Clinical communication — Kliniese mededeling

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater ( ruminantium)

S Pfitzera*, R Lastb and D T De Waalc

northern KwaZulu-Natal. It was the 2nd ABSTRACT buffalo calf found dead within 2 months Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heart- in this reserve. In addition, 1 blue wilde- water) were demonstrated in brain smears and formalin-fixed brain sections derived from a beest and one impala ram (Aepyceros buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility melampus) had died of unknown causes. that the -free environment of a quarantine boma may have affected the calf’s immunity, The dam of this buffalo calf came from the is discussed. These findings suggest that monitoring heartwater in wild ruminants and tuberculosis (TB) eradication programme making brain smears as a routine during post mortem evaluations of wild ruminants, should carried out by KwaZulu-Natal Conserva- be encouraged. tion Service (KZNCS) in quarantine Key words: buffalo, Cowdria ruminantium, cowdriosis, Ehrlichia ruminantium, heartwater, bomas at Phinda Resource Reserve. South Africa. The dam had initially originated from the Pfitzer S, Last R, De Waal D T Possible death of a buffalo calf (Syncercus caffer) due to sus- Hluhluwe–Umfolozi Game Reserve and pected heartwater (Ehrlichia ruminantium). Journal of the South African Veterinary Association had been introduced onto the property (2004) 75(1): 54–57 (En.). Pongola Animal Clinic, PO Box 682, Pongola, 3170 South Africa. about 1 year before this investigation. The calf was born in the private game reserve, which had been free of domestic rumi- INTRODUCTION deaths due to infection with E. ruminan- nants for more than 10 years. Cowdria ruminantium,4 the causative tium have been reported. To date, no organism of heartwater,has recently been deaths due to heartwater have been Pathology reclassified as Ehrlichia ruminantium based documented in the . How- on genetic analysis as well as biological ever, African buffalo2,24 as well as eland, Post mortem and protein characteristics.6 Ehrlichia kudu (Tragephalus strepsiceros strepsiceros), The calf died during the morning of ruminantium is transmitted by 3 host blue wildebeest, giraffe (Giraffa camelo- 5 June 2000. The carcass was kept cool belonging to the genus Amblyomma5. pardalis)15, sable (Hippotragus equinus) and until the post mortem could be carried out hebraeum, the vector of E. tsessebe (Damaliscus lunatus)16 are known at 15:00 that afternoon. ruminantium in South Africa, occurs to seroconvert after artificial infection and Gross post mortem findings revealed the mainly in the Limpopo, KwaZulu-Natal become carriers of the parasite. One animal to be in good condition with a low and in the Eastern Cape provinces of African buffalo showed signs of heart- tick burden. Although ticks were not South Africa23. water after artificial infection with E. specifically identified, some Amblyomma Heartwater is one of the most important ruminatium, but it recovered spontane- were present. Rigor mortis was still tick-borne diseases of domestic rumi- ously24. Different strains of E. ruminan- apparent. No signs of struggling or inju- nants in South Africa18. Mortality rates are tium have the ability to grow in ries were evident. The peripheral blood high in susceptible , and endothelial cell lines of sable antelope, smear was negative for anthrax and blood introduced into endemic regions and buffalo and eland20. These findings parasites. The rumen contained some peracute, acute, subacute and clinically suggest the possibility that wild rumi- roughage; milk was found in the aboma- inapparent forms of heartwater have nants play an important role in the sum. Mild splenomegaly and hepato- been reported in these domestic rumi- epidemiology and spread of heartwater. megaly was present, as well as ascites. nants21. In wild ruminants, such as the This hypothesis is strengthened by the Whether hydrothorax was present, could blue wildebeest (Connochaetes taurinus), results of a study in the Kruger National not be determined, as the pericard was springbok (Antidorcas marsupialis), Park of South Africa where it was shown damaged when skinners opened the bushbuck (Tragelaphus scriptus)13, black that a wildlife cycle can maintain the thorax. Lungs revealed a foamy, frothy wildebeest (Connochaetes gnu), blesbok transmission of E. ruminantium inde- material in the major bronchi and distal (Damaliscus albifrons)11, steenbok (Raphi- pendent of domestic ruminants17. 3rd of the trachea. A few lobules in the cerus campestris)8, sitatunga (Tragelaphus Numerous E. ruminantium isolates have cranial lobe appeared hepatized. Accen- spekei)14 and eland (Taurotragus oryx)25 been made, and named after the farm or tuation of lobulation due to interstitial a country of origin. These strains differ in oedema and emphysema in the lung was Pongola Animal Clinic, PO Box 682, Pongola, 3170 3,7 South Africa. E-mail: [email protected] their pathogenicity for ruminants . visible. Atrial petechiae were observed bVetdiagnostix – Veterinary Pathology Services, PO Box on the heart. In the brain blood vessels 13624, Cascades, 3202 South Africa. appeared engorged. A brain smear was cParasitology Division, ARC-Onderstepoort Veterinary CASE REPORT Institute, Private Bag X05, Onderstepoort, 0110 South performed and stained with Kyro-Quick Africa. Present address: Department of Veterinary Micro- 19 biology and Parasitology, Faculty of Veterinary Medicine, History stain (Kyron Laboratories) . Numerous University College Dublin, Belfield, Dublin 4, Ireland. On 5 June 2000, a dead buffalo calf of rickettsial-like organisms were observed E-mail: [email protected] approximately 5 months of age was in the endothelial cells of the capillary *Author for correspondence. Received: July 2003. Accepted: January 2004. discovered on a private game reserve in blood vessels, which was also seen in

54 0038-2809 Tydskr.S.Afr.vet.Ver. (2004) 75(1): 54–57 Giemsa-stained brain smears (Fig. 1). Un- stained brain smears as well as samples of frozen brain were taken. Additional samples of brain, bone marrow, heart, kidney, liver, spleen, lymphonodes and lung were preserved in 10 % formalin for further histopathological investigation.

Microscopic examination Brain. Severe congestion of meningeal and parenchymal vessels was evident. Prominent vasogenic oedema with widening of Virchow-Robin spaces, peri- neural spaces and dilatation of myelin sheaths in the central white matter neu- trophils were present as well as diffuse gliosis with scant perivascular infiltrates of individual mononuclear cells. Distinct intracytoplasmic colonies of rickett- sial-like organisms resembling E. rumi- Fig. 1: Many large colonies of small Ehrlichia ruminantium-like organisms, intra- nantium were identified within the endo- cytoplasmically within endothelial cells of brain capillaries. Giemsa; ×1000. thelial cells (Fig. 2). Spleen. Diffuse congestion of splenic rhages were scattered throughout the double-unit membrane were found in sinuses was present while lymphoid myocard. Interstitial oedema was present capillary endothelial cells, features that follicles had a washed-out appearance with teasing apart of myofibres. Individ- are characteristic of the Ehrlichia group. with depletion of lymphocytes from the ual myofibres appeared amorphous and germinal centres (lympholysis). eosinophilic (myofibre hyalinization). DNA probes Lung. Severe diffuse congestion was Frozen brain tissue was also subjected evident with engorgement of bronchial Immunohistochemistry to PCR and DNA probe examination in an vasculature and alveolar wall capillaries. Immunohistochemical (immunoperox- attempt to confirm the identity of the Effusion of fibrinous fluid had resulted idase) stains for heartwater were per- organism. The pCS20 probe22 is known to in interstitial distension (interstitial formed on formalin-fixed brain sections discriminate between E. ruminantium and oedema). There was scant interstitial infil- and revealed positive-staining granules other Ehrlichial organisms1. Unfortu- trates of mixed mononuclear cells and in many endothelial cells of parenchymal nately, the sample was in a too-advanced individual leukocytes. In individual capillaries. Confirmed cases of Coxiella stage of decomposition and the results lobules a severe inflammatory reaction burnetti and Chlamydia psittaci were used were inconclusive. was visible; bronchi were plugged with as negative controls and showed no posi- necrotic plugs consisting of fibrin, tive staining. Diagnosis necrotic cell debris and infiltrating leuko- The cause of death was suspected heart- cytes (necrotic bronchitis). Peribronchial Transmission electron microscopy water with complicating acute bacterial and intra-alveolar infiltrates contained in- On transmission electron microscopic bronchopneumonia. numerable neutrophils accompanied by (TEM) examination, pleomorphic, coc- proliferating alveolar macrophages coid, ovoid, filamentous, horseshoe- DISCUSSION (alveolar pneumonia). Large bacterial shaped and polygonal organisms, 0.49– Demonstration of rickettsial organisms, colonies consisting of bipolar, Gram-neg- 2.7 µm in diameter and enclosed by a resembling E. ruminantium, in brain ative organisms resembling Pasteurella were observed in many of these necrotic areas. Liver. The liver showed diffuse conges- tion with engorgement of hepatic sinu- soides and portal vessels, and mixed mononuclear cell infiltrates in the portal areas (triaditis). Sinusoidal leukocytosis with individual neutrophils were present in hepatic sinusoides. Kidney. The kidneys showed diffuse con- gestion with engorgement of glomerular and intertubular capillaries. Increased granularity of proximal convoluted tubular epithelial cells with increased eosinophilia and basally placed, hyper- chromatic nuclei (tubular nephrosis) was evident. Heart. Severe, diffuse congestion with engorgement of the myocardial capillar- Fig. 2: Formalin-fixed brain section showing a large colony of Ehrlichia ruminantium-like ies were apparent. Multifocal haemor- organisms within the cytoplasm of an endothelial cell. H&E; ×1000.

0038-2809 Jl S.Afr.vet.Ass. (2004) 75(1): 54–57 55 smears, formalin-fixed brain sections ously and the temperatures returned to 4. Cowdry E V 1925 Studies on the etiology of stained with haematoxylin and eosin, normal. This confirms the susceptibility heartwater 1. Observation of a , Rickettsia ruminantium, in the tissues of positive immunoperoxidase staining of of buffalo to heartwater. infected animals. Journal of Experimental organisms with E. ruminantium antibody Animals in disease-free breeding Medicine 42: 253–274 and identification of rickettsia in the brain programmes are kept in quarantine 5. Cowdry E V 1925 Studies on the etiology of sections on TEM, strongly support a bomas, where ticks are absent. This can heartwater 2. Rickettsia ruminantium, in the diagnosis of heartwater.Whether this was interfere with immunity to tick-borne tissues of ticks transmitting the disease. Journal of Experimental Medicine 42: 231–252 the primary cause of death or whether diseases. Maternal immunity might be 6. Dumler J S, Barbet A F, BekkerCPJ,Dasch death was the result of complicating adversely affected in dams kept for long G A, Palmer G H, Ray S C, Rikihisa Y, bacterial pneumonia is debatable. periods in tick-free bomas. Calves born in Rurangirwa F R 2001 Reorganization of To the best of our knowledge, no such bomas could be at risk as colostral genera in the families Rickettsiaceae and mortalities as a result of heartwater have antibodies are reported to be important in Anaplasmaceae in the order : 12 unification of some species of Ehrlichia with been reported in buffalo. This can be the protection calves from heartwater . Anaplasma, Cowdria with Ehrlichia and ascribed to several factors. If one consid- In addition, limited cross-immunity Ehrlichia with Neorickettsia, description of ers buffalo to be at least as heartwater- between the different strains of E. rumi- six new species combinations and designa- resistant as the indigenous breeds of live- nantium has been reported10. Therefore, tion of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocyto- stock, it is not surprising that relatively relocation of animals over large distances philia. International Journal of Systematic few fatalities due to heartwater have been to other facilities could result in exposure Evolution and Microbiology 51: 2145–2165 reported from the field. Only small num- to different strains of the organism and 7. Du Plessis J L, Bezuidenhout J D 1979 Inves- bers of buffalo probably succumb to the consequently susceptibility to the disease. tigations on the natural and acquired resis- disease and it is hardly possible to pick up This particular buffalo herd had been tance of cattle to artificial infection with sick or dying animals in the veld. The translocated 100 km from the Hluhluwe– Cowdria ruminantium. Journal of the South African Veterinary Association 50: 334–338 decomposed condition of most buffalo Umfolozi Game Reserve, where the herd 8. Jackson J J, Andrew H R 1994 Cowdria-like carcasses found in the wild often renders originated, before being kept in quaran- organisms in a steenbok. Zimbabwe Veteri- an investigation impossible. In addition, tine bomas. nary Journal 25: 123 buffalo are not known to be susceptible to The herd had spent about 9–12 months 9. Keffen R H 1995 Heartwater challenge of two heartwater and therefore this disease Cape buffalo (Syncercus caffer). Journal of the in the quarantine bomas, and protection South African Veterinary Association 56: 55 might have been ignored as a possible of this weaner buffalo by maternal anti- 10. Logan L L, Birnie E F,Mebus C A 1987 Cross- differential diagnosis, especially in the bodies might have been low. The animal immunity between isolates of Cowdria rumi- absence of lesions typical of heartwater. was also being weaned, which no doubt nantium. Onderstepoort Journal of Veterinary To date, only small numbers of buffalo placed it under stress, and, in addition, it Research 54: 345 have been artificially infected with heart- was suffering from pneumonia. All these 11. Neitz W O 1935 The blesbuck and the black-wildebeest as carriers of heartwater. water to examine their resistance/suscep- stress factors together could have led to a 9 Onderstepoort Journal of Veterinary Science tibility. Keffen reported on the artificial higher susceptibility to heartwater and and Animal Industry 5: 35–40 infection of two 10-month-old buffalo subsequently to death. 12.NorvalRAI,Donachie P L, Meltzer M I, calves. Both animals came from a heart- These findings show that the epidemi- Deem S L, Mahan S M 1995 The relationship between tick () infes- water-free area and were the 5th genera- ology of heartwater in buffalo is in urgent tion of animals bred in captivity. Neither tation and immunity to heartwater (Cowdria need of further investigation. It is recom- ruminantium) in calves in Zimbabwe. Veteri- of these buffalo showed any clinical signs. mended that buffalo herds translocated nary Parasitology 58: 335–352 There was, however, no control group to heartwater-endemic areas should be 13. Oberem P T, Bezuidenhout J D 1987 and the strain used for artificial infection monitored more closely. Game farmers Heartwater in hosts other than domestic 2 ruminants. Onderstepoort Journal of Veteri- is not mentioned. Andrew and Norval should be encouraged to report all fatali- described the infection of 4 susceptible nary Research 54: 271–275 ties. In heartwater areas, brain smears 14.OkohAEJ,Oyetunde I L, Ibu J O 1986 Fatal weaner buffaloes with various isolates of should be taken as a routine from all heartwater in a captive sitatunga. The Veteri- heartwater. No clinical signs were de- mortalities in wild ruminants. nary Record 21: 696 tected in these animals but transmission 15. Peter T F, Anderson E C, Burridge M J, of the disease to susceptible sheep via Mahan S M 1998 Demonstration of a carrier ACKNOWLEDGEMENTS Amblyomma ticks was possible for 161 state for Cowdria ruminantium in wild rumi- We thank Dr Heinz Kohrs of the Pongola nants from Africa. Journal of Wildlife Diseases days. 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