J. Comp. Path. 1994 Vol. 110, 237-252

The P a t h o l o g y o f E x p e r i m e n t a l Trypanosoma evansi Infec t ion in the I n d o n e s i a n Buffalo (Bubalus bubalis)

R. Damayanti, R. J. Graydon and P. W. Ladds* Research Institute for Veterinary Science ( Balitvet), Post, O~ce Box 52, Bogor, Indonesia and

*Department of Biomedical and Tropical Velerina~y Sciences, James Cook University, Townsville, Ojleensland 4811, Australia

Summary Six Indonesian buffaloes (Bubalus bubalis) were inoculated intravenously with l05 Trypanosoma evansi, examined clinically, haematologically and serologically, and then killed 1, 2, 3, 4, 8 or 12 weeks after infection tbr detailed pathological study.

Relapsing fever was related to the waves of parasitaemia and fluctuations of pulse and respiration rates. Anaemic mucous membranes, depression, weakness, refusal to walk, loss of appetite and emaciation were seen. Body weight, packed cell volume, total platelet and red cell counts, and haemoglobin values were below those of two uninfected control buffaloes, as well as below the normal range; on the other hand antibody titres against 7-. evansi in infected animals were all above those in controls.

Emaciation, serous atrophy of fat, hydropericardium, petechial to larger haemorrhages in the pericardium, pneumonia, congested liver and spleen, oedematous enlargement of the superficial lymph nodes and hyperplastic bone marrow were the major gross pathological changes. Histologically, the severity of the disease increased from 1 to 7 weeks after infection and became less obvious at 12 weeks. The most consistent lesions were interstitial pneumonia, interstitial myocarditis, splenic muhifocal necrosis, interstitial myositis and hyperplastlc bone marrow. The last three lesions appear not to have been reported previously in T. evansi infection in buffaloes or other animals.

The clinicopathological findings in this study show that 7-. evansi is both an intravascular and extravascular parasite.

Introduction

Trypanosomiasis in Indonesia is one of the most economically important diseases of horses, buffaloes and cattle (Dieleman, 1986) and its prevalence in buffaloes is higher than in cattle (Payne, 1989). Impor ted Australian buffaloes affected by the disease suffered significant mortal i ty (Unruh et al., 1988; Sudar to et al., 1990).

Trypanosoma evansi infection (surra) has been less thoroughly studied than African trypanosomiasis. The aim of this study, therelbre, was to describe the sequential pa thology and the pathogenesis of T. evansi infection in local Indonesian buffaloes, part icularly as an aid to diagnosis based on histopatholo- gical examination.

0021-9975/94/030237 + 16 $08.00/0 © 1994. Academic Press Limitcd

23B R. Darnayanti e t a l .

Materials and Methods

Animals

Eight buffaloes, two male and six female, aged 6-8 months were used. They were selected from/~ number of smallholder farms located in the Jonggol area of Bogor, West Java.

Before any buffalo was purchased, it had to be clinically sound, have a packed cell volume (PCV) of more than 30%, and be negative for T. evansz by the microhaematocrit technique (MHT) (Woo, 1969) and by the enzyme linked immunosorbent assay (ELISA) (Luckins, 1977).

The animals were kept in individual insect-proof pens during the experimental period. All were treated with ivermectin (Ivomec, MSD-AGVET, Haarlem, The Netherlands) at 200 I-tg/kg body weight to rid them of internal and external parasites. The two buffaloes which were to be the negative controls were also treated with suramin (Naganol, Bayer, West Germany) 10 mg/kg bodyweight to prevent them from acquiring trypanosomiasis.

Preparation of the Inoeulum

The inoculum was derived from a stabilate (Iso 77 Bakit 311) originally collected from a female buffalo, aged two years, from West Java.

The stabilate was diluted in 3 ml phosphate buffered saline (PBS) and 0"5 ml was then injected intraperitoneally into each &six mice. The mice were examined daily for the presence of parasites by making wet smears from the tail blood. When the number of parasites was scored as + 4 (> 20 trypanosomes/high power field), 5 gl of tail blood were diluted in 5 ml PBS, and one drop was then introduced into a Neubauer haemocytometer. The number of viable trypanosomes was counted by the method used for counting white blood cells. The mouse was then killed by ether inhalation, and blood was taken directly from the heart after thoracotomy.

The number oftrypanosomes required was 10~/buffalo. The amount of mouse blood needed for each buffalo was calculated as follows:

Number oftrypanosomes required Number trypanosomes/ml counted in haemocytometer"

The appropriate amount &mouse blood was then further diluted with PBS to give an injection volume of 2 ml.

Experimental Design

The buffaloes were divided randomly into two groups, six to be infected with 105 trypanosomes/buffalo intravenously, and the other two as uninfected controls.

Because the major interest in this experiment concerned the early events in the pathology and pathogenesis of the disease, the infected buffaloes were scheduled to be killed 1, 2, 3, 4, 8 or 12 weeks post-inoculation (PI). The control buffaloes were killed at 4 and 8 weeks. One buffalo in the infected group which was scheduled to be killed at 8 weeks died 7 weeks PI.

Daily Clinical Observations

Clinical observations (made at 9.00 a.m. daily) included the measurement of rectal temperature, pulse and respiration rates and estimation of the parasitaemia. Any other clinical signs observed were also recorded.

After collection of blood from the auricular vein into a capillary tube (see below) the presence of trypanosomes was detected and the degree of parasitaemia estimated by microscopical (x 100) examination of the plasma-buffy coat interphase. After

T. evansi in Buffaloes 239

breaking the tube at the buffy coat surface (Woo, 1969), one drop of plasma was placed on a slide, covered with a coverslip and examined at x 400 magnification by dark ground illumination (Murray et al., 1974). The log. equivalents (LEs) of the average number (X) of trypanosomes per microscope field represented the degree of parasitaemia. Twenty fields were examined for every case. The log. base 10 of X + 2 = parasitaemia in LEs. By means ofa Neubauer haemocytometer it was observed that 100 parasites per field (4LEs) was equivalent to c. 2"5 x 106 parasites/ml.

Weekly Clinical Observations

Haematological Examination. The tests included the direct estimation of PCV, total red cell count (RCC), total leucocyte count, haemoglobin (Hb) concentration, total plasma protein and differential white cell count, and the indirect estimation of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Blood samples were collected fi'om the jugular vein into ethylene diamine tetra-acetic acid (EDTA) tubes. For PCV estimation, a disposable stainless steel blood lancet (Cheil Medical Co, Korea) was used to obtain blood from the auricular vein directly into a heparinized capillary tube (Terumo Corp., Tokyo, Japan). Samples were spun in a microhaematocrit centrifuge (Haemofuge-A, Heraeus Christ, Germany) for 5 min at 12 000 rpm and measured with a microhaematocrit reader (Clay Adams, Becton Dickinson and Co., Parsippany, NY, U.S.A.). RCC and total leucocyte counts were measured with a Coulter counter (Model ZF; Coulter Electronic Ltd, Northwell Drive, Luton, Beds, U.K.). Platelet counts were estimated by an indirect method (Jain, 1986).

Body l/IZeight iVIeasurement. Animals were weighed with all electronic scale (Ruddweigh KM I Electronicweigh System; Ruddweigh Pry Ltd, Guyra, Australia).

Serological Examination. The ELISA method described by Luckins (1977) was used to detect serum antibodies to 7-. evansi. Antigen was prepared by filtration of rodent blood containing T. evansi through DEAE cellulose, centrifugation of the eluate to produce a pellet of trypanosornes, dilution of this pellet in PBS, ultrasonic disintegration, re-centrifugation and then resuspension to a chosen protein concentration by rneans of spectrophotometry (Luckins, 1977). Negative serum samples were collected frm'n newly imported buffaloes and Sahiwal-cross cattle from northern Australia and New Zealand, respectively--countries where T. evansi does not occur. Known positive serum samples were obtained fi'om cattle and buffaloes infected with the parasite at the Research Institute of Veterinary Science (RIVS), Bogor, West Java. Before the test, positive and negative control sera were assayed on a number of separate occasions in order to standardize their reactivity by the assay procedure. The readings obtained were used as day 0 values for calculating the correction factor, F. This was important to give an indication of the degree of variation between assays performed at different times. According to Luckins (1983), limits of between 0"85 and i'5 are considered acceptable. Optical density (OD) values were corrected for day-to-day variations by means of the F factor.

Factor (F) = P o - N o PL--N,

Adjusted value = F( S , - Nt) + No,

where Po = positive control serum on day 0; No=negative control serum on day 0; Pt=positive control serum on test day; Nt=negative control serum on test day; S t = unknown sample.

Test samples which, after correction, gave a reading of >/0"37, twice the reading obtained from the negative control serum, were considered positive (Luckins, 1983). The significance of differences between OD readings in infected buffaloes, and

240 R. Damayantl e t aL

uninfected controls at 2 to 8 weeks PI was examined by application of Student's t-test (Daniel, 1991).

Pathological Examination The buffaloes were killed by exsanguination, and at necropsy tissues were collected and fixed in 10% buffered neutral formalin (BNF) for 24 to 72 h for histopathological examination. Included were brain, spinal cord, pituitary gland, long-hone marrow, lung, heart, spleen, liver, gall bladder, pancreas, kidney, adrenal gland, urinary bladder, abomasum, duodenum, jejunum, ileum, colon, gluteal muscle and representative superficial lymph nodes (parotid, superficial-cervical and sub-lilac). Other tissues were also collected if any macroscopical changes were observed. Representative sections from each tissue were embedded in paraffin wax by routine methods. Sections were cut at 5-7 gm and stained with haematoxylin and eosin (HE).

Resul t s

Daily Clinical Findings

Body Temperature and Parasitaemia. Fluctuating pyrexia commenced 5-8 days PI. The maximum temperatures recorded were 40.0-40.6°C and pyrexia persisted for 1-2 days. A wave ofparasitaemia, reaching 3-4 LEs (c. 2"5 x l0 G organisms/ml of blood) preceded the onset of pyrexia by 1-4 days. The LE values dropped as the body temperatures returned to normal.

Respiration and Pulse Rates. These were increased while the animals were febrile but dropped again as the body temperatures returned to normal. The highest respiration and pulse rates, recorded in a single animal for a period of 3 days, were 49-59/rain and 92-96/rain, respectively.

Other Clinical Signs. No clinical signs other than fever and increased respi- ration and pulse rates were observed in the buffalo killed 1 week PI. Anaemic mucous membranes were first noticed on PI days 8-13 or as late as 32 days PI and remained throughout the observation period. Loss of appetite was first observed 13-16 days PI in all buffaloes and, except in one animal, persisted. Depression and weakness were observed in the buffaloes killed 2 and 3 weeks PI, and in the animal that died 7 weeks PI. Reluctance or refusal to walk were observed in several animals. The two uninfected control buffaloes remained apparently healthy.

Weekly Clinical Findings

Bodyweight Changes. All infected buffaloes lost weight during the observation period, whereas weight gains were recorded for both of the uninfected ones.

Packed Cell Volume. The PCV of the infected animals steadily decreased to levels below those in uninfected controls and also below the accepted normal range for cattle (24-46%; Jain, 1986). PCV values had dropped within 2

T. evansi in Buffaloes 241

weeks from the pre-infection values of 30-36% to 10-19%, and remained at these low .levels until the time of slaughter.

Total Platelet and Total Leucocyte Counts. The platelet counts declined sharply to below the normal range at 1-3 weeks PI but then returned to normal. Clumping of the platelets in the blood smear preparations was often observed.

Leucocyte counts were within the normal range (4000 to 12 000/gl; Jain, 1986), except for one buffalo which had elevated counts 6-12 weeks PI.

Differential Leucocyte Counts. The lymphocyte counts of the infected buffaloes were greater than those in the single remaining control buffalo at 5, 7 and 8 weeks PI, and were also higher than the normal range (Jain, 1986). Neutrophil counts also varied; the mean count for infected buffaloes was above that of the control buffaloes at 2, 3 and 4 weeks PI, but below that of the single remaining uninfected control at 8 weeks. The values were normal at 8, 9, 11 and 12 weeks PI. The eosinophil counts were below the values of the uninfected control buffalo(es) at 3-5 weeks PI but still within the normal range.

Red Cell Counts and Haemoglobin. Essentially, the RCC and Hb values of the infected buffaloes dropped as the PI interval increased. The RCC values &the infected buffaloes were below the normal range from 2 weeks PI to the end of the observation period. Haemoglobin values dropped gradually and all animals except one had Hb values below the normal range.

MCV, MCH and MCHC. No clear differences in these parameters between infected and control animals were apparent.

Total Plasma Protein. The total plasma protein values of the infected buffaloes were above those of the control buffalo(es) from 5 weeks PI, except at 11 and 12 weeks PI, but were still within the normal range.

ELISA Optical Density Values. Details of antibody titres against T. evansi as measured weekly are summarized in Fig. 1, which shows that the mean ELISA values fluctuated but that positive results began at 2 to 3 weeks PI and continued thereafter. Mean OD in affected animals at 2 weeks (0.47) was not significantly greater (P= 0.1) than in the controls (0.19) but attained signific- ance (P<0"01) at PI week 3. Differences beyond PI week 3 were not significant, essentially because of the small number of animals remaining.

Gross Pathological Findings

In general the gross changes were non-specific. Emaciation, with serous atrophy of fat, hydropericardium, and oedema of lymph nodes, was seen to a varying degree in naost animals. In the spleen a pronounced white pulp was

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Fig. 1.

R. D a n a a y a n t i et al.

1.0 I" !

0 1 2 3 4 5 6 7 8 9 10 11 12

Week (s) post infection

O ~ O - - O ~ O ~ O O. • I 6 5 4 3 2 1

• .o .o II 2 1

I: Number of animals (infected) II: Number of animals (uninfected)

Enzyme linked immunosorbent assay optical density values (means) at 4.90 nm in cxperlmcntal Trypanosoma evansi infection in buffaloes,

apparent in the buffalo killed 12 weeks PI. All of the long bones examined in the infected buffaloes had obvious red marrow whereas in the uninfected controls the proportion of red marrow to yellow inactive marrow was smaller. Apart from a subdural haemorrhage c. 2 x 6 cm 2 in size in the cerebral cortex of the buffalo killed 12 weeks PI and considered to be due to terminal trauma, no brain lesions were observed.

Histopathological Findings

No significant lesions were found in tissues from the uninfected control buffaloes. Lesions in infected animals appeared to increase in severity from 1 week to 7 weeks PI, but were not pronounced in the buffalo killed at 12 weeks PI.

Heart. Mild to severe interstitial non-suppurative myocarditis was observed in the buffaloes killed at 2, 3, 4 and 12 weeks PI and in the animal that died 7

T. evansi in Buffaloes 243

weeks PI, in which the inflammatory cells were mainl~r lymphocytes with a few macrophages and plasma cells (Fig. 2). The latter buffalo also had a large multifocal area of haemorrhage accompanied by neutrophils and mononuc- lear cells in the epicardium.

Lung. Focal thickening of alveolar walls was observed in all infected animals. The degree of infiltration, with mononuclear cells particularly, increased from 1 week to 7 weeks PI, as did interstitial oedema and capillary congestion (Figs 3 and 4). The lesions were less severe in the buffalo killed 12 weeks PI. Mild haemosiderosis was present in the lungs of all infected animals.

Liver. Venous congestion was the main feature observed in the buffaloes killed 2 and 3 weeks PI and in the animal that died 7 weeks PI. Haemosiderosis of varying degree was also a feature in all of the infected buffaloes. Trypanosomes were often seen in the lumina of blood vessels in the buffalo killed 2 weeks PI (Fig. 5).

Kidney. Slight tubular degeneration was observed in the buffaloes killed 1 week and 4 weeks PI and in the animal that died 7 weeks PI. Mild non- suppurative interstitial nephritis was seen in the buffaloes killed at 1, 3 and 4 weeks PI.

Spleen. Mild to severe congestion was observed in the spleen of the buffaloes killed at 1 week and 4 weeks PI and in the animal that died 7 weeks PI. The most striking feature, however, was the presence of severe disseminated necrotic loci in the red pulp areas and also in the peri-arteriolar lymphoid sheaths of the buffaloes killed at 1, 2, and 3 weeks PI 'and in the animal that died 7 weeks PI. The necrotic areas varied in size, consisted of poorly defined but massive accumulations of nuclear debris, and were associated with haemorrhage and fibrinous material. The areas were surrounded by histio- cytes, many of which had nuclei of bizarre shape (Figs 6 and 7). Splenic haemosiderosis was very marked in all of the infected buffaloes, regardless of the length of the PI interval.

Bone Marrow. A distinctive feature of bone marrow observed in buffaloes killed from one week PI onward was the obvious formation of lymphoid follicles (Fig. 8) and increased erythroid cells and megakaryocytes.

Lymph Nodes. No consistent changes were observed microscopically in lymph nodes from the infected animals but most of the germinal centres were prominent and active and there was moderate parafollicular hyperplasia.

244 R. D a m a y a n t i et al .

Fig. 2.

Fig. 3.

Mononuclear cell infiltration in the infected heart of a buffalo killed 4 weeks after infection. Most of the infiltrating cells are lymphocytes. HE. x 136. A non-suppurative, interstitial pneumonia, associated with oedema, in a buffalo that died 7 weeks after infection. HE. x 41.

Skeletal Muscle. The skeletal muscle lesions were the most consistent finding observed in this study. Their severity increased from 1 week to 7 weeks PI but was less pronounced in the buffalo killed 12 weeks PI. The characteristic change was essentially a multifocal non-suppurative interstitial myositis which

T. evans i i n B u f f a l o e s 245

Fig. 4.

Fig. 5.

A higher magnification of an oedematous area of the same section as in Fig. 3. Most of the infiltrating cells are lymphocytes. HE. x 131. Liver of a buffalo killed 2 weeks after infection, showing numerous trypanosomes scattered in the lumen of a blood vessel (arrows). HE. x 544.

was observed in all of the infected buffaloes but not in the controls. The mononuclear cellular infiltration was predominantly lymphocytic, with a few macrophages and plasma cells between myofibres (Fig. 9).

246 R. D a r n a y a n t i et al .

Fig. 6 . . Spleen of a buffalo that died 7 weeks alter infection. The most striking feature was the presence of disseminated focal necrosis, often in the red pulp (outlined by arrows). HE. x 96.

Fig. 7. A higher magnification of Fig. 6 to show details of a necrotic area, consisting of poorly defined nuclear debris, surrounded by histiocytes, many of which have nuclei of bizarre shape (arrows). HE. x 283.

Brain. Distension of the perivascular spaces in the brain was a feature in the buffaloes killed 1 week, 2, 3 and 4 weeks PI. A solitary glial nodule was found in the medulla of the buffalo killed 4 weeks PI and mild perivascular cuffing with lymphocytes was observed in the buffalo killed 12 weeks PI.

T. evans i i n B u f f a l o e s 247

Fig. 8.

Fig. 9.

Bone marrow in a buffalo killed 4 weeks after infection. Note formation of lymphoid follicles. HE. x 62. Infected skeletal muscle of a buffalo that died 7 weeks after inl~ction, with a non-suppurative interstitial myositis. HE. x 139.

D i s c u s s i o n

The limited number of animals used in this experiment (for reasons of economy) does not permit a statistical approach to be presented. At the beginning o f the observation period, six infected and two control animals were

248 R. Damayanti et al.

used, but every week afterwards the number of the animals was reduced by one as animals were slaughtered for a sequential study of the pathology. In general, the findings confirmed the clinicopathological observations in a concurrent study of 26 spontaneous cases of surra (Damayanti, 1991).

This report is the first to describe the sequential pathology of surra in domestic animals. The severity of the disease increased from 1 week to 7 weeks PI and became less pronounced by 12 weeks PI. The most important lesions observed, namely hyperplastic bone marrow, multifocal splenic necrosis and non-suppurative interstitial myositis, have not previously been reported in T. evansi infection in buffaloes. However, the bone marrow lesion was reported in T. evansi infection in horses (Ikede et al., 1983) and dogs (Galhotra et al., 1983). Similar splenic and muscle lesions have been reported in some African trypanosomiasis cases (Van den Ingh et al., 1976; Masake, 1980) and appear to be important lesions for diagnostic purposes.

Intermittent fever and relapsing parasitaemia as observed in this study are characteristic of T. evansi infection in cattle and other domestic animals (Losos, 1980), as in African trypanosomiasis (Stephen, 1986). It seems that antigenic variation plays an important role in causing the relapsing course of the disease, which is related to the periodical fluctuation of the parasitaemia (Turner, 1982).

The formation of antigen-antibody immune complexes is a potent stimulus for pyrogen release (Stephen, 1986). The increased pulse and respiration rates observed in the present study were reported in T. evansi infections of other animals (Galhotra et al., 1983) as well as in African trypanosomiasis (Losos, 1986). They coincide with the intermittent fever (Veenendaal and Van Miert, 1976) and may be due to the increased metabolic rate in pyrexia (Stephen, 1986).

No spectacular clinical signs were observed during the experimental period in this study and the observed anaemic mucous membranes, fever, loss of appetite, depression, weakness and decreased bodyweight were all common features of T. evansi infection as seen in other species (Losos, 1980; Dieleman, 1986) and in African trypanosomiasis (Sekoni et al., 1990). The course of an infection and the severity of the disease depends on the host and the strain of T. evansi (Losos, 1980). Cattle and buffaloes are usually very tolerant to 7-. evansi infection and the disease is often sublinical in nature (Hoare, 1972).

The observed decreases in PCV, Hb and RCC values in the infected animals at 2 weeks PI are in agreement with published findings on T. evansi infection and African trypanosomiasis (Stephen, 1986).

The MCV, MCH and MCHC values of the infected animals fluctuated, but on average they were still within the normal ranges; the type of anaemia can therefore be categorized as normocytic normochromic. This has been reported in 7". evansi infection in horses (Ng and Vanselow, 1978) and in African trypanosomiasis (Losos and Ikede, 1972; Murray and Dexter, 1988). However, Valli and Mills (1980) found that T. congolense infection in cattle produced a macrocytic normochromic anaemia, and Fiennes (1954) observed that al- though macrocytic anaemia was normochromic in acute cases it became microcytic in the chronic stages.

7". e v a n s i in Buffaloes 249

Platelet counts in the infected animals in this study decreased below the normal range during the first 3 weeks of infection, and then increased. It is possible that after initial damage to the platelets by T. evansi products, thrombocytopenia is maintained by immunological reactions in the form of trypanosome-generated antigen-antibody complexes or by auto-antibodies to platelets (Murray and Dexter, 1988).

Thrombocytopenia and platelet clumping are characteristic features in trypanosomal infection, resulting from disseminated intravascular coagulation (not observed here) or from enhanced activity of the reticuloendothelial system such that platelet life span is shortened to 7-60% of the normal (Jenkins and Facer, 1985).

Total plasma protein values of the infected buffaloes were mostly within the normal range. This was also found in T. evansi in camels (Jatkar et al., 1973). The increased concentrations observed 11 and 12 weeks PI may have reflected elevated anti-trypanosomal antibody (Ng and Vanselow, 1978). The ELISA results (Fig. 1) support this possibility.

The observation that antibody titres against T. evansi fluctuated but that positive results were observed from 2 to 3 weeks PI and remained high thereafter, was similar to that of Luckins (1983) in other T. evansi infections in cattle and buffaloes. Moreover, similar findings in African trypanosomiasis showed that antibody titres fluctuated during the course of the infection and that the antibody response to the same strain of parasite differed in each individual host (Luckins, 1977).

The severity of the disease appeared to increase from 1 to 7 weeks PI and became less pronounced in the buffalo which was infected for 12 weeks. No pathognomonic gross pathological changes were observed in this study, or in the studies of Losos (1980) and Dieleman (1986). The gross lesions ofsurra are said to be similar to those in other forms of trypanosomiasis, consistent findings being marked anaemia, emaciation, enlargement of superficial lymph nodes and spleen, and a congested liver and spleen (Ikede et al., 1983). Most of these features were found in this study.

There are some consistent histological findings in 7". evansi injected animals. A mild to moderate non-suppurative interstitial myocarditis, which was observed in this study, has been reported in other animals (Seller eb al., 1981). It has also been observed in T. brucei infection in cattle (Moulton and Sollod, 1976) and rabbits (Van den Ingh et al., 1976), as well as in T. congolense infection in cattle (Losos and Ikede, 1972). A focal interstitial pneumonia found in this experiment is also consistent with the findings of Sinha et al. (1971) in T. evansi infection in other species. This feature is also found in African trypanosomiasis (Van den Ingh et al., 1976).

The oedema of the lungs, lymph nodes and pericardial sac may be due to the release of kinins and related substances into the blood circulation (Goodwin, 1970), or to vascular damage or the direct action of the parasites on the interstitial connective tissues (Moulton and Sollod, 1976). A further possibility is that it was due to the trypanosomal myocarditis, with subsequent interfer- ence with normal circulation. In the present study, however, myocarditis was mild. The anaemia may also have contributed (Ikede and Losos, 1975a).

250 R. Damayanti et al.

Of the three most striking lesions found in this study (see above), hyperplas- tic bone marrow, characterized by lymphoid follicle formation, has been reported in T. congolense infection in calves (Valli and Forsberg, 1979) and 7". vivax infection in goats (Van den Ingh et al., 1976). Anoxia due to the severe anaemia as well as the immune-mediated destruction of the erythrocytes in the reticuloendothelial system, leading to bone marrow hyperplasia, may explain this finding (Ng and Vanselow, 1978). Moreover, in cases in which thrombocy- topenia was due to excessive destruction of platelets in the blood circulation, normal or increased numbers of megakaryocytes would be expected to be present in the bone marrow. In contrast, this would seem unlikely if thrombo- cytopenia existed because of the impairment of the marrow function (Stephen, 1986).

A non-suppurative interstitial myositis, as found in the gluteal muscle in this study, was also found in T. brucei infection in sheep and rabbits, in which species it was associated with extravascular trypanosomes, degeneration of myofibres, interstitial oedema and hypercellularity of the stroma (Ikede and Losos, 1972). Moreover, T. vivax infection may cause degeneration of myo- fibres in goats (Van den Ingh et al., 1976) and intravascular trypanosomes in the atrophic muscle are seen in T. congolense infection in calves (Valli and Forsberg, 1979). It seems that trypanosomal multiplication occurred at the sites of localization and that the local response by mononuclear cells played a role in the destructive process caused by the organisms (Losos and Ikede, 1972). Although only one muscle group (the gluteal) was sampled in the present study it seems likely that all skeletal muscles were similarly affected.

Multifocal necrosis of the infected spleens observed in this study was also seen in T. vivax infection in cattle (Masake, 1980), sheep (Anosa and Isoun, 1983) and goats (Van den Ingh et al., 1976). It has also been seen in the spleens of horses infected with T. evansi (R. Graydon, unpublished observation). Most of the tissue damage may be directly attributed to extravascular invasion by the trypanosomes (Ikede and Losos, 1972). In T. brucei infection in sheep it has been suggested that necrosis is associated with ischaemia and the invasion of the parasites (Ikede and Losos, 1975b).

Acknowledgments We thank Drs R. C. Payne, I. Prasetyawaty, N. Ginting, P. Ronohardjo, P. Daniels and A. Wilson of the Research Institute for Veterinary Science, Bogor for their help and encouragement. We also acknowledge the excellent technical assistance of Mr L. Reilly, Mr O. Sajeli and Ms Murniati.

Financial support for the senior author was provided by the Australian International Development Assistance Bureau, and the Government of Indonesia.

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Received, January 20th, 1993 Accepted, November 18th, 1993]