Group Title: Immunologic responses in Florida native sheep experimentally infected with Haemonchus contortus /
Title: Immunologic responses in Florida native sheep experimentally infected with Haemonchus contortus
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Title: Immunologic responses in Florida native sheep experimentally infected with Haemonchus contortus
Physical Description: x, 162 leaves : ill. (some col.) ; 28 cm.
Language: English
Creator: Klein, Jay Barry, 1946-
Publication Date: 1976
Copyright Date: 1976
 Subjects
Subject: Sheep -- Diseases   ( lcsh )
Haemonchus contortus   ( nal )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 151-161.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Jay B. Klein.
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Bibliographic ID: UF00098293
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000008227
oclc - 02172708
notis - AAA9882

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Immunologic Responses in Florida Native Sheep
Experimentally Infected with Haemonchus contortus
















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
















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. Bazer, 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 Chaffin 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 T01

AI00383-04 from the National Institute of Allergy and Infectious

Diseases.
















TABLE OF CONTENTS


Page
ACKNOWLEDGEMENTS ........................................ i

LIST OF TABLES .......................... ............... vi

LIST OF FIGURES ................... ..................... vii

ABSTRACT ....... ...................... ..... ............. ix

INTRODUCTION ....................... .................... 1

LITERATURE REVIEW ..................................... 5

MATERIALS AND METHODS .................................. 21

RESULTS ....................... ......................... 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

Nematode Recovery in Lambs Experimentally Infected
with Haemonchus contortus from Scheduled
Necropsy ....... .... .................. ...........30

Changes in Packed Cell Volume and Blood Hemoglobin
Levels in Florida Native Lambs During Experimental
Infection with Haemonchus contortus ............ 31

Changes in the Serum Proteins in Florida Native
Lambs Infected and Non-Infected with Haemonchus
41
contortu .................... ... ................

Proteins in Abomasal Mucous Exudate from Lambs
Infected and Non-Infected with Haewonchus
48
contorts ............................* **..........

Antibody Evaluation in Serum from Lambs Infected
and Non-Infected with Haeionchius contort .....48









Page
Antibody Evaluation in Abomasal Mucous Exudate from
Lambs Infected and Non-Infected with Haemonchus
contortus ....................................... 56

Comparison of Mean Percentages of Proteins in Serum
and Abomasal Mucous from Florida Native Lambs
Infected and Non-Infected with Haemonchus
contortus ...................................... 72

Comparison of Antibody Titers in Serum and Abomasal
Mucous from Florida Native Lambs Infected and
Non-Infected with Haemonchus contortus ......... 72

Immunoelectrophoretic Characterization of Antibody
and Proteins in Serum and Abomasal Mucous from
Florida Native Lambs Infected and Non-Infected
with Haemonchus contortus ...................... 72

DISCUSSION ............................................ .. 88

Relationship of Blood Hemoglobin Types to Blood
Hemoglobin Levels and Natural Infection with
Haemonchus contortus 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 with Haemonchus
contortus ...................................... 90

Discussion of the Changes in Packed Cell Volume,
Blood Hemoglobin Level and Serum Proteins in
Florida Native Lambs Associated with Haemonchus
contortus ...................................... 90

Discussion of Abomasal Mucous Proteins from Lambs
Infected and Non-Infected with Haemonchus
contortus ...................................... 91

Discussion of Antibody Activity in Serum and
Abomasal Mucous from Florida Native Lambs
Infected and Non-Infected with Haemonchus
contortus ..................................... 92

Discussion of Immunoelectrophoretic Characterization
of Antibody and Proteins in Serum and Abomasal
Mucous from Florida Native Lambs Infected and
Non-infected with Haemonchus contortus ......... 92

Suggestions for Future Work ....................... 100









Page

APPENDICES

I. Hemoglobin Levels and Haemonchus contortus Egg
Counts from Florida Native Ewes Prior to
Lambing .................. .................. 102

II. Packed Cell Volumes (PCV) of Lambs Pre- and Post-
Infection with Haemoncius contortus ........ 105

III. Hemoglobin Levels of Lambs Pre- and Post-
Infection with Haemonchus contortus ........ 107

IV. Changes in Serum Proteins in Florida Native Lambs
Infected and Non-Infected with Haemonchus
contortus .................................. 110

V. Serum Antibody Titer and Egg Counts from Lambs
Infected and Non-Infected with Haemonchus
contorts (Pre- and Post-Infection) ........ 133

VI. Protein Levels in Abomasal Mucous Exudate from
Lambs Infected and Non-Infected with Haemonchus
contortus with Regard to Hemoglobin Type ... 149

BIBLIOGRAPHY .......................................... 151

BIOGRAPHICAL SKETCH .................................... 162
















LIST OF TABLES


Table Page


1. Scheduled Necropsy of H. contortus Infected Lambs
Controls ....................................... .. 22

2. Statistical Comparison of Packed Cell Volume,
Hemoglobin Level, Serum Albumin, Peta-Globulin,
Gamma-Globulin and Total Serum Protein Between
Blood Hemoglobin Types in Lambs ................ .. 30

3. Nematode Recovery from Scheduled Necropsy of Lambs
Infected with Haemonchus contortus ............... 32

4. Differences Between the Changes of Serum Proteins of
Lambs Infected and Non-Infected with Hlaemonchus
contortus ........................................ 44

5. Mean Percentages of Serum Protein from Lambs Infected
and Non-Infected with Haemonchus contortus ....... 47

6. Mean Percentages of Proteins in Abomasal Mucous Exudate
from Lambs Infected and Non-Infected with Haemonchus
contortus ........................ ................. 49

7. Antibody Titer Against Haemonchus 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
Ilaemonchus contortus ............................. 74

10. Immunoelectrophoretic Analysis of Abomasal Mucous from
Sequential Necropsy of Lambs Infected and Non-
Infected with Haemonchus contortus ............... 75
















LIST OF FIGURES


Figure Page


1. Sequential Changes in Packed Cell Volume in Florida
Native Lambs Infected and Non-Infected with
Hlaemonchus contortus Divided by Hemoglobin Types .. 34

2. Sequential Changes of Packed Cell Volume in Lambs
Infected and Non-Infected with Haemonchus contortus
Without Regard to Hemoglobin Types ................ 36

3. Sequential Changes in Hemoglobin Levels in Florida
Native Lambs Infected and Non-Infected with Haemonchus
contortus Divided by Hemoglobin Types ............. 38

4. Sequential Changes in Hemoglobin Levels in Florida
Native Lambs Infected and Non-Infected with Haemonchus
contortus Without Regard to Hemoglobin Types ...... 40

5. Changes of Serum Gamma-Globulin in Florida Native
Lambs Infected and Non-Infected with Haemonchus
contortus Without Regard to Hemoglobin Types ...... 43

6. Differences in the Changes of Albumin-to-Globulin Rato
of Lambs Infected and Non-Infected with Haemonchus
contortus ......................................... 46

7. Characteristic Electrophoretic Patterns of Abomasal
Mucous Exudate from Lambs Infected and Non-Infected
with Haemonchus contortus ......................... 51

8. Protein Content in Abomasal lucous Exudate from Lambs
Infected and Non-Infected 'ith i'aemonchus contortus 55

9. Serum Antibody Against Haemonchus contortus Larvae and
Adults from Sequential Necropsy of Infected and
Non-Infected Lambs ................................. 58

10. Mucous Antibody Against Haemonchus contortus Larvae and
Adults from Sequential Necropsy of Infected and Non-
Infected Lambs .................................... 65











Figure


11. Abomasal Mucous Antibody Levels from Florida Native
Lambs Infected and Non-Infected with Haemonchus
contorts 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 Haemonchus
contortus ......................................... 80

14. Immunoelectrophoretic Patterns from Abomasal Mucous
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 Haemonchus contortus 96

17. Identified Proteins in Abomasal Mucous from Florida
Native Lambs Infected with Haemonchus contortus ... 98


viii


Page
















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




IMMUNOLOGIC RESPONSES IN FLORIDA NATIVE SHEEP EXPERIMENTALLY-INFECTED
WITH Haemonchus contortus

By

Jay B. Klein

March, 1976

Chairman: Richard E. Bradley, Sr.
Major Department: Animal Science



The host responses of worm-free Florida Native lambs to infection

with Haemonchus contortus were investigated for the elucidation of

"resistance factors" which 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 showed 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. The 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 H. contortuzs. The identification of the

immunoglobulins (IgA, IgG and IgM) responses against the parasite pre-

sented evidence that IgM and IgG were most prominent in serum,and IgA

and IgG were the most prominent in mucous. Complement proteins were

also shown to increase substantially after parasitic infection.

The 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


Iforbidity and mortality losses caused by trichostronzylid parasites

are of major economic importance in worldwide 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 trichostrongylids 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. lie also found on the Georgia Coastal Plain region an

average loss of $289 per farm on 23 sheep farms.

Haenonchus contortus, according to Wiitlock (1955a), is the only

gastrointestinal nematode which causes a primary disease. This recogniz-

able disease, called haemonchosis, produces a hemorrhagic anemia

(Richard et al., 1954, Campbell and Gardiner, 1960). Blood loss caused

by H. contortus was determined by Baker et al. (1959) who tagged sheep

erythrocytes with radioactive chromium 51. They determined that H. contortus

removed an average of 0.08 ml of blood per worm per day. 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 worm 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 growth

reduction, permanent stunting (Spedding, 1956), weakness, muscular










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). Animals less se-

verely infected have lowered resistance and may be susceptible to secon-

dary infection. These animals are unthrifty, listless and have dry,

harsh wool (Levine, 1968; Tetzlaff, 1970).

T'e 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 ewes experimentally

infected with H. contorts. Spedding (1955) and Spedding et al. (1958)

have shown a reduction in growth rate as much as 30% in ewes having

normal pasture parasite burdens. Brunsdon (1963) found that lambs

treated with thiabendazole weighed 30 pounds more than controls at

slaughter and produced 49% more wool.

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. Anthelmintic 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 (Theodorides et al., 1970). The appearance of these

strains can cause the need of increased frequency of treatments and, with

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










et al. (1963), Evans and Whitlock (1964), Loggins et al. (1965a, 1965b),

Jilek and Bradley (1969), Radhakrishnan et al. (1972) and Bradley et al.

(1973) suggest a correlation between hemoglobin type, hematocrit value

and severity of infection with H. contortus infections. Evans and

Whitlock (1964), Jilck and Bradley (1969) and Radhakrishnan et al. (1972)

found consistently higher hematocrit values in hemoglobin type A. This

physiologic factor (greater erythrocyte volume) may allow these sheep a

better chance of surviving parasite challenge. Loggins et al. (1965b)

Jilek and Bradley (1969), Radhakrishnan et al. (1972) and Bradley et al.

(1973) found Florida Native sheep were more resistant to infection with

H. contortus than Rambouillet sheep. Florida Native sheep appear to have

some type of "resistance factor" due to the fact that they are able to

undergo self cure more readily than Rambouillets and had significantly

more larval forms of TH. cotnortus than adult worms (Radhakrishnan et al.,

(1972).

Arrested development of N. contortus has been well documented,

although the factors governing this phenomenon are poorly understood.

Dineen et al. (1965), Dineen and ,!agland (1966), Soulsby (1966). Wagland

and Dineen (1967), Michel (1968) and Donald et al. (1969) attribute the

arrested development of H. 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).

The objectives of the present investigation were to characterize

the host responses of worm-free Florida Native lambs to infection with

11. contortus and to elucidate tie "resistance factors" that have a role

in maintainkn,, lower adult worm burdens. Hematological data, serum

collection and abomasal mucous extraction were the main considerations





' 4


in the determination and interpretation of these "factors." Sequential

necropsy of infected worm-free lambs from pre- through post-patency

gave an unique insight into the immunologic mechanism occurring in lambs,

in both tile humoral and mucous secretary systems, while the parasitic

infection proceeded. Tile following physiological and immunological

measurements were performed: (a) hematocrits, (b) hemoglobin levels,

(c) fecal ova counts, (d) electrophoresis of serum and mucous,

(e) immunoelectrophoresis of serum and mucous using anti-sera and

H. contorts antigen and (f) indirect hemagglutination. Through the

analyses of lamb serum and abomasal mucous, additional information was

gained concerning which protein fractions and immunoglobulin classes they

contained, how they changed with age and whether there were statistical

relationships to environment, hemoglobin types or other genetic factors.

















LITERATURE REVIEW


Life Ccle, orphology and Metabolism of Haemonchus contortis

Haemonchus contortus (Rudolphi, 1803), commonly known as the

"barber pole worm," is found principally in the abomasum of ruminants

worldwide. The males are 10-20 mm long and are reddish brown in color.

Females range from 18-37 mm in length and have characteristic white

ovaries spiralled around their reddish blood-filled intestine giving the

"barber pole" appearance.

The male H. contortus 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. This vulvular flap usually

will be large and conspicuous (linguiform), but can be diminished to a

knob-like structure in some specimens (Levine, 1968).

The mature female produces from 4,000 to 10,000 ova daily. These

ova measure 70-851 by 41-48p 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 followed 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 endodermm, ectoderm and

mesoderm), germ cells, gut and stonodeum. The first stage larvae of

iH. contortus hatch within 14-19 hours. This first stage is rhabditiform

and actively feeds on bacteria and other microorganisms. The second

stage larvae, which appear within 1-2 days, are also rhabditiform. The

third stage (L3) or infective stage is strongyliform and sheathed, and

is found 4-7 days after hatching.

The infective stage is characteristically 682-780u in length, has

16 intestinal cells and a globular buccal cavity. The tip of the tail

to the end of the sheath is 65-78p with a kink found in the sheath tail.

The intestinal cells contain granuoles which will 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. The enzyme acts on an area

about 20u back of the anterior end for release of the L3 larva (Lapage,

1968; Levine, 1968; Rogers, 1962; Silverman, 1965).

The 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. The 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 pathtrays 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 phosphorvlating glycolysis.

Phosphorylation occurs as in vertebrate tissue but neither arginine

phosphate 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 which 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 show respiratory quotients of 0.58-0.60 and

0.64, respectively. Adult H. contcrtus are found to contain a type of

hemoglobin which transports oxygen (Lapage, 1968; Levine, 1968).

Pathogenesis

llaemonchus contortus causes primary damage by sucking blood. Both

tne fourth stage larvae and adults suck blood, consequently damaging the

abomasal mucosa by their attachment and piercing activities. iThe 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 et al., 1962). Boughton 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 approximately 12 minutes after

which detachment occurred leaving wounds which continued to hemorrhage

for an additional 7 minutes.

Ilematological changes associated with anemia caused by H. contortus

may include erythrocytes showing anisocytosis, polychromasia, Howell-

Jolly bodies and punctate basophilia (Levine, 1968). Serum proteins and

albumin in parasitized lambs may show a decrease, while the alpha-l-,

alpha-2-, beta- and gamma-globulins may all be increased (Kuttler and

Marble, 1960; Leland et al., 1960). Wilson and Turner (1965) noted that

even moderate mixed nematode infections (mainly H. contortus) 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 were also seen in Florida Native and

Rambouillet lambs infected with H. contortus (Bradley et al., 1973).

Eosinophil and lymphocyte infiltration in abomasal tissue of lambs was

observed several weeks after infection with H. contortus (Bradley et al.,

1973; Malczewski, 1971). Increased lymphocytes and lymphoid hyper-

plasia were also observed in sheep resistant to H. contortus (Silverman,

1965).

Anemia as low as 3-4 grams of hemoglobin per 100 ml of blood can

be present in H. contortus infected animals (Levine, 1968). Clinically,

the gums, conjunctiva and mucous membranes are pale. Edematous swelling










under the jaw ("bottle jaw"), constipation, weakness, cold extremities,

listlessness, dull, dry, harsh wool and unthriftiness may be seen (Levine,

1968; Tetzlaff, 1970). Parasitized animals may die suddenly, symptoms

may persist for weeks before dying, or recovery may occur leaving

stunting or reduced muscle growth (Spedding, 1956).

Epidemiology and Ecology of H. contorbus

The mode of infection of sheep with H. contortus infective larvae

is by grazing on infected pastures or ingestion with their feed or

water. When pasture conditions are favorable, the infective stage (L3)

is reached within 2.5 days to 2 weeks (Levine, 1968; Levine et al., 1975).

These 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 Whitlock, 1967) and for egg development to

occur on the ground (Shorb, 1944). Optimal temperature for development

(60 hours)-is 33.3C (Berberian and iizelle, 1957). Levine (1968)

states that Hsu in 1967 studied the effects and relationships of temper-

ature and relative humidity to the development of Tichostrongylus

colubriformis and H. contortus. He found that H. contortus 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










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 weather and climate on

the bionomics of ruminant nematode larvae. lie considered the potential

transmission period of Haemronchuls to lie between mean monthly tempera-

tures of 15 to 370C when the soil water deficiency was not more than 2.0 cm.

l onnig (1956) reported that infective larvae are active climbers and

can withstand desiccation and freezing. They 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 H. contortus and other trichostrongyles might

appear to be the 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.

Crofton (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 et al. (1974),in contrast, have reported that under favorable

conditions H. contorts infective stage larvae could survive 1.5-3.5

months.

Immunology and Resistance

The idea that metazoan parasites stimulated an immune response was

first reported by Stoll (1929), using H. contortus. lie infected lambs

with the parasite and observed that after placing them on pasture










their fecal egg counts rose to high levels. After several weeks he

found a dramatic fall in egg counts, some to negative values. He

correlated this phenomenon to a loss of adult worms. Even after

subsequent reinfection with 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 was humoral, based on intradermal reactions to saline

extracts of H. conlortus. Stumberg (1933) substantiated this fact by

using a cutaneous anaphylactic test in which he detected antibody

against H. contorts in dilutions of 1:50,000.

Hawkins and Cox (1945) found that serum obtained from sheep that

had undergone a natural infection with the trichostrongyles (mainly

H. contortus) caused precipitates around the mouth, excretory pore,

anus or cuticle of exsheathed larvae. There were 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

Strong loides. Silverman (1965) also obtained similar results. Antibodies

that cause these precipitates at the physiological orifices were believed

to be "functional" by 01iver-Conzales (1946), meaning that they contribute

directly to resistance.

Stewart (1950a,b,c) has demonstrated that a complement fixing

antibody response occurs after infestation with H. contortus or

TrichostrongylZus spp. 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 Deriods of "self cure." On each occasion

most sheep which had a drop in ova counts also showed a rise in serum










titer. This occurrence was similar to the result when infective larvae

of H. eontortus were superimposed upon an existing infection producing

"self cure This was contrary to the reports by Ross and Gordon (1933)

and Gordon (1948) in which they concluded that acquisition of resistance

to H. 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 grazing flocks after rain

was because this caused large doses of infective larvae to mature and

be ingested.

The immunological 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 heavy 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 "self cure" in lambs cause a response by the host

which results in the loss of part or the whole 1H. contortus burden.

These mechanisms have been postulated and the causes have been shown

to be varied in nature. Stewart (1953) found that at the time of

'eelf cure there was a significant rise in blood histamine as well as










antibody level. If antihistamine 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

with an edematous condition of the mucous membrane of the abomasum.

This was substantiated by Stewart (1955) who reported that abomasums

of previously non-exposed lambs remain flaccid and normal when large

doses of exsheathed larvae were injected into the abomasum. In hyper-

sensitized and resistant lambs, the abomasum had increased peristalsis

and segmentation in 10 minutes. withinn 1 hour the abomasum was pale

and edematous. Histological examination of the mucosa of animals that

undergo self cure show edema and aggregation of eosinophiles (Soulsby,

1958).

Soulsby (1965) suggests resistance to parasites may be due to a

change at the environmental site caused by the parasite itself. An

alteration of oxygen tension associated with pH could be such a product

of infection which is seen in inflammatory reactions. Christie (1970)

found the activity of fourth stage AH. contortus larvae damage the function

of the cells of the gastric epithelium. 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 H. contortus in temocrate regions

(Blitz and Gibbs, 1972a). Large numbers of fourth stage larvae and low










numbers of adults are frequently observed during the winter months

(Blitz and Gibbs, 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 which depressed their immunological capacity resulting in

a stimulation of dormant larval stages to mature. This is only part of

the answer 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, whether 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 would 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 will prevent worms from developing as

demonstrated by Dineen and co-uorkers (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.









It is believed the larval stages are the important immunizing agents

inducing "self cure and protection" (Soulsby, 1965). Reports 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 et 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. contortus in sheep has also been studied. Manton et al. (1962)

found lambs infected with larvae of H. contortus at 2-4 months of age to

be unable to develop immunity while lambs 10-12 months of age could.

Urquhart et al. (1966a, 1966b) in vaccination studies against H. contortus

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) between 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

et 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, which could be










transmitted by the ram in Targhee sheep. Emik (1949), Warwick et al.

(1949) and Whitlock (1955a, 1955b, 1958) reported resistance to the

trichostrongyles (mainly H. contortus) jas genetically transmitted.

Loggins et al. (19653, 1965b) believed that genetic factors were

responsible for parasitic resistance in Florida Native sheep in compar-

ison to the Southdoxrn, Hampshire or Rambouillet sheep.

Jilek and Bradley (1969) found high frequencies of hemoglobin type

a (Hb A) in Florida Native sheep which were believed to be more resistant

to H. contorts. This was in agreement with Evans et al. (1963) who

reported that sheep with Hb A were infected with fewer adult H. contortus

than other hemoglobin types. Evans and Whitlock (1964) found sheep with

hemoglobin type A had higher total volume of circulating erythrocytes

and iiematocrit values than either types B or AB. Padhakrishnan et al.

(1972) confirmed that Florida Native sheep with lib A had consistently

higher packed cell volume (PCV) values than other hemoglobin types but

reported no data that would confirm that hemoglobin types were indicators

of resistance against H. oontortus or thlt Hb A sheep were less suscep-

tible to such infections. If sheep with Hb A do have more circulating

erythrocytes, they might be better able to withstand the effects of

H. contortus.

Radhakrishnan et al. (1972) did find significantly lower numbers

of adult worms in Florida Native lambs in comparison to Rambouillet

lambs. This observation was substantiated by Bradley et al. (1973)

who gave 1 or 2 oral doses of H. contortus. They reported Florida

Native lambs had higher 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 davs) in










resistant sheep as compared to susceptible sheep (15 days). Florida

Native sheep may possess "resistance factors" which enable them to be

resistant toward II. contortus infection without prior exposure or to

initiate a more rapid "self cure" (Bradley et al., 1973).

Natural resistance to Haemonchus 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. Urquhart et al. (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 with 1000,000 larvae but found high numbers

of worms in only 1 animal. Bitakaramire (1966) 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 Haemonchus sp. by adminis-

tering 3,000,000 infective larvae to 3 resistant and 2 worm-free lambs.

Christie (1970) believed age was a very important factor in classifying

natural resistance. This was substantiated by Dineen et al. (1965) and

Wagland and Dineen (1967) where 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









Mendelian relationships, Hb A and lib 3 being allelic and co-dominant

(Evans et al., 1956; iuisman et al., 1965). Another hemoglobin type,

Hb C (first designated lb N) has been reported, but was usually found

in either young lambs (less than 1 month of age) or animals which are

severely anemic (Efremov and Braend, 1966; Vliet and Huisman, 1964).

Evans and Whitlock (1964) correlated a relationship between hemoglobin

types and packed cell volume; hb A being greater than Hb P and Hb AB

being intermediate. This observation was 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 et al. (1963). Immunoelectrophoresis with

rabbit anti-whole adult sheep serum yielded 21 arcs of precipitation.

These 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. The beta-2- and gamma-globulin arcs

were similar to those seen in other mammalian sera and were designated

beta-2N- (later changed to IgM[; W.H.O., 1964), beta-2A- and gamma-globulin.

Jonas (1969) examined the immunoglobulin response of sheep to Salmonella

typhimurirum or human erythrocytes by immunoelectrophoresis using antisera

from guinea-pigs which had been injected with suspensions of the above

antigens treated with sheep sera or various body fluids. He reported

two gamma-globulins (fast and slow) IgM', two arcs parallel to the two

gamma-globulins, 1 beta-globulin arc and 2 weak alpha-globulin arcs.

Preliminary evidence indicates that the last 3 proteins may be components

of complement. Subsequent work by Jonas in 1972 using third stage

H. contortus larvae treated with serum from parasite free or parasitized

sheep to produce antisera in rabbits found 3 additional beta-globulins










and 1 additional alpha-globulin. Evidence was presented to indicate that

the beta- and alpha-globulins may be components of the sheep complement

system.

Silverstein et al. (1963) observed that the typical gamma-globulin

showed a "gull wing" appearance, indicative of a fast and slow protein

which are different but cross reacting. Leland et al. (1960) after

examining sera by electrophoresis from lambs infected with Trichostrongylus

axei reported various changes in the gamma-l- and 2-globulins associated

with the parasitic infection. Jonas (1969) and Jonas et al. (1972)

found separate fast and slow gamma-globulins in sheep serum. Tomasi and

Bienenstock (1968) reported fast gamma-l- and slow gamma-2- immunoglobulins

in bovine colostrum. Jonas (1969) also reported fast and slow gamma-

globulin in sheep synovial, pericardiac and Graafian follicle fluid,

colostrum and 4-day milk.

Dobson (1966) using sheep infected with Oesophagostomum col2unbianum

found intestinal nucous 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. columbianum relative concentrations of the

mucoprotein diminished because of increased alpha- and beta- proteins.

When a second infection was administered, decreasing mucoprotein was

caused mainly by an increase in gamma-globulin.

Serum protein changes in sheep with natural or experimental nematode

infections (especially H. contortus) are frequently recorded. Endrejat






20



(1956) first compared serum proteins from parasitized and nonparasitized

sheep. He reported marked increases in gamma-globulin and decreases in

albumin. Shumard et al. (1957) reported an increased albumin to glob-

ulin ratio (designated A/C) in lambs with mixed parasite infections.

Kuttler and Marble (1960) reported similar results in lambs infected

with T. axei. Turner and Wilson (1962), Wilson and Turner (1965) and

Bradley et al. (1973) also reported decreased A/Cl ratios in parasitized

sheep.

















TIATERIALS AND IETHIODS


Experimental Design

This investigation used 47 Florida Native lambs reared worm-free.

They were divided into 3 groups according to hemoglobin type (Hb A, lib B,

Hb AB) determined by electrophoresis using cellulose-acetate membranes

with tris-ethylenediamine-tetra acetic acid-borate buffer (.13:1, pH 8.9-

9.3) at 400V for 50 minutes. Each hemoglobin group was randomly divided

into 2 sub-groups, an infected and control (non-infected). The Ilb A

group consisted of 20 animals, 9 infected and 11 controls. The Hb B had

17 animals, 9 infected and 8 controls. The lb 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 wonn 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.

Twelve 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. Sampling began at 2.5 months of age and continued until




1Microzone Electrophoresis System, Beckman Instruments Inc.,
Fullerton, California.










5.5 months of age when experimental infection occurred. The 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

was intended to produce an infective dose which caused a decrease of

hematocrit values at 10 days post-infection (Tetzlaff, 1970).

On day 0 (day of infection) 1 lamb (Hh A) was 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 HI. contorts Infected Lambs and Controls.


Day 1 Day 7 Day 12

Lamb No. lib Type Lamb No. Hb Lype Lamb No. Hb Type
14* A 132* A 10* A
19 A 120 A 17 A
147* B 109* B 117* B
128 B 122 B 11 B
15* AB 27* AB
124 AB 139 AB


Day 16 _

Lamb No. Hb Tye
118* A
110 A
21 A
127* B
134 B
137* AB

Day 30

Lamb No. Hb Type
111* A
9 A
121* B
30 B
130* AB
112 A.


Day 21**

Lamb No. lb Type
114* A
24 A
113* B
126 B

patencycy occurs

Day 33


Lami No.
12*
13
28*
29
119*


AH Type
A
A
B
B
AR


Day 26

Lamb No. Hb Type
115" A
23 A
22* B
141 B
16* AB


Day,38


Lamb No.
123*
25
31*
136*


Nb Type
A
A
B
AB


- 1


*Infected with H. contortus.


---t----------------------










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 washed (washings 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 40C for 12 hours.

This temperature causes expulsion of the mucous from the goblet cells

(Dobson, 1966). The tissues were then placed in HC1-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 ii. contortu3 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 refractometerl

analysis. Mucous and serum were all stored at -200C which gives no

serum protein changes (Kuttler and Marble, 1959).

Experimental Animals

Florida Native lambs were raised under worm-free conditions in

concrete-floored pens. The ewes and their lambs were placed in pens

within 24 hours of lambing. Pens and feed and water troughs were washed




IAO T/C Refractometer, American Optical Instrument Co., Buffalo,
New York.










daily to avoid fecal contamination. Individual fecal samples were

examined weekly by a modified McMaster technique (Whitlock, 1948) to

verify nematode parasite control. After weaning at 60 days of age,

blood samples were taken weekly by jugular vein puncture from each lamb.

Five ml of blood were collected in a VacutainerR tube containing EDTA

as an anti-coagulant and 5 ml collected into a Vacutainer tube without

anti-coagulent for serum collection. Ewes and lambs were fed according

to National Research Council standards.

Fecal samples of several lambs at 1 month of age revealed

Strongyjoides sp. ova. Therapeutic doses of thiabendazole were given

to all lambs. No additional ova were detected until experimental

infection. Infection with this parasite war believed not to be through

contamination but by pre-natal infection (Pfeiffer, 1962) or through the

colostrum or milk as reported in swine (Batte and Moncol, 1966).

Haemonohzu contortus Inoculum

Infective larvae of H. contortus for use in experimental infections

were initially isolated from the University of Florida sheep flock

using ova identification techniques (Monnig, 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 which had recently undergone anthelmintic treatment with

thiabendazole. Subsequent collection of ova for culturing purposes

to obtain larvae for antigen was derived from these rams.

Infective larvae were obtained by fecal-vermiculite cultures at




1Vacutainerp" Becton, Dickinson and Co., Rutherford, New Jersey.










270C for 7 days. These cultures were then put into cheesecloth and

placed in a Baermann apparatus which consists of a clamped funnel and

wire mesh sieve. Warm water is added to the funnel until contact with

the cultured material. After several hours, the larvae attracted to

the warm 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 10OC. The larvae were washed and allowed to

settle, and the dilution adjusted so that each ml of fluid contained

1000 larvae. The lambs were infected with a 40 ml syringe equipped

with an 8 inch flexible metal tube that was rubber coated. The tubing

was inserted into the esophagus of the lamb and the correct larval dose

was expelled. This was followed by passing 1 washing of distilled water

through the syringe.

Hematology and Immunology

Hematocrit values were obtained using a microcapillary technique.

tlicrocapillary 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

15 centrifuge.1 Values were obtained using a microcapillary tube reader2

which gave tie packed cell volume measured as per cent (%).

For hemoglobin concentration determination, the cyanmethemoglobin

method was employed (Anonymous, 1965a). This technique employs the use

of 5.0 ml of cyanmethemoglobin reagent mixed with 0.02 ml of blood by




IInternational Equipment Co., Needham Heights, Massachusetts.

Ibid.










inverting several times. The contents are transferred to a cuvette and

read against a reagent blank using a spectrophotometer. The wave-

length used is 540 mu and the reading converted into grams of hemoglobin

per 100 ml (Hh grams%) of blood using a standard curve.

Nucoprotein and serum protein fractionation was carried out by

electrophoresis2 on cellulose acetate membranes using a barbital buffer

(plH 8.6) at 300V for 30 minutes. After a staining and clearing process

(Anonymous, 1965b), membranes were scanned on a densitometer3 which

produced density curves. These curves were divided into areas repre-

senting discrete fractions (mucoprotein, albumin, alpha-, beta- and

gamma-globulins) in which the area under the curve could be determined,

giving relative percent (%) of these proteins. Total protein was

determined by refractometer,4 thus giving relative amounts of the

fractions (mg/ml).

Antibody titers in serum and mucous exudate were measured by

indirect hemagglutination (IHA). This test involves the use of

erythrocytes coated with the antigen for which 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




1G. K. Turner Associates, Palo Alto, California.

2licrozoneR Electrophoresis System, Beckman Instruments, Inc.,
Fullerton, California.

Model R-110, Beckman Instruments Inc., Fullerton, California.

4AO T/C Refractometer, American Optical Instrument Co., Buffalo,
New York.










settle to the bottom of the test wells indicating positive or negative

reaction. This test is sensitive (0.003 Ug antibody/ml) and can be

used in conjunction with other tests (Kagan and Norman, 1974). IHA

microtiter test (Kagan and Norman, 1974) was done using MicrotiterR

equipment.

Immunoelectrophoretic analysis of lamb serum and mucous exudate

was performed on electrophoretic apparatus. Samples were tested for

their activity for immunoglobulins (IgG, IgA and IgM), gamma-globulin,

beta-globulins, alpha-globulins, albumin and H. contortus antigen.

Rabbit anti-sheep IgG, gamma-globulin and serum;3 Rabbit anti-ovine

globulins and serum;4 and Rabbit anti-bovine IgG, IgA and IgM5 were

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 was obtained from pooled larvae and fresh H. 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.

2LicrozoneR Electrophoresis System, Beckman Instruments, Inc.,
Fullerton, California.

ICN Pharmaceuticals, Inc., Cleveland, Ohio.

Colorado Serum Co., Denver, Colorado.

5'iles Laboratories, Inc., Kankakee, Illinois.










R I1
on a Vortex Genie Mixer Volume was brought to 5 ml in physiological

saline. Similar procedures were used with 200 adult worms brought to

2 ml volume.

Statistical Analysis

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. The "Z" two-tailed test

was also used (4landenhall, 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 were 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) Hb type by time (d) time by infection and

(e) lib type by infection.


1Scientific Products, Inc., vanston, Illinois.
Scientific Products, Inc., Evanston, Illinois.
















RESULTS


Hemoglobin Levels and Haemonohus contortus Ova Counts from Florida
Native Eves Prior to Lambing

Hemoglobin Levels (gms. %) and ova counts (eggs per gram) of

H. contortus (based on morphology, Skerman and Hillard, 1966) were

taken on 114 ewes divided by hemoglobin type (Hb rype). The data

is presented in Appendix I. Average values for hemoglobin levels and

ova counts, respectively, were Hb A, 10.6 gms. % and 352.6 e.p.g.,

Hb B, 10.3 gms. % and 324.3 e.p.g. and Hlb AB, 10.1 gms. % and 312.8

e.p.g. Statistical comparison between Hb type using the Z test

(ilendenhall, 1971) showed no differences in hemoglobin levels or ova

counts.

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 Exoerimental 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 H. contortus is summarized in Table 2.











Table 2. Statistical Comparison of Packed Cell Volume, Hemoglobin
Level, Serum Albumin, Beta-Globulin, Gamma-Globulin and Total Serum
Protein Between Blood Hemoglobin Types in Lambs.

meanss
No. of PCV Hb Level Beta- Gamma- Tot
Samples (%) (gms. %) Albumin Globulin Globulin Prot

19 36.9 15.9 3.0 0.43 1.4 6.
17 30.5 13.3 2.9 0.40 1.2 5.1
10 34.1 15.1 2.9 0.42 1.3 6.'


Overall
Means 46


33.9 14.8


al
ein

1
8
0


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 was 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.

Analysis of the serum proteins (albumin, beta and gamma) showed

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 between types 4 and AB or B and AB.

Nematode Recovery in Lambs Experimentally Infected with Haemonchus
contortus from Scheduled Necropsy.

The 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 shown in


Hb
Type

A
B
ABl











Table 3. Ova were first detected on the 21st day post-infection (see

Appendix V). Adult worms were first recovered in low numbers (8 total)

on 6/18 which corresponds to 21 days post-infection. Early 5th stage

larvae were seen until the end of the experiment period.

Changes in Packed Cell Volume and Blood Hemoglobin Levels in Florida
Native Lambs During Experimental Infection with Haemonchvus contortus.

Mean packed cell volumes and blood hemoglobin levels based on

sampling data prior to experimental infectionrmre compared to values

taken at necropsy in infected and control animals (see Appendices IIand

III). Tuc differences between infected and controls were contrasted with

and without regard to hemoglobin types.

Figure 1 shows 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 between blood hemo-

globin types but found statistical differences at day 26 and 33 post-

infection. These days 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

with respect to hemoglobin type but produced "F" values showing a signifi-

cant difference (p < 0.04) between infected and non-infected animals.

Figure 3 illustrates the changes of the hemoglobin levels in the lambs

infected and non-infected with H. contortus with respect to hemoglobin

type. The variability of the levels at each collection period is very

marked. Figure 4 shows these shifts without regard to blood hemoglobin




















Ce,







CI I
gvo1. .2i..













Mt 1


0 H. o 41 000 i' 3
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Cd UJ
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ro


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E0






0 0
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t o



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0 0 0 0 0 0 0 0 0
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Figure 3. Sequential Changes in Hemoglobin Levels in Florida
Native Lambs Infected and Non-Infected with Haemonchus
contortus Divided by Hemoglobin Types.



















O= Infected Hb Type A
l= Non-Infected Hb Type A
a= Infected Hb Type B
V= Non-Infected Hb Type B
-= Infected Hb Type AB
+= Non-Infected Hb Type AB


+5.0


+4.0


+3.0


+2.0


+1.0



a
0


-1.0
E







-2.0


-3.0



-4.0


-5.0


0 2 4 6 8 10 12 14 6 118 20 22 24 26 28 30 32 34 36 38
Days Post-Infection








































CO



S0*

* C





AC
0 t
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x c


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type. This figure also illustrates that the infected animals had more

lower values throughout the infection period. The 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-Infected iuth flaermon/chus contortus

Serum collected prior to infection was compared to serum at the day

of necropsy. This data is presented in Appendix IV. The serum protein

levels (gms. %) of albumin, alpha-l-globulin, alpha-2-globulin, beta-

globulin and gamma-globulin were then statistically analyzed for differ-

ences between the infected and control lambs after infection with

H. contortus.

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. The infected lambs had consistently higher

values than the controls as shown 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 shows this ratio decrease by hemoglobin type along with

the average values (without regard to hemoglobin 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.







































01





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A I I i fii
0 0 0 0 0 0 0 0 0 0
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Table 4. Differences Between the Changes of Serim: Prcteius of
Lambs Infected and Noa-Infected with .one'.r.c:s cotor.us


Sheep Post-Infection
Typel (Days)


Electrophoresis Vales (V s .) CGamma A/G A/G
Albumin Alpha-] Alpha-2 Beta Gam3a Average Ratio Average

+0.51 +0.96 -0.32 +0.01 +1.07 +1.68
-0.43 -0.18 -0.26 +0.52 +0.03 -0.25 -0.37 +0.31
-0.34 -0.28 -0.29 -0.05 +0.26 -0.38


A 7 -0.23 -0.08 -0.10 -0.03 +0.22 -0.72
B 7 40.04 +0.09 -10.02 +0.2 +0.09 +0.03 -0.20 -0.12
AB 7 +0.40 -0.14 +0.16 -0.08 -0.21 +0.55

A 12 +1.31 +0.09 +0.43 -0.71 +0.57 40.22 +0.06 +072
B 12 +0.24 +u.16 -0.25 +0.03 -0.14; +1.49

A 16 -0.68 -0.15 +0.60 -0.02 +0.54 +0.24 -0.90 -0.3
8 16 +0.37 -0.16 +0.25 40.C3 -0.07 +0.31

A 21 -0.81 +0.15 +0.23 +0.53 -0.22 -0.18 +0.53 44.25
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 -0.06 +0.08 +0.22 -0.04 +0.60 -0.73
B 30 -0.37 -0.15 +0.13 +0.21 +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 +0.14 -0.59
B 33 -0.81 -0.03 -0.19 -0.10 +0.09 +0.35 -0.66 -0.76
AS 33 -0.18 -0.15 +0.30 -0.18 +0.82 -1.04

A 38 +0.21 +0.02 -0.46 -0.41 +0.70 -0.51
B 38 -0.81 +0.10 +0.03 +0.05 +0.11 +0.32 -0.88 -0.63
AB 38 -0.69 +0.10 +0.06 +0.05 +0.16 -0.49


1
Hemoglobin type







































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T-1
0,


0U











dm
bO






- u
H I


ro
oJnl
n-ai


















cico
0-, 1
H dO

'0








































































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0D "3 N 0 Ni- '1-D CO 0
5 d o d d d d d d -
LIOIID!JDA 10 sa3uaJa@i!a


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0
ro








CD







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(D




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Table 5. Mean Percentages of Serum Proteins from Lambs
Infected and Non-Infected with Haemonchus contortus.


Infected
Control


No. of Day
Lambs Post-Infection


3 1
3 1


Infected 3 7
Control 3 7


Infected 2 12
Control 2 12


Infected 3 16
Control 3 16


Infected 2 21
Control 2 21


Infected 3 26
Control 2 26


Infected
Control


Infected
Control


Infected
Control


Total
Infected

Total
Control


Electrophoresis Values (%)

Albumin Alpha Beta Gamma


42.0 23.9 11.8 22.3
45.7 26.6 7.3 20.4


49.8 20.4 8.4 21.4
53.9 21.2 7.2 17.8


50.2 19.2 9.2 21.3
48.1 19.8 12.4 19.6


48.2
46.3


21.0 8.1 22.7
20.3 10.2 23.3


45.8 22.5 8.4 23.1
45.6 19.7 7.1 24.1


54.6 19.1 7.5 18.8
50.7 20.5 7.7 21.0


41.7 22.8 9.2 26.3
52.0 20.6 9.4 18.0


42.2 20.7 8.5 28.8
49.9 20.5 8.8 20.8


44.4 17.8 7.6 27.0
38.0 24.0 9.0 29.0



46.4 22.0 8.7 23.6


48.6 21.5 9.0 20.9


i-----


---- --t-


i











Proteins in Abomasal Mucous Exudate from Lambs Infected and Non-Infected
with Haemonchus contortus

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-globulins. Common characteristic

electrophoretic patterns of the abomasal mucous are shown 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

the 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,

wiile 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. There was

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 contorts

Serum obtained at necropsy during the course of infection was tested

by indirect iemagglutination (IHA). Sheep erytbrocytes used in the test

were either coated with antigen derived from adult or larval H. contortus.

Signigicant changes in antibody titer between the controls and infected

animals were noted using the test with adult antigen (p < 0.08) and










Table 6. Mean Percentages of Proteins in Abomasal Mucous
Exudate from Lambs Infected and Non-Infected with
Haemonchus contortus.


Day
Post-Infection


1
1


7
7


Infected 2 12
Control 2 12


Infected 3 16
Control 3 16


Infected 2 21
Control 2 21


Infected 3 26
Control 2 26


Infected 3 30
Control 3 30


Infected 3 33
Control 2 33


Infected 3 38
Control 1 38


Total
Infected 25

Total
Control 21


Electrophoresis Values (%)

1Micoprotein & Mucoprotein & Gamma-
Albumin Alpha-Globulin Beta-Globulin Globulin


39.4
38.9


38.3
40.4


6.3 37.1 17.2
9.1 38.6 12.2


10.1 36.6 15.0
17.5 21.2 20.9


t -________________________________________________


31.2
33.9


36.9
38.7


27.3
31.7


21.8
38.9


12.0 40.7 16.0
6.9 43.5 16.1


10.4 30.8 21.9
8.0 34.5 18.8


27.6
23.3


20.9
10.1


17.6 11.3
38.0 9.3


34.1 10.7
34.5 12.8


26.8 7.3
34.4 6.1


34.4 22.9
32.2 18.7


50.2 20.9
28.2 24.5


29.2 26.0
31.1 21.5


37.9 27.9
34.1 25.4


33.0 19.7


No. of
Lambs


Infected 3
Control 3


Infected 3
Control 3


1 ~




































Figure 7. Characteristic Electrophoretic Patterns of Abomasal Mucous
Exudate from Lambs Infected and Non-Infected with
Haemonchus controtus.










1-













17
T





K






7 TT




L1-1
*L



I -n


-* _: - e -





































Figure 7. "continued"












































Figure 8. Protein Content in Abomasal Mucous Exudate from
Lambs Infected and Non-Infected with Haemonchus
contortus.



















- o= Infected
------o = Control

-~O--~-?s- -----


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 20 32 34 36 38
Days Post-Infection


Sc
-0 =40
S0;30

0 zi20
o ElO
10






c
c 50

-c -40





> 30
0 20
C- a

a)



>9










larval antigen (p < 0.008). The test using 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,

Hb 8 and Hb AS (Figures9a, 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 showing early response,

Type B varied response and Type AB having a later response.

Antibody Evaluation in Abomasal 'lucous Exudate from Lambs Infected and
Non-Infected with Haemonchus contorts

Abomasal mucous which had been concentrated to within ranges of serum

protein levels was tested by THA. Significant changes in antibody titer

between the controls and infected animals were noted using adult 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 IHiA test

but had some differences at the 89% level using larval antigen. Mean

responses of type 8 were slightly better than types A and AS which were

very similar. As with serum antibody, Figures 0a, b and c (Hb A, Hlb B

and lib AB, respectively) show similar responses between 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 were not statis-

tically significant.

Figure 11 plots mucous antibody titer without regard to blood

hemoglobin types. Good responses were shown throughout the infection

period particularly at day 12 and day 30. Indirect hemagglutination

testing using adult antigen gave higher titer response.






































cu






9t 0
CO)


0
Li a
mu








So
D-I


SCO
301




c0

ZrI
ac
E-U






U I
c- n


cc3 -
1H

















< ro




-o o



C)

0-

I // O
Us 0 ---


,, I \ O
I <






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> I N
S3 -0 '/-(0











fo
1 10
I I / aI





I /I




_ 1 --L---- -o
O


-j@._L Apoq!IuV IDoojdioo9

























































































C





C)







44





60








C) / 0

0a /
QO.- / ro



. a /



z j <

1 II I*
SI 0-









S (- O0
C\J

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C\ 0 cO
f a





I 0

(. @Ii A poqjuv) IDDOid!iD
j. OJ 00d

















































































a







CC

















-ro
c- o




CJ e
T- O o. T
S > 0 C\1




Z .< .




-)
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I .IO _.















ol
/\;




___- L I J0

ia!l Apoq!!uv' lDOOl!oa












Table 7. Antibody Titer Ag~;ist acrr.cj:i c:ony-g'r;s in Serum and Aboasal
Mucous Extracti;n frcm 'a-uet a ::ecrosvy uf Infected and No n-Infected Iambus.


Shoep No.
(Typl)


14* A
29 A
147* R
128 B
15* AB
124 AC

132* A
120 A
109* B
122 B
27, AB
139 AB

10* A
17 A
117* B
11 B

1186 A
110 A
21 A
127* B
134 B
137* Al


Eaysb P CPva | -n lTiter2
Trfecr'o. [Court Larval Adult
S I- .An.'ie Pnttien

1 L' 32 64
1 0 8 16
1 0 16 256
1 0 6 8
S0 8 32
1 0 32

7 0 8 61
7 0 8 8
7 0 32 128
7 0 16 16
7 128 64
7 0 8 16

12 0 32 256
12 0 16 16
12 0 16 32
12 C 32 8

16 0 8 64
16 16 32
16 0 32 32'
16 0 64 128
16 0 16 16
16 0 64 64


21
21
21
21

26
26
26
26
26

30
30
30
30
30
30


33
33
33
33
33

38
38
38
38


6/18 114* A
24 A
113* B
126 B

6/23 115* A
23 A
22* S
141 B
16* AB

6/27 111* A
9 A
121" B
30 B
130* AB
112 AB

7/2 12* A
13 A
28* B
29 B
119* AB

7/7 123* A
25 A
31* B
136* AB


1
Hemoglobin Type

2Reciprocal titers using IIA

*Infected Lambs


400
0
200
0

2,200
3
1,400
0
1,200

4,400
0
5,600
0
6,000
0


7,600
0
5,800
0
8,200

4,200
0
8,800
5,600


16
16
32
8

32
4
64
16
64

8
1,;
123P
16
128
8


32
32
64
16
64

128
8
16
32


64
16
8
8

16
8
128
16
64

16
16
128
16
256
8


32
32
32
16
64

64
16
16
8


Serum Titer
Larval AdulE
Ant .Ienn Antir-e

8 16
4 I1
4 64
4 4
4 8
4 64

4 16
8 4
8 32
8 8
16 4
S 4-

8 16
8 4
4 8
8 2

4 32
8 8
8 64
16 32
8 2
8 16

16 32
8 8
8 4
8 8

8 8
4 4
32 32
8 4
16 16

8 8
8 8
32 16
8 4
32 64
4 4

16 16
8 4
16 16
8 8
16 4

16 8
8 8
8 4
16 8


h!


" --- -L----"~1


II


"_- /


I










































4-



0 U
3W












r
0 .
Si
0 1-








to
0 01
0 -





j 0-







0















H1
It



















































































- Jal!jL poq!tuv ID30oJd!i3o


4)
a.


a
zci: -1
r- o -
"0 .. 0



-S-o-


ro

1-


ro

CM


0
CM'


C"
c- 0


-o




0
-0



-~Q


(0''l
10
NU
























































































ci
4r
H
4-
C


C
C



0r


ci
Cr





































--




I I




















/
^^^^ !




gf! ,

*^^3~~~--


o

co
(0



a,
CM
(0






0
N -





Co
(I0

- Q



0,

(0

1-
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Nr
jol!l Apoq!IuV loowdioaa


Lo O




















































































C)
Ut
U
H





ai
U


C0

C)l
Ur


















256 = Infected Hb Type AB
2(C) += Non-Infected Hb Type AB I
-= Lorval Antibody _L L
T- --= Adult Antibody T V


14 16 18 20 22 24 26 28
Days Post-Infection










































0G








> cj


04-i


4- J






ot
>0





41 0
ri 0




Ccl
Z E


.0 (0

0-1 -

(u
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,0 40
0 1
4-





C



Ge o




































U)
.0



E





xo 0 0.













x
E a ci
o a 0
Oi" = <


(0
.oi!l Xpoq!iuV [IDo3d!oad l


r')
rC



I-
rr)
N

ro
0

00


(0
C
(-
.1-


C
Oi
0-



-o






0
0U


(- c0
~J


I0 a0









Comparison of Mean Percentages of Proteins in Serum and Abomasal Mucous
from Florida Native Lambs Infected and Non-Infected with Haemonchus
contortus

Iean 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 Hacemonchus contortus

The results of IIIA testing of serum and abomasal mucous exudate

from lambs infected and non-infected with H. contorts 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 upswing 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 Haemonchus contortus

The 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.

contortus adults or larvae. There were responses shown in mucous against







73













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I Em. 0 .

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O o *J -- -



C C"







I n
0 0) 4j ml 1
So j* o
CQ) m




u 'p I I O U I n



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4
co
c .J ^
C 'NN O C

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O J u +1 +1 u I











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o 0 *H
00) C 3
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HO a 0 u
U( 4 Q) Q
C0Z T

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j~iO






























0 OO 1
H 0-' C U




CU CU- C



4'C CC 0 H
HU 'i I- (C



CMU C1)
C) QJ C CT'
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Table 9. Immunoelectrophoretic Analysis of Serum from Sequential
Necropsy of Lambs Infected and Non-Infected with aemrlon~chet con3trotus.




Date Sheep No. Gammna- IgG IgA IgMI H. contortus atntigen
(Typel) Globulin Adult Larvae

5/29 14* A +H -H -- +
147* B +++ ++4 4 -- + -
15* AB +++ 4-+ -- + -
19 A 44+ -+++ -- + -
128 B 4++- -+ -- + -
124 AB +4-+ +-+ -- +

6/4 132* A ++4 +-H+ -- 4++
109* B +-+ +++ -- + -
27* AB +++ +++ -- + -
120 A +4-+ +- -- +
122 B +4- +-+ -- -++
139 AB +-++ 4-+ -- +

6/9 10* A H++ 4-++ -- + --
117* B 4-+I +++ +
17 A +++ +4+ -- + --
11 B +- +- -- + -

6/13 118* A +4-+ +- -- ++ -
127* B +++ ++ -- + -
137* AB +++ +4- -- + -
110 A +- +4++ -- + -
21 A +++ ++- -- 4+ +
134 B +++ ++ -- 4++ -


6/18


6/23






6/27


7/2






7/7


114* A
113* B
24 A
126 B

115* A
22* B
16* AB
23 A
141 B

111* A
121* B
130* AB
9A
30 B
112 AB


12* A
28* B
119* AB
13 A
29 B

123" A
31* B
136* AB
25 A


+++ +++ -- + -
+++ +-+ -- + -
+- +- -- +


+4+ +++ -- + .
4-+ 4+4-+ -- + .--
+++ +4-+ -- + -
+- ++- -- .++.
--++ ++-+ -- + ...
+H- -r4- -- ++ _


44-


4-++

H-
-+++
+4-+
S4++-
S+1++


+H-
+++
H+4
-4++
+4-

4+-
+++
+++
+-+


lHemoglobin type

+++ = strong precipitation line
++ = good precipitation line
+ detectable precipitation lirn
- = no detectable reaction













Table 10. Irmunoelectrophoretic Analysis of Abomasal Mucous from Sequential
Necropsy of Lambs Infected and Non-Infected with Haemanchus con torture.



Date Sheep No. Ganma- H. c-atortus Antigen
(Typel) Clobulin IgG IgA IgM Adult Larvae

5/29 14* A + +++ ++ -
147* B +++ +++ -
15* AB ++-- +++- + -
19 A +++ +++ + -
128 B +++ ++4 + -
124 AB +++ + +

6/4 132* A 4++ +4+ ++ + +
109* B +++ +++ ++ + +
27* AB +++ +++ + -
120 A +++- ++- -
122 B +++ + -
139 AB +- +++ 4+ -

6/9 10* A +++ + + --
117* B +++ +-+ 1-+ +
17 A +4+ +4-- ++ + -
11 B ++ 4++ + + -

6/13 118* A +++ + -
127* B +++ + -
137* AB +4+ +++ -
110 A ++ + -
21 A +++ -- -
134 B +44 ++ ++ +

6/13 114* A +++ -++ + + -
113* B ++ ++ + -
24 A +++ +++ +4 -- + +
126 B +4+ +++ + + +

6/23 115* A ++4 + + -
22* B +- ++ + -
16* AB +++ + + -
23 A +4- ++ + -
141 B +++ + -
_-4 ....--- -


6/27


7/2






7/7


111* A
121* B
130* AB
9 A
30 B
112 AB


++F
+++

+4+
+++
-+-4


12* A +4-
28* B +4-
119* AB +++
13 A +++
29 B 4++


123* A 4++
31* B +44-
136* AB +++
25 A -:++


+4+ +


+++ +
4++ ++

4++ ++
-H-- +

+++ +
+++ +
+4+ +



+++ ++
4+4- ++
4++ +4
+4+ +4
-4--4 +


.H-
+





+
+





4-
+


+












4-
+
-


IHemoglobin type
++ = strong precipitation line
++ = good precipitation line
+ = detectable precipitation line
= no detectable reaction










adult and larval antigen though more reactions were seen using adult

antigen.

Typical immunoelectrophoretic patterns from control lambs are shown

in Figure 12. Tests for responses against TgA, IgM, IgG, larval IH.

contortus (L.HC) and sheep serum fractions (..S.) were developed using

anti-sera to the above immunoglobulin classes or proteins. Figure

12a demonstrates that there are good IgH and IgG responses. Arnti-sheep

serum revealed: the immunoglobulins-G (1) and M (2), 1 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 H. contortus. The 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 (4).

The patterns in Figure 13a from infected Florida Native lamb sera

demonstrated good response to IgC. This IgC arc also revealed slow

IgG (1) and fast IgG (2). There was 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 Igl response (5) and two additional beta-proteins (3) two

alpha proteins (4) and albumin (8). There were no detectable reactions

to either larval or adult H. contortus 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 H. contortus.





78







SER

A.I



A.S




A.--g



































Figure 13. Characteristic Immunoelectrophoretic Patterns of Serum
from Florida Native Lambs Infected with Haemonchus
contortus.

Abbreviations used:

A.IgG=anti-immunoglobulin G; A.IgM=anti-immunoglobulin M;
L.HC=larval H. contortus antigen; A.G.=anti-sheep globulins
(serum minus albumin); A.S.=anti-sheep serum; A.HC=adult
H. contortus antigen.





80



























Figure 14. Immunoelectrophoretic 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. contortus antigen; L.HC=larval H. contortus
antigen.




82





muci
A.IgA


A. Iqq


A.G.


a.



MUC

A.HC


A.IgA_ ,


A.S.





































Figure 14. "continued"






































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 H. contortus antigen;
L.HC=larval H. contortus antigen.










MUC

A.GA

A. G


L.HC





87




(5) were characteristic of infected lambs. Similar patterns were seen

in non-infected lambs (Figures 15a and b) except that only 1 beta protein

was likely to be seen. Besides TgG, IgA response was excellent and

produced an identity with a spur arising from the gamma protein area

(Figures 14b, 7 and 15a, 7). An IgM response could not be detected

with anti-IgM. Infected lambs also showed response against adult

H. contortis antigen (Figure 14c, 8).
















DISCUSSION


Relationship of Blood Hemoglobin Types to Blood Hemoglobin Levels and
Natural Infection with Haemonchus contorts in Florida Native Ewes

Evidence of hemoglobin type differences in natural infection of

Florida Native Ewes with H. contortus 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. contortus

in Hb A than other hemoglobin types. This was in agreement with

Radhakrishnan et al. (1972) whose data did not suggest any differences

in infection rates by hemoglobin type in Florida Native or Rambouillet

sheep. In fact, Radhakrishnan et al. (1972) and Bradley et al. (1973)

reported lower adult populations and egg counts in Hb AB than Hb A or Hb B

lambs experimentally infected with H. 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 et al. (1975) in which

sheep kept on the same pasture where 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
to Hemoglobin Types in Worm-Free Lambs

Packed cell volumes between blood hemoglobin types showed significant

differences which is in agreement with reported literature. Hemoglobin

type A had the highest erythrocyte volume, lib B the least and lib AB was

intermediate between the other two types. The significance level of

the PCV observation is considerably higher than the reports of

Radhakrishnan et al. (1972) and Bradley et al. (1973). Reports by

Evans and IJlitlock (1964) and Jilek and Bradley (1969) indicating lower

infection rates in sheep with HbA are contradictory to the reports of

Radhakrishnan et al. (1972) and Bradley et al. (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 Ilb A was significantly greater than lb B, Hb AB

was greater than Hb B, but differences between Hb A and Hb AB iere

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

llb types. The total protein data did give an indication that Hb A was

higher in total protein content than H!b B, though 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. These physiologic factors alone might give

Hb A sheep the capacity to withstand the effects of H. contortus










infection as reported by Evans and Whitlock (1964), Jilek and Bradley

(1969) and Bradley et al. (1973). The 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 Hb type and individual animals were noted,

experimental data collected after infection must take these facts into

consideration. Therefore, a double control system was used. Collected

data after infection with H. contortus was compared to sampling data

prior to infection. These changes in themselves give significant data

but to give further creditability, comparisons were also made to non-

infected animals handled in a similar manner.

Nematode Recovery in Florida Native Lambs Experimentally Infected with
Haemonchus contortus

The lower recovery rates in Florida 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, Blood Hemoglobin Level
and Serum Proteins in Florida Native Lambs Associated with Haemonchus
contortus 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.




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