Pathogenesis of Trypanosoma brucei infection in sheep

J. COMP.

PATH.

1975. VOL.

23

85

PATHOGENESIS

OF INFECTION I.

B. 0. Department

TRYPAflOSOMA IN SHEEP

CLINICAL

IKEDE

of Veterinacv Pathology,

BRUCEI

SIGNS*

and G. J. Losos University

of Ibadan, Ibadan, Nigeria

INTRODUCTION

Following our earlier studies on the pathology of irrypanosoma brucei infection in sheep in which some of the animals developed secondary bacterial infections (Ikede and Losos, 1972a), we decided to re-investigate the pathogenesis of the disease in a larger group of sheep in which secondary infections were controlled. In this communication, the clinical signs observed up to the time of death are described. MATERIALS

AND

METHODS

The strain of T. brucei used (Strain 8/18) was described previously (Ikede and Losos, 1972a). It was maintained as a stabilate for 1 year and then passaged 4 times in rats before the sheep were inoculated. Thirty-three West African Dwarf sheep, approximately 2 years old, were purchased from local markets. They were housed in fly-proof pens and observed for 2 months during which time they were shown to be free from trypanosomiasis and other blood parasites by routine blood examination, including sub-inoculation into rats, as described by Godfrey and Killick-Kendrick (1961). About a month before the experiment started, the sheep were treated for coccidiosis for 4 days, using sulphadimidinef orally at 120 mg./kg. body weight. They were wormed with pyrantel tartrate,: before the start of the experiment and on 13 days and 9 1 days after infection. The feeding and management of the sheep was adapted from the standard practice in the Ibadan University Farm. A mixture of cut giant star grass (Cynodon plectostachyus) and legume (Centrosema pubiscens) was supplemented with approximately 0.5 kg. of concentrate per head each day. The concentrate was maize, guinea corn and groundnut cake. Mineral licks and water were supplied ad lib. The 33 sheep were randomly divided into 3 equal groups A, B and C and housed in adjacent fly-proof pens. Animal5 in groups A and B were inoculated i.v. with 5 x lGs trypanosomes in 0.5 ml. of rat blood. Group C animals (control) were injected with O-5 ml. of normal rat blood. It had been observed earlier that Pasteurella haemolytica and Streptococcus spp. were the commonest complicating bacterial agents in sheep infected with T. brucei (Ikede and Losos, 1972a). To suppress these complications in one group (B) penicillin and streptomycin were given to provide broad-spectrum antibiotic cover. Neither antibiotic is known to have a direct effect on 1: brucei. The antibiotic cover was started in * From a thesis presented by the senior author to the University Philosophy. f “Sulphamethazine,” May and Baker, Lagos, Nigeria. 1 “Barnninth”, Pfizer Agricultural Division, Lagos, Nigeria.

of Ibadan

for the degree

of Doctor

of

24

B.

0.

IKEDE

AND

G. J.

LOSOS

the 3rd month after infection when bacterial complications were first manifested clinically, and the course was repeated every 3 weeks or earlier, depending on the clinical presentation. Each course consistedof 500 000 i.u. of penicillin and 0.5 g. of

streptomycin given intramuscularly for 2 days as prophylaxis or for 4 days as treatment. Animals that survived longer received up to 5 courses of antibiotics, but the average for the group was 2.5 courses. Animals in groups A and C were not treated with antibiotics.

Rectal temperatures were taken daily between 07.00 and 08.00 h. Body weights were recorded weekly before the morning feed. The animals were examined clinically several times during the day and at night. Diseased animals which were moribund for more than 12 h. were killed to obtain fresh specimens. At 150 days, the experiment

was terminated and the 3 surviving animals in groups A and B were killed. Two control animals were also killed every 2 weeksfrom 105 days onwards. At necropsy, specimenswere taken from diseasedlungs for routine bacteriological examinations. RESULTS

General The main clinical signs are summarized in Table 1. In general, both groups of infected animals reacted in the same way, but the course of the disease in 4 animals in group A appeared to have been shortened as a result of untreated bacterial complication. The mean survival was 92.9f29.6 days for group A and 110.9 f28.6 days for B. Apart from the sudden onset of fever on the first day, the other clinical signs developed gradually and were noticeable from the 6th week onwards in both infected groups. These signs were loss of weight, dullness, weakness, palpable enlargement of the peripheral lymph nodes, oedema of the head, scrotum, limbs, tail and ventral wall of thorax and abdomen, progressive anorexia, and moderate pallor of the mucous membranes (Table 1). The animals were frequently in sternal recumbency with the neck flexed so that the head was turned into the flank. Oedema of the head affected the lips, bridge of the nose, eyelids and ears, but was occasionally generalized. Facial oedema was so severe in Nos 47, 53 and 70 as to interfere with their chewing and breathing (Figs 1 and 2). Neurological signs were observed in 9 of the 11 animals in group A and 6 of the 11 animals in B. Paresis of the limbs, especially the hind limbs, was evident from the 5th week onwards in some animals. It was followed by tremor and choreiform movements of the head and neck, twitching of the eyelids and ears, and shivering. In the last 1 to 3 weeks before death 4 animals in group A and 2 in B showed severe nervous signs characterized by chewing movements, foamy mouth, grinding of teeth, walking backwards, staggering, somnolence and incoordination. Three of these animals had torticollis and hyper-excitability. Paddling movements of the limbs were observed in animals recumbent for some time before death. There was unilateral and sometimes bilateral photophobia and lachrymation in 5 infected animals 1 to 3 weeks before death. These signs were associated with recurrent opacity of the cornea, inflammation of the conjunctiva and hypopyon, and in one sheep, complete blindness. Two pregnant ewes in group A and 1 in B had late abortions on 4, 46 and 55 days respectively after infection. The aborted foetuses were about 15 to 20 cm. long from crown to rump.

T.

b?XCei

INFECTION

IN

SHEEP:

I. CLINICAL

SIGNS

25

26

Fig.

B. 0. IKEDE

1. Subcutaneous

Fig. 2. Facial

oedema

oedema,

AND

of the head and limbs

with

marked

G. J. LOSOS

and ruffled

involvement

coat of sheep infected

of the lips, jaws,

eyelids

with

and ears.

1. brucei.

?,-. brUCei

INFECTION

IN SHEEP:

I. CLINICAL

27

SIGNS

From the 3rd month, coughing and dyspnoea were observed in 5 animals in group A and 4 in B. The respiratory signs subsided in 3 of the 4 animals in group B following antibiotic therapy. The remaining animal died of respiratory insufficiency despite repeated treatment. Dyspnoea in group A animals usually progressed rapidly and resulted in death of 4 of the 5 within 2 days. In the 5th animal, normal breathing was restored in 3 weeks without treatment. At necropsy, Pasteurella, Staphylococci, Streptococci and Klebsiella were isolated from the bronchopneumonic lungs. One animal in group A also had streptococcal dermatitis secondary to chronic leg oedema. Control animals remained clinically healthy and did not develop bacterial infections. Changes in Rectal Temperatures

The mean temperatures of all the animals before infection and of group C animals throughout the experiment were usually below 39 “C. From 1 to 5 days after infection, mean temperatures varied from 39 “C. to 41 “C. in groups A and B (Fig. 3). During the following 10 to 14 days, there was fluctuation of the I

I

I

I

I

,

I

41 -

4l-

I

I

Group

A (Infected)

Group

B (infected)

,

I

I

I

I

I

4

I

7

---

40-

-

/

f

Group

C (control) I

-5

0

5

IO

15

20

a-30

31-40

41-50

51-60

M-70

71-80

a-90

91-100

Days

Fig. 3. Mean

rectal

temperatures

of 7. brucei-infected

and control

sheep.

temperatures in the infected animals. Thereafter, a sustained fever was present for several weeks in most animals in groups A and B. When periods of remission occurred, they were short, infrequent and irregular. A terminal hypothermia lasting for about 24 h. was observed in 3 animals in group A and 2 in B. Changes in Body Weights

Changes in the body weights of the infected and control animals were evident from about the 6th week (Fig. 4). Whereas the control animals continued to gain weight, those in groups A and B lost weight. Their mean body weights

28

B.

0.

IKEDE

AND

G. J. LOSOS

decreased from 15.5,lr 1.8 kg. and 16.2 f 1.6 kg. respectively, to 13*8f2-7 kg. and 13.7-&2*4 kg. respectively on the 16th week. At death, the weight loss in individual animals in group A varied from 10 to 30 per cent. with a mean of 21.5 per cent. Corresponding values for group B were 10 to 30 per cent. with a mean of 2 l-2 per cent. On the other hand, control animals gained an average of 24-O per cent. of their body weights during the experiment. The changes in the body weights of the infected animals were highly significant (P
-l

20 -

a 5

17-

f .” f

16-

2

15-

II 1

Fig. 4. Mean body treated, (0) untreated.

weights Group

I 0

I

I a

4

I 12

of T. brucei infected and control sheep. R = infected and treated with antibiotics,

I

J

16

(A) Group A = infected and un( n ) Group C = uninfected and

DISCUSSION

Edwards, Judd and Squire (1956a) classified the disease in 2 sheep and 4 goats experimentally infected with 1: brucei into acute, subacute, chronic and cryptic forms, based on the duration of the disease and the frequency of detectable parasitaemia. The clinical signs observed in our 22 infected sheep could not be so divided. The disease was essentially progressive and the outcome appeared to be influenced by the time the trypanosomes invaded vital organs such as the brain, eyes, skin and bone marrow, as has been shown previously in sheep and other animals (Losos and Ikede, 1970; Goodwin, 1971; Ikede and Losos, 1972a, b), rather than their scanty and irregular appearance in the peripheral circulation. Our results support the observations of Hornby (1952) who stated that 1. brucei produced a severe disease in sheep which was characterized clinically by fever, anaemia, weakness, anorexia, keratoconjunctivitis, facial oedema and terminal pronounced neurological signs. On the other hand, Stephen (1970) suggested that reports of lachrymation, salivation and facial oedema in sheep

T.

hCei

INFECTION

IN

SHEEP:

I. CLINICAL

SIGNS

29

(and goats) infected with Z-. brucei were probably due to intercurrent parasitic infections such as haemonchosis, fascioliasis and nasal myiasis. Subcutaneous oedema of the head and dependent parts of the body was observed in 18 out of 22 infected sheep (82 per cent.) in the absence of helminthiasis. The pathogenesis of oedema in TT. brucei infections has been attributed to the direct action of trypanosomes in the interstitial tissues (Hornby, 1952) and to vascular damage and release of kinins and related substances (Boreham, 1967). It may also be due to trypanosomal myocarditis (Ikede and Losos, 1972a) or to myocardial weakness related to chronic anaemia. Lachrymation and photophobia were evident in 5 infected sheep (24 per cent.). Details of the ocular lesions have been described separately (Ikede, 1974), and trypanosomes were found at necropsy in the aqueous and vitreous humours in association with the eye lesions. Furthermore, the sheep were free from nasal myiasis at necropsy. Specific trypanosomal ophthalmitis has been reported previously in natural and in experimental infections with the TT. brucei complex in the equine (Yorke, 1910)) feline (Davis, 1931; Hill, 1955) and bovine species (Ikede and Losos, 1972d). There is no direct evidence that the abortions observed in 3 infected ewes were due entirely to the infection, as neither trypanosomes nor other pathogens were isolated from the decomposed foetuses. On the other hand, the pregnant ewe in the control group lambed normally during the experiment. Abortions have been reported in both animal and human trypanosomiasis (Hornby, 1921; Apted, 1970) and congenital infections can occur (Dikmans, Manthei and Frank, 1957; Ikede and Losos, 1972c). In the present studies, autolysis of the foetuses was so marked that even if there had been intrauterine infection, the parasites would have lysed and escaped detection. Hornby (1952) noted that in sheep and goats infected with T. brucei, fever was usually high and often continuous, and might set in before parasites were demonstrable in the blood. On the other hand, Edwards, Judd and Squire (195613) did not observe any correlation between temperature peaks and high parasitaemia in sheep and goats infected with T. vivax, 1: congolense and ir. brucei. One possible explanation for this discrepancy might be that the latter authors grouped together the responses of the animals to the three different species of trypanosome, whereas, their modes of action might be different (Losos and Ikede, 1970). In our experiments, high fever was a prominent and early feature. It was very marked on the first day after inoculation, even when parasites were not detectable in the blood of some infected sheep. The fever became sustained for several weeks from the third week, with only occasional, short and irregular periods of remission until the animals died 3 to 5 months later. The sustained fever despite antibiotic therapy could be of some significance in clinical diagnosis, especially in enzootic areas. The blood should then be examined repeatedly to demonstrate the trypanosomes. The mechanism of the thermal response in animal trypanosomiasis has not yet been determined. There was no significant difference in the clinical response of the two groups of infected animals with and without antibiotics. This shows that although the general debility and possible immune suppression of the host frequently predispose to secondary bacterial infections, the primary disease caused by

30

B.

0.

IKEDE

AND

G. J.

LOSOS

1. brucei in the sheep can be severe and often terminates fatally, as was emphasized by Hornby (1952). Intercurrent disease can hasten the fatal termination. SUMMARY

Sheep inoculated with Tryfianosoma bruceihad prolonged fever after two weeks, dullness, weakness, progressive anorexia, loss of weight, moderate pallor of mucous membranes, palpable enlargement of the peripheral lymph nodes and oedema of the head, scrotum and limbs from the 6th week onwards. Later, there were pronounced neurological signs and in some cases recurrent keratoconjunctivitis and hypopyon. All but 3 of the 22 infected sheep died by the time the experiment was terminated on the 150th day. Cases of secondary bacterial infection were effectively controlled in one group of sheep with antibiotic treatment. ACKNOWLEDGMENTS

This work was supported by grant 66024, allocation 74, from the Rockefeller Foundation. The authors are grateful to Professor D. H. Hill, Head of the Department of Veterinary Medicine and Surgery, for his advice and interest during the work; to Mr Daniel Ejeko, the animal attendant; to Mr L. A. Orobator for the bacteriological examinations; and to Mr Isaac Obiwale who typed the manuscript.

REFERENCES

Apted, F. I. C. (1970). Clinical Manifestations and Diagnosis of Sleeping Sickness. In African Trypanosomiases, H. W. Mulligan and W. H. Potts, Eds. George Allen and Unwin, London. Boreham, P. F. L. (1967). The possible role of kinins in the pathogenesis of chronic trypanosomiasis. Transactions of the Royal Society of Trofiical Medicine and Hygiene, 62, 120-121. Davis, L. J. (1931). Experimental feline trypanosomiasis with especial reference to the effect of splenectomy. Annals of Tropical Medicine and Parasitology, 25, 79-90. Dikmans, G., Manthei, C. A., and Frank, A. H. (1957). Demonstration of Tvpanosoma theileri in the stomach of an aborted bovine foetus. Cornell Veterinarian, 74, 344353. Edwards, E. E., Judd, J. M., and Squire, F. A. (1956a). Observations on trypanosomiasis in domestic animals in West Africa I. The daily index of infection and the weekly haematological values in goats and sheep infected with Trypanosoma vivax, 1: congolense and T. brucei. Annals of Tropical Medicine and Parasitology, 50,. 223-240. Edwards, E. E., Judd, J. M., and Squire, F. A. (1956b). Observations on trypanosomiasis in domestic animals in West Africa. II. The effect on the erythrocyte sedimentation rate, plasma protein, bilirubin, blood sugar, red celi osmotic fragility, body weight and temperature in goats and sheep infected with Trypanosoma vivax, T. congolense and T. brucei. Annals of Tropical Medicine and Parasitology, 50, 242-25 1. Godfrey, D. G., and Killick-Kendrick, R. ( 196 1) . Bovine trypanosomiasis in Nigeria. I. The inoculation of blood into rats as a method of survey in the Donga Valley, Benue Province. Annals of Tropical Medicine and Parasitology, 55, 287-297. Goodwin, L. G. (1971). Pathological effects of Trypanosoma brucei on small blood vessels in rabbit ear-chambers. Transactions ofthe Royal Society of Tropical Medicine and Hygiene, 65, 82-88. Hill, D. H. (1955). Trypanosoma brucei in the cat. British Veterinary Journal, 3, 77-80.

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Hornby, H. E. (1921). Tryp anosomes and trypanosomiasis of cattle. Journal oj Comparative PatholoQ, 34, 2 1 l-240. Hornby, H. E. (1952). Animal Tryfianosomiasis in Eastern Africa, 1949. H.M. Stationery Office, London. Ikede, B. 0. (1974). Ocular lesions in sheep infected with Trypanosoma brucei. Journal of Comparative Pathology, 84, 203-2 13. Ikede, B. O., and Losos, G. J. (1972a). Pathology of the disease in sheep experimentally produced by Trypanosoma brucei. Veterinary Pathology, 9, 278-289. Ikede, B. O., and Loses, G. J. (197213). Spontaneous canine trypanosomiasis caused by T. brucei. Meningoencephalomyelitis with extravascular localization of trypanosomes in the brain. Bulletin of Epizootic Diseases of Africa, 20, 221-228. Ikede, B. O., and Loses, G. J. (1972c). Hereditary transmission of Trypanosoma vivax in sheep. British Veterinary Journal, 128, i-ii. Ikede, B. O., and Losos, G. J. (1972d). Pathology of experimental disease in cattle produced by Ttypanosoma brucei. Veterinary Pathology, 9, 272-277. Losos, G. J., and Ikede, B. 0. (1970). Pathology of experimental trypanosomiasis in albino rats, rabbits, goats and sheep-A preliminary report. Canadian Journal of Comparative Medicine and Veterinary Science, 34, 209-2 12. Stephen, L. E. (1970). Clinical Manifestations of the Trypanosomiases in Livestock and other Domestic Animals. In African Trypanosomiases, H. W. Mulligan, and W. H. Potts, Eds. George Allen and Unwin, London. Yorke, W. (1910). A note on the pathology of Iesions of the cornea and skin in animals experimentally infected with Trypanosoma rhodesiense. Annals of Tropical Medicine and Parasitology, 4, 385-394. [Receivedforpublication,

February 27th, 19741