STATION BULLETIN 416 Avian Mass Immunization for Infectious Bronchitis and Newcastle Disease W. R. Dunlop R. G. Strout AGRICULTURAL EXPERIMENT STATION UNIVERSITY OF NEW HAMPSHIRE DURHAM, N. H. ACKNOWLEDGMENTS The authors wish to express their appreciation to Professor W. C. Skoglund, Head of the Department of Poultry Husbandry at the University of Xew Hampshire, for his guidance in the re- search program and the preparation of this liulletin. We are also grateful to Mr. Robert E. Leventhal, Graduate Research Assistant in Poultry, and to Mr. Donald E. Coller, Senior Laboratory Technician, both of the Poultry Department. University of New Hampshire. Thanks also are extendetl to the many poultrymen in New Hampshire who cooperated in this study. The Poultry Disease Research Committee of the New Hampshire Poultry Growers Association was instrumental in organizing the poultrymen. With- out their financial help and willingness to provide experimental flocks this study would not have been possil)le. Acknowledgement is also made to the Lederle Laboratories Division of the American Cyanamid Co. for providing a research grant to make part of this study possible. In addition, thanks are also extended to the following poultry students at the University who did the actual spra}'ing in the field : Edwin Antz, Harold Albin, Charles Avery, Andrew Brochu, John Dodge, Fred Jennings, Roger Laber, Jerald Uuimby, and Dean Towle. COVER: One method of mass immunization by spray. JANUARY 1955 Avian Mass Immunization for Infectious Bronchitis and Newcastle Disease By W. R. Du nlop and R. G. Strout=^ Introduction THE PHENOMENAL growth of the poultry industry in the last decade, manifested in size of flocks rather than in number of flocks, pre- sents the need for an entirely new approach to poultry disease control. Areas of heavy poultry populatidu lend themselves, unfortunately, to rapid spread of acute diseases. An example of this is readily observed in the prevalence of respiratory conditions, such as infectious bronchitis and New- castle disease. The control of the diseases over the entire country presents a very pressing problem. In spite of the highly specialized technicjues of vac- cinations carried on today, this problem is increasing in magnitude. It is apparent that in keeping pace with the industry of today, a method of mass immunization is needed. The arduous task of vaccinating individual birds not only presents an expensive labor problem but also invites a factor known as human error. This factor is considered by some to be largely re- sponsible for the inadequacy of avian immunization programs. Mass immunization, whether a dust or a spray, is based on one of the soundest principles in the study of the spread of virus diseases. Specifically, it is understood that most virus diseases of the respiratory tract are spread by means of the virus particles riding on dust particles or moisture droplets in the air. Review of Literature In 1948, Hitchner and Johnson (7) reported the use of the Bj strain of Newcastle virus in the intranasal vaccination of day-old chicks. While the duration of immunity was not established, day-old vaccinated chicks were immune at 4 months of age. It was also demonstrated that the vaccinatio;i of laying birds did not produce any marked drop in egg production. At- tempts to immunize baby chicks by atomization of the Bj virus resulted in a high morbidity and mortality, but there was no evidence of paralytic symptoms. Later, Bankowski ( 1 ) concluded from laboratory and field vaccination trials with an attenuated Newcastle virus that the length of exposure to V:W air-borne virus and the age of the chickens being vaccinated are important considerations. The results were of a highly encouraging nature. * Dr. Dunlop is Research Poultry Pathologist and Mr. Strout is Assista-it Pathologist, New Hampshire Agricultural Experiment Station. Hitchner in 1950 (5) ohserved that ])ai"fntal iniunniit\ nuiy he over- come in day-old chicks, and that imninnit\' from da\-iild vaccination mav he ohtained even in the face of parental immnnitw Hitchner and Keising in 1952 (6j, however, demonstrated hy labo- ratory and field trials that atomization of the H, strain of virns offered a l)ractical method for vaccinating and revaccinating hirds ^ weeks old or older. By such administration, successful revaccination was accomplished in l)roiler flocks 4 weeks old after intranasal vaccination at one day of age. Re- placement flocks revaccinated at 15 to 20 weeks of age demonstrated a good antigenic res])onse. It was noted also hy Hitchner and Keising in this same study that the Bj strain of virus produced a greater reaction when administered to chicks as a spray than when administered intranasally. The respiratory reaction varied from flock to flock with negligi])]e mortality exce])t in those cases where it appeared that a concurrent infection was |)resent. Luginhuhl {^>) was one of the first workers to combine the vaccinations of Newcastle and infectious l)ronchitis at an early age. The administration of this vaccine involved the intranasal route applied to each bird. Crawley in 1953 (2) and 1954 (3) has demonstrated that chicks could ])c inoctflated by a spray technique from one da_\" of age up to 20 weeks. It was observed that the com])ination of infectious bronchitis virus and New- castle disease virus into one vaccine produced less interference in growth when the 2 vaccines were applied at the same time than if given separately In regards to Crawley's work it has been found by many investigators dif- ficult to duplicate Canadian results under American field conditions. Candling embryos in preparation for vaeoine prodiu'lion. 4 Experimental Materials Newcastle Disease Virus I'he strain oi Xewcastle disease virus used was the Wi or Blacksburg strain. In the field trials (with one exception, in which a commercial vaccine was used), the avian embryo titre of this virus was 10 "''-^''. This strain was obtained directly from llitclmer in 1951. Infectious Bronchitis Virus From observations of held outbreaks of infectious l>ronchitis, it was quite ap])arent that variations in strains occurred. In 1951, fifteen strains of infectious l)ronchitis were isc^lated, and for the remainder of that year and up to 'the fall of 1952, repeated screenings of these strains were carried out. This work resulted in tlie selection of one strain which appeared to be adaptable to mass imnuuiization procedures. This strain was found to have high antigenic properties. Screenings of embryo passages were carried out and it was found that the twentv-fourth and twenty-fifth embryo passages were desirable for the field trials. The avian enil)ryo titre was 10 ■'•^. using one tenth ml. of inocuhun per embryo. Sprayers Four different s]M"ayer> were used in the field trials. 1. Binks power spra}er (electric) 2. Aerosol hand sprayer 3. Common hand fly sprayer 4. Z & \\' hand spraxer The type of sprayer first mentioned was used in tlie application of the commercially dehydrated Xewcastle vaccine and, later, in the application of the experimental combination of Newcastle and infectious bronchitis viruses under the direction of a commercial concern. The three latter types oi sprayers were used in conjunction with the experimental vaccine prepared in the University of New Hampshire lab- oratories. Diluents The diluents used in the experiments were modified l)uffered saline solutions. Chicks The chicks u.^ed in the experimental lal)oratory trials were obtained from the poultry department at the University of New Hampshire. The chicks used in tlie field trials were provided 1)y cooperating pouhrynien in the state of Xew llam]xshire. Experimental Procedures Field Trial Procedure Due to the fact that the University of Xew Hami)shire did not have sufficient facilities for Ivophili/.ation of the viruses involved, the vaccines used were niaintaiued in a frozen state until time of use. Commercial de- hydration of similar strains of infectious In-onchitis did not materially afTect the response to the vaccination. In the maj(jrity of trials the vaccine was mixed at the farm h}- a representative of the poultr}- department. As the \'(jlume increased in the state, however, it was necessary in manv instances to mix the vaccine beforehand. Such vaccine was picked up at the laboratory by the farmer or field service man. Xo difference in results was noted be- tween those flocks sprayed by a member of the University or those sprayed l)y tlu' ])oultrvman himself. Due to the ease of uKjre complete coverage, the majority of flocks spray-vaccinated were done so at night when the chicks were bedded down. Night administration was later observed not to be necessarv providing complete coverage of the chicks could be accomplished. This factor of suc- cessful daytime inoculation depended carried mit 1)_\ injecting" .5 CO. of 10 nild's (oljtained by l)ird titre ) of the Honey strain of virus in- tramuscnlarlv. l^iirds showing s\mptoms of paralysis were considered as dead. Preliminary Laboratory Results 'liiiKiv CHICKS, parentalh' immune to Newcastle disease, were u>ed in the first laboratory trial. The chicks were sprayed on the second day. A respir- atory "take" occurred 5 days later which lasted for an additional 5 days. At 5 weeks, blood samples were taken for an H. I. and S. X. test, and the en- tire group was challenged with 10 mld's (»f the P)oney challenge strain of Newcastle virus. Two birds died and one showed paralysis. Seventy and one-half (70.5) per cent of these birds showed positive Td. I. tnres while 79.3 per cent showed positive neutralization litres. Diu'ing this same period laboratory trials were being run on the se- lected strains of infectious bronchitis mentioned previously. .\t this juncture it was decided to combine Newcastle and infectious bronchitis vaccines. The Blacksburg strain of Newcastle \irus and selected strains of infectious bron- chitis were used in these trials. Four parallel groups of 30 susce])tible 4- week-old chicks were sprayed with combination Newcastle and infectious bronchitis strains. In addition. 2 groui)s of 30 chicks, 3 and 4 days of age. i)arentally imnnnie to Newcastle and infectious bronchitis were sprayed. Recording sterility aiul pott'iu-v, 8 The respiratory take was apparent in the \oiinger chicks on the third or fourth dav, l)eing quite mild until the fifth day. The 4-week-old birds showed a take on the second day. The length of respiratory take in all groups was about 15 days. Xn mortality appeared in the older birds during this time while one chick was lost in the younger group. All birds were challenged 4 weeks later. One half were challenged by the intratracheal route with 1000 infective chick doses of a field strain of in- fectious bronchitis virus. 'Vhe second half were challenged with the Boney challenge strain of Newcastle virus as previously described. Two birds died as a result of the Newcastle challenge and one showed paralysis. Three birds showed mild respirator)- symptoms for two days when challenged with in- fectious bronchitis. Following this trial a further laboratorv test was conducted on some 600 chick divided into 6 pens. The parent stock was vaccinated with a field strain of infectious bronchitis virus and with wing web vaccine for New- castle disease just as they came into production. Random blood samples were taken from all groups of chicks and it was apparent that parental im- munitv existed for Newcastle disease and infectious bronchitis. The birds were vaccinated with combination Newcastle and infectious bronchitis vaccine by the spvay technique. The take was mild on the second and third days. About the fifth day the take was quite apparent. Respiratory take had disappeared from all groups by the 17th day. At 8 weeks random blood samples were drawn for serological test. On Hemagglutination Inhibition test 63.3 per cent showed positive H. I. titres. while on serum neutralization test for infectious bronchitis 76.8 per cent showed positive titres. At 16 weeks onlv 55.1 per cent showed positive H. I. titres for New- castle disease; 68.8 per cent were capable of neutralizing better than 100 embryo lethal doses of infectious bronchitis virus. Experimental Results Spraying with UNH Newcastle Vaccine During a three-month period. 25 flocks involving 67.280 birds were inoculated in the field at 2 to 3 days of age with the Bi strain of Newcastle virus. The spray technique, utilizing the Z & W type sprayer, as well as others, was used in the administration of this vaccine. The size of the flocks ranged from 550 to 10.000 chicks. Brooding systems of all types were en- countered. In all cases, respiratory symptoms were observed around the fifth or sixth day post inoculation. The average length of response was 6.5 days. The mortality from all causes averaged 1.8 per cent for the first two weeks fol- lowing vaccination. Blood samples, taken at random, were withdrawn at 4. 8, 12, and 16 weeks, respectively. Geometric mean titres are shown in Table 1. (In the compilation of geometric mean titres for this bulletin, serum samples in the H. I. test were diluted no higher than 1 :128. This explains the relatively low figure obtained. It must be remembered here that the geo- metric mean titre is merely a value obtained for the use of comparison purposes.) 9 8oo Figure I RESULTS OF INFECTIOUS BRONCHITIS VACCINATION BV SPRAY (weeks) C/O CO O CD Zi < I— z> LU UNH SPRAY PARENTAL IMMUNITY "— ■ PARENTAL IMMUNITY WEEKS 9 |0 II IX CO O CD —I < 900, 8oc 600 SOO 400 Figure H RESULTS OF INFECTIOUS BRONCHITIS VACCINATION BY SPRAY UNH SPRAY PARENTAL IMMUNlTy 3 ■^ S 6 7 6 9 lo II la. i3 14- 15 PARENTAL IMMUNlTy REVACCINATIOM MONTHS 10 Table 1. Results of UNH Newcastle Spray Vaccine Age 4 weeks 5-8 weeks 9-12 weeks 13-16 weeks Geometric mean titre ~.7S 6.94 7.11 5.582 Serum neutralization tests indicated that y^KS i)er cent of the samples neutralized 100 or more neutralizing doses. Random samples of l)irds. withdrawn at the same time for challenge, exhibited a resistance of d7 .S per cent, with 98.8 per cent of the susceptible controls dying. Spraying of Commercial Newcastle Vaccine During a period covering approximately 3 months, experiments were undertaken using the Binks power paint spraver in the administration of a commercially dehydrated Newcastle vaccine. Approximately 45.750 birds were sprayed at 2 to 3 days of age with this equipment. The mortality from all causes for 2 weeks following vac- cination was 1.7 per cent. The fluration of the respiratory symptoms was an average of 9 days. Blood samples, taken at random, were withdrawn at 4. 8. and 12 weeks, respectively. The geometric mean titres are recorded below. Table 2. Results of Spraying a Commercial Newcastle Vaccine Age 4 weeks 8 weeks 12 weeks Geometric mean titre 5.821 5.743 5.133 Unfortunately, due to the lack of adequate space for challenge, this process was omitted. Serum neutralization tests showed that 80.9 per cent of the samples tested neutralized 100 or more neutralizing doses. Spraying of Combination Newcastle- Infectious Bronchitis Vaccine (UNH) Field trials were carried out utilizing the Bi strain of Newcastle virus in combination with the strain of infectious bronchitis previously described. The first spray vaccination utilizing the combination of Newcastle and I)ronchitis vaccines took place on a farm where 100.000 broilers are raised every 12 weeks, and 50.000 breeders are kept each year. Newcastle disease was a very serious problem on this farm, for it had experienced outbreaks of 31 per cent and 85 per cent losses in the past. Infectious bronchitis and chronic respiratory diseases were also a definite problem. Blood samples, taken at random on 2- or 3-day-old chicks, showed a significant H.I. titre in 88.7 per cent of the samples. The inoculations took place on either the second or third day of age. Respiratory symptoms were observed on the second day post inoculation and lasted approximately 16.5 days. Random samples taken at intervals of 4 weeks presented the following geometric mean titres. 11 Table 3. Results of Newcastle Spray at Farm No. 1 Age 4 weeks 8 weeks 12 weeks Geometric mean litre 1.111 7.464 7.127 At ten weeks of age, two groups of ten Ijirds were withdrawn for chal- lenge purposes. One group was challenged with 1000 infective doses of a held strain of infectious bronchitis, employing .2 cc. intratracheally. One bird showed respiratory symptoms against challenge. In the other group, wdiich was challenged with the Boney strain of Newcastle virus, two birds showed paralysis. The mortality from all causes on the 100.000 liroilers was 2.3 per cent for three weeks following vaccination. According to serological test, adequate parental immunity was present in chicks hatched from the spray-vaccinated parents. These chicks were spray-vaccinated, showing a geometric mean titre of 16.14 at day-old, with 95.0 per cent of the samples tested for bronchitis antilxxlies. neutralizing 100 or more neutralizing doses of an embryo-adapted bronchitis virus. During the past two years over 3,000,000 birds have been vaccinated in the State of New Hampshire, with over 200 individual farms having been involved. The size of the flocks ranged from 100 to 100,000 chicks. Brooding systems of various types were used on these farms. Management conditions of all types were also encountered. The age of the chicks ranged from 1 to 7 days at the time of vaccination. The mortality figures on the entire number of birds sprayed averaged 2.4 per cent. This figure includes losses from all causes, not merely losses resulting from the vaccination alone. At one period during the fall and early winter of 1953, the average mortality was 3.1 per cent, but a change in the technique at that time lowered the mortality figures. The figures for the three months following presented an average mortality from all causes of 1.9 per cent. The symptoms appeared on an average of three days post inoculation. Such symptoms were generally mild and, in isolated instances, went un- noticed. The average length of symptoms was approximately 17 days. Blood samples, taken at the time of vaccination, and at 4-week intervals thereafter, exhibited the following geometric mean ])attern. (Samples labeled as day-old may actually be from 2- to 3-day-old chicks, but in all instances they are pre-inoculation titres.) Table 4. Results of Newcastle Spray When in Combination with Infectious Bronchitis (Pre-Inoc.) Age Day-Old Weeks 4 5-8 9-12 13-16 16-19 20-f Gcometric mean titre 15.32 7.52 6.97 8.82 121 4.34 18.24 Positives 86.8% 76.7% 72.57^ 73.0% 75.67c 70.5% 91.8% 'o Serum neutralization tests for Newcastle disease antibody, while of necessity very limited, demonstrated that 76.8 per cent neutralized 100 or more neutralizing doses of an embr}o adapted Newcastle virus strain at 16 weeks of age. 12 Fi 2£ a* gu iqure m 111 2i QC h- ao l— ^ le <: lij 5: 16 0 14- a- f- 1^ LU 2: 0 10 LU ^ RESULTS OF NEWCASTLE VACCINATION By SPRAY (months) UNH SPRAY PARENTAL IMMUWIT/ — ^ MO REVACCI NATION MONTHS O Ct: O Li_) Figure GZ ! RESULTS OF NEWCASTLE VACCINATION i BY SPRAY (weeks) UNH SPRAY — COMMERCIAL SPRAY — - PARENTAL INMUNiry - — 1 a. PAPENT/M- ■^' WEEKS 13 The serology on the infectious bronchitis presented the data indicated in Figures 3 and 4. Individual blood samples were used. The results on the graphs show that 74.2 per cent of the samples neu- tralized 100 neutralizing doses or better at day-old: 85.7 per cent at 4 weeks, 84.2 per cent at 5-8 weeks, 89.0 per cent at 9-12 weeks, 73.5 per cent at 13-16 weeks, 96.6 per cent at 17-20 weeks, and 96.7 per cent at 20 weeks or more. The reason for the higher percentage of positives at 16 weeks and beyond is the result of revaccinations carried out from 16 to 20 weeks. At the time blood samples were withdrawn for serological purposes, birds were also withdrawn for challenge. The ages of the challenge birds ranged from 4 to 16 weeks. The results are shown in Table 5. Table 5. Results of Bronchitis Challenge Vaccinates Number Birds Symptoms Controls Number Birds Symptoms Group Group Group I II III 25 0 20 1 25 0 2 2 2 2 2 2 Table 6. Results of Newcastle Challenge Vaccinates Number Birds Died Controls Number Birds Died Group Group Group I II III 25 2 20 2 25 3 2 2 2 2 2 2 One hundred thirty-eight chicks, found to be susceptible to Newcastle disease at 4 weeks of age by random sampling, were spray-vaccinated at the age of 4 weeks. Two birds died during the period of symptoms. Twelve random samples taken at 6 weeks of age exhibited a geometric mean titre of 14.25. confirming previous work (8) that older birds develop higher levels of antibody than young chicks. Six samples run on S. N. tests for infectious bronchitis antibody showed that one sample neutralized 10 neutralizing doses, one sample 100 neutralizing doses, and 4 samples 1000 neutralizing doses. The numbers, while of necessity small, indicate susceptible birds pro- duce a higher titre upon vaccination. Discussion There is one period in the life of a chicken when carefully controlled factors result in optimum conditions for life and growth. This period, when variables are more constant than at any other time, is when the chicks are being brooded under the hover. It is at this time, when the optimum con- ditions exist, for any early age vaccinations to be carried out. With this factor in mind, early field trials of spray vaccination of New- castle vaccine, or Newcastle in combination with infectious bronchitis vaccine, were carried out from 2 to 3 days of age in some cases. Recent work indicates that any age prior to production may be satisfactory for vac- cination, however. 14 The data presented in this hnlletin was gathered entirely from the State of New Hampshire, a state on the eastern seal)oard, in which consid- erable poultry population exists at the present time. Birds raised on some farms, according to random samples, possess im- munity to infectious bronchitis at all times. This situation exists because of a constant exposure to the virus. The majority of breeding farms do not find this situation to exist, and as a result, a field strain of bronchitis is given to the birds prior to coming into egg production. The general picture of breeding flocks in Xew Hampshire is that practically all flocks have been exposed to bronchitis either by artificial means or by naturally occurring field strains. Consequently, with very few exceptions, chicks hatched from these breeding flocks possess a high parental immunity during the first week of age. Furthermore, it must be kept in mind that such immunity has been stimulated by an unmodified strain. Luginbuhl demonstrated immunity to infectious bronchitis at 5 weeks when the chicks were vaccinated at day-old intranasally with modified infectious bronchitis. As may be seen from the tal)le. the majority of chicks hatched from Xew Hampshire breeders possess high parental immunity to Newcastle disease. The practice of applying Newcastle vaccine by the wing web method has been a favorite of the breeders of this state. In the presence of this high parental immunity, however, it has been shown to be possible to infect chicks with the Bj strain. Hitchner (5) showed that it was possible to do so at an early age. and Markham and Bottorff (TO) proved that it was possible to immunize chicks by use of the intranasal or intraocular route in tlie presence of parental immunity. Another important factor to be mentioned in connection with the pre- vious results is that of chronic resi)iratory disease. It has l)een determined by the oljservation and study of pipped embryos which showed enlarged hocks and caseous material in the air sacs, also by isolation of CRD agents from day-old chicks, that the incidence of CRD in the chicks hatched in X"ew Hampshire is exceedingly high. In those instances in which high mor- tality was encountered in the field trials, CRD was found to be present in over 90 per cent of the cases. While it was originally felt that the chicks to be spray vaccinated must possess parental immimity to infectious bronchitis, results of field trials and laboratory trials on susceptible chicks would seem to indicate that parental immunity, while desirable, is not necessary. From all observations, the in- dications are that the CRD complex presents the biggest problem in af- fecting the results of any mass immunization or other vaccination pro- cedures todav. IS Summary 1 . It has been demonstrated that mass immunization of young chicks with the l^i strain of Newcastle virus, employing spray techniques, is a safe method, and will produce sufficient immunity to withstand 10 min- imum lethal doses of Boney Newcastle disease challenge virus at approx- imately 12 to 16 weeks of age. 2. It has been demonstrated that there is no apparent interference in- volved in the combination of Blacksburg strain of Newcastle and the UNH strain of infectious bronchitis viruses when such a combination vaccine is administered to young chicks utilizing the spray method of mass im- munization. 3. It has been demonstrated that mass immunization by the spray methods of a combined Newcastle-bronchitis vaccine is a safe method of vaccination and will produce sufficient immunity to withstand 10 minimum lethal doses of Boney Newcastle challenge and 1000 infective doses of field strain of infectiou.s bronchitis up to approximately 12 to 16 weeks of age. 4. It has been demonstrated that the spray method of mass immun- ization is an easy and rapid method of vaccination. One man may vaccinate up to 10.000 chicks per hour, using this method, depending on brooding conditions. References 1. Bankowski, R. A. 1950. Further Studies /;/ vitro Cultivated Pheuinoenoeqhalitis (Newcastle Disease) Virus and Its Use as a Vaccine, Vet. Med. 45: 322-327. 2. Crawley, J. F. 1953. Spray Immunization of Broiler Chicks with Combined Bronchitis — Newcastle Disease Live \'irus Vaccines. 25th .Annual Conf. Lab. \\'orkers Pullorum Disease Control. 3. Crawley, J. F. and Fahey, J. E. 1954. The Spray Method for Bronchitis and Newcastle Disease Vaccination. Southwest Vet. 7. 4. Fabricant, J. 1951. Studies on the Diagnosis of Newcastle Disease and In- fectious Bronchitis of Fowls. IV. The Use of the S N Test in Diagnosis of Infectious Bronchitis. Cornell Vet. 41: 68-80. 5. Hitchner, S. B. 1950. Further Observations on a Virus of Low Virulence for Immunizing Fowls .Against Newcastle Disease (Avian Pneumoencc- phalitis). Cornell Vet. 40: 60-70. 6. Hitchner, S. B. and Reising, G. 1952. Flock Vaccination for Newcastle Di- sease by Atomization of the Bi Strain of Virus. Proc. Bk. Am. Vet. Med. Assoc: 258-264. 7. Hitchner, S. B., and Johnson. E. P. 1948. A Virus of Low Virulence for Immunizing Fowls .\gainst Newcastle Disease (Avian Pneumoeno- cephalitis). \'ct. Med. 43: 525-530. 8. Jungherr, E. L. and Terrell, N. L. 1948. Naturally Acquired Passive Ini- munity to Infectious Bronchitis in Chicks. Am. Jour. Vet. Res. 9:201-205. 9. Luginbuhl. R. E. 1952. Studies on Serologic Diagnosis and Immunization of Avian Infectious Bronchitis. Master's Thesis, University of Connecticut, Storrs, Connecticut. 10. Markham. F. S., Bottorff, C. A., and Cox, H. R. 1951. The Conjunctival .Application of Newcastle Disease Vaccine (Intranasal Type) in Paren- tally Immune and Susceptible Chicks. Cornell Vet. 41: 267-282. 16