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The writers (Son and Nining) have known Dr. Ramachandran since 1975. He was one of the FAO experts who helped the Indonesian Veterinarians to strengthen the capacity of a newly built Disease Investigation Centre in Bali, Indonesia. As young veterinarians, we were very enthusiastic to learn many aspects of laboratory diagnostic techniques. To us, Dr. Rama was a very good “Guru’. A disease that kept us very busy was Jembrana disease. Efforts were made to explore the picture of the disease especially the causal agent. In memoriam of Dr. Ramachandran, the literature dealing with historical aspects of Jembrana disease either published in International Journals or in the Indonesian language in national journals and laboratory reports between 1970 and 2000 is reviewed in this paper.
Jembrana disease
Jembrana disease when first observed in domesticated banteng (Bibos javanicus), colloquially called Bali cattle, in the Jembrana district of Bali island in 1964, was misdiagnosed as rinderpest (1). The disease is now endemic in Bali and elsewhere in Indonesia affecting the cattle industry significantly. There is general agreement that Jembrana disease develops clinical signs only in Bali cattle; other breeds are susceptible and may become viraemic inappearently (2, 3).
Bali cattle are about 1.3 to 1.5 meters high and have the general appearance of other types of beef cattle. In both sexes a striking white oval patch covers the rump, and all 4 legs have appearance of ‘white stockings”. The coat colour is sex dependent: calves and females are usually light brown with a thin black line along the middle of the back; bulls are of the same colour as females and calves when young, but turn a dark brown to black colour at maturity, unless castrated (4). Cross-breeding between bantengs and Bos indicus is possible. Cross-breeding of Bali cattle with Java Ongole cattle is commonly practiced in East Java, the progeny of which are referred to as Rambon cattle; the female Fl progeny of such crossbreeds are fully fertile whereas the Fl and F2 male progeny are infertile (5). Crosses between banteng and European taurine cattle (Bos taurus) have been less successful than those with Bos indicus; female progeny are fertile but male progeny are always sterile (4).
Although there are many advantageous properties of Bali cattle, they also have disadvantages. The cattle are very susceptible to bacterial and viral diseases including Pasteurella infections, malignant catarrhal fever and Jembrana disease. A potential problem is that they are timid and need constant contact with humans to remain tame; they are easily upset by rough handling and react like deer, crashing blindly into fences or sinking into a state of shock which renders them comatose (4).
Historical aspects of Jembrana disease in Bali cattle
Since December 1964, several groups have had responsibility for investigating the Jembrana disease. These investigations have occurred in 5 principal phases. First phase was the earliest investigation of the first outbreak of Jembrana disease in early 1965. These investigations were conducted by a team from Lembaga Virologi Kehewanan (LVK), Surabaya, Indonesia, led by Professor Tandjung Adiwinata in association with a Japanese veterinarian, Professor Sonoda. In the second phase from 1972 to 1974, further investigation of Jembrana disease was conducted by a team from Bogor Agricultural University, Bogor, Indonesia, which included Dr. Iwan Budiarso and Dr. Soehardjo Hardjosworo. They investigated an outbreak of the disease designated Tabanan disease which occurred in the Tabanan district of Bali. The third phase from 1974 to 1982 was an investigation by a Food and Agriculture Organization (FAO) sponsored group that included Dr. Ramachandran, Dr. H.P. Harding, Dr. E. Teuscher, Dr. Dennig, Dr. Sweatman and veterinarians from the Disease Investigation Centre, Denpasar, Bali. In the fourth phase from 1982 to 1986, investigations were conducted by the Bali Cattle Disease Investigation Unit (BCDIU), established specifically by the Ministry of Agriculture to investigate diseases of Bali cattle, especially Jembrana disease. This group was led initially by Professor A.A. Ressang and concentrated on attempts to establish the aetiology of the disease. In the fifth phase, the research was continued by the same Unit but under the supervision of Dr. G.E. Wilcox from Murdoch University, Australia.
Jembrana disease was first recognized in December, 1964, in the village of Sangkaragung in the Jembrana district of western Bali (1,2). The disease spread to all parts of Bali island within 9 months. It was estimated that approximately 60,000 cattle and possibly some buffaloes out of a total cattle population in Bali of about 300,000 died as a consequence of the disease. Other animals such as pigs, sheep and goats were unaffected (2). In the district of Jembrana, more than 19,000 cattle out of a total population of 26,000 died during 1965.
Initial investigations determined that the disease was transmissible to healthy cattle by parenteral inoculation of blood and suspensions of lymph nodes from affected animals. Investigations conducted by the staff of LVK Surabaya in 1967 did not result in any conclusive diagnosis at the time but based on the clinical signs, postmortem findings of field as well as experimental cases, the high morbidity and mortality and the high infectivity of the disease, it was suspected that the disease was rinderpest (2). Mass vaccination with lapinized-avianized attenuated rinderpest virus vaccine, imported from Japan, was carried out in 1967. There were no further reports of the disease after September 1967(1).
In 1972 an outbreak of a disease similar to Jembrana disease but with lower mortality rates than in the previous epizootic, occurred in Tabanan, a district adjacent to the Jembrana district where the original outbreak had occurred in 1964. During this outbreak, the disease also occurred in cattle which had received rinderpest vaccine. The morbidity rate was lower than in the first outbreak and the mortality was estimated at 13% (3). Unlike the 1964 outbreak, there were no reports of the disease in buffaloes. Although there were many similarities between the clinical and pathological features of the disease in Bali cattle affected during this outbreak with those reported in the initial outbreak, the disease in this outbreak was designated as “Tabanan disease” and not Jembrana disease.
Though the export of cattle from Bali to other areas of Indonesia was banned following the occurrence of Jembrana disease in 1964, a Jembrana-like disease in cattle outside of Bali island was reported in May, 1976, in a Balinese transmigration settlement in Rama Dewa, a subdistrict of Lampung province, Sumatra. This disease was designated as “Rama Dewa Disease” even though it was clinically and pathologically similar to Jembrana disease and occurred only in Bali cattle and did not affect Ongole cattle or buffaloes or sheep and goats (6). The exact number of affected animals was not determined but was evidently high and the mortality was significantly high. In the period 1980 to 1984, following the initial outbreak in Lampung, approximately 200 out of 500 known affected cattle died in the affected area (7). Anecdotal but unconfirmed evidence indicated that this outbreak was associated with the illegal importation of cattle to Rama Dewa from Bali (Prabowo, personal communication). A further epizootic of a Jembrana-like disease was reported in the Banyuwangi district, East Java (Jawa Timur), in November 1978, in 14 of the 18 kecamatan (sub-districts), and the disease was designated as “Banyuwangi disease”. In this area, clinical disease was reported in Bali cattle, in Rambon cattle in first generation Bali x Ongole cattle, Madura cattle and buffaloes, while Ongole cattle, sheep and goats were not affected (8,9). The investigations were limited and the involvement of non-Bali cattle was not confirmed. The outbreak was presumed to be due to the illegal movement of cattle from Bali to East Java. Over the ensuing years, presumably due to a prohibition on the movement of cattle from areas where the disease was known to occur, the disease remained localized to the 3 provinces i.e. Bali, Jawa Timur and Lampung.
In April 1992, another epizootic of Jembrana disease occurred in Sawahlunto in West Sumatra. This was a transmigration area where Bali cattle had been distributed by an International Fund for Agriculture Development (IFAD) project to assist small farmers in Indonesia. The first case was recognized on 7 April 1992, in village of Timpeh in cattle owned by two farmers. Within one week, 14 affected animals (11 adult and 3 calves) were reported to the regional veterinary office. By May, 1992, 124 cattle had been affected; 44 of these died, 76 recovered, and 4 cows, 6 to 8 months pregnant, aborted. The abortions were not proved to have been due to Jembrana disease as antibodies against Brucella abortus were detected in sera taken from the affected animals (Mulyawan, personal communication). In total, at least 168 cattle out of 398 were affected and 105 of the affected cattle, died.
Animal host range
Clinical Jembrana disease has been confirmed only in Bali cattle (3). There has been general agreement by all investigators that the disease can be readily reproduced in Bali cattle, but not in Ongole cattle, Friesian (Bos taurus) cattle, sheep, goats, pigs, guinea pigs, rabbits or mice, by the parenteral injection of blood, plasma and tissue suspensions from affected cattle. Experimentally inoculated Bali cattle developed clinical disease and pathological lesions similar to those detected in natural cases (2,9,10). There have been reports, during the initial outbreak in 1964-65 (2) and in the outbreak in East Java; of Jembrana disease in buffaloes (8,9). However, subsequent investigation indicated that buffaloes and Ongole cattle also could be infected and became viraemic, but they did not develop clinical signs of Jembrana disease as seen in Bali cattle (6,9). There was some disagreement about the consequences of artificial infection of guinea pigs. Budiarso and Hardjosworo (11) reported that infection of guinea pigs resulted in swelling of the scrotum, an extensive haemorrhagic reaction and peritonitis. Ramachandran et al.(12), however, did not detect any lesions in infected guinea pigs and they were unable to reproduce the disease in Bali cattle by subinoculation of scrotal suspensions of infected guinea pigs. Adiwinata (1) also reported that he was unable to induce lesions in guinea pigs, rabbits or unweaned and adult mice.
Clinical signs
The common signs of both natural and experimental cases reported by the investigators were sudden depression, anorexia, fever up to 41.5C, enlargement of superficial lymph nodes, progressive anaemia and leukopenia (1,4,6,7,8,9,10). This acute phase of the disease is transient in animals that survive the initial clinical signs. In the 1964 outbreak the fever was reported to persist approximately for 7 days (1) and for 3 to 6 days in natural and experimental cases observed by Ramachandran (9). Other signs less frequently observed were hypersalivation, nasal discharge, and diarrhoea with blood in the faeces. During the acute febrile phase some animals develop erosions on the buccal mucosa and a mild serous ocular discharge (11). “Blood sweating” or haemohydrolysis are terms given to the appearance of pinpoint drops of blood on the cutaneous surfaces which may be observed during the acute febrile phase of the disease (2,10,11). Pregnant animals may abort (13).
Haematological aspects
In the early investigations the haematological changes in Bali cattle with Jembrana disease were difficult to describe. The limited reports available have indicated that leukopenia and thrombocytopenia were constant features in the early febrile period (1,9) and that the leukopenia was due primarily to a lymphopenia (10). Ramachandran (9) reported that lymphopenia was followed by a significant lymphocytosis but this has not been mentioned in other reports. Anaemia was noted by Teuscher et al. (10). During the acute febrile phase, the peripheral blood cytology was dominated by an increase in the number of abnormal types of lymphocytes (10,14,15). Abnormal peripheral blood cells, including large monocyte-like cells which contained acidophilic inclusions, considered to be rickettsia-like organisms by Ressang et al. (16) but not by Teuscher et al. (10), and lymphocytes with mitotic figures were detected (15). Electron microscopic examination of the buffy coat indicated that some of the large monocyte-like cells contained phagocytosed thrombocytes, which were morphologically similar to inclusions by light microscopy (9,10).
Aetiological studies
The definitive cause of Jembrana disease is now believed to be a virus belonging to the family Retroviridae, sub-family Lentivirinae. It is interesting to know the history of research approaching the aetiology of Jembrana disease when Dr. Ramachandran was involved.
Since, Jembrana disease was first recognized in 1964, several theories had been proposed as to the aetiology of the disease. The theories had been mainly based on the similarity of the clinical, pathological and histopathological features of the disease to other diseases. This is not surprising since these criteria were the only tools in the early years of the investigations. The first investigators proposed rinderpest virus as the cause of the 1964 disease outbreak, based on the epidemiological features and severe haemorrhagic lesions, which were considered typical of rinderpest (1). Following the second outbreak of the disease in the Tabanan district of Bali in 1972, when the disease was also detected in Bali cattle, which had been vaccinated with rinderpest vaccine, this hypothesis was abandoned. Budiarso and Hardjosworo (11) proposed a rickettsia as a possible cause of the disease. This hypothesis was based on the reported presence of intracellular rickettsia-like organisms in cells of smears and biopsies of lymph nodes and spleen of all cattle, which died (17). During an international seminar held on Jembrana disease in Denpasar in 1975, there was general support for the rickettsial theory based on continued reports of the presence of the rickettsia- like organism in the cytoplasm of monocytes and lymphocytes of affected animals and in swollen scrotal contents of inoculated guinea pigs, and the clinical similarity to the pathology of Jembrana disease to Ondiri disease, a disease of cattle in Kenya caused by the rickettsial agent Ehrlichia ondiri and to bovine ehrlichiosis caused by Ehrlichia bovis.
In late 1970s, the FAO sponsored group which studied the disease when they established a veterinary diagnostic laboratory in Denpasar, found evidence that a virus and not a rickettsia-like agent was the causative agent of the disease. They determined by exclusion membrane filtration studies that the infectious agent present in plasma of affected animals was between 100 and 200 nm in size, too small for any known rickettsia. They also determined that the infectious agent in plasma could be destroyed by low and high pH (pH 3.0 and 11.2 respectively), and that treatment of animals with tetracyclines did not inhibit the development of Jembrana disease in Bali cattle, did not prevent the persistence of the infectious agent in the blood of recovered Bali cattle, or reduce the infectivity of the agent present in plasma (6,9,10). Ramachandran (9) considered that the probable viral aetiological agent was not rinderpest virus or antigenically related to rinderpest virus.
The theory that a virus was the aetiological agent was disregarded and not supported by a subsequent research group based at the Bali Cattle Disease Investigation Unit in Denpasar, a research unit established specifically to conduct research on Jembrana disease. They believed that a rickettsia-like agent and not a virus was involved, confirmed reports of a rickettsia-like organism in the cytoplasm of monocytes and lymphocytes of affected animals, and reported a serological cross-reaction between the infectious agent associated with Jembrana disease and bovine ehrlichiosis (16). They were, however, unable to repeat the detection of rickettsia-like organisms in guinea pigs infected with tissue from affected cattle (Budiarso, personal communication).
A hypothesis based on the ability of the infectious agent to pass through a 220 nm membrane filter, the resistance of the agent to antibiotics and the nature of the histopathological changes, was that the disease was caused by a virus (9,10). Difficulty in characterising the infectious agent associated with Jembrana disease has been caused by the inability to culture the agent in any in vitro system. However, studies showed there was a high titre of the infectious agent in the blood plasma of infected Bali cattle during the acute stage of the disease (18). This suggested blood plasma from such cattle could be used as a source of the infectious agent and enable its identification and characterisation. On the basis of reverse transcriptase (RT) activity, virus morphology and C type budding from cell membrane, the infectious agent that caused Jembrana disease was characterised as a retrovirus. The complete nucleotide sequence of the RNA genome of Jembrana disease virus (JDV) has been obtained (19). The genome was that of a typical lentivirus. In addition to the gag, pol and env genes and flanking long terminal repeats (LTRs) that characterise all retroviruses, a number of accessory genes were identified. The nucleotide sequence was deposited in GenBank (Acession number U21603).
Culture of the infectious agent in vitro
There have been many efforts to culture the causative agent of Jembrana disease in in vitro systems but these attempts were not repeatable. In the belief that the aetiological agent was a rickettsia-like agent, attempts were made to culture the agent in embryonated eggs. Hardjosworo et al. (20) reported that they were able to induce embryo death in 13 of 23 infected embryonated eggs and were able to detect haemorrhages and dullness in infected embryos. Ramachandran (9) was unable to repeat these results. Attempts to culture the aetiological agent in various types of cell cultures have been tried with no conclusive results. Calf kidney cell cultures infected with tissues of affected cattle were reported to develop multinucleated giant cells (1). Hardjosworo et al. (20) reported that a cytopathic effect (CPE) developed in African green monkey kidney (Vero) cells 96 hours after infection. Ramachandran (9) failed to detect replication of the agent in chicken embryo fibroblasts or pig kidney cells; he was unable to detect a CPE in the cell cultures or to reproduce the disease in Bali cattle by sub-inoculation of the cultures back into Bali cattle. However, he was able to maintain the infectious agent in cultures of blood leukocytes derived from infected cattle for 8 days but not for 14 days. These cultures did not show any CPE for periods up to 42 days. Additionally, he was unable to reproduce the disease in cattle with lymph node cells from infected cattle after they were maintained in culture for 6 days. Ressang et al. (16) reported that they were able to maintain the infectious agent in macrophage cultures and reproduce Jembrana disease in cattle sub-inoculated with macrophage cultures derived from infected cattle and maintained for 28 days. Hartaningsih et al. (21) reported inconsistently detecting a CPE in chicken and quail embryo fibroblasts, but this was not confirmed by subinoculation of the cultures back into Bali cattle.
Although details of the technical procedures in the above reports are limited, there is some support for the possible replication or persistence of the infectious agent in cultures of blood leukocytes (9), and possibly macrophages, and these cell types could be of value in further attempts to culture the infectious agent associated with Jembrana disease.
Persistence of the infectious agent in recovered cattle
Recovered animals develop a persistent infection with the infectious agent. By subinoculation of blood from recovered cattle into susceptible Bali cattle, the infectious agent was demonstrated in blood for periods up to 448 days after experimental infection (9). There is only limited information on the immune status of recovered cattle but the few reports have indicated that animals, which survive the initial infection, develop immunity and resist further challenge with the infectious agent associated with the disease. Ramachandran and Madlangbayan (22) were unable to induce clinical disease by reinfection of recovered animals, and were also unable to induce disease in a splenectomized animal 5 to 6 weeks after it had recovered from experimental infection. There have been no reports of recurrence of disease in cattle, which have survived the initial acute febrile disease.
The immune mechanism involved in the recovery process was investigated to a limited extent by Ramachandran (9) who analyzed immunoglobulin levels in acute phase and convalescent sera of 5 experimentally infected Bali cattle. No abnormal changes in IgM values were detected, but elevated levels of IgG were detected in the convalescent sera. An attempt to detect neutralizing antibody in sera of recovered animals was unsuccessful (9); one volume of undiluted plasma obtained from an infected animal was mixed with 9 volumes of pooled concentrated convalescent serum from 5 animals, held at 4ºC for 24 hours and then at 37ºC for 1 hour, and was still infectious when inoculated into a healthy Bali animal. The absence of a significant follicular response and the scarcity of plasma cells in lymphoid tissue until the fifth week after infection was suggested by Dharma et al. (23) to indicate there may be a delayed antibody response, possibly a transient immunosuppression. A concurrent Pasteurella pneumonia is commonly detected in animals, which die during or following the acute phase of Jembrana disease, and this may be related to immunosuppression (23).
Age susceptibility
Ramachandran in 1981 reported that most affected animals were in the 6 month to 2 year age group and mortalities were also higher in this group. Putra et al. (24) reported that during, a small outbreak of Jembrana disease in Karangasem, Bali in 1981 that most affected animals were 4 to 6 years of age. In the Lampung outbreak, most cases were 2 to 7 years of age (7). In the 1972 Tabanan outbreak, the youngest animal affected was 18 months of age (3) but Ramachandran (9) reported disease in a 4 week-old calf. During the study on Rama Dewa disease in Lampung in 1980-1983 an animal of 2 months of age was affected (7).
Transmission of the disease
Due primarily to the hypothesis that the disease was of rickettsial aetiology and the known role of arthropods in the transmission of rickettsia, Jembrana disease has been suspected to be transmitted by blood sucking arthropods. Denning and Ramachandran (25) conducted limited experiments, which indicated Boophilus microplus to transmit the infection but these results were not confirmed. However, spraying of cattle with insecticides has been practiced as a form of control during small outbreaks of the disease in Bali and has also been conducted as a routine control procedure in the endemic area in Lampung by the Animal Health Services since the late 1970’s (Hassan, personal communication).
Therapy and control of Jembrana disease
Since the first outbreak of Jembrana disease in 1964, there have been many efforts to treat the disease with antibiotics but there is no substantiated evidence to indicate that this has been effective. Adiwinata (1) was unable to alter the normal course of the disease when tetracycline was administered. Treatment trials conducted by Soeharsono and Darmadi (6) in Lampung, using several types of broad spectrum antibiotics such as Ertilen (chloramphenicol, corticosteroid and antihistamine), Oxyvet (oxytetracycline), Tetrachlorine (chloramphenicol, oxytetracycline and ascorbic acid) and Leucomycin, did not detect any significant differences between the treated and untreated groups.
References
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