Studies on Bunostomum trigoncephalum, The Hookworm of Sheep
A Thesis submitte.d in partial fulfilment of the requirements for the Degree of Master of Veterinary Science at Massey Un.iversity.
J.M. GRAHAM, 1969
ABSTRACT
This thesis records a series of experimental observations on Bunostomum trigonocephalum. The studies were made under the following broad headings:
a) pre-parasitic development
b) comparison of routes of infection and studies of the mechanism of infection including exsheathment of the infective larvae c) the pathogenesis and patnogenicity of the parasite for the sheep
host.
The morphology and development of the first1 second and third larval stages was examined, The moult of the first to the second stage was observed and the first stage cuticle appeared to disintegrate rather than be cast as an intact sheath. This moult occurred after about 5 days incubation at 21°c.
Little larval growth occurred after the first moult and the second moult follow- ed comparatively quickly, 80% of the larvae were in the infective stage after 7 days incubation.
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Survival of infective larvae was compared at temperatures of 0 C, 10 c,
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20 C, 27 C and 37 C. Larvae survived longest at 10 C.
Infective larvae were administered to sheep intravenously, subcutaneously, intraruminally, orally and percutaneously. The largest infections were est- ablished after percutaneous administration.
Infective larvae applied to the skin of sheep exsheathed rapidly and penetrated the keratinised layers of the skin. They were also found to ex- sheath on and penetrate into the skin of calves, gtli.nea pigs and rabbits.
There was evidence that larvae migrated to the lungs in the guinea pigs and patent infections were established in some calves,
During exsheathment the anterior portion of the sheath appeared to disin- tegrate allowing the larvae to emerge, This is unlike the processes which have been described in other strongylate nematodes.
A variable proportion of the infective larvae were found to exsheath
"spontaneously" in faecal cultures,
Attempts were made to find what stimulates the infective larvae to ex- sheath, The results were inconclusive. The highest rate of exsheathment was obtained by·exposing larvae to buffer of pH 2. The addition of
co
2 en-hanced the exsheathment rate but only at pH 2. Even so, the results were variable and difficult to reconcile with conditions on the skin surface.
After infection of sheep the prepatent period was found to range from 54 to 60 days. The 24 hour egg-output per female worm was estimated in 5 infected sheep and was found to range from 2579 to 10480 eggs. Most of the Bunostomum were found in the intestine between 5 and 35 feet posterior to the gastric pylorus. Numbers of Bunostomum in excess of 2-300 caused a significant and progressive decline in haemoglobin, packed cell volume and total serum protein levels over observation periods of up to 16 weeks after infection, The serum protein decline was primarily due to a fall in albumin concentration. The animals so affected also showed evidence of anorexia, retarded growth rate and loss of weight, Diarrhoea occurred sporadically.
Acknowledgements
I would like to thank my Supervisor, Dr. W.A.G. Charleston, for the stimulation, guidance and sincere contructive criticism that he has given me during this study. I am also especially indebted to the help and encouragement of Miss Kerry McRae for assisting me with many of the
experiments in this thesis.
I would also like to thank Miss Anne Woodward for the careful and thorough manner in which she typed both the original draft and final script.
The assistance of Mrs. Fitch and Mrs. Montgomery of Tasman Vaccine Laboratory's Library and the staff of the Massey University Library in locating many
references is much appreciated.
Thanks are due to Mr. A. Finn and Miss S. Jackson who were
responsible for the management of the experimental animals and to Mr. R. Dwight and Mr. D. Liardet for their advice on the statistical calculations and
photography respectively.
I would like to express my gratitude to the Directors of
Tasman Vaccine Laboratory, especially Mr. Fitch for granting me the opportunity and financial assistance to undertake this work.
Finally my sincere thanks to my wife Jennifer for her support and encouragement throughout the whole course of this study.
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Index to Figures Figure
1
Facing page Graph showing egg production of Donor sheep 272 6 2 G,raph showing egg production of Donor sheep 273 7 3 Photomicrograph of the anterior end of a 1st stage larva
of B. trigonocephalum.
4 Photomicrograph of the posterior end of a 2nd stage larva of B. trigonocephalum.
5 Photomicrograph of the anterior end of a 2nd stage larva of B. trigonocephalum.
6 The growth pattern of .the preparasitic stages of the larvae of B. trigonocephalum.
17
18
19
21 7 Photomicrograph of a late 3rd stage larva of B. trigonocephalum
recovered from the lung of a sheep.
8 Photomicrograph of the buccal capsule of an adult B. trigonocephalum.
22
24 9 Male and female B. trigonocephalum in p~ocess of copulation. 25 10 Stages in the development of the free living stages of
B. trigonocephalum.
11 The effect of temperature on the survival of the 3rd stage larvae of B. trigonocephalum.
12 Colour photograph of the lung of a sheep showing focal traumatic damage caused by the migrating larvae of
13
14
B. trigonocephalum.
Sheep skin tensed across an Agee lid.
Water bath with funnels containing skin in contact with 0. 85% saline.
15· Petri dish being placed in
a
pocket under the skin of an anaethetised sheep.26
28
35 38
39
40
Figure Facing page 16 (a and b)
Photomicrograph of 3rd stage larvae of B. trigonocephalum migrating through the epidermis and superficial layers of the dermis.
17 Colour photograph of the lungs of a guinea pig showing focal traumatic damage caused by migrating larvae of B. trigonocephalum.
18 Photomicrograph of the anterior end of a 3rd stage larva of B. trigonocephalum showing anterior portion of the sheath disintegrating.
19 Photomicrograph of an empty cast sheath of a 3rd stage larva of B. trigonocephalum.
20 Photomicrograph of the anterior end of a 3rd stage larva of B. trigonocephalum showing the cuticle markedly
distended in the region of the excretory pore.
21 Photomicrograph of the 3rd stage larva of B. trigonocephalum retracting within the sheath.
22 Test tubes containing ensheathed larvae of B. trigonocephalum being gassed with 20% C02 and 80% air.
23 Electrophoretic patterns of serum samples taken from sheep before and after infection with B. trigonocephalum.
24 The haemoglobin level in blood samples taken from sheep in Experiment II, Chapter 9.
25 The packed cell volume percentage of blood samples taken from sheep in Experiment II, Chapter 9.
26 The estimated total serum protein in samples taken from sheep in Experiment II, Chapter 9.
42
46
1 '
72
73
74
81
105
106
107
108
Figure
27 The haemoglobin level in blood samples taken from sheep in Experiment III, Chapter·9.
28 The packed cell volume percentage of blood samples taken from sheep in Experiment III, Chapter 9.
29 The estimated total serum protein in samples taken from sheep in Experiment III, Chapter 9.
30 Photograph showing 3 sheep from Group A, Experiment III together with one control sheep from Group C, Experiment III, Chapter 9.
31. Colour photograph of the small intestine showing
B. trigonocephalum attached to the mucosa and subsequent petechiae on the surface of the epithelium.
Facing page
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110
111
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