The effect of Trypanosoma evansi infection on the oestrous cycle of Friesian Holstein heifers

Veterinary Parasitology, 51 ( 1993 ) 1-11 0304-4017/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved

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The effect of Trypanosoma evansi infection on the oestrous cycle of Friesian Holstein heifers R. C. Payne a, I. P. Sukanto b, Katrine Bazeleyb, T. W. Jones*,a aCentre for Tropical Veterinary Medicine, Easter Bush, Roslin, EH25 9RG, UK bResearch Institute for Veterinary Science, Bogor, West Java, Indonesia (Accepted 8 April 1993)

Abstract

The effect of Trypanosoma evansi infection on oestrous cycling was studied in 12 Friesian Holstein heifers. In the Phase 1 of the investigation, six heifers were infected with T. evansi, the remaining six acted as uninfected controls. Daily body temperature, packed red cell volume (PCV) and parasitaemia measurements were obtained from each animal for 90 days. The animals were examined for external signs of oestrous activity twice daily, blood samples were taken three times a week and subjected to an enzyme-linked immunosorbent assay to detect plasma progesterone. Body weights were measured weekly. Parasites were eliminated by trypanocidal drug treatment 90 days after infection. In Phase 2 of the trial, the uninfected heifers were injected with a different stock of parasites and monitoring was continued as before. Infection with T. evansi resulted in a marked reduction in the rate of weight gain, an increase in body temperatures and a fall in PCV values. Eleven of the heifers continued to cycle normally for the duration of the study, irrespective of their infective status. One animal which stopped cycling lost 16.2% of its pre-infection body weight as a result of the infection and cessation of oestrous activity was considered to have been due to weight loss. Key words: Cattle-Protozoa; Trypanosoma evansi; Pathology-Protozoa

Introduction

Trypanosome infections have been associated with impairment of reproductive efficiency (Stephen, 1966) and in an investigation on the effect of Trypanosoma congolense infection on oestrous cycling of Boran cows, Llewelyn et al. ( 1988 ) concluded that cycling was disrupted as a result of interference with the endocrine control system. Trypanosomosis caused by Trypanosoma evansi is widespread in Indonesia (Adiwinata and Dachlan, 1969; Dennig, 1976; Payne et al., 1991a) and is characterised by sporadic outbreaks of disease involving cattle, buffalo and horses (Adiwinata and Dachlan, 1969; Anonymous, 1985; Payne et al., 1990). *Corresponding author.

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R. C Payneet aL/VeterinaryParasitology51 (1993) 1-11

Little is known of the sub-clinical effects of T. evansi such as the effect on the reproductive ability of its hosts. This communication describes an investigation, conducted at the Research Institute for Veterinary Science, Bogor, to determine the effect of T. evansi on oestrous activity of non-pregnant Friesian Holstein (FH) heifers. Materials and methods

Twelve FH heifers 15-18 months of age and weighing between 182 and 270 kg, judged by rectal palpation of the ovaries to be cycling normally, were obtained from local dairy farms. The animals were parasitologically negative for T. evansi by the microhaematocrit test (MHCT) performed according to Woo (1970) and negative for trypanosomal antibodies by the enzyme-linked immunosorbent assay (ELISA) as described by Payne et al. (1988). The animals were treated against fasciolosis using Fasinex (Ciba Geigy, Basle, Switzerland) and against gastrointestinal parasites with ivermectin (Merke, Sharpe & Dohme, Rahway, N J). Heifers received a daily ration of cattle concentrate ( 1.5 kg per 100 kg body weight (BT)) and elephant grass (Pennisetum purpureum, 6.5 kg per 100 kg BW). Before infection, heifers were kept in flyproof accommodation for 90 days (the first day of this observation period designated Day 0). Body temperature and packed cell volume (PCV) were monitored daily for each animal during this time to provide baseline data; body weights were measured each week. Blood samples were taken three times a week and subjected to an ELISA to detect plasma progesterone in order to establish patterns of cyclicity. Additional blood and serum samples were screened on a weekly basis by MHCT and ELISA for evidence of T. evansi infection. After 90 days the animals were stratified according to body weight and divided into two groups of six according to a randomised block design (Bancroft et al., 1970). Experimental design Phase I In Phase 1 of the investigation, six heifers were infected with T. evansi (Group A), the remaining animals acted as uninfected controls (Group B ). Body temperature, packed cell volume, parasitaemia and oestrous activity were monitored in all animals until Day 180, when parasites were eliminated using Cymelarsan® (mel-Cy, Rhone Merieux, Lyon, France) at a dose rate of 0.75 mg kg-1 i.m.

R.C. Payne et al./Veterinary Parasitology 51 (1993) 1-11

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Phase 2 Two days following the treatment of Group A animals, the six uninfected animals in Group B were injected with a different isolate of T. evansi. Monitoring was continued as before and 90 days later (Day 272 ), parasites were eliminated. The heifers were kept in single stalls for the duration of the study. Parasites In Phase 1, a population of T. evansi (BAKIT 230), stored in liquid nitrogen was expanded in outbred white laboratory mice. Each of the six heifers in Group A ( 300, 304, 305,308, 310 and 311 ) were injected intravenously with 2 X 10 7 parasites. In Phase 2, a second stock of T. evansi (BAKIT 362) was used at the same dose to infect the six heifers in Group B (301,302, 303,306, 307 and 309).

Body temperature and weight The rectal body temperature of each animal was recorded daily between 08:00 and 09:00 h and the animals were weighed once a week between 09:00 and 10:00 h before feeding.

Parasitaemia Daily blood samples were collected from each of the heifers for 90 days during the first infection and for a further 90 days when the second group was infected. Samples of blood were collected in heparinised haematocrit capillary tubes (Hawksley & Sons, Lancing, UK) by venepuncture of a marginal ear vein and monitored for parasitaemia and PCV according to Payne et al. (1991b).

Oestrous activity Observations for oestrous behaviour were carried out twice a day for 15 rain at 08:00 and 14:00 h. These observations were limited to the behaviour of individual animals and included checking for vaginal mucous discharge, bellowing and restlessness.

Palpations The reproductive tract of each animal was examined by rectal palpation three times weekly. The uterus was examined for tone and ovarian size and structure were recorded according to Noakes ( 1986 ).

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Measurement of plasma progesterone Blood samples for progesterone measurements were collected three times weekly from a marginal ear vein using 0.75 ml microvettes containing EDTAK2 as an anticoagulant (Sarstedt, Numbrecht, Germany). Immediately after collection, microvettes were centrifuged at 3000 rev m i n - l for 15 min, the plasma was removed and stored at - 7 0 ° C until it could be subjected to a progesterone ELISA (Ovucheck kits, Cambridge Veterinary Sciences, Cambridge, U K ) . Data collected during the investigation were processed and summary statistics obtained using PANACEA, a database management system developed by Pan Livestock Services, Reading University, UK. Comparisons of parasitaemia, body temperature and PCV parameters between groups of infected animals were undertaken on summary statistics using a two sample t-test as implemented on the STAT-SAK statistical analysis software package (STATOOLS, Malden, MA ). Results

Parasitaemia The pattern of parasitaemia was similar in both groups of infected animals. Trypanosomes were detected within 48 h of infection and parasitaemia levels fluctuated with an approximate frequency of 6-7 days between the onset of paroxysms. Infected heifers remained MHCT positive throughout each of the observation periods. There was no significant difference ( P > 0.05 ) between the two infected groups as judged by the mean of the number of days parasites were observed (Tables 1 and 2).

Body temperature The mean daily body temperatures measured over each 90 day infection period are shown in Tables 1 and 2. In both phases of the investigation, the mean temperature of the infected group was above that of the non-infected animals ( P < 0.05 ), similarly the number of days on which temperatures exceeded 39 °C was higher in the infected animals.

Packed cell volume The Group A mean PCV value measured during the 90 day period following infection (Days 90-180) was not significantly different ( P > 0.05 ) from the mean PCV of the non-infected animals (Table 1 ). In Phase 2, the mean PCV of the infected Group B animals (Table 2) was significantly lower

Mean

38.56

38.48 38.50 38.57 38.61 38.59 38.60

301 302 303 306 307 309

Control heifers (group B )

38.64 38.72 38.57 38.74 38.77 38.72

38.69

300 304 305 308 310 311

Mean BT (°C)

Mean

Infected heifers (Group A)

Calf no.

2.5

1 3 3 2 6 0

10

5 10 5 17 15 8

No. days BT > 39 ° C

31.0

30.7 31.1 30.5 30.5 30.6 32.7

29.8

27.1 28.4 28.2 30.5 32.2 32.4

Mean PCV

15.5

15 16 20 27 15 0

46.5

83 71 70 30 23 2

No. days PCV < 30%

0

0 0 0 0 0 0

0.8

1 1 3 0 0 0

No. days PCV < 25%

665

622 578 756 511 744 778

461

578 489 478 378 356 489

Body wt. gain ( g d a y -1 )

21.5

17.3 15.7 24.3 18.7 23.8 29.2

13.8

17.9 15.4 12.6 13.4 11.5 11.8

%Body wt. gain

49

52 55 55 49 41 40

No. days MHCT+

Table 1 Body temperature (BT), packed cell volume (PCV), weight gain and parasitaemia measurements for uninfected heifers and heifers infected with Trypanosoma evansi (BAKIT 230) in Phase 1 of the trial

~

~.

r~

'~

Mean

38.86

38.85 39.25 38.83 38.72 38.83 38.69

301 302 303 306 307 309

Infected heifers (Group B)

38.57 38.59 38.61 38.53 38.60 38.66

38.59

300 304 305 308 310 311

Mean BT (°C)

Mean

Control heifers (Group A)

Calf no.

20.3

18 44 19 10 21 10

5

4 3 7 3 5 8

No. days BT > 39°C

27.2

25.4 22.8 27.1 29.9 27.5 30.4

30.9

28.2 29.8 28.6 31.8 34.0 33.1

Mean PCV

64.3

88 88 80 31 70 29

32.8

78 37 74 8 0 0

No. days PCV < 30%

21.7

38 61 14 3 13 1

0

0 0 0 0 0 0

No. days PCV < 25%

148

67 -689 476 333 322 378

506

511 378 611 444 489 600

Body wt. gain (g day-~)

4.5

1.6 - 16.2 12.1 10.3 8.3 11.0

13.2

13.5 10.3 14.3 13.9 14.2 12.9

%Body wt. gain

51

59 63 58 45 44 35

No. days MHCT+

Table 2 Body temperature (BT), packed cell volume (PCV), weight gain and parasitaemia measurements for uninfected heifers and heifers infected with Trypanosoma evansi (BAKIT 263) in Phase 2 of the trial

C7~

R.C. Payne et al./VeterinaryParasitology51 (1993) 1-11

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(P< 0.05 ) than the mean obtained from the same animals when they were not infected (Table 1 ). Tables 1 and 2 show that the PCV values of infected animals fell below 30% and 25% more frequently in Group B (BAKIT 362) than in Group A (BAKIT 230).

Body weight The body weight gains of individual animals during Phase 1 (Days 90180) are shown in Tables 1 and 2 in absolute terms and as a percentage increase. Body weights at the beginning and end of each phase of the investigation are shown in Table 3. In both phases of the trial, the growth rates of infected animals were less than those achieved by the non-infected animals ( P < 0.05 ). The mean growth rate of infected animals in Group B (Table 2) was significantly lower than that of the uninfected group ( P < 0.05 ) and it was also lower than the mean weight gain achieved in Phase 1 of the study by the same animals (Table 1 ). One heifer in Group B (302) lost 16.2% of its pre-infection body weight (Table 2). Weight losses were not correlated significantly to changes in PCV values.

Oestrous activity."physical signs Vaginal mucous discharge was the only external sign of oestrus observed during the study. The appearance of a mucous discharge always coincided Table 3 Heifer body weights (kg) Animal no.

Phase 2

Phase 1

Day 0

Day 90

Day 180

Day 270

GroupA 300 304 305 308 310 311

232 214 276 196 228 284

290 286 342 254 278 374

342 330 385 288 310 404

388 364 440 328 354 472

Group B 301 302 303 306 307 309

258 260 205 190 204 198

324 332 280 246 282 240

380 384 348 292 349 310

386 322 390 322 378 344

Day 0, start of the observation period; Day 90, first infection (Group A); Day 180, treatment of infected animals and 2 days before the second infection (Group B); Day 270, 2 days before the treatment of second infection.

2 3 0 0 1 3

4 3 3 1 2 2

Incidence of oestrous detection

Phase 1

4 5 5 5 5 5

5 4 4 5 5 5

No. of cycles detected by ELISA

21 21 21 21 18 19

21 23 23 20 20 24

Average cycle length (days)

2 1 1 1 3 3

2 2 2 3 5 0

Incidence of oestrous detection

Phase 2

4 4 4 4 5 5

4 4 5 5 5 4

No. of cycles detected by ELISA

21 21 20 20 19 19

21 21 20 19 18 23

Average cycle length (days)

1In Phase 1, Group A animals were infected and Group B animals acted as controls; in Phase 2, Group B animals were infected on Day 182 and Group A animals acted as controls, having been treated on Day 180.

301 302 303 306 307 309

GroupB

300 304 305 308 310 311

GroupA

no.

Animal

Table 4 Incidence of external signs of oestrous activity, number of cycles detected by progesterone ELI SA and average cycle length during Phases 1 and 21 of the experiment

3.

f~

oo

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with the detection of basal progesterone levels as measured by ELISA. The frequency of oestrous detection by physical signs is shown in Table 4. No differences were observed between the behaviour of infected and non-infected animals.

Palpation Palpation of the ovaries indicated normal follicular development associated with oestrous cycling in 11 of the 12 heifers with no differences being detected between infected and non-infected animals. One heifer (302) in Group B appeared to be cycling normally until Day 262 (80 days post-infection), after which time developing follicles were not detected for a further 2 l days until palpation was discontinued.

Progesterone The number and duration of cycles detected by the progesterone ELISA are shown in Table 4. All the heifers were cycling at the beginning of the trial and cycling continued in 11 animals throughout the study. A prolonged period of basal progesterone levels was observed in one infected heifer ( 302, Group B ) beginning 85 days after infection, indicating a cessation of ovarian activity. Monitoring of oestrous activity continued in this animal after parasites had been eliminated and normal cycling recommenced 60 days later. Discussion

This study has demonstrated that infection with T. evansi can impair body weight gain in Friesian Holstein heifers and furthermore, that a substantial reduction of body weight was associated with cessation of oestrous activity in one animal. Although 11 of the 12 experimental animals continued to cycle normally for the duration of the study, irrespective of their infection status, results from the progesterone ELISA indicated that one heifer (302, Group B) which lost 16.2% of its pre-infection body weight stopped cycling 84 days after infection. The progesterone ELISA proved to be a simple and effective method for oestrous detection. External observations for oestrous activity were limited to detection of vaginal mucous discharge; behavioural aspects, such as mounting, could not be included in the study because heifers were isolated in individual stalls. Dairy cattle are kept in individual stalls on the majority of smallholder dairy operations on Java and inefficient heat detection is a common problem. A similar system was adopted in the present study in order to mimic the farming situation as closely as possible. Changes in body temperature, PCV and rate of body weight gain are fea-

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tures of infection with T. evansi which have been described in Friesian Holstein calves (Payne et al., 1992). The pathological changes observed after infection in the present study suggest that the second parasite population (BAKIT 362) was more virulent in its effect than the first (BAKIT 230), with reduced body weight gain being the most significant feature of infection. Cessation of oestrus appeared to be associated with loss of body weight, the most severely affected heifer (302) stopped cycling after losing 16.2% of its pre-infection body weight. An association between weight loss and disruption of ovarian activity has been well established in dairy cows (Heinonen et al., 1988) and Teleni et al. (1988) found that a loss of approximately 17% of liveweight was detrimental to reproductive function in cattle with a medium body condition. In the present trial, the most severely affected heifer (302) began to gain weight immediately after parasites had been eliminated by trypanocidal drug treatment and oestrous cycling, detected by vaginal mucous discharge and progesterone activity, was re-established 60 days after treatment, by which time the animal had regained its pre-infection body weight. These results suggest that weight loss was the most significant factor in the cessation of oestrous activity. There was considerable heterogeneity of responses to infection observed in individual animals during the present trial and the animal that ceased oestrous activity was affected much more severely, as shown by body weight loss and anaemia, than any of the others. Variation in the clinical manifestations of T. evansi infection has previously been demonstrated in Friesian Holstein calves (Payne et al., 1992) and in water buffalo (Payne et al., 1991b). Although these results make it difficult to gauge the importance of T. evansi infections as a cause of infertility of cattle under field conditions in Indonesia, our results suggest that in areas where T. evansi is endemic, and the frequency of transmission is likely to be high, prophylactic intervention or chemotherapeutic elimination of parasites from closed herds is indicated. Such an approach would remove the negative effect on weight gain imposed by T. evansi infection and minimise the possibility of reduced fertility occurring as an indirect consequence of infection.

Acknowledgements This study was supported financially by the Overseas Development Administration of the Foreign and Commonwealth Office, London. The authors would like to thank Dr. Ginting, Director of the Research Institute of Veterinary Science, for the support of the Institute during the investigation.

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Anonymous,, 1985. Buku Statistik Peternak Direktorat Bina Program. Direktorat Jenderal Peternakan, Indonesia, 140 pp. Bancroft, H., Ipsen, J., Feigl, P. and Bancroft, S., 1970. Introduction to Biostatistics, 2nd edn. Harper and Row, New York, pp. 175-176. Dennig, H., 1976. Final report of the FAO/UNDP project (INS/72/008) in Indonesia. FAO, Rome, 18 pp. Heinonen, K., Ettala, E. and Alanko, M., 1988. Effect of post partum live weight loss on reproductive function in dairy cows. Acta Vet. Scand., 29 (2): 249-254. Llewelyn, C.A., Munro, C.D., Luckins, A.G., Jordt, T., Murray, M. and Lorenzini, E., 1988. The effects of Trypanosoma congolense infection on the oestrous cycle of the Boran cow. Br. Vet. J., 144(4): 379-387. Noakes, D.E., 1986. Fertility and Obstetrics in Cattle. Library of Veterinary Practice. Blackwell Scientific, Oxford, UK, pp. 16-20. Payne, R.C., Ward, D.E., Usman, M., Rusli, A., Djauhari, D. and Husein, A., 1988. Prevalence of bovine haemoparasites in Aceh Province of northern Sumatra: Implications for imported cattle. Prev. Vet. Med., 6: 275-283. Payne, R.C., Sukanto, I.P., Graydon, R., Saroso, S. and Jusuf, H., 1990. An outbreak oftrypanosomiasis caused by Trypanosoma evansi on the island of Madura, Indonesia. Trop. Med. Parasitol., 41: 445-446. Payne, R.C., Sukanto, I.P., Djauhari, D., Partoutomo, S., Wilson, A.J., Jones, T.W., Boid, R. and Luckins, A.G., 1991a. Trypanosoma evansi infection in cattle, buffaloes and horses in Indonesia. Vet. Parasitol., 38: 109-119. Payne, R.C., Djauhari, D., Partoutomo, S., Jones, T.W. and Pearson, R.A., 1991b. Trypanosoma evansi infection in worked and unworked buffaloes (Bubalus bubalis) in Indonesia. Vet. Parasitol., 40:197-206. Payne, R.C., Sukanto, I.P., Partoutomo, S. and Polytedi, F., 1992. Experimental infection of Friesian Holstein calves with an Indonesian isolate of Trypanosoma evansi. Trop. Med. Parasitol., 43: 115-117. Stephen, L.E., 1966. Observations on the resistance of West African N'dama and Zebu cattle to trypanosomiasis following challenge by wild Glossina morsitans from an early age. Ann. Trop. Med. Parasitol., 60: 230-246. Teleni, E., Boniface, A.N., Sutherland, S. and Entwistle, K., 1988. The effect of depletion of body reserve nutrients on reproduction ofBos indicus cattle. Draught Animal Power Project Bull. No. 8. ACIAR Publ., Canberra, Australia, p. 10. Woo, P.T.K., 1970. Evaluation of the haematocrit centrifuge and other techniques for field diagnosis of human trypanosomiasis and filariasis. Can. J. Zool., 47:921-923.