Immunologic Responses in Florida Native Sheep
Experimentally Infected with Haemonohus aontortus
By
Jay B. Klein
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1976
CD
ACKNOWLEDGEMENTS
The author wishes to extend his sincere gratitude to the members
of the Supervisory Committee: Dr. Richard E. Bradley, Sr. , Chairman,
for his time, energy and concern; Dr. F. W. Bazar, Dr. H. L. Cromroy
for their guidance and suggestions during this study and in the
preparation of this dissertation and Mr. P. E. Loggins for his
assistance in providing the animals and suggestions for completion of
this dissertation. Additional thanks are also extended to Dr. A. C.
Warnick and Dr. E. M. Hoffmann for their assistance; and to Dr. R. C.
Littell who acted as statistical consultant.
The assistance and support of Mr. Louis N. Ergle is gratefully
acknowledged, as was the help by Mr. James Chaff in for animal care
and data collection.
Special thanks are due Ms. Velma Mitchell, secretary for her
perseverance, Mr. W. F. Randell and Mr. R. B. Grieve for their
acrimonious remarks.
The author is grateful for the support of the Animal Science
and Veterinary Science Departments. This study was also supported
by Hatch Project 1419 (W-102) and NIH Training Grant no. 5 TOl
AI00383-04 from the National Institute of Allergy and Infectious
Diseases.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES . .
LIST OF FIGURES .
ABSTRACT
INTRODUCTION
LITERATURE REVIEW
RESULTS
Nematode Recovery in Lambs Experimentally Infected
with Haemonchus contortus from Scheduled
Necropsy
Changes in the Serum Proteins in Florida Native
Lambs Infected and Non-Infected with Haernonchus
contovtus
Proteins in Abomasal Mucous Exudate from Lambs
Infected and Non-Infected with HacnDnchur.
contortus
Antibody Evaluation in Serum from Lambs Infected
and Non-Infected with Haemonahus contovtus
lii
Page
ii
MATERIALS AND METHODS 21
29
Hemoglobin Levels and Haemonchus contortus Ova Counts
from Florida Native Ewes Prior to Lambing 29
Comparison of Packed Cell Volume, Hemoglobin Level,
Albumin, Beta-Globulin, Gamma-Globulin and Total
Serum Protein Between Hemoglobin Types in Worm-
Free Lambs Prior to Experimental Infection
29
30
Changes in Packed Cell Volume and Blood Hemoglobin
Levels in Florida Native Lambs During Experimental
Infection with Haemonchus contortus 31
41
48
48
Page
Antibody Evaluation in Abomasal Tlucous Exudate from
Lambs Infected and Non-Infected with Haemonahus
contortus 56
Comparison of Mean Percentages of Proteins in Serum
and Abomasal Mucous from Florida Native Lambs
Infected and Non--Infected v;ith Haemonahus
acntortua 72
Comparison of i\ntibody Titers in Serum and Abomasal
Mucous from Florida Native Lambs Infected and
Non-Infected v/ith Haemonahus aontor-tus 72
Immunoelectrophoretic Characterization of Antibody
and Proteins in Serum and Abomasal Mucous from
Florida Native Lambs Infected and Non-Infected
with Haemonahus aontortus 72
DISCUSSION
Relationship of Blood Hemoglobin Types to Blood
Hemoglobin Levels and Natural Infection with
Haemonahus aontortus in Florida Native Ewes .... 88
Relationships of Packed Cell Volume, Hemoglobin
Level and Serum Proteins to Hemoglobin Types
in Worm-Free Lambs 89
Nematode Recovery in Florida Native Lambs
Experimentally Infected v\?ith Haemonahus
aontortus 90
Discussion of the Changes in Packed Cell Volume,
Blood Hemoglobin Level and Serum Proteins in
Florida Native Lambs Associated with Haemonahus
aontortus 90
Discussion of Abomasal Mucous Proteins from Lambs
Infected and Non-Infected with Haemonahus
aontortus 91
Discussion of Antibody Activity in Serum and
Abomasal Mucous from Florida Native Lambs
Infected and Non-Infected with Haemonahus
contortus 92
Discussion of Immunoelectrophoretic Characterization
of Antibody and Proteins in Serum and Abomasal
Mucous from Florida Native Lambs Infected and
Non-Infected with Haemonahus aontortus 92
Suggestions for Future Work 190
Page
APPENDICES
I. Hemoglobin Levels and Haemonchns aontovtus Egg
Counts from Florida Native Ev;es Prior to
Lambing 102
II. Packed Cell Volumes (PCV) of Lambs Pre- and Post-
Infection v;ith Haemonohus aontovtus 105
III. Hemoglobin Levels of Lambs Pre- and Post-
Infection \\7ith Haemonohus aontovtus 107
IV. Changes in Serum Proteins in Florida Native Lambs
Infected and Non-Infected v/ith Haemonohus
aontovtus 1 10
V. Serum Antibody Titer and Egg Counts from Lambs
Infected and Non-Infected with Haemonohus
aontovtus (Pre- and Post-Infection) 133
VI. Protein Levels in Abomasal. Mucous Exudate from
Lambs Infected and Non-Infected with Haemonohus
aontovtus with Regard to Hemoglobin Type ... 149
BIBLIOGRAPHY 151
BIOGR^\PHICAL SKETCTl 1 62
LIST OF TAJ5LES
Table Pae:e
1. Scheduled Necropsy of H. contortus Infected Lambs
Controls 22
2. Statistical Comparison of Packed Cell Volume,
Hemoglobin Level, Serum Albumin, F-eta-Globulin,
Gamma-Globulin and Total Serum Protein Betveen
Blood Hemoglobin Types in Lambs 30
3. Nematode Recovery from Scheduled Necropsy of Lambs
Infected v/lth tlaemonahus contortus 32
4. Differences I'etween the Changes of Serum Proteins of
Lambs Infected and Non-Infected with Haer:onchus
contortus 44
5. Mean Percentages of Serum Protein from Lambs Infected
and Non-Infected v;ith Haernonchus contortus 47
6. Mean Percentages of Proteins in Abomasal Mucous Exudate
from Lambs Infected and Non-Infected with Haernonchus
contortus 49
7. Antibody Titer Against Haernonchus contortus in Serum
and Abomasal Mucous Extraction from Sequential
Necropsy of Infected and Non-Infected Lambs 63
8. Mean Percentages of Proteins in Serum and Abomasal
Mucous Extraction from Sequential Necropsy of
Infected and Non-Infected Lambs 73
9. Immunoelectrophoretic Analysis of Serum from Sequential
Necropsy of Lambs Infected and Non-Infected with
Haernonchus contortus 74
10. Immunoelectrophoretic Analysis of Abomasal Mucous from
Sequential Necropsy of Lambs Infected and Non-
Infected V7ith Haernonchus contortus 75
LIST OF FIGURES
Figure Page
1. Sequential Changes in Packed Cell Volume in Florida
Native Lambs Infected and Non-Infected \7ith
Haemonohus oontortus Divided by Hemoglobin Types . . 34
2. Sequential Changes of Packed Cell Volume in Lambs
Infected and Non-Infected with Eaemonchus oontortus
Without Regard to Hemoglobin Types 36
3. Sequential Changes in Hemoglobin Levels in Florida
Native Lambs Infected and Non-Infected with Haemonohus
oontortus Divided by Hemoglobin Types 38
4. Sequential Changes in Hemoglobin Levels in Florida
Native Lambs Infected and Non-Infected with Haemonohus
oontortus Without Regard to Hemoglobin Types 40
5. Changes of Serum Gamma-Globulin in Florida Native
Lambs Infected and Non-Infected v;ith Haemonohus
oontortus Without Regard to Hemoglobin Types 43
6. Differences in the Changes of Albumin-to-Globulin Rato
of Lambs Infected and Non-Infected with Haemonohus
oon tortus 46
7. Characteristic Electronhoretic Patterns of Abomasal
"Mucous Exudate from Lambs Infected and Non-Infected
with Haemonohus oontortus 51
8. Protein Content in Abomasal IIucous Exudate from Lambs
Infected and Non-Infected with Uaem.onchus oontortus 55
9. Serum Antibody Against Haemonohus oontortus Larvae and
Adults from Sequential Necropsy of Infected and
Non-Infected Lambs 58
10. Mucous Antibody Against Haemonohus oontortus Larvae and
Adults from Sequential Necropsy of Infected and Non-
Infected Lambs 65
Figure Page
11. Abomasal Mucous Antibody Levels from Florida Native
Lambs Infected and Non-Infected v;ith Hae'monchus
oontovt'us at necropsy 71
12. Characteristic Immunoelectrophoretic Patterns of Serum
from Worm-Free Florida Native Lambs 78
13. Characteristic Immunoelectrophoretic Patterns of Serum
from Florida Native Lambs Infected with Jlaemonchus
oontovtus 80
14. Immunoelectrophoretic Patterns from Abomasal '^'ucous
Exudate in Parasitized Florida Native Lambs 82
15. Immunoelectrophoretic Patterns from Abomasal Mucous
Exudate in Non-Parasitized Florida Native Lambs ... 86
16. Identified Proteins in Serum from Florida Native Lambs
Infected and Non-Infected with Uasmonahus contortuo 96
17. Identified Proteins in Abomasal Mucous from Florida
Native Lambs Infected with Haemonolius cantortus ... 98
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
IMMUInIOLOGIC RESPONSES IN FLORIDA NATIVE SHEEP EXPERIMENTALLY- INFECTED
WITH Haemonahus cofitortus
By
Jay "B. Klein
March, 1976
Chairman: Richard E. Bradley, Sr.
Major Department: . /\iiimal Science
The host responses of v.'orm-free Florida Native lambs to infection
with Haemonchus contovtus were investigated for the elucidation of
"resistance factors" wliich may play a role in maintaining lower worm
burdens. Physiological and immunological measurements were made on
worm-free lambs divided by blood hemoglobin type prior to infection
and post-infection during a sequential necropsy. Immunoelectrophoresis,
electrophoresis and indirect hemagglutination for antibody titer, dem-
onstrated which protein fractions and antibodies predominated in the
serum and abomasal mucous, how they changed with the parasitic infection
and the relationship to blood hemoglobin type. Physiological factors
monitored included PCV, blood hemoglobin levels and fecal egg count.
Infected animals shov/ed maximum blood loss, demonstrated by lowered
PCV's and hemoglobin level, at 26 days after infection. Subsequently,
increased gamma-globulin and a decreased albumin-to-globulin ratio were
observed. Tae increased gamma-globulin fraction may be related to the
antibody activity (exhibited by indirect hemagglutination testing) in
both serum and abomasal mucous which increased significantly after
experimental infection with //. oontovtus . The identification of the
immunoglol)ulins (IgA, IgG and IgM) responses against the parasite pre-
sented evidence tliat IgM and IgC, were most prominent in serum, and IgA
and IgG were the most prominent in mucous. Complement proteins were
also sho^'m to increase substantially after parasitic infection.
Tlie percentages of the abomasal mucous and blood serum proteins
were established. Characterization of these proteins, including the
immunoglobulins, by immunoelectrophoretic techniques, revealed a maximum
of 8 and 10 proteins in serum from worm-free and parasitized lambs,
respectively. Mucous extracted from the abomasum exhibited 5 to 7
proteins regardless of infection status.
INTRODUCTION
uorbidity and mortality losses caused by trichostronpylid parasites
are of major economic importance in v/orldwide sheep production. Figures
by the United States Department of Agriculture (U.S.D.A., 1965) estimated
annual losses of $21 million in sheep due to the trichostron[^ylids which
include: $7 million due to deaths, $11 million to morbidity and $3
million to wool loss. Becklund (1961) found economic losses due to
parasite mortality of sheep on 15 farms in southern Georgia averaged
$1,233 per farm. He also found on the Georgia Coastal Plain region an
average loss of $289 per farm on 23 sheep farms.
HaemonchiiS contovtus, according to vriiitlock (1955a), is the only
gastrointestinal nematode which causes a primary disease. This recogniz-
able disease, called haemonciiosis , produces a hemorrhagic anemia
(Richard e^ _a_l. , 1954, Campbell and Gardiner, 1960). Blood loss caused
by H. aontortus was determined by Baker ejt aj^. (1959) who tagged sheep
erythrocytes with radioactive chromium 51. They determined that H. contovtus
removed an average of 0.08 ml of blood per worm per dav. Clark et al .
(1962), also using radioactive chromium 51 and iron 59, found blood loss
in experimentally infected lambs to be 0.049 ml per v/orm per day.
Clinical symptoms of haemonchosis vary from the peracute form where
the parasite causes rapid death, to a sub-clinical form which is essen-
tially asymptomatic. Heavily parasitized lambs may exhibit grov7th
reduction, permanent stunting (Spedding, 1956), vjcakness, muscular
1
trembling, pale mucous membranes, cold extremities, rapid weak pulse,
increased respiratory rate and edema with accompanying "bottle jaw"
swelling under the jaw (Levine, 1968; Tetzlaff, 1970). Mimals less se-
verely infected have loV.'ered resistance and may be susceptible to secon-
dary infection. These animals are untnrifty, listless and have dry,
harsh wool (Levine, 1968; Tetzlaff, 1970).
Tae losses incurred in sheep carrying worm burdens at the sub-
clinical level have been reported to be substantial. Gordon (1958)
demonstrated a drop in milk production in lactating ev/es experimentally
infected with H. contortus . Spedding (1955) and Spedding et al. (1958)
have sho\-7n a reduction in growth rate as much as 30% in ewes having
normal pasture parasite burdens. Brunsdon (1963) found that lambs
treated with thiabendazole v/eighed 30 pounds more than controls at
slaughter and produced 49% more v7ool.
Due to the short period from uptake of infective larvae to ova
production (14-21 days) and the fact that infective larvae are resistant
to freezing and drying (Monnig, 1956), control of haemonchosis by
management practices (pasture rotation or dry lot feeding) may not be
effective. i\nthelmintic drug control is, at present, the most effective
and economical means to reduce worm burdens. Although, where these
compounds are under continuous use, there have appeared resistant strains
of H. contortus (Taeodorides e_t a_l . , 1970). The appearance of these
strains can cause the need of increased frequency of treatments and, v.rith
the rising costs of anthelmintics, this is of economic concern in sheep
production.
Immunity and genetic resistance in sheep against internal parasites
have been reported frequently in the literature. Observations by Evans
e_t al . (1963), Evans and Whitlocl: (1964), Loggins e_^ al. (1965a, 1965b),
Jilek and Eradley ( 1969) , Radliakrlshnan e^ al. (1972) and Rradlay et al.
(1973) suggest a correlation between hemoglobin type, hematocrit value
and severity of infection with //. contovtus infections. Evans and
Wiitlock (1964), Jilek and Bradley (1969) and Radhakrishnan e_t al. (1972)
found consistently higher hematocrit values in hemoglobin type A. This
physiologic factor (greater erytlu-ocyte volume) may allov? these sheep a
better chance of surviving parasite challenge. hoggins e_t £1. (1965b)
Jilek and Bradley (1969), Radhakrishnan et_ al . (1972) and Bradley e_t al^.
(1973) found Florida Native sheep were more resistant to infection with
H. aontoi'tus than Rambouillet sheep. Florida Native sheep appear to have
some type of "resistance factor" due to the fact that thev are able to
undergo self cure more readily than Rambouillets and had significantly
more larval forms of H. contortus than adult vrorms (Radhakrishnan et al. ,
(1972).
Arrested development of U. contovtus has been well documented,
although the factors governing this phenomenon are poorly understood.
Dineen et al. (1965), Dineen and Wagland (1966), Soulsby (1966), Wagland
and Dineen (1967), Michel (1968) and Donald e_t a 1 . (1969) attribute the
arrested development of //. contortus to resistance of the host while
Blitz and Gibbs (1972a) ascribe it, at least in part, to an environmental
diapause-inducing condition (decreasing photoperiod) .
Tlie objectives of the present investigation v;ere to characterize
the host responses of worm-free Florida Native lambs to infection v/ith
U. contortus and to elucidate t.ie "resistance factors" that have a role
in maintain in,; lov.'cr adult v;orm burdens. Hematological data, serum
collection and abomasal mucous extraction were the main considerations
in the determination and interpretation of these "factors.'' Sequential
necropsy of infected worm-free lambs from pre- through post-patency
gave an unique insip,ht into the immunologic meciianism occurring in lambs,
in botu the humoral and mucous secretory systems, while the parasitic
infection proceeded. Tixe following physiological and immunological
measurements \-iere performed: (a) hematocrits, (b) hemoglobin levels,
(c) fecal ova counts, (d) electrophoresis of serum and mucous,
(e) immunoelectropiioresis of serum and mucous using anti-sera and
H. contortu3 antigen and (f) indirect hemagglutination. Through the
analyses of lamb serum and abomasal mucous, additional inform.ation was
gained concerning which protein fractions and immunoglobulin classes they
contained, how they changed with age and v/hether there v/ere statistical
relationships to environment, hemoglobin types or other genetic factors.
LITERATURE REVIEW
Life Cycle, _ I lorpliolo[;y an d_ J'fe_tab_oJ. is_m oF Haernon oliii.i a on tor tu s
Haewonohus contoptus (Rudolphi, 1803), commonly known as the
"barber pole V\?orm^" is fonnd principally in the abomasum of ruminants
v/orldwide. The males are 10-20 min long and are reddish brovm in color.
Females range from 18-37 mm in length and have characteristic v/hite
ovaries spiralled around their reddish blood-filled intestine giving the
"barber pole" appearance.
The male H. contoi'tus is identified by a bursa having elongated
lateral lobes and long slender rays. A small dorsal lobe of the bursa
is asymmetrically located around the left lateral lobe supported by a
Y-shaped dorsal ray. Its spicules are 0.46-0.51 mm long. In the female,
the vulva is covered by an anterior flap. Tl.iis valvular flap usually
will be large and conspicuous (linguiform) , but can be diminis'ned to a
knob-like structure in some specimens (Levine, T968) .
The mature female produces from 4,000 to 10,000 ova daily. These
ova measure 70-85li by 41-48^ and are expelled in the feces of the host
as a 16 to 32-cell embryo. Continual mitosis of the embryo is dependent
on temperature, moisture and metabolic oxygen (Jilek, 1968; Levine, 1968;
Tetzlaff, 1970). Cleavage is highly determinant, meaning germ cell lines
are segregated and can be follo\;ed as to which structures they may become.
Morphologically, the cells within the egg form a morula, blastula, gastrula
and then elongate to a vermiform embryo (Levine, 1968). The elongated
embryo shows defined organs, three cell layers (endoderra, ectoderm and
mesoderm), germ cells, gut and stomodeum. The first stage larvae of
//. aontortus hatch within 14-19 hours. This first stage is rhabditifom
and actively feeds on bacteria and other microorganisms. The second
stage larvae, which appear within 1-2 days, are also rhabditif orm. The
third stage (L-) or infective stage is strongyliform and sheathed, and
is found 4-7 days after hatching.
The infective stage is characteristically 682-780p in length, has
16 intestinal cells and a globular buccal cavity. The tip of the tail
to the end of the sheath is 65-78^ with a kink found in the sheath tail.
The intestinal cells contain granuoles which \i±ll be used to maintain
the infective larvae (Lapage, 1968). These larvae are negatively
geotropic and positively phototropic except for strong sunlight and can
exist for months if temperature and moisture conditions are adequate
(Levine, 1968; Soulsby, 1965). In the morning or evening hours, infec-
tive larvae may migrate up blades of grass where they are available to
be ingested by the grazing animal. Exsheathment is triggered after inges-
tion by physical and chemical components such as temperature and CO2-
carbonic acid concentrations (Rogers, 1962). This triggering mechanism
causes secretion of exsheathing fluid containing leucine aminopeptidase
from a region around the excretory cell. Tlie enzyme acts on an area
about 20u back of the anterior end for release of the L, larva (Lapage,
1968; Levine, 1968; Rogers, 1962; Silverman, 1965).
rne released third stage larvae migrate to the paramucosal lumen
at the surface of the mucosa or become lodged in the epithelial processes
of the mucosa (Silverman, 1965; Tetzlaff, 1970). Ecdysis occurs after
3 days and a fourth stage attaches to the mucosa with its buccal capsule
and ingests blood. In 9 to 11 more days, after growth and development,
another ecdysis occurs and fifth stage or immature adults emerge. They
attach to the abomasal mucosa where they also ingest blood. Tlie mouth
of the fifth stage now has a dorsal lancet which has two thorn-type points.
Within 6 to 8 more days, morphological and physiological development is
finished and the parasite is a functional blood sucking adult.
Metabolic pathirays of nematode ova, larvae and adults have been
studied extensively (Smith, 1965). Cheng (1973), Levine (1968) and Rogers
(1962) present chemical formulas of these pathways. Glycogen is the main
source of stored energy in the ova and sheathed infective larvae. Energy
is released by the Embden-lleyerhof route of phosphorylating glycolysis.
Phosphorylation occurs as in vertebrate tissue but neitlier arginine
pJiosphate or creatine phosphate have been detected (Rogers, 1962). Lactic
acid may not be the end product of anaerobic metabolism and pyruvic acid
may also be metabolized v;hich suggests that it may be involved in produc-
tion of lower fatty acids which are secreted (Rogers, 1962). Data for
H. contortus ova and larvae shov; respiratory quotients of 0.5 8-0.60 and
0.64, respectively. Adult //. contortus are found to contain a type of
hemoglobin which transports oxygen (Lapage, 1968; Levine, 1968).
Pathogenesis
Haemonclius contor-tus causes primary damage by sucking blood. Both
tiie fourth stage larvae and adults suck blood, consequently damaging the
abomasal mucosa by their attachment and piercing activities. llie anemia
produced is proportionally related to the numbers of adult worms present,
yet cannot be correlated to the numbers of eggs per gram of feces
(Andrews, 1942; Kingsbury, 1965). Blood first appears in the feces 6-12
days after infection (Clark e_t al . , 1962). Roughton and Hardy (1935),
using an abomasal fistula, observed varying degrees of petechiation on
the abomasal mucosal surface indicating sites of recent attachment. They
observed the parasites attach themselves by a striking motion of the
head and neck. Attachment lasted for approximatelv 12 minutes after
whicli detachment occurred leaving wounds which continued to hemorrhage
for an additional 7 minutes.
Hematological changes associated with anemia caused by 11. aontortus
may include erythrocytes showing anlsocytosis , polychromasia , Howell-
Jolly bodies and punctate basophilia (kevine, 1968). Serum proteins and
albumin in parasitized lambs may shov; a decrease, v/hile the alpha-1-,
alpha-2-, beta- and gamma-globulins may all be increased (Kuttler and
Marble, I960: keland e^ al. , 1960). VJilson and Turner (1965) noted that
even moderate mixed nematode infections (mainly //. aontortus) caused a
decrease in the serum albumin to globulin ratio and an increase in serum
gamma-globulin. Decreased total serum protein, albumin concentrations
and albumin to globulin ratios v/ere also seen in Florida Native and
Rambouillet lambs infected v/ith //. aontortus (Bradley e^ al_. , 1973).
Eosinophil and lymphocyte infiltration in abomasal tissue of lambs was
observed several weeks after infection with //. aontortus (Bradley et al . ,
1973; Malczewski, 1971). Increased lymphocytes and lymphoid hyper-
plasia v^7ere also observed in sheep resistant to .7. aontortus (Silverman,
1965) .
Anemia as lov; as 3-4 grams of hemoglobin per 100 ml of blood can
be present in //. aontortus infected animals (Levine, 1968). Clinically,
the gums, conjunctiva and mucous membranes are pale. Edematous swelling
under the jaw ("bottle jaw''), constipation, xjeakness, cold extremities,
llstlessness , dull, dry, harsh v;ool and unthrif tiness may be seen (Levine,
1968; Tetzlaff, 1970). Parasitized animals may die suddenly, symptoms
may persist for vjeeks before dying, or recovery may occur leaving
stunting or reduced muscle growth (Spedding, 1956).
Epidemiology and Ecology of //. contortuQ
llie mode of infection of sheep v/ith //. contovtus infective larvae
is by grazing on infected pastures or ingestion with their feed or
v/ater. When pasture conditions are favorable, the infective stage (Lo)
is reached within 2.5 days to 2 weeks (Levine, 1968; Levine e^ al . , 1975).
Tliese conditions are complex in nature. Levine (1968) describes them
to be a combination of climatic and micrometeriologic. Additional
conditions such as the terrain and soil type, nature and type of vege-
tation, degree of stocking and number of nematode species competing
for space are important factors.
Oxygen has also been found to be obligatory for the adult female
to lay eggs (LeJambre and IvTliitlock, 1967) and for egg development to
occur on the ground (Shorb, 1944). Optimal temperature for development
(60 hours) is 33.3°C (Berberian and Mizelle, 1957). Levine (1968)
states that Hsu in 1967 studied the effects and relationships of temper-
ature and relative humidity to the development of TiH-chostrongijlus
colubvifoimis and H. aontortus . He found that H. aontortus needed rela-
tive humidities above 85%. Rose (1963) reported that desiccation
severely reduced Haemonchus larvae developing from sheep feces.
The habitat of the larvae is in a thin layer at the surface of
the ground. Conditions can be different in this microhabitat than
10
above ground where weather is usually measured. Gordon (1948) introduced
bioclimatographs to help recognize the relationship of temperature and
precipitation to the epidemiology of gastrointestinal parasites of
ruminants. Levine (1963) introduced parasite profiles of geographic
regions and discussed in detail the effects of v/eather and climate on
the bionomics of ruminant nematode larvae. He considered the potential
transmission period of Ilaemonchus to lie between mean monthly tempera-
tures of 15 to 37°C when the soil water deficiency was not more than 2.0 cr
Monnig (1956) reported that infective larvae are active climbers and
can withstand desiccation and freezing. Thev are negatively geotropic and
positively phototropic to soft light, a state seen after sunrise and
before sunset (Rees, 1950; Soulsby, 1965).
A means of control of //. contortus and other trichostrongyles might
appear to be trie elimination of the larvae and eggs on the ground.
Several authors have described methods for soil treatment but no satis-
factory method is mentioned that is safe, efficient, and inexpensive.
Trofton (1949) found that removal of sheep for at least 12 days reduced
the number of larvae on a pasture. He found reductions of 57% when
sheep were removed 3 weeks and 90% at 4 weeks. He believed plowing and
reseeding would eliminate larvae from a pasture. Soulsby (1965) and
Levine e_t al. (1974), in contrast, have reported that under favorable
conditions //. oontortus infective stage larvae could survive 1.5-3.5
months .
Imiriunol_ogy and Resistance
Tlie idea that metazoan parasites stimulated an immune response was
first reported by Stoll (1929), using //. contortus . He infected lambs
with the parasite and observed that after placing them on pasture
11
their fecal egg counts rose to high levels. After several x/eeks he
found a dramatic fall in egg counts, some to negative values. He
correlated this phenomenon to a loss of adult x^forms . Even after
subsequent reinfection v;ith large numbers of infective larvae, the lambs
remained refractory to reinfection. Stoll termed this phenomenon "self
cure and protection." In 1930, Stoll and Nelson reported that this
resistance v/as humoral, based on intradermal reactions to saline
extracts of //. oontortus . Stumberg (1933) substantiated this fact by
using a cutaneous anaphylactic test in which he detected antibody
against H. aontovtus in dilutions of 1:50,000.
Ilav/kins and Cox (1945) found that serum obtained from sheep that
had undergone a natural infection v/ith the tricliostrongyles (mainly
H. oontortus) caused precipitates around the mouth, excretory pore,
anus or cuticle of exslieathed larvae. There v/ere no precipitates in
suspensions of ensheathed larvae in immune sera or larvae in sera of
lambs that had been raised parasite free except for coccidia or
Stronayloides. Silverman (1965) also obtained similar results. Antibodies
that cause these precipitates at the physiological orifices '/ere believed
to be "functional" by Oliver-Gonzales (1946), meaning that they contribute
directly to resistance.
Stewart ( 1950a, b,c) has demonstrated that a complement fixing
antibody response occurs after infestation with //. oontortus or
Triohostmngytus son. He found a correlation between the fall of ova
counts and the rise in antibody titer in experimentally infected sheep.
In field studies he observed 7 periods of "self cure." On each occasion
most sheep \diicn had a drop in ova counts also sho^/ed a rise in sen.
'um
12
titer. This occurrence uar, similar to t'le result when infective larvae
of //. aontoT'tUiJ were superimposed upon an existing infection producing
"self cure/' This was contraty to the reports by Ross and Gordon (1933)
and Gordon (1948) in v;hich they concluded that acquisition of resistance
to //. contortus by previous infestation was uncertain; that evidence
did not indicate that "self cure" was a manifestation of resistance and
that it occurred close to periods after rain. Stewart (1950c) found the
reason "self cure" takes place in naturally e,razin,'> flocks after rain
was because this caused large doses of infective larvae to mature and
be ingested.
T.ie imp.iunQlogical reaction and subsequent protection depends on
the availability of the infective larvae (Stewart, 1950a). Even though
infective larvae are continuously available, infection is maintained
at a low level (Soulsby, 1958). If non-immune sheep were placed on
this type of pasture they would probably acquire iieavy burdens of
gastrointestinal parasites. In Florida, weather conditions are such to
allow the above situation to be maintained year round or for at least
longer periods of time. In areas where conditions become unfavorable
for larval development there is a depression of the immune status due to
lack of stimulation by infective larvae. This is confirmed by a
persistent fall in antibody titer in which "Spring rise" (characteristic
increase of ova count) occurs (Soulsby, 1957).
The mechanisms of "sel.f cure" in lambs cause a response by the host
which results in the loss of part or the whole //. contortus burden.
Tliese mechanisms have been postulated and the causes have been shown
to be varied in nature. Ste\jart (1953) found tliat at the time of
'ielf cure there was a significant rise in blood histamine as well as
13
antibody level. If antiliistamine drugs were given at this time the
phenomenon did not occur, yet there was still an increase in antibody
titer. This reaction was characteristic of an allergic sensitization
v/ith an edematous condition of the mucous membrane of the abomasum.
This was substantiated by Stev7art (1955) who renorted that abomasums
of previously non-exposed lambs remain flaccid and normal \/hen large
doses of exsheathed larvae \7ere injected into the abomasum. In hyper-
sensitized and resistant lambs, the abomasum had increased peristalsis
and segmentation in 10 minutes. V.'ithin 1 hour the abomasum was pale
and edematous. Histological examination of the mucosa of animals that
undergo self cure shov-/ edema and aggregation of eosinophiles (Soulsby,
1958).
Soulsby (1965) suggests resistance to parasites may be due to a
cUange at the environmental site caused by the parasite itself. An
alteration of oxygen tension associated with pH could be such a product
of infection vjhich is seen in inf laminatory reactions. Christie (1970)
found the activity of fourth stage H. oontortus larvae damage the function
of the cells of the gastric epitiielium. Hydrogen ion concentrations
are increased and the pH of these cells which is near neutral drops
dramatically to pH 1.8 to 3.5. This is unfavorable to the development
and persistence of the adults. Ejection of adult worm populations and
"self cure" could be explained because of these changes after intake of
large doses of larvae.
Arrested development of larvae is an important immunologic-related
occurrence. It is of particular significance, for it depicts the
primary means of overwintering for //. oontortus in temoerate regions
(Blitz and Gibbs, 1972a). Large numljers of fourth stage larvae and low
14
numbers of adults are frequently observed during the winter months
(Blitz and Gibbc, 1972b; Gibbs, 1967). It is maturation of these larvae
in the spring that contributes to the characteristic increase in the
number of ova at this time called "Spring rise" (Field et^ al^. , 1960;
Gibbs, 1967; Parnell, 1962; Procter and Gibbs, 1968). O'Sullivan and
Donald (1970) hypothesized on the importance of hormonal changes in
lactating ewes \7hich depressed their immunological capacity resulting in
a stimulation of dormant larval stages to mature. This is only part of
the ansvjer as Spring rise is also observed in wethers and young virgin
ewes (Brunsdon, 1964; Croften, 1958).
The factors initiating arrested larvae are generally felt to be
from high levels of resistance, \jhether from previous exposure or inher-
ent mechanism (Dineen et^ al^. , 1965; Dineen and Wagland, 1966: Wagland
and Dineen, 1967: Donald et^ al . , 1969). Blitz and Gibbs (1972a) have
also added another dimension to the mechanisms of arrested development
by presenting evidence that showed if larvae were cultured in the
laboratory at constant temperature and in darkness, and then exposed for
4 to 6 weeks to environmental conditions similar to those prevailing
during September they v;ould become inhibited following ingestion by worm-
free lambs. They believe two factors are operating: that preinfective
and infective H. contortus are sensitive to diapause-inducing stimuli
(decreasing photoperiod or temperature) causing the inhibition and
that resistance from the host V7ill prevent worms from developing as
demonstrated by Dineen and co-VJorkers (1965) . Bradley et al. (1973)
reported significantly higher levels of larvae in Florida Native lambs
than Rambouillet lambs indicating that either one or both of the afore-
mentioned mechanisms may be in operation.
15
It is believed the larval stages are the important immunizing agents
inducing "self cure and protection" (Soulsby, 1965). "eports by Silverman
(1965) and Silverman and Patterson (1960) point to the antigenicity of
the fourth and early fifth larval stages and that the antigens are released
during growth and development. Soulsby e_t al^. (1959) and Soulsby and
Stewart (1960) obtained serological evidence of a noticeable reaction to
exsheathing fluid at the time of "self cure."
The relationship of age to the production of immunity against
H. aontortus in sheep has also been studied. Manton e_t al^. (1962)
found lambs infected V7ith larvae of H. aontortus at 2-4 months of age to
be unable to develop immunity while lambs 10-12 months of age could.
Urquhart e_t aj^. (1966a, 1966b) in vaccination studies against H. aontortus
found lambs 1-3 months of age unable to develop immunity and lambs 7
months old produced a high degree of protection. Tetley (1959) found
no differences in susceptibility between worm-free Romney lambs 6-10 or
3-6 months of age. Similar results were reported by Dineen and Wagland
(1966) betv/een sheep 320 and 455 days of age, though Silverman (1965)
and Silverman and Patterson (1960) reported laboratory infected sheep
aged 8-12 months showed longer parasite life cycles than sheep aged 4-6
months, indicating some resistance.
Resistance to parasitic infections has also been correlated with
genetic factors. As early as 1932 Clunies-Ross reported observations
on genetic resistance of sheep to infections with stomach worms. Stewart
e_^ al. (1937) reported that the Romney Marsh sheep were more resistant
to Ostertagia sp. than other breeds they used. Scrivner (1964a, 1964b,
1967) found genetic resistance to ovine ostertagiasis , x-zhich could be
16
transmitted by the ram in Targhee slieep. Emik (1949), IJar^./ick e_t al.
(1949) and Vfaitlock (1955a, 1955b, 1958) reported resistance to the
trichostrongyles (mainly //. aontortus) was genetically transmitted.
Loggins et_ al. (1965a, 1965b) believed that genetic factors were
responsible for parasitic resistance in Florida Native sheep in compar-
ison to the Soutiido\m, Hampshire or Uarabouillet sheep.
Jilek and Bradley (1959) found high frequencies of hemoglobin type
a (Hb A) in Florida Native sheep \;hich \iere believed to be more resistant
to //. aontortus . This was in agreement with Evans e_t al . (1963) who
reported that sheep with Hb A v/ere infected with fewer adult tl . aontortus
than other hemoglobin types. Evans and IJ^iitlock (1964) found sheep with
hemoglobin type A had higher total volume of circulating erythrocytes
and iiematocrit values tlian either types B or A.B . lladhakrishnan e_t al .
(1972) confirmed that Florida Native sheep with hb A had consistently
higher packed cell volume (PCV) values than other hemoglobin types but
reported no data that \-7ould confirm that hemoglobin types were indicators
of resistance against H. aontortus or tint }!b A sheep vjere less suscep-
tible to such infections. If sheep vjith Hb A. do have more circulating
erythrocytes, they might be better able to v/ithstand the effects of
H. aontortus.
Radhakrishnan Qt_ al_. (1972) did find significantly lov/er numbers
of adult vjorms in Florida Native lambs in comparison to '^.ambouillet
lambs. Tnis observation was substantiated by Bradley e^ al. (1973)
who gave 1 of 2 oral doses of H. aontortus. They reported Florida
Native lambs had liigher levels of larval stages, prolonged prepatent
period (21 days) and a more rapid "self cure" than Rambouillet lambs.
Silverman (1965) also reported a delayed prepatent period (20 days) in
17
resistant sheep as compared to susceptible sheep (15 days). Florida
Native sheep mav possess "resistance factors" which enable them to be
resistant toward H . contoT'tus infection v/ithout prior exposure or to
initiate a more rapid "self cure" (Bradley et ad^. , 1973).
Natural resistance to Uaemonchus spp. in animals not previously
exposed, at- least post-natally , has been noted. Fourie (1931) reported
a great deal of difficulty in producing a sufficient number of typical
cases of pure haemonchosis . Urquliart et_ ajl. (1962) reported low peak
egg counts and low numbers of adult worms at slaughter 30 to 40 days after
challenge in 50 per cent of sheep given 10,000 larvae. Brambell et al.
(1964) dosed 6 young sheep vjith 1000,000 larvae but found high numbers
of worms in only 1 animal. Bitakaramire (1956) found low numbers of worms
in 5 sheep challenged with 50,000 larvae. Christie (1970) demonstrated
the ability of sheep to resist large doses of Uaemonchus sp . by adminis-
tering 3,000,000 infective larvae to 3 resistant and 2 worm-free lambs.
Christie (1970) believed age Mas a very important factor in classifying
natural resistance. This v?as substantiated by Dineen e_t aJ. (1965) and
Wagland and Dineen (1967) v/here 27 total deaths among 58 lambs aged 2
to 4 months were attributed to haemonchosis. Dineen and Wagland (1966)
had no deaths among 40 lambs aged 7 months given similar doses of infec-
tive larvae.
Blood, Serum, 'lucous and Immunoglobulin Proteins
Harris and Warren in 1955 described 3 types of hemoglobin proteins
in ewes: 1) a fast moving hemoglobin, 2) a slow moving hemoglobin and
3) a combination of the faster and slower hemoglobins. Subsequently,
Evans et al. (1956) labeled these types as Hb A, Hb B and Hb AB , respec-
tively. These hemoglobin types are genetically determined by simple
18
Mendelian relationships, Kb A and lib 1 being allelic and co-dominant
(Evans e_t al^. , 1956; Tiuisman e_t a_l. , 1965). Another hemoglobin type,
Hb C (first designated Hb N) has been reonrted, but was usually found
in either young lambs (less than 1 month of age) or animals \jhich are
severely anemic (Efremov and Rraend, 1966; Vliet and Huisman, 1964).
Evans and Whitloclc (1964) correlated a relationship betvjeen hemoglobin
types and packed cell volume; Hb A being greater than Hb F. and Hb M
being intermediate. Tliis observation i;as also substantiated in this
study (see Results Table 2) .
The first report of the serum proteins found in the normal adult
sheep was by Silverstein ejt al^. (1963). Immunoelectrophoresis with
rabbit anti-whole adult sheep serum yielded 21 arcs of precipitation.
Tiiese consisted of prealbumin, albumin, 3 alpha-1 proteins, 6 alpha-2
proteins, 9 arcs in the beta-1 protein area and 4 arcs in the beta-2
protein and gamma-globulin region. Tlie beta-2- and gamma-globulin arcs
were similar to tliose seen in other mammalian sera and were designated
beta-2M- (later changed to IgM; W.H.O., 1964), beta-2A- and gamma-globulin,
Jonas (1969) examined the immunoglobulin response of sheep to Satmonelta
typhimurlum or human erythrocytes by Immunoelectrophoresis using antisera
from guinea-pigs which had been injected with suspensions of the above
antigens treated with slieep sera or various bodv fluids. He reported
tV7o gamma-globulins (fast and slow) , Ig''i, tv7o arcs parallel to the two
gamma-globulins, 1 beta-globulin arc and 2 weak alpha-gloinilin arcs.
Preliminary evidence indicates that the last 3 proteins may be components
of complement. Subsequent work by Jonas in 1972 using third stage
H. aontortus larvae treated with serum from parasite free or parasitized
sheep to produce antisera in rabl'its found 3 additional beta-gloliulins
19
and 1 additional alpiia-globulin . Evidence A-zas presented to indicate that
the beta- and alpha-globulins may be components of the sheep complement
system.
Silverstein £t al. (1963) observed that the typical gamma-globulin
showed a "gull vzing" appearance, indicative of a fast and slow protein
which are different tut cross reacting. Leland et_ al. (1960) after
examining sera by electrophoresis from lambs infected with Trichostvongylus
axei reported various changes in the gamma-1- and 2-globulins associated
with the parasitic infection. Jonas (1969) and Jonas e^ si. (1972)
found separate fast and slow gamma-globulins in sheep serum. Tomasi and
Bienenstock (1968) reported fast gamma-1- and slow gamma-2- immunoglobulins
in bovine colostrum. Jonas (1969) also reported fast and slov; gamma-
globulin in sheep synovial, pericardiac and Graafian follicle fluid,
colostrum and 4-day milk.
Dobson (1966) using sheep infected with Oesophagostomim aolimbianum
found intestinal mucous exudate to contain gamma-, alpha- and beta-
globulins, albumin and mucoprotein. Antibody titer determined by passive
hemagglutination was low in control and high in infected animals
especially when mucous came from areas of infection. Electrophoretic
patterns from non-infected sheep showed high levels of mucoprotein.
After first infected with 0. aolimbianum relative concentrations of the
mucoprotein diminished because of increased alpha- and beta- proteins.
VJhen a second infection v^/as administered, decreasing mucoprotein was
caused mainly by an increase in gamma- globulin.
Serum protein cnanges in sheep with natural or experimental nematode
infections (especially H. contortus) are frequently recorded. Endrejat
20
(1956) first compared serum proteins from parasitized and nonnarasitized
sheep. He reported marked increases in gamma-globulin and decreases in
albumin. "^humard e_t £l. (1957) reported an increased albumin to glob-
ulin ratio (designated A/H) in lambs uith mixed parasite infections.
Kuttler and Marble (1960) reported similar results in lambs infected
with T. axei . Turner and K'ilson (1962), Wilson and Turner (1965) and
Bradley e_^ a_l. (1973) also reported decreased A/''; ratios in parasitized
sheep.
IIATERTALS AND tCTllODS
Experimental Desi;.',n
This investigation used 47 Florida Native lambs reared worm-free,
Tney were divided into 3 groups according to hemoglobin type (Hb A, Hb B,
Hb AB) determined by electrophoresis^ using cellulose-acetate membranes
with tris-ethylenediamine-tetra p.cetic acid-borate buffer (.13M, pH 8.9-
9.3) at 400V for 50 minutes. Each hemoglobin group v/as randomly divided
into 2 sub-groups, an infected and control (non-infected). The lib A
group consisted of 20 animals, 9 infected and 11 controls. Tae Hb B had
17 animals, 9 infected and 8 controls. The Mb AB group had 10 lambs,
7 infected and 3 controls.
Prior to lambing, 114 ewes in the flock from which the lambs would
be selected were examined for worm burdens and tested for hemoglobin type
and hemoglobin level. Correlations between the amount of worms, hemo-
globin type and hemoglobin levels are presented in the results and
discussion sections.
T^Afelve weeks prior to experimental infection with infective larvae
of H. contortus , weekly fecal, blood and serum samples were collected
for ova examination, hematological observation and serum analysis,
respectively. Samoling began at 2.5 months of age and continued until
fiicrozone Electrophoresis System, Beckman Instruments Inc.
"ullerton, California.
21
22
5.5 months of age when experimental infection occurred. Tlie infection
dose was given each lamb based on the equation (110 + body weight of
lamb) X body weight of lamb = number of larvae to use. The equation
xjas intended to produce an infective dose v;hich caused a decrease of
hematocrit values at 10 days post-infection (Tetzlaff, 1970).
On day 0 (day of infection) 1 la'iib (Hb A) \ias euthanized and
necropsied for base line study. Subsequent scheduled necropsy of
infected lambs and controls began 1, 7, 12, 16, 21, 26, 30, 33 and 38
days post-infection (Table 1). Blood, serum and fecal samples were
Table 1. Scheduled Necropsy of //. aontovtus Infected Lambs and Controls,
'Infected with H. aontortiis .
23
collected on necropsy days. At necropsy, animals were inspected grossly
for any abnormalities. The abomasums were then ligated, separated and
collected.
Abomasums were opened and waslied (vjashings collected for parasite
examination), with all adult parasites collected and counted. Abomasums
were then placed in 50 to 100 ml of 0.85% saline at 4°C for 12 hours.
This temperature causes expulsion of tlie mucous from the froblet cells
(Dobson, 1965) . Tne tissues were then placed in HCl-pepsin solution
for digestion of tissue and recovery of larval parasite stages (Herlich,
1956) . The washings and dissolved tissues were washed through an 100
mesh screen sieve (0.149 mm openings) to collect larvae and adult para-
sites. Immature parasitic stages of H. contortus were identified
according to Douvres (1957).
Mucous extracts were concentrated with vacuum dialysis. This pro-
cess uses 1/4 inch dialysis tubing attached to a funnel placed into a
filtering flask and put under vacuum for 24 hours . Measured protein
concentrations similar to that of serum were attained by ref ractometer^
analysis. Mucous and serum v/ere all stored at -20°C which gives no
serum protein changes (Kuttler and Marble, 1959).
Experimental Animals
Florida Native lambs vrere raised under vjorm-free conditions in
concrete-floored pens. The ev/es and their lambs were placed in pens
within 24 hours of lambing. Pens and feed and water troughs were washed
AG T/C Refractometer, American Optical Instrument Co., Buffalo,
New York.
24
daily to avoid fecal contamination. Individual fecal samples were
examined \7eekly by a modified McMaster technique (U^hitlock, 1948) to
verify nematode parasite control. After ueaning at 60 days of age,
blood samples uere taken v/eelcly by jugular vein puncture from each lamb.
Five ml of blood v;ere collected in a Vacutainer^ ^ tube containing EDTA
as an anti-coagulant and 5 ml collected into a Vacutainer tube without
anti-coagulent for serum collection. Ev7es and lambs were fed according
to National Research Council standards.
Fecal samples of several lambs at 1 month of age revealed
Stroncruloides sp. ova. Therapeutic doses of thiabendazole were given
to all lambs. No additional ova v.'ere detected until experimental
infection. Infection wit'n this parasite was believed not to be through
contamination but by pre-natal infection (Pfelffer, 1962) or through the
colostrum or milk as reported in swine (Batte and Moncol, 1986).
Haernonchus contovtus Inoculum
Infective larvae of H. aontortus for use in experimental infections
were initially isolated from the University of Florida sheep flock
using ova identification techniques C^Ionnip, 1956) and infective larvae
identification (Keith, 1953; Skerman and Hillard, 1966). Infective
larvae were tested for viability by first giving them to 2 Finnish
Landrace rams 'jhich had recently undergone anthelmintic treatment with
thiabendazole. Subsequent collection of ova for culturing purposes
to obtain larvae for antigen V7as derived from these rams.
Infective larvae were obtained by fecal-vermiculite cultures at
n'acutainer'' Recton, Dickinson and Co., Rutherford, Nev; Jersey.
25
27 C for 7 days. Tliese cultures were then put Into cheesecloth and
placed in a Baermann apparatus which consists of a clamped funnel and
v.'ire mesh sieve. Warm water is added to the funnel until contact with
the cultured material. After several hours, the larvae attracted to
the v/arm water moved through the cheesecloth and collect at the bottom
of the funnel where they are drained off into shallow petri dishes for
pooling and storage at 10°C. The larvae were washed and allo\;ed to
settle, and the dilution adjusted so that each ml of fluid contained
1000 larvae. Tlie lambs v/ere infected with a 40 ml syringe equipped
with an 8 inch flexible metal tube that v;as rubber coated. The tubing
was inserted into the esophagus of the lamb and the correct larval dose
was expelled. Tliis was followed by passing 1 v/ashing of distilled water
through the syringe.
Hematology and rmmunology
Hematocrit values were obtained using a microcapillary technique.
Microcapillary tubes were filled with blood and sealed at one end with
plastic clay and centrifuged at 11,500 r.p.m. for 5 minutes in a Model
1 9
I-IB centrifuge. Values were obtained using a microcapillarv tube reader
which gave the packed cell volume measured as per cent (%) .
For hemoglobin concentration determination, the cyanmethemoglobin
method V7as employed (Anonymous, 1965a). Tills technique employs the use
of 5.0 ml of cyanmethemoglobin reagent mixed with 0.02 ml of blood by
International Equipment Co., Needham Heights, Massachusetts.
^Ibid.
26
inverting several times. The contents are transferred to a cuvette and
read against a reagent blank using a spectrophotometer. Tlie wave-
length used is 540 niM and the reading converted into grains of hemoglobin
per 100 ml (lib grams%) of blood using a standard curve.
Hucoprotein and serum protein fractionation was carried out by
electrophoresis^ on cellulose acetate membranes using a barbital buffer
(pll 8.6) at 300V for 30 minutes. After a staining and clearing process
(Anonymous, 1965b), membranes were scanned on a densitometer"^ which
produced density curves. These curves were divided into areas repre-
senting discrete fractions (mucoprotein, albumin, alpha-, beta- and
gamma-globulins) in v;hich the area under the curve could be determined,
giving relative percents (%) of these proteins. Total protein was
determined by refractometer, thus giving relative amounts of the
fractions (mg/ml) .
z\ntibody titers in serum and mucous exudate were measured by
indirect hemagglutination (lllA) . This test involves the use of
erythrocytes coated with the antigen for \/hich the animal has made
antibodies or with which the antibodies will cross-react. If the
serum has activity through a series of dilutions, the erythrocytes will
G. K. Turner Associates, Palo Alto, California.
^Microzone Electrophoresis System, Beckman Instruments, Inc.,
Fullerton, California.
3
Model R-110, Beckman Instruments Inc., Fullerton, California.
AG T/C Refractometer, American Optical Instrument Co., Buffalo,
New York.
27
settle to the bottom of the test wells indicating positive or negative
reaction. This test is sensitive (0.003 yg antibody/ral) and can be
used in conjunction with other tests (Kagan and Norman, 1974) . IHA
microtiter test (Kagan and Norman, 1974) was done using Microtiter
equipment .
Immunoelectrophoretic analysis of lamb serum and mucous exudate
9
was performed on electrophoretic apparatus. Samples were tested for
their activity for Immunoglobulins (IgG, IgA and Ig>t) , gamma-globulin,
beta-globulins, alpha-globulins, albumin and H. contortus antigen.
Rabbit anti-sheep IgG, gamma-globulin and serum ;-^ Rabbit anti-ovine
globulins and serum;'^ and Rabbit anti-bovine IgG, IgA and IgM v/ere
used in test analysis. Rabbit anti-bovine immunoglobulins were found
to be cross reactive with sheep serum.
Antigen for use in IHA and the diffusion phase of Immunoelectro-
phoresis v/as obtained from pooled larvae and fresh //. contortus adults
from necropsied lambs by a modified method described by Dobson (1966).
One ml of centrifuged (2000 r.p.m. for 15 minutes) packed larvae were
disintegrated using a tissue grinder and then transferred into 5 dram
containers with 3 mm glass beads and shaken for three 15-minute intervals
Cook Engineering Co., Medical Research Division, Alexandria,
Virginia .
9 F
""llicrozone ' Electrophoresis System, Beckman Instruments, Inc.,
Fullerton, California.
3
ICN Pharmaceuticals, Inc., Cleveland, Ohio.
4
Colorado Serum Co., Denver, Colorado.
Ililes Laboratories, Inc., Kankakee, Illinois.
28
on a Vortex ("tenie ^lixer' . Volume v;as broup^ht to 5 ml in p'aysiological
saline. Similar procedures were used vjith 200 adult worms brought to
2 ml volume.
Statistical Analysi s^
Analysis of variance, regression coefficients program and statistics
of fit for dependent variables was carried out with the aid of the IBM
360-65 computer at the University of Florida. Tlie "Z" two-tailed test
was also used (M-.ndenhall , 1971). Variables analyzed include PCV, blood
hemoglobin levels, serum proteins (albumin, beta-globulin and gamma-
globulin) , abomasal mucous proteins (albumin and gamma-globulin) , ova
counts, total protein, serum antibody (larval and adult antigen test).
Comparisons made v;ere pre-infection by blood hemoglobin type and pre-
and post-infection with regard to infected or non-infected status by
(a) Hb type (b) infection (c) lib type by time (d) time by infection and
(e) lib type by infection.
Scientific Products, Inc., Evanston, Illinois.
RESULTS
Hemoglobin Levels and Haemonchus oantortuG Ova Counts from Florida
Native E\7es Prior to Lambing
Hemoglobin Levels (gms. %) and ova counts (eggs per gram) of
H. contortus (based on morphology, Skerman and Hillard, 1966) v/ere
taken on 114 ewes divided by hemoglobin type (Hb type). The data
is presented in Appendix I. Average values for hemoglobin levels and
ova counts, respectively, v;ere Hb A, 10.6 gms. % and 352.6 e.p.g.,
Hb B, 10.3 gms. % and 324.3 e.p.g. and Hb AB, 10.1 gms. % and 312.8
e.p.g. Statistical comparison between Hb type using the Z test
(Mendenhall, 1971) showed no differences in hemoglobin levels or ova
counts .
Comparison of Packed Cell Volume, Hemoglobin Level, Albumin, Reta-
Globulin, Gamma-Globulin and Total Serum Protein Between Hemoglobin
Types in Worm-Free Lambs Prior to Experimental Infection
Sampling data for mean packed cell volume (Appendix II) , hemo-
globin level (Appendix III) albumin, beta-globulin, gamma-globulin
and total serum protein (Appendix IV) prior to experimental infection
with U. contovtus is summarized in Table 2.
29
30
Table 2. Statistical Comparison of Packed Cell Volume, Hemoglobin
Level, Serum Albumin, Beta-Globulin, Gamma-Globulin and Total Serum
Protein Betv/een Blood Hemoglobin Types in Lambs.
Hb
N
0. of
PCV
Hb
Level
Beta-
Gnmma-
Total
Type
S
amples
(%)
(P
ms. %)
Albumin
Globulin
Globulin
Protein
A
19
36.9
15.9
3.0
0.43
1.4
6.1
B
17
30.5
13.3
2.9
0.40
1.2
5.8
AB
—
10
34.1
15.1
2.9
0.42
1.3
6.0
Overall
Me ans
46
33.9
14.8
2.9
0.42
1.3
6.0
Analysis of variance of the packed cell volumes based on the "F '
test found that the blood hemoglobin types were significantly different
(p < 0.01). Type A was significantly greater than type B (p < 0.01),
type A v;as greater than type AB (p < 0.01) and type B is less than type
AB (p < 0.01) .
Analysis of blood hemoglobin levels in the lambs, based on the F
test, found the blood hemoglobin types to be significantly different
(p < 0.01). Type A was significantly greater than type B (p < 0.01),
type AB was greater than B (p < 0.01), but types A and AB were not
different .
Malysis of the serum proteins (albumin, beta and gamma) shov;ed
no differences between the blood hemoglobin types. Analysis of variance
of total serum proteins showed types A larger than B (p < 0.05) but
no differences betv/een types ^ and AB or B and AJ? .
Nematode P.ecovery in Lambs Experimentally Infected with Haemonchus
contortus from Scheduled Necropsy.
Tlie inoculation doses of infective larvae, recovery of larvae,
early 5th stage and adults from abomasal contents, abomasal digest and
mucous exudate, the total recovery and percent recovery are shoim in
31
Table 3. Ova were first detected on t!ie 21st day post-Infection (see
Appendix V) . Adult v/orms \;ere first recovered in low numbers (8 total)
on 6/18 which corresponds to 21 days post-infection. iRarly 5th stage
larvae were seen until tne end of the experiment period.
Ciianpcs in Packed Cell Volume and Blood Hemoglobin Levels in Florida
Native Lambs During Experimental Infection \7ith Haerronchiis aontortus .
Mean packed cell volumes and blood liemoglobin levels based on
samplinr; data prior to experimental infection i.^ere compared to values
taken at necropsy in infected and control animals (see Appendices II and
III). Tiie differences between infected and controls were contrasted with
and without regard to liemoglobin types.
Figure 1 siiot/s the sequential changes in the packed cell volume in
infected and non-infected lambs with regard to hemoglobin type. Testing
by use of regression coefficients, analysis of variance and statistics
of fit for dependent variables found no differences betv^^een blood hemo-
globin types but found statistical differences at day 26 and 33 post-
infection. These davs correspond to a period of marked variation of
these points in Figure 2. The "T" test using the sum of squares from
the above test found a significant change at day 26, (p < 0.05) and at
day 33, (p < 0.1).
Statistical analysis of blood hemoglobin levels found no difference
V7ith respect to iiemoglobin type but produced "F" values shov7ing a signifi-
cant difference (p < 0.04) between infected and non-infected anim.als.
Figure 3 illustrates the changes of the hemoglobin levels in the lambs
infected and non-infected v;ith //. aontovtus with respect to hemoglobin
type. The variability of the levels at each collection period is very
marked. Figure 4 shows these shifts v/ithout regard to blood hemoglobin
32
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I
Figure 3. Sequential Changes in Hemoglobin Levels in Florida
Native Lambs Infected and Non-Infected with Haemonahus
aontortus Divided by Hemoglobin Types.
38
+ 5.0 -
+ 4.0 -
0 2 4 6 8 10 IJ
14 16 10 20 22 24 2S 28 30 32 34 36 38
Days Post- Infection
^ I m
o
C
ffi
M
C)
60 EJ;
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n
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40
o
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+
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+
+ I
■%suj6) 95uDqo
41
type. This figure also illustrates that the infected animals had more
lower values througiiout the infection period. T"ie packed cell volumes
and hemoglobin levels both increased after day 30 (see Figures 2 and 4) .
Changes in the Serum Proteins in Florida Native Lambs Infected and
Non-In fee ted with Uaerioncnus contortuc
Serum collected prior to infection '7as compared to serum at the day
of necropsy. This data is presented in Appendix IV. Tae serum protein
levels (gms. %) of albumin, alpha-1-globulin, alpha-2-globulin, beta-
globulin and gamjTia-globulin were then statistically analyzed for differ-
ences betv/een the infected and control lambs after infection with
H. contoY'tus .
No significant differences between the infected and control lambs
were apparent for albumin, alpha-globulins or beta-globulin. However,
there was statistical significance (p < 0.1) in the differences of the
amounts of gamma- globulins . Tlie infected lambs had consistently higher
values than tiie controls as snown in Figure 5. This can also be seen
in Table 4 by looking at the differences between the changes of serum
protein values. The analysis also indicated a decrease in the albumin-
to-globulin ratio in infected lambs, with no relationship to hemoglobin
type. Figure 6 sliows this ratio decrease by hemoglobin tyne along with
the average values (\.7ithout regard to hemoglol^in type) .
The mean percentages of the serum proteins at each necropsy period
are presented in Table 5. Significant trends are masked since comparisons
of the animals prior to infection are not included as above and the
differences in total proteins are not taken into account. The infected
animals overall had lower albumin and higher gamma-globulin percentages.
g CTJ D
c
S
m
M
-u
<u
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^
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m
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43
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4-
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O
+
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O CM *^- <1> CO O
d d a d d -f
till
(% sujD) obuoqo
44
Table 4. Differencf.s Between the Changes of Serur.i Proteins of
Lambs Infected and Moa-Infected with Saernor.ch::S aontortus
Sheep
Type!
Post-Infection
(Days)
Electrophores
is Values
(p=u %
)
Ganma
Average
A/G
Rstio
A/G
Average
Albumin
Alpha-1
.(L;.pha-2
Beta
Ganda
A
1
+0.51
+0.96
-0.32
+0.Q7
+1.07
+1.63
B
1
-0.43
-0.18
-0.26
+0.52
+0.03
-0.25
-0.37
+0.31
AB
1
-0.34
-0.28
-0.29
-0.05
+0.26
-0.33
A
7
-0.23
-0.08
-0.10
~0.C3
+0.22
-0.72
B
7
+0.04
+0.09
HO. 02
+0.25
+0.09
+0.03
-0.20
-0.12 -
AS
7
+0.40
-0.14
+0 . 16
~Q.03
-0.21
+0.55
A
B
12
12
+1.31
+0.24
+0.09
+U.16
+0.43
-0.25
-0.71
+0.03
+0.57
-0 . 14
-to. 22
+0.0t)
+1.'^,9
+0.72
A
B
16
16
-0.63
+0.37
-0.15
-0.16
+0.60
+0.25
-0.02
+0.C3
+0.54
-0.07
+0.24
-0.90
+0.31
-0.30
A
21
-0.81
+0.L5
+0.23
+0.53
-0.22
-0 . 18
+0.53
H-0,25
B
21
-0.40
+0.10
+0.03
+0.17
-0.14
-0.04
A
26
-0.11
+0.04
0.00
+0.03
+0.43
-0.90
B
26
+0.72
-0.25
-0.15
+0.20
+0.17
+0.32
+0.20
-0.35
AB
26
-0.42
-0.10
+0.10
-0.19
+0.35
-0.46
A
30
-O.OG
+0.08
+0.22
-0.04
+0.60
-0.73
B
3G
-0.37
-0.15
+0.13
+0.2]
+0.77
+0.29
-2.00
-0.63
AB
30
+0.34
-0.10
-0.54
+0.07
-0.50
+0.85
A
33
-0.74
-0.03
-0.15
-0.27
+0.1/4
-0.59
B
33
-O.Sl
-0.03
-0.19
-0.10
+0.09
+0.35
-0.6S
-0.76
AS
33
-0.18
-0.15
+0.30
-0.]S
+0.82
-1.04
1 -
38
+0.21
+0.02
-0.46
-0.41
+0.70
-0.51
^
38
-0.81
+0.10
+0,C3
+0.05
+0.11
+0.32
-0.88
-0.63
1 -
j8
-0.69
+0.10
+0.06
+0.05
+0.16
-0.49
Hemoglobin type
3
3 C
w
c
0)
n)
ri
Tl
0)
4J
b
O
CO
ti)
^•^
01
M-l
+i
r;
ri
i*
u
M
-1^
c
en
S'
H
r",
u
0)
O
o
M-l
•H
£.-
M-t
■U
w
•H
crt
fi
H
pi
i-c;
46
OQ
< OQ
<
CU 0)
a. a.
>. >«
1- H
Q.
o
J3 XI
X X
J3
X
^
II II
II
II
0<3
>
o
o o
<4- CJ
o o o o o o
O 00 CO ^. CM. O
— ~ d d d o a
(% SLu5) uoipuDA jO saDuajo^^iQ
o o
d d
o o o o' o
U3 CO O <^l CD
do — " —
47
Table 5. Mean Percentages of Serum Proteins from Lambs
Infected and Non-Infected v.'ith Haernonahus contortiis .
Electro
phoresis
Values
(%)
No. of
Day
Lambs
Post-Infection
Albumin
Alpha
Beta
Gamma
Infected
3
1
42.0
23.9
11.8
22.3
Control
3
1
45.7
26.6
7.3
20.4
Infected
3
7
49.8
20.4
8.4
21.4
Control
3
7
53.9
21 .2
7.2
17.8
Infected
2
12
50.2
19.2
9.2
21.3
Control
2
12
48. 1
19.8
12.4
19.6
Infected
3
16
48.2
21.0
8.1
22.7
Control
3
16
46.3
20.3
10.2
23.3
Infected
2
21
45.8
22.5
8.4
23.1
Control
2
21
45.6
19.7
7.1
24.1
Infected
3
26
54.6
19.1
7.5
18.8
Control
2
26
50.7
20.5
7.7
21.0
Infected
3
30
41.7
22.8
9.2
26.3
Control
3
30
52.0
20.6
9.4
18.0
Infected
3
33
42.2
20.7
8.5
28.8
Control
2
33
49.9
20.5
8.8
20.8
Infected
3
38
44.4
17.8
7.6
27.0
Control
1
38
38.0
24.0
9.0
29.0
Total
Infected
25
—
46.4
22.0
8.7
23.6
Total
Control
21
—
48.6
21.5
9.0
20.9
48
Proteins in Abomasal Mucous Exudate from Lambs Infected and Non-Infected
with Haemo'nahus aontortus
Mean electrophoretic values of proteins found in abomasal mucous
exudate during sequential necropsy are presented in Table 6. Total
data is recorded in Appendix VI. Areas corresponding to albumin, alpha-
globulin, beta-globulin and gamma-globulin were observed as was a large
protein area designated as mucoprotein. This mucoprotein migrated
within the area of the alpha and beta-globnlins . Common characteristic
electrophoretic patterns of the abomasal mucous are shovm in Figure 7.
The pattern in Figure 7a represents the albumin (1), alpha (2), beta (3),
and gamma (4) protein areas. Note the migration of mucoprotein into
tlie beta area. The pattern in Figure 7b exhibits migration of the muco-
protein past the beta area where it infringes on the alpha protein area,
waile Figure 7c shows a pattern with low amounts of mucoprotein.
The albumin, mucoprotein and alpha-globulin, mucoprotein and beta-
globulin and gamma-globulin levels are presented graphically in Figure 8.
Statistical analysis produced a significant "F" value (p < 0.04) for
albumin comparison between the infected and control animals. Tliere V7as
a particular significance of albumin differences at day 30 (p < 0.08).
No significant differences in gamma-globulin levels of infected versus
control lambs were noted.
Antibody Evaluation in Serum from Lambs Infected and Non-Infected with
Haemonchus aontortus
Serum obtained at necropsy during the course of infection was tested
by indirect hemagglutination (IHA) . Sheep erythrocytes used in the test
were either coated with antigen derived from adult or larval H. aontortus.
Sisnigicant changes in antibody titer between the controls and infected
animals were noted using the test v/ith adult antigen (p < 0.08) and
49
Table 6. Mean Percentages of Proteins in Abomasal Mucous
Exudate from Lambs Infected and Non-Infected with
Haemonchus contortus.
N
0 . of
Day
Electrophoresis Values (%)
Gamma -
Mucoprotein &
Mucoprotein &
Lambs
Post-Tnfcctlon
Albumin
Alpha- Global in
P.eta-Clobulin
Globulin
Infected
3
1
39.4
6.3
37.1
17.2
Control
3
1
38.9
9.1
38.6
12.2
Infected
3
7
38.3
10.1
36.6
15.0
Control
3
7
40.4
17.5
21.2
20.9
Infected
2
12
31.2
12.0
40.7
16.0
Control
2
12
33.9
6.9
43.5
16.1
Infected
3
16
36.9
10.4
30.8
21.9
Control
3
16
38.7
8.0
34.5
18.8
Infected
2
21
27.3
4.9
40.1
27.6
Control
2
21
31.7
6.3
38.6
23.3
Infected
3
26
21.8
20.9
34.4
22.9
Control
2
26
38.9
10.1
32.2
18.7
Infected
3
30
17.6
11.3
50.2
20.9
Control
3
30
38.0
9.3
28.2
24.5
Infected
3
33
34.1
10.7
29.2
26.0
Control
2
33
34.5
12.8
31.1
21.5
Infected
3
38
26.8
7.3
37.9
27.9
Control
1
38
34.4
6.1
34.1
25.4
Total
Infected
25
—
30.5
10.6
37.2
21.7
Total
Control
21
—
37.2
10.0
33.0
19.7
Figure 7. Characteristic Electrophoretic Patterns of Abomasal Mucous
Exudate from Lambs Infected and Non-Infected with
Haemonahus aon tro tus .
51
;
;:-;:m--:
rim
pl=t--:
- ' . o
k:-.:
— —
i i : \ :
— 4— -
" '■- '-'■■-^\
■5ii -
- : : ,-
A
— :--^
— I — .
-lirr
— t-nS
1 . , ■ ;
- 1 ■ ■ "
; ::
1 \ '
__;__
v
'f 1 :
- '-^
— 1 — —
H
:-: : ;
— —
+
; _t
- — -
'-- ^
:- J
f^--
' ' ■ '■ i! : ; ;
: : ; :
_^^:..^
fW
-
^
\
4
—4
' ; .0
^ : : ;
\
■^
'/}:'■-['-
: !■!
V
■ i
/:" i VI
: i
- - o
i-' ; •
^
Htl
■ li-
-Mi^i.H
-^-P
H-l-t
■ ■ ; ;
i^
'yj
^"'IT
==^
^-oi
4=H-.t;
zi-iii:
mf-i+M
mi.
S ir
Figure 7. "continued"
53
Figure 8. Protein Content in Abomasal Mucous Exudate from
Lambs Infected and Non-Infected with Eaemonchus
contovtus .
55
40
30 -
:20
10
-1— c
■o e'140
:3 0)0
m 2? 30
3 ':^f 20
o s<
O -"
2
10
50
o
n
= 540
«) o
y?3o
o
§"2
{/)
'icD20
iO
5
u>
Q.
-40
J.-3
S30
j:s o
cr
E20
E
a
o 10
-o = Infected
--« = Control
J L
0 2 4 6 8 10
12 14 16 18 20 22 24 26 28 'dO 32 34 36 38
Days Post- Infection
56
larval antigen (p -^ 0.008). The test usinc larval antigen gave better
responses than did the adult antigen. Differences in blood hemoglobin
type approaching a significant level of 87%, and a level approaching 81%
considering type by time from the tests using larval antigen were noted.
Looking at these variations in Figure 9, the differences between lib A,
ilb B and hb AB (Figures 9a, 9b and 9c, respectively) are actually a type
by time relationship not due to one type having a better response. These
responses occurred at different times; Type A shov/ing early response,
Type B varied response and Type AB having a later response.
Antibody Evaluation in Abomasal Mucous Exudate from Lambs Infected and
Non- Infected with Haemonohus oontovtiis
Abomasal mucous v/hich had been concentrated to within ranges of serum
protein levels vjas tested by IHA, Significant changes in antibody titer
bet'jeen the controls and infected animals were noted using adtalt antigen
(p < 0.00) and larval antigen (p < 0.004 Table 7). Differences between
blood hemoglobin types were not shown using adult antigen in the IHA test
but had some differences at the 89% ].evel using larval antigen. Mean
responses of type K \-;ere slightly better than types A and AB ^^'hich were
very similar. As \-]xt\\ serum antibody, Figures 10a, b and c (Hb A, Hb B
and Hb AB , respectively) show similar responses betxv'een all types of
lambs. Hemoglobin type A had an earlier response, type B had a varied
response and type AB had a later response though these '-/ere not statis-
tically significant.
Figure 11 plots mucous antibody titer wit'nout regard to blood
hemoglobin types. Good responses v;ere shown throughout the infection
period particularly at day 12 and day 30. Indirect hemagglutination
testing using adult antigen gave higher titer response.
a)
ri
m
(fl
>
V-i
X)
tfl
fl)
h-i
•u
CJ
CO
0)
.N
LH
4^
a
i^H
M
(.■^
-tJ
4-1
r
0
u
O
>,
I/I
w
ti
n
rS^
u
<:•,
u
■s-
a)
'-•^
^
w
•H
«
cfl
U3
■H
00 C/2
43
<;
H
m
H
cfl
>^ o
1-1
• »v
Td M
<^
o m
X)
rCl
(1)
QJ
•H m
4-1
O.
■U 4-1
CJ
>.
^-5
CU
H
XI
n
,a
B <
M
13
1
!-i X)
c
58
Q.
X
>«
T3
>,
•X3
O
JQ
T3
O
J_
0)
C
'^
<l)
<T
c
•o
c
<
o
o
->
QJ
c
k.
3
o
n
■n
z
_J
<
II
SI
II
11
O
n
1
1
1
00
<M
-^ C\J
to ro
J8;!X Apoqijuv iDOOJdioa^
<jO CO
60
9|ij_ Apoqijuv |D30Jd!09y
62
<
<^
-O >s
O "O
£i O
< c
<
~> — >
S '^ -^ <
II II >i 11
!>+ '
>.
X
H
-o
.a
CJ
X.
o
o
T7
H,-
CJ
c
•9—
o
1
o
c
o
PC.
jaill Apoqiiuv/
IDOOJdlOD^J
Table 7. Antibo.'y TiLer A-;i;iast raemor.ahy^ contjrz-^s in S.?rviEA aiid Aboaasal
Mucous Ixtra^Cion frc-n C .quon'.: .fJ llecropsy uf Infected and Non-Infected Lambs
63
Dale
Shee.p No.
(Type-)
1
Tr-fecr-'ou i
...
Ova
Cour.c
Scrun Titer
La:val
Adult
Larval Adulc
. 1
1
.Antioeu
Antieen
Antisen Ant:?en
5/29
14* A
1
0
22 j
64
S
16
;9 A
1
0
8
16
4
16
147* B
1
0
16
256
4
64
128 B
1
0
8
8
4
4
15* AS
1'
0
8
32
4
S
124 AE
.1
G
8
32
6
64
6/4
132* A
7
0
8
64
A
16
120 A
7
0
8
8
S
4
109* B
7
0
32
123
8
32
122 B
7
0
16
16
S
8
27* AB
1
0
128
64
16
4
139 A3
1
0
8
16
S
4
6/9
10* A
12
0
32
256
8
16
17 A
12
0
16
16
8
4
117* B
12
0
16
32
4
o
11 B
12
c
32
8
3
z
6/13
118* A
16
0
8
64
4
32
110 A
16
0
16
32
8
8
21 A
16 1
a
32
37-
8
64
127* B
16
0
64
128
16
32
134 3
16
0
16
16
8
2
137* kZ
16
0
64
64
8
16
6/18
114* A
21
400
16
64
16
32
24 A
21
c
1 16
16
8
8
113* B
31
200
32
8
8
4
126 B
21
0
8
8
8
8
6/23
115* A
23 A
26
2,200
32
16
8
8
26
0
4
8
4
4
22*- 3 i
26
1,400
64
123
32
32
141 B ,
26
0
16
16
S
4
16* AB
26
1,200
1 6'^
64
16
16
6/27
111* A
30
4,400
8
16
8
8
9 A
30
0
16
16
8
3
121* B
30
1 5,600
12S
128
32
16
50 B
30
0
16
16
8
4
130* AB
30
6 , 000
128
256
32
64
112 AB
30
0
8
8
4
4
7/2
12* A
33
7,600
32
32
16
16
13 A
33
0
32
32
8
4
28* B
33
5,800
64
32
16
16
29 B
33
0
16
16
8
8
119* A3
33
8,200
64
64
16
4
7/7
123* A
38
4,200
128
64
16
8
25 A
38
0
8
16
8
8
31* E
33
8,800
16
16
8
4
136* AB
38
5,600
32
8
16
^
Heaoglobin Type
^Reciprocal tltars using IlIA
*Infected Lambs
0) T3
CO C
t ""
CD -d
1-4 tU
j-i
CO cj
M,
S 0)
<i
-^i M-l
Sh C
CU
O M
P-
+i
S M-i
H
Q O
o,
(IJ
i~-
k-.
^^^
c^
rH
&:
cfl
^
>, e CTJ
■:l
m
tn
x)
C
^
3
<;
M
m
o
1
o
•r)
C
3
a
o
•
65
ja|i_L Apoqijuv poojclioay
67
j8;!l Apoquuv
to
Doojdioavj
69
256r
128
(c)
>^
-a
o
JO
<
a 64
o
o
Q.
'o
CD
« = Infected Hb Type AB
+ = Non-Infected Hb Type AB
— = Larval Antibody
— = Adult Antibody
l\
32
16-
0 2 4 6 8 10 12 14 16 IS 20 22 24 26 28 30 32 34 36 38
Days Post-Infection
O Q^
iH 0)
0) 4-1
> a
1-1 H-l
d
o a
q
T)
o
<rl
c
M
CO
CJ
tn
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d
t:!
r,
()
(1)
o
u
■M
1
C)
n1
(1)
M-l
fo
iH
n
r-N
CTl
h- 1
-t-i
(/I
^1
crt
m
v^
p:
.n
+i
u
Fi
r
JJ
tH
u
<;
1-1
o
71
.JOiix Apoqjiuv iDOOJdiOoy
72
Comparison of Mean Percentages of Proteins in Serum and Abomasal Mucous
from. Florida Native Lambs Infected and Non-Infected v/ith Haemonohus
contortus
Mean levels of the serum proteins as compared to abomasal mucous
proteins are presented in Table 8. Substantial differences were seen
in the albumin levels, but gamma-globulin was not substantially different.
Alpha and beta proteins could not be compared due to the presence of
mucoprotein.
Comparison of Antibody Titers in Serum and Abomasal Mucous from Florida
Native Lambs Infected and Non-Infected with Haemonohus aontortus
The results of IIIA testing of serum and abomasal mucous exudate
from lambs infected and non-infected with H. aontortus are presented in
Table 7. Higher titers were observed in the mucous than serum. Indirect
humagglutination testing using adult antigen gave better responses than
did larval antigen. There was no substantial upsv/ing of titer levels
at any point during infection, but there did appear to be a grouping
of slightly higher responses at days 26 and 30. This time period
corresponds to the time just after the parasite reaches patency.
Immunoelectrophoretic Characterization of Antibody and Proteins in
Serum and Abomasal Mucous from Florida Native Lambs Infected and Non-
Infected with HaemonchMS aontortus
Tlie results of the immunoelectrophoretic analysis for immunological
responses: gamma-globulin, IgG, IgA and IgM in serum and abomasal mucous
are given in Tables 9 and 10, respectively. In both the sera and mucous,
a strong reaction to anti-gamma-globulins and IgG was noted. The sera
had no detectable reaction against IgA but good response for IgM, while
the mucous had good IgA response and no detectable IgM response. In
these instances responses appeared stronger in infected animals. There
were no detectable responses in serum against antigen made from H.
aontortus adults or larvae. There were responses shown in mucous against
73
E
Cr,
u
;^;
1-1
4^
M-(
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ri
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-tJ
3
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1.^
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0)
CJ
01
(11
OC 14-1
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a)
rt
n.
,-1
.^ o
CO ,-1
■i:
Ol ^
LO
r^
h- 1
M
c^t — (
OJ
c--)
u
+1 -t-l
u
+ 1
+1
CO
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E
r~~.
r^
1
c^ O
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a-'.
CO
c^i r-j
CO
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cn
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r~~
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+ 1
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+ 1
c
a
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in
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D
3
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vO <Xi
jn
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r-i
<} <t
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ro
ro
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4-1 cn
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(» ol
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a
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c
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74
Table 9. Inmiuiioelectrophoretic Analysis of Serum from Sequential
Necropsy of Lambs Infected and Non-Ir.f ecced with Haemanah-:^ aontvotus.
Date
Sheep No.
Ganmia—
IgG
IgA
XgM
H. eontovtvs antigen
(Typel)
Globulin
Adult
Larvae
5/29
14* A
■H-l-
-H-f
+
.._
~
147* B
+++
+4-f-
—
4
—
—
15* AB
+++
44-+
—
+
—
—
19 A
+++
4-H-
—
+
—
—
128 B
-{-++
-H-+
—
+
—
—
124 A3
-l-H-
4-^+
—
+
—
—
6/A
132* A
4-H-
4-H-
—
++
—
~
109* B
+++
+++
—
+4-
—
—
27* AB
+-H-
+4-+
—
+
—
—
120 A
+++
4-4+
—
+
—
—
122 B
+++
4-H-
—
++
—
—
139 AB
+++
+++
—
+
—
—
6/9
10* A
f-H-
4-H-
+
—
—
117* B
+-H-
+4-+
—
+
—
—
17 A
+++
+44-
—
+
—
—
11 B
+++
4-H-
~
+
—
—
6/13
118* A
-l-H-
4-H-
—
++
—
—
127* B
+++
+++
—
+
—
—
137* AB
+++
+++
—
+
—
—
110 A
+-H-
+++
—
+
—
—
21 A
+++
4-t4-
—
44-
—
—
13A B
+++
4-++
—
++
—
—
6/18
114* A
+4+
+++
—
+
~
—
113* B
+-H-
4++
—
+
—
—
24 A
+++•
44->-
—
+
—
—
126 B
+++
+++
—
+
—
—
6/2?,
115* A
+++
+++
+
—
—
22* B
+-H-
44-f
—
+
—
—
16* AB
+++
+4-+
—
++
—
—
23 A
-f-H-
+++
—
4-
—
—
141 B
+++
4-H-
—
++
—
—
o'll
111* A
-H-)-
+++
—
+
—
—
121* B
-H-f
+++
—
+
—
—
130* AB
-H-+
+44-
—
+
—
—
9 A
+4-1-
44-+
—
+
—
—
30 B
H-H-
-l-t-f
—
+
~
—
112 AB
+4+
+++
—
+
—
—
111
12* A
+++
4-H-
—
44-
—
—
28* B
+44
-H-+
—
+
—
—
119* AB
-l-H-
+++
—
+
—
—
13 A
4-++
+++
—
+
—
—
29 6
+4-+
4-H-
—
+
—
—
7/7
123* A
-K4-
+44
—
++
.-_
~
31* B
44-4
+4 +
—
+++
—
—
136* AB
4-H-
+4+
—
++
—
—
25 A
+4 +
4-14-
1
+
—
^Heiaoglobi a type
+++ = strong precipitation line
44- = good precipitation line
+ = detectable precipitation lint
= no detectabJe reaction
75
Table 10. Iramunoelectrophoretic Anilysis of Abonasal Mucous from Seq-aential
Necropsy of Lambs Infected and Non-Infected with 'dasrjcnahus aontovt^ua.
Date
Sheep No.
Ganna-
a. aor.tortu
s Antigen
(TjTJel)
Clobuliu
IgG
4
IgA
IgM
Adult
Lar/ae
5/29
14* A.
-H-f
-H-f
++
—
147* B
-HH-
+++
4
—
—
—
13* AB
++(-
+++
-H-
—
4-
—
19 A
++-(-
-H-l-
+
—
—
—
128 B
+++
•1-H-
—
—
4-
—
124 AB
+++
+++
+
—
—
~
6/4
132* A.
-t-f+
+++
++
4-
4-
109* B
+++
+++
++
—
4-
4-
27* AB
+-H-
+-H-
+
—
—
—
120 A
-H-t-
-m-
•H-
—
—
—
122 B
+++
+++
+
—
—
—
139 AB
+-H-
+++
■\-¥
—
—
—
6/9
10* A
+-H-
+++
+^
—
—
117* B
+++
+H-
-H-
—
4-
—
17 A
+++
4-H-
++
—
4-
—
11 B
+++
+++
+
—
4-
—
6/13
118* A
-t-H-
4-H-
•r
127* B
-H-l-
+++
+
—
—
—
137* AB
-H-l-
+++
■H-
—
—
—
110 A
+++
-M-)-
+
—
. —
—
21 A
+++
-m-
-H-
—
—
—
134 B
-m-
-H-f
-H-
—
+■
—
5/13
114* A
-m-
+++
+
4-
113* B
+++
+++
4+
—
4-
—
24 A
4-H-
+++
+4-
—
4-
+
126 B
+++
+++
4-
—
4-
+
6/23
115* A
+++
-H-f
4-1-
_
22* B
-H-l-
+++
4-
—
—
—
15* AB
+-H-
+++
■H-
—
—
—
23 A
+-H-
+++
+
—
—
—
141 3
+++
+-H-
4-
—
—
—
6/27
111* A
+-H-
+++
4-
—
121* B
-H-f
+++
l-(-
4-)-
44-
130* AB
+++
+++
44-
4-
+
4-
9 A
+++
+-H-
4-
—
—
—
30 B
-H-l-
+++
4-
—
—
—
112 AB
+++
+++
4-f
—
~
~
7/2
12* A
+++
■H-+
44-
+
_
23* B
•1-H-
-H-!-
4-
—
4-
—
119* AB
+++
+++
4-
—
—
—
13 A
+++
+++
4-
—
—
—
29 B
+++
+++
4-4-
—
—
—
111
123* A
4-H-
-H+
4-
4-
^
31* B
+++
+++
4-f
—
4-
4-
136* AB
-H-f-
+++
44-
—
4-
4-
25 A
-;-H-
+++
4-
—
—
—
Heaoglobln type
4-1-4- = strong precipitation line
4-4- = good precipitation line
4- = detectable precipitation line
- = no detectable reaction
76
adult and larval antigen though more reactions v/ere seen using adult
antigen.
Typical immunoelectrophoretic patterns from control lambs are shovm
in Figure 12. Tests for responses against TgA, Ig'l, TgG, larval //.
oontovtus (L.IIC) and sheep serum fractions (/>.S.) \jere developed using
anti-sera to the above immunoglobulin classes or proteins. Figure
12a demonstrates that there are good IgM and IgG responses. Anti-sheep
serum revealed: the immunoglobulins-G (1) and M (2), I additional beta
protein, 2 alpha protein arcs (3) and albumin (4) . Figure 12b from
another non-infected lamb revealed no immunoglobulin-A response or reaction
to larval //. aontortiuJ . Tne anti-sheep globulins (sheep serum minus
albumin) responses revealed: good immunoglobulin-G (1), 3 arcs in the
beta protein area (5), one arc being an identity with IgM, 3 alpha protein
arcs (3) and albumin (A) .
The patterns in Figure 13a from infected Florida Native lamb sera
demonstrated good response to IgG. Tliis IgG arc also revealed slow
IgG (1) and fast IgG (2). There v.'as an increase to 5 arcs in the beta
protein area (3) and only 1 alpha arc (4). Although an increase in
precipition arcs were noted in most Infected lambs, some lambs had
similar patterns to non-infected lambs (Figure 13b) . This figure reveals
excellent IgN response (5) and two additional beta-proteins - (3) two
alpha proteins (4) and albumin (8) . Tliere uere no detectable reactions
to either larval or adult H. contovtuo antigen.
Immunoelectrophoretic patterns developed from abomasal mucous
exudate are presented in Figures 14 and 15. Slow (1) and fast (2) IgG,
1 or 2 beta proteins arcs (3) , 1 or 2 alpha protein arcs (4) and albumin
Figure 12. Characteristic Immunoelectrophoretic Patterns of Serum
from Worm-Free Florida Native Lambs .
Abbreviations used:
A.IgA=anti-immunoglobulin A; A. IgG=anti-immunoglobulin
G; A. IgM=anti-immunoglobulin M; A. S.=anti-sheep serum;
L.HC=larval E. contortus.
78
•I
r •
Figure 13. Characteristic Immunoelectrophoretic Patterns of Serum
from Florida Native Lambs Infected with Haemonahus
oontortus.
Abbreviations used:
A.lgG=anti-immunoglobulin G; A. lgM=anti- immunoglobulin M;
L.HC=larval E. oontortus antigen; A.G.=anti-sheep globulins
(serum minus albumin); A.S.=anti-sheep serum; A.HC=adult
H. oontortus antigen.
80
Figure 14. Innnunoelectrophoretic Patterns from Abomasal Mucous Exudate
In Parasitized Florida Native Lambs.
Abbreviations used:
A. IgA=anti-immunoglobulin A; A. IgG=anti- immunoglobulin G;
A.G.=anti-sheep globulins; A.S.=anti-sheep serum;
A.HC=adult H. aontortus antigen; L.HC=larval H. oontortus
antigen.
82
Figure 14. "continued"
84
Figure 15. Immunoelectrophoretic Patterns from Abomasal Mucous Exudate
in Non-Parasitized Florida Native Lambs.
Abbreviations used:
A. IgA=anti-immunoglobulin A; A.G.=anti-sheep globulins;
A. S.=anti-sheep serum; A.HC=adult B. aontortus antigen;
L.HC=larval H, aontortus antigen.
86
87
(5) \;ere characteristic of infected Iambs. Similar patterns were seen
in non-infected lambs (Figures 15a and b) except that only 1 beta protein
V7as likely to be seen. Besides TgG, IgA response \ias excellent iind
produced an identity ^/ith a spur arising from the gamma protein area
(Figures 14b, 7 and 15a, 7). An IgM response could not be detected
with anti-IgH. Infected Iambs also showed response against adult
H. oontoY't'iia antigen (Figure 14c, 8).
DISCUSSION
Relationship of Blood Hemoglobin Types to Blood Hemoglobin Levels and
Natural Infection with Haemonohus oontortus in Florida Native Ewes
Evidence of hemoglobin type differences in natural infection of
Florida Native Ev/es with H. oontortus as determined by ova counts was
not shown in sampling data taken on 114 ewes. This was in disagreement
with Evans et_ al^ . (1963) and Jilek (1968) who reported fewer H. oontortus
in Hb A than other hemoglobin types. Tnis was in agreement with
Radhakrishnan e^ al^. (1972) whose data did not suggest any differences
in infection rates by hemoglobin type in Florida Native or Rambouillet
sheep. In fact, Radhakrishnan e_t al_. (1972) and Bradley e^ al. (1973)
reported lower adult populations and egg counts in Hb AB than Hb A or Hb B
lambs experimentally infected with E. contortus . The average blood
hemoglobin levels in the three hemoglobin types revealed no differences,
suggesting an even distribution of infection among the adult sheep.
Perhaps, over time, immunologic factors initially different during first
exposures become similar due to constant reexposure . This observation
is substantiated by observations reported by Soulsby (1958), Levine et al .
(1956), Bradley and Levine (1957) and Levine e_t £l . (1975) in which
sheep kept on the same pasture v/here infective larvae are continuously
available have lower worm populations than sheep that are rotated to
different pastures or have a non- immune status.
Relationships oF Packed Cell Volume, Hemoglobin Level and Serum Proteins
t(j Hemoglobin Types In Worni--Free Lambs
Packed cell volumes betv/een blood hemoglobin types showed significant
differences which is in agreement with reported literature. Hemoglobin
type A had tlic highest erytlirocyte volume, lib B the least and Hb A15 was
intermediate betv/een the other two types. Tne significance level of
the PCV observation is considerably higher than the reports of
Radhakrishnan e_t al. (1972) and Bradley e_t jd . (1973). Reports by
Evans and Uliitlock (1964) and Jilek and Bradley (1969) indicating lower
infection rates in sheep with Hb ^ are contradictory to the reports of
Radhakrishnan e_t al . (1972) and Bradley e_t a_l . (1973) indicating lower
helminth egg counts and fewer adult worms in Hb AB . These authors did
report higher weight gains in Hb A though the statistical test was
ambiguous. Both of these points will be discussed in more detail in a
later section.
The blood hemoglobin values substantiate the report by Jilek and
Bradley (1969) that Hb A was significantly greater than Hb B, Hb AB
was greater than Hb B, but differences between Hb A and Hb AB \/ere
slight. Large fluctuations in hemoglobin levels were observed over
the periods prior to and after infection (see Figure 3) .
Differences in the blood serum proteins could not be correlated to
Hb types. Tne total protein data did give an indication that Hb A was
higher in total protein content than Hb 15, thougli no differences were
seen between Hb A and Hb AB or Hb B and Hb AB . Possible relationships
may exist between higher PCV and hemoglobin levels and the higher total
serum proteins in Hb A. Tliese physiologic factors alone might give
Hb A sheep the capacity to withstand the effects of H. contovtus
90
infection as reported by Evans and l-Jliitlock (1964), Jilek and Bradley
(1969) and Bradley e_t al • (1973). Tne ability of certain breeds of
sheep to resist parasitic infections is considered to be immunologic as
well as physiologic in nature. These factors (immunologic and physio-
logic) are discussed in further detail using the Florida Native Sheep
as a model .
Since differences in lib type and individual animals were noted,
experimental data collected after infection must take these facts into
consideration. TTierefore, a double control system v;as used. Collected
data after infection with //. oontovtus was compared to sampling data
prior to infection. Tliese changes in themselves give significant data
but to give further creditability , comparisons V7ere also made to non-
infected animals handled in a similar manner.
Nematode Recovery in Florida Native Lambs Experimentally Infected with
Haemonohus co'ntortus
The lower recovery rates in i^lorida Native lambs (Table 3)
initially establishes that some factors are acting to keep infection
at a lower than expected level. This is in agreement with similar
results reported by Radhakrishnan et_ al . (1972) and Bradley et al.
(1973) in Florida Native lambs.
Discussion of the Changes in Packed Cell Volume, B lood Hemoglobin Level
and Serum Proteins in Florida Native Lambs Associated with Haemonohus
aontortus Infection
Maximum blood loss appeared at approximately 26 days after infection
according to the results of the packed cell volumes and hemoglobin
levels (Figures 2 and 4). Brambell et al_. (1964) reported first blood
losses in the feces of sheep 6 to 10 days after infection with H.
contortus , with most blood loss occurring at 22 days. Bradley et al .
91
(1973) reported maximum blood loss at 24-26 days post-infection. Blood
hemoglobin V7as a more sensitive test for the determination of blood
loss since significant statistical differences (p < O.OA) were observed
between infected and non-infected animals. The increase in PCV and
hemoglobin after day 30 post-infection is believed to be a recovery due
to increased liemopoiesis due to stimulation from the blood loss.
The increase in gamma-globulin and decreased albumin-to-globulin
ratio (Figures 5 and 8) in the infected Florida Native lambs verify
similar reports in the literature. Turner and Wilson (1962) and Wilson
and Turner (1965) presented evidence that increased gamma-globulin
content may be related directly to the degree of resistance. Identifi-
cation of tlie serum proteins, immunoglobulins and antibodv activity as
determined by THA will be discussed furtlier.
Discussion of Abomasal >fucous Proteins from Lambs Infected and Mon-
Infected v/ith Uaenonchus contovtus
T!ie percentages of the abomasal mucous proteins are presented for
the first time in sheep. These values were similar to those presented
by Dobson (1966) for intestinal mucous exudate in Border Leicester x
Merino sheep infected and non-infected with 0. oolimbicoium.
Tiie largest differences between infected and control (non-infected)
animals were noted with the albumin protein values. At the 30th day
post-infection a significant decrease was noted in infected animals.
This time sequence corresponds to shortly after the parasite matures
and begins increased blood sucking activity (verified by PCV decrease) .
This response, coupled v;ith increases in gamma-globulin, is a
characteristic response seen in serum and can nov; be said to also
occur in abomasal mucous in sheeiJ.
92
Discussion of Antibody Activity in Serum and Abomasal Mucous from Florida
Native Lambs Infected and Non-Infected \7it.'i Hae'nonchiiS oontortiis
Tlie activity of antibody in both serum and abomasal mucous increased
significantly after experimental infection with H. oontovtus . Tliis is
similar to reports in the literature. Indirect hemmagglutlnation testing
used antigen derived from larvae and adult worms. The larval antigen
gave higher statistically significant results, although adult antigen
gave higher titer responses. Differences in blood hemoglobulin type
vjere not observed in analysis of serum antibody activity but significant
differences were approached using larval antigen in abomasal mucous titer
analysis. The pattern being that Hb A had the earliest response, Hb B
was varied and lib AB was seen later in the infection. Responses verified
the presence of antibody activity directed against the parasitic
infection. Hucous had significantly higher titers than serum, especially
after patency occurred (Figure 11). The area at the site of infection
in the abomasum had the highest antibody activity, similar to the results
of Dobson (1966) v;ho found highest titers at the site of infection in
the small intestine with 0. aolurnhianitn.
Discussion of Immunoelectrophoretic Characterization of Antibody and
Proteins in Serum and Abomasal Mucous from Florida Native Lambs Infected
and Non-Infected v/ith Hae'nonchns contovtus
In order to identify gamma -globulin responses against H. contovtus
immunoelectrophoretic patterns were developed using antigen (adult and
larval) as well as anti-sheep serum, anti-sheep globulins and anti-
immunoglobulins (IgA, IgC. and IgM) . Tlie lack of precipitin reaction
from the serum demonstrates that infected animals v/ere not hyperimmunized
by their contact with tlie parasites. This does not mean responses did
not occur in t'ne serum as antibody titer reveals reaction did occur.
93
Silverstein (1963) reported anti-antigen precipitin activity to
occur principally in the 7S gamma-globulin (both fast and slov/ regions).
Although antigen reaction was not revealed in serum, reaction was seen
in mucous (Figures 14b, 5 and 14c, 8), as was a good gamma-globulin
response (Tables 9 and 10) . Identification of the gamma-globulins in
serum revealed excellent IgO and Igil response. A spur was seen at the
junction of tiie fast and slov; IgG in the serum of some Florida Native
lambs (Figure 13a, 9). Jonas (1969) also reported spurs at the junction
of the fast and slov; gamma-globulin in serum and various body fluids.
A similar spur v/as seen in abomasal mucous (see Figures 14b, 7 and
15a, 7) which did form an identity with anti-IgA, indicating that IgA
may also be responsible for some immunological response. Tlie reason
anti-IgA in serum did not develop a reaction cannot be explained since
no immunological differences have been reported between serum and
secretory IgA (Tomasi and Bienenstock, 1968). Consequently, these spurs
from serum, mucous or body fluids may be similar in appearance, but not
identical .
Tlie anti-sheep serum or anti-sheep globulins developed with serum
from worm-free lambs revealed a maximum of 8 proteins (Figure 12) : slow
and fast gamma-globulin, Ig'!, 2 additional beta proteins, 2 alpha proteins
and albumin. Jonas (1969J reported 3 beta proteins (besides Ig'l) from
parasite-free sheep v/hich he designated beta-1 , beta-4 and beta-5, and
3 alpha proteins designated alpha-1, alpha-2 and alpha-3. Tliese iinmuno-
electrophoretic patterns that he reported deviated from the patterns
shov/n here in Florida Native lambs possibly because of age or breed
differences, making labeling of beta protein arcs from Florida Native
94
Sheep difficult. These beta proteins v;ere designated beta-1 and beta-2
(after Silverstein, 1968 and Jonas et^ al . , 1972) in descending cathode to
anode order as were the alpha proteins, alpha-1 and alpha-2 (Figure 16a).
Jonas' e_t al . (1972) beta-5 protein arc which almost falls in the alpha
protein region was not seen in either infected or non-infected lambs.
Tlie 10 protein arcs developed from the serum of parasitized Florida
Native lambs are similar to those reported by Jonas e_t jal • (1972). The
alpha-3 and beta-5 proteins as Jonas e_t al^. (1972) describes them could
not be detected in the Florida Native lamb. The proteins detected in these
lambs were again labeled in a similar fashion as Jonas e^ al^. (1972) but
in descending (cathode to anode) order (see Figure 16b) . This increase in
the beta proteins in the parasitized lambs is attributed to an increase
of the sheep complement system (Jonas, et al., 1972).
The immunoelectrophoretic patterns from abomasal mucous developed
with anti-sheep serum or anti-sheep globulins revealed 5 to 7 proteins.
Mucoprotein, as demonstrated in electrophoretic patterns, v;as not detected
by this method. Tne proteins are identified and labeled in Figure 17
from cathode to anode. The IgA and IgG immunoglobulin reaction was the
most prominent in mucous. The lack of increase in the beta proteins
(complement) plus the increase in antibody activity (IHA) leads to the
conclusion that much of the antibody activity is IgA, a non-complement
fixing antibody.
In the higher vertebrates, IgM response accounts for most of the
initial reaction (humoral) to foreign invasion (Weinstein, 1967) . Jones
ej^ al . (1970) reported protective antibodies against Nippostrongylus
brasiliensis in rat serum that contained IgM and other immunoglobulins
Figure 16. Identified Proteins in Serum from Florida Native Lambs
Infected and Non-Infected with Haemonahus aontortus .
a. infected lamb b. non-infected lamb
Abbreviations used;
A=alpha proteins; B=beta protein; A.IgA=anti-immuno-
globulin A; A.IgG=anti-irimunoglobulin G; A.G.=anti-sheep
globulins; A.S .=anti-sheep serum; L.HC= larval H. aontortus
antigen.
96
Figure 17. Identified Proteins in Abomasal Mucous from Florida Native
Lambs Infected with Haemonchus contortus.
Abbreviations used:
A=alpha protein; B=beta protein; A.G.=anti-sheep globulins;
A.S.=anti-sheep serum; A.HC=adult H. contortus antigen.
98
99
(7S gamma-l and 7S gamma-2) . This observation appears to be in part one
of the reactions from Florida Native sheep serum directed to^zards the
parasitic invasion. The role of complement in producing protective
immunity against helminths has not been shown in the literature, but
IgM has been demonstrated to be highly effective in cell lysis
(Humphrey and Dourmashkin, 1965; Hoesler, 1972) and most efficient in
binding complement (Glynn and Medhurst, 1967: Hoesler, 1972). Other
actions of complement v;hich may play a role in helminth immunity are
the adherence of polymorphonuclear leucocytes to larval helminths
(Morseth and Soulsby, 1969) and in phagocytosis (Ogilvie, 1970).
Tomasi and Bienenstock (1968) have suggested that IgA may be an
important factor in the defence of the host in the intestine. Douvres
(1962) and Oobson (1965, 1966) have demonstrated antibody against
helmintns in the intestinal mucous of sheep and cattle. In these
reports the immunoglobulin classes were not determined. The major im-
munoglobulins demonstrated in abomasal mucous from Florida Native sheep
were IgA and IgG.
Tlie IgG antibody was found in both sera and mucous of Florida Native
lambs. Evidence that this immunoglobulin has protection capacity has
been reported against Diatyoaaulus viviparus (Wilson, 1966) and
Nippostrongylus brasiliensis (Jones e_t al^. , 1972) who found this
immunity to be associated with the 7S immunoglobulin fraction, predom-
inantly the 7,S-gamma-l fraction. Tnis protection can be in the form
of direct action, probably by neutralization of enz^Tnes needed by the
parasite (Weinstein, 1967), complement fixation (Humphrey and Dourmashkin,
1965) or enhanced phagocytosis.
100
Suggestions for Future Work
Immunoelectrophoretic precipitating arcs were not observed in
serum against parasite antigen (larval or adult H. aontortus) , but
increasing beta proteins (complement system) v/ere seen. Tais phenomenon
lends credence to the premise that the active serum immunoglobulin was
principally Igll. Tliis can be verified by treating samples with 2-
mercaptoethanol followed by IHA testing. Reduction in antibody titer
activity would indicate inactivation of Ig'I immunoglobulin.
Reaginic antibodies (IgE) have been identified following helminth
infections in several animals including the sheep (Ogilvie, 1970). One
of the most potent stimulus known for production of IgE is parasite
infection (Jarrett, 1973). "Self cure" or sudden elimination of para-
sites (well documented in sheep with //. aontortus) lias been suggested
to be related to reagin antibody (Jarrett, 1973). Characterization of
IgE in the serum and abomasal mucous of sheep would add to the knowledge
in this area and the role it plays in H, aontortus infection. Since IgE
is present in the humoral circulation in very small quantities but
plasma cells that produce IgE are found in large numbers in tissues
close to mucous surfaces (Ishizaka e_t al^. , 1969) , it f ol lows that its
detection in mucous may demonstrate additional evidence as to why Florida
Native sheep shov/ resistance toward parasitic infection. Detection would
be accomplished by either passive cutaneous anaphylaxis reaction or the
radio-allergo-sorbent technique .
APPENDICES
APPENDIX I
Hemoglobin Levels and //. oontovtus Egg Counts from
Florida Native Ewes Prior to Lambing
Hb level
Egg counts
iheep No.
Typel
(gms. %)
(per gm. feces)
on
A
9.5
0
013
A
10.9
0
014
A
9.5
800
017
A
12.6
0
018
A
6.7
400
026
A
10.6
200
027
A
8.5
0
031
A
10.6
0
039
A
9.5
0
047
A
9.5
0
053
A
10.6
400
070
A
6.7
200
101*
A
4.0
3,600
132
A
12.1
800
135
A
6.7
0
154
A
13.3
400
157
A
9.5
0
205
A.
9.2
400
227
A
9.2
400
235
A
11.8
600
245
A
12.6
0
250
A
5.7
0
302
A
12.1
0
305
A
15.8
0
307
A
13.0
1,200
308
A
13.0
0
310
A
9.2
0
319
A
11.8
0
323
• A
12.1
0
324
A
12.3
0
325
A
12.3
0
326
A
11.3
0
331
A
10.6
1,600
332
A
5.7
2,400
334
A
14.1
0
338
A
13.0
0
353
A
13.0
0
357
A ■
14.7
0
X, = 10.6
A
X, = 352.6
A
102
Hemoglobin and Egg Counts (Continued)
103
HI) level
Egg Counts
Sheep No.
Type^
(gms. %)
(per gm. feces)
010
B
6.7
0
016
3
7.8
0.
022
B
10.1
0
025
B
10.6
0
032
B
6.0
0
051
B
10.2
0
O&l
B
10.1
0
067
B
10.2
400
063
B
9.5
0
252
B
10.6
0
312
B
10.9
600
313
B
12.1
2,600
320
B
10.9
0
321
B
15.8
0
322
B
12.1
400
329
B
10.2
0
333
B
12.1
0
337
B
11.8
0
341
B
10.2
1,400
343
B
10.6
0
351
B
11.3
1,200
352
B
13.7
0
354
B
11.3
0
355
B
12.3
200
358
B
10.6
800
359
B
10.9
600
360
B
11.3
0
363
B
12.3
600
367
B
12.1
0
369
E
12.6
0
601
B
8.5
200
715
B
7.8
1,600
755
B
5.3
0
778
B
7.4
1,400
822
B
8.5
0
844
B
, 9.5
0
951
B
8.8
0
? =10.3
B
X = 324.3
B
104
Hemoglobin and Ova Counts (Continued)
Hb level
Egg Counts
Sheep No.
Type^
(RHIS. %)
(per gm. feces)
029
A3
12.1
0
0/^2
AB
8.8
0
048
AB
9.2
0
049
AB
9.5
200
052
AB
10.2
0
059
AB
12.1
200
065
AB
14.0
0
112
AB
7.4
0
141
AB
13.3
0
156
AB
10.1
0
, 160
AB
8.1
0
207
AB
9.5
400
217
AB
7.8
1,200
224
AB
10.2
400
234
AB
10.2
3,600 ■
239
AB
6.0
0
301
AB
10.9
200
303
AB
12.3
0
318
m
12.3
0
327
AB
11.8
0
323
A3
11.3
0
330'
AB
13.3
0
336
AB
11.3
200
344
AB
12.3
200
349
AB
13.0
200
368
AB
11.3
600
764
AB
6.0
400
788
AB
10.1
200
813
AB
12.1
0
819
AB
7.8
400
820
AB
7.1
200
825
AB
7.1
0
832
AB
10.6
0
849
AB
5.0
2,200
853
PS
8.1
200
856
AB
9.5
200
909
AB
10.9
0
7125
AB
12.3
200
7155
AB
8.1
800
X =10.1
AB
X = 312.8
AB
Hemoglobin type
*Died one week later
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APPENDIX V
Serum Antibody Titer and Egg Counts from Lambs Infected and
Non-Infected with Haemonchus aontortus (Pre- and Post-Infection) .
Date
Sheep No.
Age
Egg
Reciprocal
Antibody Titer-^
(Ty.
36^
(Months)
Count
Larvae
Adult
2/28
14*
A
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Negative
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
'2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
5/29*''>-
5.5
0
8
16
2/28
19
A
2.5
0
Negative
^ 4
3/7
0
Negative
2
3/14
3.0
0
Negative
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
4
2
5/16
5.0
0
4
2
5/29**
5.5
0
4
16
2/28
147*
B
2.5
0
4
4
3/7
0
4
4
3/14
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
2
4
5/16
5.0
0
2
4
5/29**
5.5
0
4
64
133
134
Serum Antibody Titer (Continued)
Date
Sheep No.
Age
Egg
Reciprocal
Antibody Titer2
(Typel)
(Months)
Count
Larvae
Adult
2/28
128 B
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2 •
4
5/9
0
2
4
5/1&
5.0
0
2
4
5/29''""
5.5
0
4
4 •
2/28
15* AB
2.5
0
2
4
3/7
0
4
4
3/14
3.0
0
2
,4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
5129**
5.5
0
4
8
2/28
124 AB
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Negative
2
3/21
0
Negative
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
4
5/9
0
2
4
5/16
5.0
0
2
4
5/29^-'*
5.5
0
4
64
135
Serum Antibody Titer (Continued)
Date
Sheep No.
Age
Egg
Reciprocal
Antibody Titer^
(Typel)
(Months)
Count
L
arvae
Adult
2/28
132'V A
2.5
0
2
8
3/7
0
2
8
3/14
3.0
0
2
4
3/21
0
2
4 ,
3/28
3.5
0
2
4
4/4
0
4
4
4/14
4.0
0
4
4
4/21
0
4
4
4/25
4.5
0
4
4
5/2
0
4
4
5/9
0
4
4
5/16
5.0
0
4
4
6/4'VA
5.5
0
4
16
2/28
120 A
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
4
4
6/4-^*
5.5
0
8
4
2/28
109" B
2.5
0
Ne
gative
,8
3/7
0
Ne
gative
4
3/14
3.0
0
Ne
gative
4
3/21
0
Ne
gative
2
3/28
3.5
0
Ne
gative
2
4/4
0
2
2
4/14
4.0
0
Ne
gative
2
4/21
0
Ne
gative
2
4/25
4.5
0
2
2
5-2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
6/4''^*
5.5
0
8
32
136
Serum Antibody Titer (Continued)
Date
Sheep No.
Age
Egg
Reciprocal
Antibody Titer2
(Typel)
(Months)
Count
Larvae
Adult
2/28
122 B
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
4
3/21
0
2
2
3/28
3.5
0
Negative
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
b/h**
5.5
0
8
8
2/28
27* AB
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
6/4^'=^=
5.5
0
16
4
2/28
139 A3
2.5
0
2
2
3/7
0
2
2
3/14
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
4
2
5/2
0
4
2
5/9
0
2
2
5/16
5.0
0
4
4
6/4"=*
5.5
0
8
4
137
Serum Antibody Titer (Continued)
Date
Sheep No.
Age
Egg
Reciprocal
Antibody Titer2
(Typel)
(Months)
Count
Larvae
Adult
2/28
10* A
2.5
0
Negative
4
3/7
0
Negative
2
3/14
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
-5/9
0
2
2
5/16
5.0
0
2
2
6/9**
5.75
0
8
16
2/28
17 A
2.5
0
Negative
4
3/7
0
Negative
4
3/14
. 3.0
0
Negative
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
4
4
5/16
5.0
0
4
4
6/9**
5.75
0
8
4
2/28
117* B
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Negative
2
3/21
0
Negative
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
2
8
5/16
5.0
0
2
8
6/9**
5.75
0
4
8
138
Serum Ajitibody Titer (Continued)
Date
Sheep
No.
Age
Egg
Reciprocal
Antibody Titer^
(Typel)
(Nonths)
Count
Larvae
Adult
2/28
11
B
2.5
0
Negative
4
3/7
0
Negative
4
3/14
3.0
0
Negative
4
3/21
0
Negative
4
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
4
5/16
5.0
0
2
4
6/9*vv
5.75
0
8
2
2/28
118*
A
2.5
0
Negative
4
3/7
0
Negative
4
3/14
3.0
0
Negative
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
4
4
5/2
0
4
4
5/9
0
4
4
5/16
5.0
0
8
8
6/13-;:*
6.0
0
4
32
2/28
110
A
2.5
0
2
2
3/7
0
2
2
3/14
3.0
0
2
2
3/21
0
2
'2
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
4
4
5/16
5.0
0
4
8
6/13**
6.0
0
8
8
139
Serum Antibody Titer (Continued)
Date
Sheep No.
Age
Egg
Reciprocal
Antibody Titer-^
(Typel)
(Months)
Count
Larvae
Adult
2/28
21 A
2.5
0
2
16
3/7
0
2
8
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
4
4
4/21
0
4
4
4/25
4.5
0
8
4
5/2
0
8
4
5/9
0
8
4
5/16
5.0
0
8
16
6/l3>'<A
6.0
0
8
64
2/28
127='= B
2.5
0
2
4
3/7
0
4
4
3/14
3.0
0
4
4
3/21
0
4
4
3/28
3.5
0
4
4
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2 ■
2
4/25
4.5
0
2
2
5/2
0
4
2
5/9
0
4
2
5/16
5.0
0
2
4
6/U**
6.0
0
16
32
111%
134 B
2.5
0
Negative
8
3/7
0
Negative
4
3/14
3.0
0
Negative
4
3/21
0
2
4
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5 •
0
2
2
5/2
0
2
2
5/9
0
4
2
5/16
5.0
0
8
2
biu-^*
6.0
0
8
2
140
Serum Antibody Titer (Continued)
Date
Sheep No .
Age
Egg
Reciprocal
i^jitibc
dy Titer'-^
(Typel)
(Months)
Count
Larvae
Adult
2/28
137* AB
2.5
0
4
8
3/7
0
2
8
3/14
3.0
0
Negative
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
4
6/13**
6.0
0
8
16
2/28
114* A
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Negative
Ne
gative
3/21
0
Negative
Ne
gative
3/28
3.5
0
Negative
2
4/4
0
Negative
Ne
gative
4/14
4.0
0
Negative
Ne
gative
4/21
0
Negative
Ne
.gative
4/25
4.5
0
Negative
Np
-gative
5/2
0
Negative
Ne
-gative
5/9
0
Negative
Negative
5/16
5.0
0
2
2
6/18**
6.0
400
16
32
2/28
24 A
2.5
0
2
2
3/7
0
2
2
3/14
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
6/18**
6.0
0
8
8
141
Serum Antibody Titer (Continued)
Date Sheep No. Age Egg Reciprocal Antibody Titer2
(Type^) (Months) Count Larvae Adu 1 1
2/28 113* B 2.5 0 2 2
3/7 0 2 2
3/14 3.0 0 2 2
3/21 0 2 2
3/28 3.5 0 2 2
4/4 0 2 2
4/14 4.0 0 2 2
4/21 0 2 2
4/25 4.5 0 2 2
5/2 0 2 2
5/9 0 2 2
5/16 5.0 0.2 2
(i/lQ** 6-.0 200 8 4
2/28 126 B 2.5 0 2 4
3/7 0 2 4
3/14 3.0 0 2 2
3/21 0 2 2
3/28 3.5 0 2 2
4/4 0 2 2
4/14 4.0 0 2 2
4/21 0 2 2
4/25 4.5 0 2 2 .
5/2 0 2 2
5/9 0 2 2
5/16 5.0 0 2 .4
(i /!?>** 6^0 0 8 8
2/28 115'^ A 2.5 0 2 4
3/7 0 2 4
3/14 3.0 0 2 4
3/21 ^02 4
3/28 3.5 ■ 0 2 2
4/4 0 2 2
4/14 4.0 0 2 2
4/21 0 2 2
4/25 4.5 0 2 2
5/2 0 2 2
5/9 0 2 2'
5/16 5.0 0 2 2
6/23^^-* 6.25 3,500 8 8
142
Serum Antibody Titer (Continued)
Date
Sheep No.
Age
Egg
Rec]
.procal
Antibody Titer2
(Typel)
(Months)
Count
Larvae
Adult
2/28
23 A
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
2
4
5/16
5.0
0
2
4
6/23'^^*
6.25
0
4
4
2/28
22* 15
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Nee
native
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
6/23**
6.25
1,400
32
32
2/28
141 B
2.5
0
Negative
2
3/7
n
Nr,c
^3t Lve
2
3/14
3.0
0
Ne[
'.ativG
2
3/21
0
Negative
2
3/28
3.5
0
Net
native
2
4/4
0
Nej
Va t ive
2
4/14
4.0
0
We^
'ative
2
4/21
0
l:ie'T,ative
2
4/25
4.5
0
2
2
5/2
0
2
2 '
5/9
0
2
2
5/16
5.0
0
2
4
6/23**
6.25
0
8
4
143
'
Serum j^jitibody
Titer
(Continued)
Date
Sliee
(TV
p No
pel)
A',e
(^'onths)
Egg
Count
Reciprocal Ant
ibodv Titer2
L
arvae
Adult
2/28
16*
AB
2.5
0
Ne
'^ative
2
3/7
0
Ne
rative
n
S/IA
3.0
0
Ne
gative
2
3/21
0
Ns
gative
2
3/28
3.5
0
Ne
gative
2
4/4
0,
Ne
gative
2
4/14
4.0
0
Ne
gative
2
4/21
0
Ne
gative
2
4/25
4.5
0
Ne
gative
2
5/2
0
Ne
gative
2
5/9
0
2
2
5/16
5.0
0
2
2
6/2 3**
6.25
1,200
16
16
2/28
HI
* A
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2 •
4
5/9
0
2
4
5/16
5.0
0
2
4
6/27**
5.25
4,400
8
8
2/28
9
A
2.5
0
2
4
3/7
0
2
4
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4 .
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
A
5/2
0
4
4
5/9
0
4
4
5/16
5.0
0
4
4
6/2 7**
6.25
0
8
8
144
Serum Ajitibody Titer (Continued)
Date
Sheen No.
(Tvpel)
A,f;e
(ilonths)
Egg
Count
"eciprocal ■^xitibody Titer-
Larvae Adult
2/28 121''; E 2.5 0 Negative Negative
3/7 0 Negative Negative
3/14 3.0 0 Net^ative Negative
3/21 0 2 2
3/28 3.5 0 2 2
4/4 0 2 2
4/14 4.0 0 2 2
4/21 0 2 2
4/25 4.5 0 2 2
5/2 0 2 2
5/9 0 2 2
5/16 5.0 0 2 2
bin*- 6.25 5, 600 32 16
2/28 30 B 2.5 0 2 4
3/7 0 2 4
3/14 3.0 0 2 4
3/21 0 2 4
3/28 3.5 0 2 4
4/4 - 0 2 4
4/14 4.0 0 2 4
4/21 0 2 4
4/25 4.5 0 2 4
5/2 0 2 4
5/9 0 2 4-
5/16 5.0 0 2 4
6/11** Jl • 25_ 0 8_ 4
2/28 130* AB 2.5 0 2 2
3/7 0 2 2
3/14 3.0 0 2 2
3/21 0 2 2
3/23 3.5 0 2 2
4/4 0 2 2
4/14 4.0 0 2 2
4/21 0 2 2
4/25 4.5 0 2 2
5/2 0 2 4
5/9 0 2 4
5/16 5.0 0 2 4
bill*" 6.25 6,000 32 ' 64
145
53erum Antibody Titer (Continued)
Date
S'lieep No.
Age
Egg
Reciurocal
Antibody Titer2
(Typel)
(Months)
Count
1,
irvae
Adult
2/28
112 AB
2.5
0
Ne
'.ative
2
3/7
0
Me
'native
2
3/14
3.0
0
Ne
^.ative
2
3/21
0
2
2
3/2ff
3.5
0
2
2
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
2
4
5/16
5.0
0
4
4
6/2 7^^*
6.25
0
4
4
2/28
12''' A
2.5
0
Ne
native
2
3/7
0
Ne
''.ative
2
3/14
3.0
0
Ne
^,ative
2
3/21
0
2
■ 2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
2
4
5/16
5.0
0
2
4
7/2>v,v
6.25
7,600
16
IG
2/28
13 A
2.5
0
4
4
3/7
0
4
4
3/14
3.0
0
4
4
3/21
0
4
4
3/28
3.5
0
4
4
4/4
0
4
4
4/14
4.0
0
4
2
4/21
0
4
2
4/25
4.5
0
4
2
5/2
0
4
2
5/9
0
4
4
5/16
5.0
0
4
4
1/ !■:<■!'
6.25
0
8
4
146
Serum Antibody Titer (Continued)
Date
• Sheep No.
(Typel)
A^e
(Months)
Count
Reciprocal
Antibody Titer2
L
irvae
Adult
2/28
28* 15
2.5
0
4
4
3/7
0
4
4
3/14
3.0
0
2
4
3/21
0
2
4
3/28
3.5
0
2
4
4/4
0
2
4
4/14
4.0
0
2
4
4/21
0
2
4
4/25
4.5
0
2
4
5/2
0
2
4
5/9
0
2
4
5/15
5.0
0
2
4
111**
6.5
5,800
16
8
2/28
29 B
2.5
0
4
4
3/7
0
4
4
3/14
3.0
0
4
4
3/21
0
4
4
3/28
3.5
0
4
4
4/4
0
4
2
4/14
4.0
0
4
2
4/21
0
4
2
4/25
4.5
0
4
2
5/2
0
4
2
5/9
0
4
2
5/16
5.0
0
4
4
111**
6.5
0
3
15
lll'd
119''^ AB
2.5
0
Ne
native
2
3/7
0
Ne
:;ative
2
3/14
3.0
0
Ne
;ative
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0 •
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
112**
6.5
8,200
16
4
147
S>3ruin Antibody Titer (Continued)
Date
Sheen
No.
A;; a
Eg;^
Reciyrocal
Aiitibody Titer2
(Type^)
(i^'ontlis)
Count
Larvae
Adult
2/28
12 3*
A
2,5
0
Ne;^,ative
2
3/7
0
Negative
2
3/14 ,
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
' 0
2
2
5/16
5.0
0
2
2
111--'*
6.75
4,200
16
8
2/28
25
A
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Negative
2
3/21
0
Negative
2
3/28
3.5
0
Negative
2
4/4
0
Negative
2
4/14
4.0
0
Negative
2
4/21
0
Negative
2
4/25
4.5
0
Negative
2
5/2
0
0
4
5/9
0
2
4
5/16
5.0
0
2
8
111**
6.75
0
8
8
2/28
31*
B
2.5
0
2
2
3/7
0
2
2
3/14
3.0
0
2
2
3/21
0
2
2
3/28
3.5
0
2
2
4/4
0
2
2
.4/14
4.0
0
2
2
4/21
0
2
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
0
2
2
111**
6.75
8,800
8
4
148
Serum Antibody Titer (Continued)
Date
Slieep Mo.
(Tvnel)
A re
(ilont^is)
Count
"ociprocal
Anti
bodv Titer2
T-arvae
Adult
2/28
136-''- Aj;
2.5
0
Negative
2
3/7
0
Negative
2
3/14
3.0
0
Neo;ative
2
3/21
0
Ne'gative
2
3/28
3.5
0
2
2
4/4
0
2
2
4/14
4.0
0
2
2
4/21
0
9
2
4/25
4.5
0
2
2
5/2
0
2
2
5/9
0
2
2
5/16
5.0
n
2
2
7/7 AA
6.75 5
,600
16
8
Mieniorjlobin Type
^Determined by THA
'-Infected
*"Day of Necropsy
Lamh
Type 2
AI^PENDIX VI
Protein Values in Abomasal ?iucous Exudate from
s Infected and Non-Infected v/ith Haemonchiis contox'tus
vjith "egard to Hemoglobin type.
Sheep
No.
Day
Post-Infection
Protein Values (%)
Mucoprotein &
A.lplia- ^!ucoprotein &
Albumin Globulin Beta-Globulin
Gamma-
Globulin
14*
19
147*
128
15*
124
A
A
15
B
AB
AB
•'■
23.8 4.5 59.5
48.5 6.1 32.2
42.3 S.3 30.3
34.1 12.6 40.4
52.1 6.1 21.4
34.0 8.5 43.1
12.2
9.2
19.1
12.9
20.4
14.4
132*
120
109*
122
27*
139
A
A
B
B
/\JB
AB
35.5 11.3 35.5
24.2 37.4 19.2
48.8 16.5 20.7
47.8 4.4 19.6
30.7 2.5 53.5
49.1 10.7 24.8
17.7
19.2
14.0
28.2
13.3
15.4
10*
17
117*
11
A
A
B
B
12
12
12
12
32.6 10.2 40.1
37.9 10.1 38.9
29.9 13.8 41.3
30.0 3.7 48.1
17.1
13.1
15.0
19.2
118*
110
21
127*
134
137*
A
A
A
B
B
AB
16
16
16 ■
16
16
16
35.8 11.1 28.5
33.4 11.0 43.0
40.0 4.7 37.5
30.5 14.7 39.0
42.7 8.3 23.1
44.5 5.5 24.8
24.6
12.6
17.8
15.8
25.9
25.2
114*
24
113*
126
A
A
B
B
21
21
21
21
29.6 5.0 31.8
30.6 3.3 36.4
25.0 4.9 48.4
32.9 9.3 40.9
33.6
29.7
21,7
16.9
115*
23
2 2*
141
16*
A
A
li
B
AB
26
26
26
26
26
27.6 23.1 29.8
36.1 11.6 34.7
18.2 27.5 34.1
41.8 8.6 29.8
19.7 12.0 39.3
19.6
17.6
20.2
19.8
29.0
149
Aboinasal Mucous (Contiinjed)
150
Protein
Values (%)
,Mucoprotein5i
Slieep
Type2
Day
Alpha-
Mucoprotein &
Gamma-
No .
Post-Infection
Albumin
Globulin
Beta-niobulin
Globulin
111--^
A
30
14.5
1.9
63.1
20.5
9
A
30
36.7
5.9
30.1
27.3
121='<
B
30
27.1
21.3
29.3
22.3
30
B
30
43.0
4.6
26.2
26.2
130*
AB
30
11.2
10.6
58.3
19.9
112
AB
30
34.3
17.3
28.3
20.1
12>'^
A
33
33.2
9.6
35.1
22.1
13
A
33
23.5
13.1
32.5
25.9
28>'=
B
33
38.7
14.5
25.3
21.5
29
13
33
40.6
12.5
29.8
17.1
119*
AB
33
30.5
7.9
27.1
34.5
123*
A
38
41.0
7.3
24.1
27.6
25
A
38
34.4
6.1
34.1
25.4
31*
B
38
27.2
9.9
40.2
■ 22.7
136*
AJi
38
12.3
4.8
49.5
33.4
^Determined by Electrophoresis
^Hemoglobin Type
*Infected Lamb
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BIOGRAPHICAL SKETCH
Jay Barry Klein was born at Miami Beach, Florida, on November 4,
1946. He received an Associate of Arts degree from Miami-Dade Junior
College in May, 1966. His Bachelor of Science degree in Chemistry
and Biology was received from the University of Miami in August, 1968.
Beginning in September, 1968 until June, 1970, Mr. Klein taught basic
science and mathematics at the Lear School in Miami, Florida. In
January, 1971, he enrolled at the University of Florida and
subsequently entered the graduate program in the Department of Animal
Science. He received a Master of Science in Agriculture majoring in
Parasitology in August, 1973.
Jay Barry Klein was awarded a National Institute of Health
Pre-Doctoral Traineeship in September, 1972. He is a member of
Alpha Zeta Fraternity and the American Society of Parasitologists.
He is married to Roberta Joyce and has a son, David and daughter,
Jennifer.
162
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of schoJarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Richard E. Bradley, Sr. , Chaii
Associate Professor of Veterinary Science
(Associate Parasitologist)
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
/Fuller W. Bazer
Associate Professor
(Associate Animal Physiologist)
I certify that 1 have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
J "t^\\j^.
-^K,V\V
Harvey L. Cromroy
Professor of Radiation Biology and
Entomology ■■'
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
I. t i-^
r*
Philip fi. Loggins
Associate Professor of Animal Science
,/
This dissertation was submitted to the Dean of the College of Agriculture
and to the Graduate Council, and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
March, 1976
n
of Agrieulti
Deap, College of Agri^eulture
Dean, Graduate School
UNIVERSITY OF FLORIDA
IllHi.
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