucleus is almost homogeneous (figs. 33-35). Much discussion has arisen recently as to the existence of specially differentiated portions of the blepharo- plast, described variously as karyosomes and centrioles. In some cases, by the use of iron-haematoxylin, some granules have been demonstrated, but it is admitted that the existence of these granules depends for its demonstration on the degree of removal of the stain by the iron alum. They are not found when other stains are employed. The basing of argument on the fleeting appearances produced by too great or too little differentiation—the degree of differentiation that is ‘accurate’ being an absolutely individual opinion—is merely vexatious, and does not tend to the advancement of knowledge on really scientific lines. B. THE HERPETOMONAD DURING PUPATION As the period for pupation of the host approaches, the post- flagellate forms of the parasite leave the gut of the larval host and pass out with the faeces. Rapid flagellation of any preflagellates that are present occurs, together with division of the flagellates. The organisms seem to collect in the middle-third of the gut and place themselves with their flagella towards the centre of its lumen, their aflagellar ends being in contact with its wall. The latter is very frail at this time, and the flagellates pass through it with ease. They thus reach the haemocoel, and there they swim for a short time in their normal position with their flagella forwardly directed. Division occurs but infrequently. The flagellates (figs. 23-25) gradually slow their movements and then rapidly concentrate their substance (fig. 27). They soon become typical, post-flagellate forms (figs. 27-31), with distinct nuclei and blepharoplasts, and very thin cyst walls. These organisms are, perhaps, somewhat more frail-looking than those found in the rectum and faeces of the larva (figs. 32-36). At first they are intermingled with the numerous wander cells found in the pupae at this time, but they seem to prefer the more fluid parts of the host. The result is that, in a late pupa 616 in which the musculature has become fairly well developed, the post- flagellates lie in the more fluid medium, internal to the region of differentiated muscles. At times, they have been found crowded together roughly at the centre of the pupa, and seem to be united temporarily by gelatinous material. | When the differentiation of the alimentary canal is in progress, the parasites in most cases become surrounded by it and can be found attached by the gelatinous secretion to the cells of the gut. They remain thus until the emergence of the imago. Four out of fifteen pupae were found infected, but in one only was the infection heavy. C. THe HERPETOMONAD IN THE IMAGO The number of infected imagines is much smaller than that of infected larvae, though neither are abundant. The occurrence of larvae in numbers in one district enables them to become infected with post-flagellates from their neighbours with a certain amount of ease, as they swallow the cysts with their food. But all infected larvae do not give rise to infected pupae, while the latter may have but a slight infection that fades out and disappears in the adult. The habits of the imagines do not readily allow of them acquiring infection ab initio, and consequently the numbers found infected have been extremely small. The parasites have been found in all stages. Non-flagellates, like those in the pupa, have been found in the gut near the junction of the thorax and abdomen. They rapidly form flagellates that spread quickly throughout the whole length of the gut, while post-flagellates occur in the rectum and faeces. No stages of the parasite have been observed up to the present in the genital organs and but few in the haemocoel of the host. When there is haemocoelic infection, there are two possible means of origin: (1) the parasites may have remained there from the pupation period, or (2) they may have penetrated from the gut as flagellates. As they are capable of moving and developing fully in the haemocoelic fluid, hereditary infection is possible, though we are not in a position to pronounce on this with certainty, owing to the small number of infected flies that have been examined and to the difficulty of obtaining eggs. We hope to continue this part of the work in the future. 617 V. MODE OF INFECTION The mode of infection is contaminative originally. The larvae acquire post-flagellate forms of the Herpetomonas with their food. The cysts may have been derived either from already infected larvae or from the dejecta of adult flies. The faeces of a larva on one occasion contained active flagellates as well as post-flagellate forms. As the flagellates lived for some hours in ordinary water, it is possible that they could do so in nature, and act as an additional source of infection of new hosts. The parasite can persist through the metamorphosis of the host and the imagines thus emerge infected. It is possible, though we think it is probably exceptional, that an adult insect may become infected by sucking plant juices contaminated with excrement from other imagines, and thus acquiring the post-flagellate stages of the parasite. VI. CONCLUDING REMARKS The flagellate parasite of Stvatzomyia chameleon and S. potamida is a member of the genus Herpetomonas as originally defined. It differs from H. pediculz (Fantham, 1912) from the body louse. Pediculus vestimentt, in its characteristic, oval, preflagellate form. Herpetomonas jaculum from Nepa cinerea is similar to A. stratiom- ytaeé in appearance, but again the preflagellates of H. stratiomyzae are distinctive, the same feature differentiating the parasite from Hi. lygaez and H. culicts. It also shows differences from H. muscae domesticae, H. ctenocephali, H. ctenophthalmi and other known Herpetomonads. In consequence of these morphological differences, and as it is the first time that such a parasite has been recorded from the Stratiomyidae, the organism has been named Herpetomonas stratiomytae. Ne believe that this account is the first detailed record of the behaviour of an insect flagellate during the pupation of its host, a feature which gives a special interest to Her petomonas strattomytae. The study of the life-cycle of such herpetomonad flagellates of insects is of the utmost importance in view of the recent experiments of Laveran and Franchini (1913) on the successful experimental inoculation of H. ctenocephali (Fantham, 1912), from the gut of the dog-flea, into mice and other mammals. Such researches suggest an experimental leishmaniasis in the making. 618 VII. SUMMARY 1. Hertetomonas stratiomytae, n. sp., 1s a parasite of the larvae, pupae and imagines of the flies, Stvatzomyza chameleon and S. potamida. 2. The herpetomonads present three stages in their life-history, (2) a preflagellate stage, (4) a flagellate stage and (c) a post- flagellate stage. 3. Preflagellate forms are characteristically oval, with elongate nucleus and distinct blepharoplast. The flagellates vary from 20°6u to 57m in total length. The post-flagellates, as found in the hind gut of the larvae, are oval, with somewhat thicker walls than the preflagellates. 4. Multiplication is by longitudinal division and can occur in any phase of the life-history of the organism. 5. Just prior to pupation, the flagellates migrate from the gut of the larva into the haemocoel where they gradually become motionless, withdraw their flagella and assume the post-flagellate form. They collect in the more fluid parts of the body of the pupa, and, ultimately, as a result of this, they become enclosed in the gut of the adult. 6. The imago, if formed from an infected pupa, usually emerges from the pupa case infected with non-flagellate forms of the herpetomonad. The development of the non-flagellate into the flagellate form is rapid, and soon all stages of the organism are present. 7. Infection of the larvae is contaminative, that of the pupa is transmitted from the larva, while the imago may retain the pupal infection or may possibly acquire it ab initio by ingesting contaminated food. 619 REFERENCES Further references will be found in some of the memoirs cited. Fantuam, H. B. (1912). Herpetomonas pedicult, nov. spec., parasitic in the Alimentary Tract of Pediculus vestimenti, the Human Body Louse. Proc. Roy. Soc., B, Vol. LXXXIV, Pp. 505-517. One plate. —— (1912). Some Insect Flagellates and the Problem of the Transmission of Leishmanta. Brit. Med. Journ., November 2, pp. 1196-1197. Laveran, A., and Francuint, G. (1913). Infections expérimentales de Mammifeéres par des Flagellés du tube digestif de Ctenocephalus canis et d’ Anopheles maculipennis. Compt. Rend. Acad. Sci., Vol. CLVII, pp. 744-747. Patton, W. S. (1908). Herpetomonas lygaet. Arch. f. Protistenkunde, Bd. XIII, pp. 1-18. One plate. —— (1912). Studies on the Flagellates of the Genera Herpetomonas, Crithidia and Rhyn- choidomonas. No. 1, The Morphology and Life-History of Herpetomonas culicis, Novy, MacNeal and Torrey. Sci. Mem. Govt. India, No. 57, pp. 1-21. One plate. Porter, A. (1909). The Life Cycle of Herpetomonas jaculum, Léger, parasitic in the Alimentary Tract of Nepa cinerea. Parasitology, Vol. IV, pp. 237-254. One plate. 620 EXPLANATION “OF PLEATED XE All the figures were outlined with an Abbé-Zeiss camera lucida, using one-twelfth inch oil immersion lens and compensating ocular No. 8. Magnification 1,500 diameters, approximately. Figs. Figs. Fig. Fig. Figs. Fig. Fig. Fig. Figs. Figs. 1-22. Illustrate the preflagellate and flagellate stages found in the larva. 1-9. Illustrate the development of Herpetomonas stratiomyiae from the preflagellate to the flagellate stage. 1. Preflagellate form with nuclear chromatin evenly distributed. 2. Preflagellate with nucleus showing a karyosome. 3-5. Stages in the division of a preflagellate. 3. Preflagellate with blepharoplast dividing. 4. Form with blepharoplast and nucleus dividing. 5. Separation of the daughter organisms. 6-9. Show the development of a flagellum and assumption of the flagellate form. 10-16. A series of flagellates showing variation in size and appearance. . II, 13. Show flagellates containing chromatoid granules. . 17. An aggregation rosette of flagellates of different ages, attached by their flagella to a piece of débris. . 18-22. Stages in the division of flagellates. . 18. Parasite showing constriction of blepharoplast. . 19. Parasite with flagellum splitting. . 20. Form with flagella and blepharoplast separate and nucleus dividing. . 21. Commencement of division of the general body cytoplasm. Daughter organisms almost separate. 2. . 23-30. Illustrate post-flagellate stages obtained from larvae and pupae. . 23-25. Transitional forms between flagellate and _post- flagellate stages, as obtained from the haemocoel of the larva. 26. Similar transitional flagellate from the hind gut of a larva. 27-31. Post-flagellates from pupae. 32-36. Post-flagellates from the hind gut of a larva. 621 THE .CULLURHim@ bt. BABESIA (PIROPLASMA) CANIS IN VITRO BY J. G. THOMSON, M.A., M.B., Cu.B. (EDIN.) (CLINICAL PATHOLOGICAL ASSISTANT, LIVERPOOL SCHOOL OF TROPICAL MEDICINE, AND PATHOLOGIST, ROYAL SOUTHERN HOSPITAL, LIVERPOOL) AND H. B. FANTHAM, D.Sc. (LOND.), B.A. (CANTAB.) (LECTURER ON PARASITOLOGY, LIVERPOOL SCHOOL OF TROPICAL MEDICINE) (Received for publication 10 December, 1913) PLATE XLII CONTENTS PAGE INTRODUCTION <6 Hee aac ees =a os ace wee 621 TECHNIQUE ... a aa Ach He aa RSS tes the 622 PROGRESS OF THE CULTURES see see Se Sis mats EB 622 MorpuHotocGy oF THE CuttTuRAaL Forms... aoe 508 ae soe 624. DIviIsIon OF PARASITES IN VITRO... Ae 45D ae bake enh AS 626 SuccessruL INOCULATION OF THE ANIMAL Host FROM A 41 HOURS CULTURE 629 SUMMARY ace ee bed oe oes Br, eee 1s oe 62 REFERENCES... anc ae ae ee eee ee ae ck 630 EXPLANATION OF PLATE ae A 8 as Ae sak Ws 632 INTRODUCTION The present paper contains the preliminary results of the authors’ investigations on the cultural forms of Babesza canis. The importance of the investigation of cultural forms of Protozoa does not need emphasis, and many practical applications are suggested, but in this paper we confine ourselves to some remarks on the morphology and life-history of the Sporozo6én as seen in the culture tube. The method of C. C. Bass (1912) has been followed, without any addition or modification, though the parasite is not so easily cultivated as the Plasmodia of man. 622 TECHNIQUE Cultivation succeeded in two out of four attempts. In each successful case the infected animal, whose blood was used, was a puppy about three months old. The blood used in the first culture was taken from the heart on the fifth day after inoculation, in the second case on the fourth day after inoculation. Ten c.c. of heart blood, drawn with aseptic precautions, was mixed with 1/10 c.c. of a 50 per cent. aqueous solution of Merck’s glucose. The blood was carefully and_ gently defibrinated by means of a rod, and the clot was removed. It was noticed that the amount of clot removed was much in excess of that taken from a similar quantity of human malarial blood. The defibrinated blood was distributed into smaller tubes, placing about one inch of liquid in each tube. No centrifugalisation was necessary. The tubes were incubated at 37°C. The corpuscles settled to the bottom in a short time, leaving a layer of serum above. Marked haemolysis was seen in all the cultures attempted. It was found advantageous to take the blood of the puppy before the crisis, that is, before too many parasites were present in the peripheral or heart blood. No sodium citrate nor ascitic fluid was added to the cultures, as was done by Ziemann (1913), nor citrate and saline as used by Toyoda (1913). PROGRESS OF THE CULTURES We will set forth in some detail the progress of events in our FIRST CULTURE, which was more successful than the second, in that more divisions occurred in it. In the original heart blood, before inoculation, the parasites were not very numerous, pairs or singles being found in the infected red blood corpuscle (Pl. XLII, fig. 1). Only one group of four parasites was seen in any corpuscle.* The Babesia were chiefly pyriform in shape, with small compact nuclei, and the secondary * Graham-Smith (1905) found that the infected red blood corpuscles containing more than four Babesta canis constituted less than 0°3 % of the total. He counted 22,589 infected corpuscles from peripheral and heart blood of dogs. | Corpuscles containing one and two parasites formed 96-4 % of the total. (Yourm. Hygiene, V, p. 252) ee ee eee 623 loose mass of chromatin was not well marked. Phagocytosis was evident, one large mononuclear leucocyte had ingested four infected corpuscles (Pl. XLII, fig. 2). After 7 hours large numbers of intracorpuscular parasites occurred in clumps, especially at the margins of the smears. The infected corpuscles usually contained four parasites each (Pl. XLII, fig. 3), a few contained eight merozoites (Pl. XLII, fig. 4). Hence the original parasites had divided once. The piroplasms, usually pyriform, now showed clearly the secondary loose mass of chromatin. Examples of division by budding and chromatin forking (Pl. XLII, fig. 5), as described by Nuttall and Graham- Smith (1907) from dog’s blood, were seen. After 15 ours numerous infected corpuscles contained four parasites, some corpuscles contained eight, while a very few showed sixteen merozoites (Pl. XLII, fig. 7). At 24 hours there was an increase in the number of infected corpuscles containing eight parasites, as well as in those containing sixteen (P]. XLII, fig. 8). Corpuscles containing four piroplasms were also present. Rounded dividing forms, exhibiting chromatinic forking were found. There was thus evidence of another division beginning. After 30 ours some parasites were seen to be degenerating. Corpuscles containing eight living piroplasms were numerous. The parasites seemed to have grown larger. After 48 Zours numerous corpuscles were found to contain sixteen merozoites (Pl. XLII, fig. 9). One cluster of 29 was found. Three of the parasites in this group each showed two chromatinic dots, and were probably about to divide, so that the cluster represented 32 daughter forms derived from one parent Babesia. The host ° corpuscle had burst, but its remains could be distinguished. At 60 hours the parasites were few and were smaller in size. Two groups of eight and one of four were noticed in one smear. These parasites had circular chromatin masses. No others were seen on this smear. Most of the cultural piroplasms were now dead, and at 68 hours none was found. Three divisions had occurred in the cultures, from twos, through fours to eights and sixteens in infected red blood corpuscles. Heart blood of the dog, kept as control, and incubated at 37° C., 624 showed a few somewhat shrunken parasites after 24 hours, and no parasites after. In our SECOND CULTURE only pairs or single pyriform parasites were found in the heart blood before incubation. This culture progressed more slowly than the former. After 6 Aours’ incubation there was more variety in the form of the parasites, some being larger and round and some amoeboid. At 7 hours a very few parasites were showing the commence- ment of gemmation. After 8! hours some free pyriforms were found and an intra- corpuscular group of four. The nuclei of these contained small dots (karyosomes) and a little loose chromatin. At 16 hours fours were more numerous, and distinct chromatinic budding was seen in several specimens. After 181 hours there were numerous groups of four pyriform parasites, a few eights and one group of twelve. The loose chromatin was well marked. There was evidence of various types of division. Some degenerating forms were now noticed. At 234 ours corpuscles were seen containing one, two and four parasites. At 244 hours groups of four merozoites were fairly common. At 30 fours many fours were present and some groups of eight. At 41 hours groups of fours were still found, and } c.c. of the culture, containing most of the corpuscles, was inoculated into a young puppy. The puppy developed piroplasmosis and succumbed five days later. The remains of the culture were examined at about 66 hours and no live parasites were seen. There is thus evidence that only two divisions occurred in this culture. It certainly did not grow so well nor so rapidly as the first. We are unable to explain the cause of this difference in the progress of the two cultures, but we may remark that the strain in the second case was of a more chronic character. MORPHOLOGY OF THE CULTURAL FORMS The piroplasms were examined fresh, and after fixation and staining. Smears were made from time to time and fixed by the wet and by the dry methods, and stained by Romanowsky, Giemsa 625 and haematin stains. Bouin’s fluid was used in some cases as a fixative. In this paper we do not propose to deal exhaustively with the morphology of the cultural forms of Aabesza canis, but to record only the more important findings. The parasites exhibited marked variation in shape. Pyriform, amoeboid, round and oval types were seen (Fig. 1, A—D). The method of gemmation and chromatinic forking was observed, as first described by Nuttall and Graham-Smith from the blood of the dog. There was also evidence of binary fission. The pyriform piroplasms (Fig. 1, A—B) usually exhibited a distinct nucleus, as a dot of chromatin, often surrounded by a clear achromatic halo. Such a nucleus is of the karyosomatic type, the chromatinic dot representing the karyosome. Such a structure has Fic. 1. Various forms of Babesta canis in culture. A, B. Two pyriform parasites showing variation in nuclear position. Loose chromatin also present. C. Amoeboid form. D. Rounded form. been recorded in Babesia canis by Schuberg and Reichenow (1912). The presence of a very thin nuclear membrane is sometimes suggested, but at other times such a nuclear membrane is certainly not well marked. A secondary mass of loose chromatin, of a reticulate or ‘woolly’ character is also seen, as described from blood by Nuttall and Graham-Smith and by Christophers (1907) in Babesia cants, and by one of us (Fantham, 1907) in Babesia bovis. The secondary mass of chromatin was well seen in 7-hour cultures. In some pyriform parasites a very small dot of chromatin was observed (Fig. 1, B), the so-called blepharoplast of Schaudinn and Liihe; 626 but this punctiform chromatinic mass is not comparable with the blepharoplast of a flagellate. Pyriform Babesia in cultures of 7 hours’ duration had the nucleus usually near the pointed end (Fig. 1, A), while after 24 hours’ culture it was sometimes seen to be near the rounded or blunt end of the parasite (Fig. 1, B). Thus the position of the nucleus may vary in different specimens. Some amoeboid forms with fine pseudopodia were present, especially in 7 hour cultures (Fig. 1, C). Such parasites were also found, motile, on examining fresh preparations. Rounded forms often showed signs of division by the method of gemmation. Fic. 2. Infected corpuscle containing four parasites. The upper one of the four is oval, and is Leishmania-like ; the lower three show masses of loose chromatin. In one case, in a 15 hours’ culture, a remarkable Leishmania-like oval form was seen, with nucleus and blepharoplast (Fig. 2, oval parasite). DIVISION OF PARASITES IN VITRO Many examples were found of the mode of gemmation with chromatinic forking (Figs. 3, 4), now made a diagnostic character of the genus Babesia. Round piroplasms protrude two small buds symmetrically arranged to one side (Fig. 3, C), which buds contain chromatinic cores connected with the main nucleus (Fig. 3, entail ee La a air 627 D—G). In some cases the buds are almost entirely composed of chromatin at first, and seem as if they are about to separate from the parent. The buds then grow at the expense of the rounded Fic. 3. Babesia showing division by gemmation, accompanied by chromatin budding and forking. A. Parasite with arcuate chromatin. B. Somewhat amoeboid parasite with chromatin fork. C. Shows small, symmetrical cytoplasmic buds and chromatin processes entering buds. D. Parasite with larger buds; main chromatin not distinguishable, but chromatin cores well marked. E. Larger buds shown. Well marked main chromatin mass and processes. F. Parasite showing commencement of fission of main chromatin mass. G. Form showing typical Y shaped chromatin bifurcation. portion of the parent parasite, and the linear processes of chromatin give rise to the loose mass of secondary chromatin seen in the daughter pyriform piroplasms. Early stages of chromatinic 628 budding and forking were seen while the parasite was still round or somewhat amoeboid (Fig. 3, B) in contour. Various stages of division may be observed concurrently in several parasites in one corpuscle (Fig. 4). Certain cases were noticed in which the chromatinic forking did not assume the typical Y form (Fig. 3, G), but was arcuate in character (Fig. 3, A). Sometimes it is difficult (vide Fig. 3, D) to observe the main chromatin mass from which the forks arise. It is unnecessary to enter into a lengthy description of this mode of division, as it has already been described by various Fic. 4. Infected corpuscle showing parasites in various stages of division. authors from the blood of dogs. The present is the first record of its occurrence in cultures, and we also succeeded in seeing the method in operation in living parasites taken from cultures. Christophers (1907) considers that, beside the mode described above, there is also division by direct binary fission. Nuttall and Graham-Smith have recorded such direct division in the case of rounded blood forms of Babesia canis. We agree with the above- mentioned workers, as parasites of various shapes are often seen with two and even four principal chromatinic masses (Fig. 5, A—B). We have also seen parasites whose nucleus exhibited a form of promitosis. Further, we have observed long, somewhat sausage- shaped parasites, containing two chromatin masses, and others OE 629 indented in the middle, apparently dividing into two pyriforms, as figured by Christophers (1907, p. 21). A. B. Fic. 5. Two parasites showing direct division. A has two nuclei, B has four chromatin masses. The free forms of the piroplasms, seen in cultures, which were attempting to enter fresh blood corpuscles, were invariably of the pyriform type, and were endeavouring to enter by the blunt or rounded ends. SUCCESSFUL INOCULATION OF THE ANIMAL HOST FROM A 41 HOURS’ CULTURE A young puppy was inoculated successfully with about } c.c. of a 41 hours’ culture of Babesia canis. The inoculation was performed intraperitoneally. The puppy showed numerous parasites on the early morning of the fifth day after inoculation, and died on the evening of the same day. Unfortunately, the removal of a series of portions of the culture for examination, has hitherto left us with insufficient material to attempt sub-cultures. We have, however, no doubt that such sub- cultures would be successful, as was shown by Ziemann (1913). SUMMARY 1. We have succeeded in cultivating Babesza (Piroplasma) canis in two out of four attempts, following the method of Bass, using blood and glucose, and incubating at 37°C. 2. In one of these cultures, starting with heart blood containing corpuscles infected with one, two or, exceptionally, four piro- plasmata, we succeeded in obtaining a maximum of 32 merozoites in a corpuscle. 630 3. Various types of Babesia were seen in these cultures, namely, pyriform, amoeboid, rounded and oval parasites. Division of rounded forms was observed, following the method of gemmation with chromatinic forking. There was evidence, in stained specimens, of direct binary fission. 4. Haemolysis occurred in all the culture tubes. 5. A puppy was successfully inoculated from a 41 hours’ culture and succumbed to piroplasmosis on the fifth day. 6. Babesia canis is not so easily cultivated by Bass’s method as the malarial parasites of man. REFERENCES Further references will be found at the ends of the memoirs cited. Bass, C. C., and Jouns, F. M. (1912). The Cultivation of Malarial Plasmodia (Plasmodium vivax and P. falciparum) in vitro. Journ. Exper. Med., Vol. XVI, pp. 567-579. Curistopuers, S. R. (1907). Piroplasma canis and its Life-cycle in the Tick. Sci. Memoirs Govt. India, No. 29, 82 pp. ‘Three plates. Fantuam, H. B. (1907). The Chromatin Masses of Piroplasma bigeminum (Babesia bovis), the parasite of Texas Cattle Fever. Quart. Journ. Microsc. Sci., Vol. LI, pp. 297-324. One plate. Nurrati, G. H. F., and Granam-Smiru, G. S. (1907). Canine Piroplasmosis VI. Journ. Hygiene, Vol. VII, pp. 232-272. ‘Two plates. Scnuperc, A., and RetcHenow, E. (1912). Uber Bau und Vermehrung von Babesia canis im Blute des Hundes. Arbeit. a. d. Kaiserl. Gesundheitsamte, Bd. 28, pp. 415-434. One plate. Tuomson, J. G., Tomson, D., and Fanruam, H. B. (1913). The Cultivation of one genera- tion of Benign Tertian Malarial Parasites (Plasmodium vivax) in vitro, by Bass’s method. Annals Trop. Med. and Parasitol., VII, pp. 153-164. One plate. Toyopa, H. (1913). Ziichtungsversuche mit Babesia canis nach der Bassschen Methode. Centralbl. f. Bakt., Abt. I, Orig., Bd. 72, pp. 76-81. One plate. Ziemann, H. (1913). Uber die Kultur der Malariaparasiten und der Piroplasmen (Piroplasma canis) in vitro. Archiv f. Schiffs-u. Tropen-Hygiene, Bd. 17, pp. 361-391. Two plates. Also in Trans. Soc. Trop. Med. and Hyg., Vol. VI, pp. 220-227. 2 = a Max” eae placa a, 3% ator} bats Pastis inlined snd oye Wee aac: wnt sand: age, Yo bool dead al ba Seischid- abt > aeorgiogeng ewore - iOlisdyani switsd bonkd teat mor spolnEnqro> = y aalaaucgian Saba giirwode aliqurgodadqorsilé 4 orellag fawoil, tise * wiged ee ie Das owt i, nn) _goliosorsint sss to. ane nore mt “ fidsmorl> oepol fede Per dies coe ont zd noiaivib ‘to. eesOuUT ft Rodiew red] om “ipvee unblr. atantouds bps” aovsnmeg ia. \ .@matlia % : - yore a ie seoloueaprionn “aword cs 8 mest restigsing: lo avo. “htitw cenlisgot = fats ~~ | ries oye bas Selah to aquow bili Peo poo Gest011 eohosoron nealing 46. quoi - 28 ewor *e Ronin) ‘eollcise an =esatvie io qucI- 6 S8iz bi, Sefsaoni ert Fig. Fig. Fig. Fig. Fig. Fig. N Ni 632 EXPLANAIION. OR (Pia ir eit Microphotograph showing pyriform Babesza canzs from heart blood of puppy before incubation. Shows phagocytosis of four infected red _ blood corpuscles. From heart blood before incubation. Microphotograph showing infected corpuscles containing two and four Babesia. Seven hours’ culture. Division rosette of eight merozoites from seven hours’ culture. Some show loose chromatin. Two parasites in process of division by the method of gemmation and chromatin forking. Seven hours’ culture. Shows mononuclear leucocytosis in _ piroplasmosis, together with groups of parasites, from a 15 hours’ culture. Groups of eight and four parasites from a 15 hours’ culture. Group of sixteen merozoites from a 24 hours’ culture. Group of sixteen merozoites from a 48 hours’ culture, showing increase in size. INDEX LENDERS ORCATEDORS ee ten eerie nso ie co SRT aerate NOR oa rte ae ones CCOTENTSRUAUTS LIN DIOXCs oe SMM OS rcs oo catcianie Sacaiacroteeie MERMES ee eens END EXT OPSSPECIES NEW EOLOCIENIGCE, « 5.c.c0s x.ccccrd eo.cfb quem contesinine tetilesce INDEX OF AUTHORS PAGE Balfour, A. . baie vices cane a Barratt, J. O. fiat eweieds bbenee 367 Blacklock, bat epee MILL TG OL Blacklock, B.; and Stephens, J. W.W. 303 Blacklock, B.; and Yorke, W. ...563, 603 Garter, H. F. SiR SE Chalmers, A. Ie; and O” Farrell, W.R. Q Chalmers, A. ie: sand Stirling, AI D. : Christophers, S. R.. Wh gita2 Les Darko Ss Loony 321 Fantham, H. B. ; and. Porter; AL "569, 609 Fantham, H. Fe and Stephens, J-eWaWs 27 Fantham, H B.; : . and Thomson, J. G. 621 Fantham, H. B.; Thomson, J G.; and Thomson, D. eg: RLS3 Kinghorn, A.; Meson wie : aon Lloyd, Ll... ss EA .« oR NTS Lloyd, Ll.; “Kinghorn, A :- and Yorke, W.. S183 Lloyd, Lies Wallace, / WA HE areas 299 Macfie, J. W. Scott... #15°339,1359 Peale ny Pe od sacha 2285s ORI AN AOS 35 inven ye te PanGrmeets To 1 teeter engl Mawsteacksie izle... t.. asenat.d. OST O’Farrell, W. R.. i545 O’Farrell, W. Be ae ena eal tthe §25 GENERAL PAGE Aedimorphus quinquepunctata, nov. sp. 598 Ankylostomiasis, prevention in the Panama Canal Zone 1S 7 Anophelinae, classification oon ‘phylo- BEMVyUOL >) ce Ret bese. OT Colour marking other variable characters of 45 x Geographical distribu- DPOF On 5 sn ois Yona deus teead: > Grouping of species ... 87 PAGE ili ill vill PAGE Porter, A. ; and pages aa Hei: oe 609 RoserssiWio xc... 25303 Ross, G. A. Park.. ee) 3 71 Scott, H. Harold.. $A GROS Seidelin, Harald . Binz Stephens, J. W. W.. 479 Stephens, J. W. Wes Meal ‘Black- Naekes Bos tre: Series Jec!:.< GOR Stephens, Je W. W. r fad Fantham, HH Bs ied 27 Stirling, Line sD; wiyends ‘Chalmers, Aq): ee egy Theobald, “Fred. V... Stas. “SOT Thomson, David.. ees eG Thomson, D.; Deatasere oe Be: and Thomson, J. G. Py, Thomson, D.; and Thomson, uh ich . 509 ities, J. G.; and Heap an. lle B. 621 ‘Thomson, J. ai and Thomson, D.... 509 Thomson, J. G.; Thomson, D.; and ae HB: Me ocads OES Todd, John L.. -» 309 Wallace, ALES saten ZOD Wallace, A. F. ; “and Lloyd, HILIED Mei 299 Yorke, W. ; and Blacklock, B. ...563, 603 Yorke; W.3)+' lloyds 7 Lib paand Kinghorn, A. SBISE SUS RED OS INDEX PAGE Babesta canis, cultivation in vitro ... 621 »» Morphology in cultures 624 Siiou A. ‘Trypanosomiasis in the Sudan and notes on tsetse fly traps and immune breed of cattle......... 113 Banksinella, scare mosquitos of ther genus? «Ci eevte nea cr ies, HOt Barracu je: W. NReCenE experi- mental research bearing upon black- WATCEPIE VET IMA. 0055 Letoen wedtercc es FSOT PAGE Blacklock, B. Posterior nuclear forms of 7. rhodesiense in rats .... Blacklock, B., and Stephens, J. W. W. Non- Jdentity of I. brucei with the trypanosome of the same name from the Uganda ox (T. ugandae, sp. nov.) Blacklock, B., and Yorke, W. Try- panosoma vivax in rabies Fes Blacklock, B., and Yorke, W. Tikes probable identity of I. congolense (Broden) and 7. nanum (Laveran) Blackwater fever, Correlation between malaria and black- water statistics... Effect of period of residence ......... » Influence of malaria Malarial parasites... Pigmented __leuco- YES aarieces oss x ,, Post-mortem exami- nasties PL. ae Recent experimental research ons papling. 3.005, .cceiee Zs re Relationship to species of malarial parasite J... Seasonal prevalence Second attacks Statistical . British Ginana; Sanitation in . Garrer; BF: Mosquitos of the genera Banksinella, Theobald, and Taenio- rhynchus, Arribalzaga Cattle breed immune to trypanoso- miasis - LKQ; Cerebro-spinal meningitis, fhe ing, in Jamaica .... : Chalmers, A. J., and O'Farrell, W.R. The Trichonocardiases ..... Chalmers, A. J., and Stirling, A. D. Epidemic Trichonocardiasis ........ Christophers, $. R. Colour marking and other variable characters of Anophelinae .. Chrysoconops nociurnus, nov. sp. Crithidia byalommace, hereditary infec- tion with, of Hyalomma aegyptium ... 593 Io! 303 563 603 499 488 486 +19 485 . 485 367 ... 486 492 504 syn thet 45 545 iv PAGE Crithidia hyalommae, Morphology Culicelsa centropunctata, Nov. sp....... Culicidae from the Sudan : Darling, S. T. Identification of the pathogenic entamoeba of Panama... Diplococcus jamatcensis, sp. nov. Causing vomi- ting sickness in Jamaica .. Domestic Animals, ‘Trypanosomiasis in Dysentery, prevention in the Panama Canal Zone . : ‘ Entamoeba, feeding and “inoculation experiments 32 33 Identification of the pathogenic, of Panama Fantham, H. B., and Porter, A. The pathogenicity of Nosema apis to insects other than hive bees......... Fantham, H. B., and Porter, 3° ” oh MENMENEISIM lylis.suteose 1O5 » Stegomyta fasciata in ......... 467 Ea Outing Sickness in .:..:...: 377 ide Clow, lever iit t.¢,..sqeeeenes 407 Kinghorn, A., Yorke, W., and Lloyd, Ll. Final report, Luangwa goes prcless(ConimissiOm...2.....80.5-.-.- 183 Kingia maculoabdominalis, nov. sp. 597 Lloyd, Ll., Kinghorn, A., and Yorke, W. Final report, Luangwa oo Sickness'Commission ........3.0....2.. 183 Lloyd, LI., and Wallaces A. F. Experiment to ascertain whether Tabanids transmit trypanosomes in GAPBES role seaiheuet ile PAGE Plague, prevention in the Panama Canal Zone. 24187 Plasmodium jalciparum, “Morphology it in culeareci.e< 513 z 6 Morphology in the human H@Ség: - 25.5 510 = vivax, Cultivation in vitro 153 $5 “5 ips cola cul- Pare” -paee 518 Rs £ Morphology i in — human host ... 519 Porter, A., and Fantham, H. B. The pathogenicity of Nosema apis to insects other than hive bees......... 569 Porter, A., and Fantham, H. B. Herpetomonas stratiomyiae, n.sp., a flagellate parasite of the flies, S. chameleon and S. potamida, with remarks on the biology of the hosts 609 Puptpara in N. Rhodesia ................ 297 Reedomyia sudanensis, nov. sp.......... 595 Rhodesia, Northern, blood-sucking insects and ticks in the Luangwa Valley 293 3 4 Meteorological observations 189, 190 Rogers, W. A note ona case of Loa DBO Se sence sae ea Uso nn once POMS Ross, G. A. P. “A fictitious native disease (Lsigwebedhla) ¥ 3yh Sanitation in the Panama Canal Zone 125 Scott, H. H. Fulminating cerebro- spinal meningitis in Jamaica......... 165 Seidelin, H. ‘ Vomiting Sickness’ in Jamaica ee) See 7 | Sleeping Sickness Commision. Final report of the Luangwa ......... 183 z Z Digaisngen of, one to I. rhodesiense 185 = : in Eket, S. Nigeria 339 a » symptomatology ... 186 9» 3 Sex and age of Africans suffering from . 309 Southern Nigeria, sleeping sickness due EO Dir WIGETIENSC, OSD sso cotencaccap sey 2990 | PAGE Sudan, cattle breed immune to try- panosomiasis ..119, 124 »» New Culicidae from the 591 a ‘Trypanosomiasis Ns wzivecak eae Stegomyia fasciata in Jamaica ...... Stephens, J. W. W. Studies in black- Water, feéver.s..; . 79 Stephens, J. W. W., aad Black laces zB Non-identity of ZT. brucei with ‘the trypanosome of the same name from the Uganda ox (T. ugandae, sp. nov.) 303 Stephens, J. W. W., and Fantham, H. B. Measurements of T. hides, tense and T. gambiense ........ 27 Stirling, A. D., and Chalmers, AM af Epidemic iichonwesediaes Sey 5: Stomoxys possibly transmitting try- panosomiasis 3s nigra, trypanosome in gut of 359 Stratiomyia chameleon, infected with Herpetomonas Stratiomy1ae = 5. DIOlOSy Ont _ potamida, infected with Herpetomonas stratiomytae a 35...) Biology: of crs. Stratiomys, vide Stratiomyia ......0++..- Tabanidae in N. Rhodesia Tabanus, trypanosome transmission EXperIMENty ius cea! . 299 Taeniorhynchus, ‘Theobald vase of the genus.. : Theobald, F. V. New Cilicidaes front the Sudan . 591 Thomson, D. Conitaeioul int dhe Panama Canal Zone, Trinidad and British Guiana.. i Thomson, D., Fag than ‘i. ipa aaa Thomson, ip G. Cultivation of Plasmodium vivax 1n Vitro..........+. Thomson, D., and Thomson, J. G. Growth and sporulation of the benign and malignant tertian malarial parasites in the culture tube and in the Iraman* host. .:...-.cec Thomson, J. G., and Faia H. B. The culture of Babeun Cae canis in vitro........... ; ge Sr seen 609 610 609 610 - 585 Tegale 153 - 509 PAGE Thomson, J. G., and Thomson, D. Growth and sporulation of the benign and malignant tertian malarial parasites in the culture tube anaeam the human host:.-.....4...0.. Thomson, J. G., Thomson, D., and Fantham, H. B. Cultivation of Plasmodium vivax in vitro.. 2153 Todd, J. L. Sex and age of Africans suffering from Trypanosomiasis...... 309 Trichonocardiasis Beh sete eS epidemic Ae acy. Trinidad, Sanitation in. 141 Trypanosomes in the gut of Stomoxy nigra =. se 359 a in wild G. rare 239 of game and domestic stock ° so eg - Transmission experi- ment with Tabanids Trypanosomiasis, Cattle breed immune to.. PLIOMIZ4 Sune wide Sleep- ing Genet = Fromunity- aot, 10 Hf In cattle, ‘ebagonal distribution ...... 7,8 fe Incubation and duration of, due to I. rhodesiense 103 99 In the Lado(Western Mongalla, Sudan) 113 a Of domestic animals in N. Nigeria...... I - Prophylaxis ....... o Transmitted possibly by Stomoxys ...... 8 Treatment ..... 9 Tsetse fly traps 118, 124 _ ane in domestic animals ...... ZS. ih >) Non-identity with the try- panosome of the same name from the Uganda ox (I. ugandae, sp. nov.) 303 ne Posterior nuclear forms...... 3 . congolense from donkey ............ 122 a Probable identity of, and TER RANE... 828 222 A OOR . gambiense, Measurements of ...... 27 NEROIMIESPATMOV... 0s... 2:2. ts2d,00 osh> 203 ~ APTOS! 5 aot. eetexs ZOO vil | PAGE T. ignotum In wild G. morsitans, 240, 242 43 Measurements 5a. 204 A Morphology sax, 203 x Pathogenicity .........:..... 265 a Transmission . at 206 . multiforme, sp. NOV.. eo wcy is Diagnosis. ae 257 oe In game in N. “Rhodesia 233 3 Measurements ...... 255, 259 ui Morphology .. wae Ly _ Pathogenicity . ee, Ss Transmission . 261 . nanum In domestic animals, 2, 3, 7, 235 et In game in N. Rhodesia, 231, ce 234 53 Measurements aut « 249 5 Morphology ..... 248 - Pathogenicity 248 Probable identity af, : and F.. concolense® .. Leek... 603 ns TPransmnsston.. Sietoese. 200. 251 T. nigeriense, n. sp. 356 i Animal reactions ...... 345 : Measurements ............ 348 Morphology 343 . pecorum from bull...... L2Z 9% In domestic animals, 2, 3, 7, 235 - In game in N. Rhodesia, 233, 234 5s In wild G. morsitans, 240, 243 Measurements <¢.0...:-..--.<. 252 #3 Morphology 2251 3 Pathogenicity” $22: -...)s-00 254 ‘Transmission = 20% S indeseniee a seek . 245 i Development in G. mor- sitans : ers - Development 1 in REN mor- sitans influenced by meteorological con- GUTIOBS! 53. pncgensinnsce5 COL a Distribution of . 185 - In domestic animals ... 235 3 In game in N. Rhodesia, 233, 234 ig In G. morsitans in nature 215, 240, 242 < Measurements of ... 27, 218 a Pathogenicity of ......... 223 ee Posterior nuclear forms IDS aet seems eo eta ae LOE peer 0 Posterior nuclear forms... 3 vill T. vivax In domestic animals, 2, 3, 6, 235 ss, In game in N. Rhodesia, 231, 233, 234 i, In rabbits een 503 ., Measurements of . 246 Su. Morphologyroitcmiss ycer. 245 ay bathogenicity Of :iJc: eens 247 Transmission ae 247 Trypanosoma, sp. (? montgomeryi), diag nosis .. i203 - role ull montgomeryi). ne domestic animals ... 235 9 sp. (? montgomery1), mor- phology tet G2 INDEX OF SPECIES PAGE ~ Aedimorphus quinquepunctata............ 598 Chrysoconops nocturnus . 593 Culicelsa centropunctata ..........0000++ 599 Diplococcus JAMAICENSIS sss see eeveerveees 465 Glosstaig StiIg GTi vein as inn saaoeen << sad endee GE Heptaphlebomyia kingi. Me fept ein sotene te TOOL Herpetomonas stratiomytae ............... 609 Kingia maculoabdominalts ............... 597 Trypanosoma, sp. (? montgomeryt), measurements ......... 3 sp. (?montgomeryt), pathogenicity ......... sp. (? montgomeryt), transmission Typhoid, oo lgaaae in the Panama Canal Zone . ‘ Vomiting sickness in “Jamaica Wallace, A. F. Attempt to transmit T. rhodesiense by means of O. mou- batgadeot es Wallace, v3 F.. ae Cad Lloyd, “Li oe periment. to ascertain whether Tabanids transmit trypanosomes in DATUTOry-.nvecicm raflewne Yellow fever in Jamaica. Sec ee Preventionin the Panama Canal. Zonep. s..:<2. Yorke, W., and Blacklock, B. Try- panosoma vivax in rabbits sisi Yorke, W., and Blacklock, B. oie probable identity of TZ. congolense (Broden) and 7. nanum (Laveran)... Yorke, W., Lloyd, LI., and Kinghorn, A. Banal report, Luangwa oe sickness commission Leaiet 3 99 99 NEW TO SCIENCE Micrococcus castellantt Wi weidais, Migerrimus: "spn. dtocoue nce gore RECAOMY1A SUAANENSIS. 6.2. sjtene eens sere Trypanosoma 1QNOtUM .. 22. se eseese cee eaees multiforme NU CEMUCMSPN | cry vccteon chennai LA RCLAPDE, «ax 5.2 shite ugandae PAGE 262 261 263 oe, 138 - 377 . 301 .. 299 . 467 136 563 603 > 183 Annals Trop. Med. & Parasitol., Vol. VII PEATEM iG C. Tinling & Co., Ltd., Imp. IIT EER IS jews ., Vol. VII Parasitol — Med. Annals Trop. ae” ot wre et IG. 4 4 I Co., Ltd., Imp. & C. Tinling Annals Trop. Med. & Parasitol., Vol. VII PEATE Tit eS, uke bat ‘wig at teh Moc Ye Mae NR op a. fe aed poe Mi es. ents Pe iy a) 2. 4" “@ } aa a2) ‘ * pn . >) @ r “f y Fe é SR ~ = , ‘ oe eg Ss NO VE ege@e, O® xs a Ad ted oe e é of *" e@ 2 * - Vf, ’ za \\ ite ® ‘“ eo 4 oe a ce Z a” g é & i %e ai 5 * @ r) e ' x F 7 i 09 I~ ee NP 0? i ote vie 25 a /% 3) of é ee. . shee e 4 = @ Sree" <: Py J & fost or 4 “so ¢ ’ ee" Ae Ee 2 et, Ee dg serge, 9/6 <7 4e Se be) g 9 wm, 2,P as ao 5 9? Pad «3 % ia Year 0 &) @ ® Se on é pi ;: é . 62 ® © : Q Lg @ }$ ° a ae Ms ? S37 e A ® 06. 09% Ae '* @ @ oT ) @oae as ®@ :) G . fal! @ 9e we Ih, ~ “e hfe 0 A a) & S ® 608 5 3 MMe 4 Margrethe Seidelin pinc. Annals Trop. Med. & Parasitol., Vol. VII PLATE XXX Margrethe Seidelin pins. ae ay “i ” hig, vine ue 8 - wie ¥ he te PLATE XXXII Annals Trop. Med. & Parasitol., Vol. VII ° 1 te = 'S ap PLATE XXXIII Annals Trop. Med. & Parasitol., Vol. VII *eh> HF) ta ' = RED otnnals Trop. Med. d. Parasitol., Vol. VII. ie Di 3 aX ~ fs A i ‘ Fy" 6. Wy e @ } doo * ‘= a @ Ea. 8 pag £ aes, 4995 8% — ies 12 dni 16. 6 as =e Ss es a 2 ‘ Nas aie ros sg) ae £3 bi, es ‘ci Nigedeg = : ise P. 21 Az J.G.Thamsan & D.Thomson, del. CULTURAL FORMS OF MALARIAL PARASITES. eo) 18. @,, BSE 20. Annals Trop. Med. & Parasitol., Vol. V11 PLATE XXXVI a Be ay 2 ry ~ ‘2 2 > “it a8 e. ee +~ Ce . e He, * ote: