A sero-survey of rinderpest in nomadic pastoral systems in central and southern Somalia from 2002 to 2003, using a spatially integrated random sampling approach.

A cross-sectional sero-survey, using a two-stage cluster sampling design, was conducted between 2002 and 2003 in ten administrative regions of central and southern Somalia, to estimate the seroprevalence and geographic distribution of rinderpest (RP) in the study area, as well as to identify potential risk factors for the observed seroprevalence distribution. The study was also used to test the feasibility of the spatially integrated investigation technique in nomadic and semi-nomadic pastoral systems. In the absence of a systematic list of livestock holdings, the primary sampling units were selected by generating random map coordinates. A total of 9,216 serum samples were collected from cattle aged 12 to 36 months at 562 sampling sites. Two apparent clusters of RP seroprevalence were detected. Four potential risk factors associated with the observed seroprevalence were identified: the mobility of cattle herds, the cattle population density, the proximity of cattle herds to cattle trade routes and cattle herd size. Risk maps were then generated to assist in designing more targeted surveillance strategies. The observed seroprevalence in these areas declined over time. In subsequent years, similar seroprevalence studies in neighbouring areas of Kenya and Ethiopia also showed a very low seroprevalence of RP or the absence of antibodies against RP. The progressive decline in RP antibody prevalence is consistent with virus extinction. Verification of freedom from RP infection in the Somali ecosystem is currently in progress.

Rinderpest seroprevalence in wildlife in Kenya and Tanzania, 1982-1993.

Eight hundred and thirty five serum samples collected from eight wild artiodactyl species in Kenya and Tanzania between 1982 and 1993 were tested for virus-neutralising (VN) antibodies to rinderpest (RP) virus. Antibodies were found in 116 of 344 buffaloes (Syncerus caffer) but not in the other species including 349 wildebeest (Connochaetes taurinus). Most of the antibody positive buffaloes were from the Maasai Mara-Serengeti ecosystem (MM-SE) and would have had opportunity for exposure to the virus during the epidemic of rinderpest in buffalo confirmed there in 1982. Buffalo born after 1985 did not have antibody indicating that virus stopped circulating in this population at or around that time. This second demonstration that RP virus disappears from the MM-SE is further evidence that these species are not permanent reservoirs of this virus. Re-infection of wildlife is transient and they remain valuable sentinels for infection in nearby domestic livestock.

Re-infection of wildlife populations with rinderpest virus on the periphery of the Somali ecosystem in East Africa.

We report surveillance for rinderpest virus in wildlife populations in three major ecosystems of East Africa: Great Rift Valley, Somali and Tsavo from 1994 to 2003. Three hundred and eighty wild animals were sampled for detection of rinderpest virus, antigen or genome and 1133 sampled for antibody in sera from Kenya, Uganda, Ethiopia and Tanzania from 20 species. This was done modifying for wildlife the internationally recommended standards for rinderpest investigation and diagnosis in livestock. The animals were selected according to susceptibility and preference given to gregarious species, and populations were selected according to abundance, availability and association with livestock. Rinderpest virus, antigen and/or genome were detected in Kenya; within Tsavo, Nairobi and Meru National Parks. Serological results from 864 animals (of which 65% were buffalo) from the region were selected as unequivocal; showing the temporal and spatial aspects of past epidemics. Recent infection has been only in or peripheral to the Somali ecosystem (in Kenya). Our evidence supports the hypothesis that wildlife is not important in the long-term maintenance of rinderpest and that wildlife are infected sporadically most likely from a cattle source, although this needs to be proven in the Somali ecosystem. Wildlife will continue to be a key to monitoring the remaining virus circulation in Africa.

Characterisation of African isolates of rinderpest virus.

Isolates of rinderpest virus (RPV) recovered from outbreaks of the disease in Kenya and Southern Sudan between 1986 and 1993 were compared to each other and to earlier isolates from East and West Africa. The recent isolates were mildly pathogenic for susceptible cattle and thus resembled other mild strains of RPV recovered from cattle and wildlife in East Africa more than 30 years ago. Monoclonal antibody analysis using a panel of 12 anti-RPV haemagglutinin protein-specific antibodies (mAbs) revealed that individual isolates were distinguishable. However, the panel of mAbs could not be used to relate the isolates on the basis of their pathogenicity or geographic origin. Immunoprecipitation of the virus-induced proteins from infected Vero cells, followed by SDS-polyacrylamide gel electrophoresis, showed that the recent mild RPV isolates from eastern Africa were closely related to each other and to their contemporary isolates from Nigeria and Egypt, but they were distinct from another mild isolate recovered from the region three decades ago. Two distinct lineages of African RPV isolates were identified by sequencing a region of the genome around the proteolytic enzyme cleavage site of the fusion protein from the old and new isolates. One lineage, which included virus isolates recovered from East and West Africa during the 1960s, showed a closer phylogenetic relationship to Asian and Middle Eastern RPV isolates. The other lineage consisted mainly of isolates recovered from East, West and North Africa between 1983 and 1993. The results showed that there was co-circulation of two different lineages of RPV in Nigeria during the epizootics of the 1980s.

Pathomorphological and immunohistological findings in cattle experimentally infected with rinderpest virus isolates of different pathogenicity.

Experimental infection of nine cattle with seven rinderpest virus strains of different pathogenicity resulted in significant variations of clinical signs, morphological lesions and distribution of viral antigen in tissues. The severity of clinical disease was correlated with the extent of tissue alterations and the amount of immunohistologically detectable viral antigen. Both mild and virulent strains of rinderpest share essentially the same tissue tropisms in vivo, i.e. epithelio- and lympho-tropism. However, rinderpest virus isolates of higher pathogenicity showed a more rapid and wider distribution with more extensive lesions than milder strains, which probably accounts for the higher mortality.

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes.

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.

Inexpensive vaccines and rapid diagnostic kits tailor-made for the global eradication of rinderpest, and technology transfer to Africa and Asia.

Rinderpest is an acute and highly contagious viral disease of ruminants, often resulting in greater than 90% mortality. We previously reported the development of first- and second-generation recombinant vaccinia virus vaccines which provide complete protection against rinderpest virus (RPV) and peste-des-petits ruminants virus (PPRV). These vaccines are safe even for immunodeficient mice and macaques with acquired immunodeficiency syndrome. We developed a third-generation recombinant vaccinia virus vaccine (v2RVFH) that expresses the fusion and haemagglutinin genes of RPV under strong synthetic vaccinia virus promoters. Cattle vaccinated intramuscularly with as little as 10(3) plaque-forming units (PFU) of v2RVFH were completely protected from rinderpest. Vaccinated animals did not develop pock lesions or transmit v2RVFH to contact animals. Cattle vaccinated with a standard dose of 10(8) PFU of v2RVFH developed long-term, sterilizing immunity against rinderpest. Thus, v2RVFH is safe, efficacious, heat stable, inexpensive, easily administered, and allows serological differentiation between vaccinated and infected animals. To aid in diagnosis and differentiation of vaccinated from infected animals, we developed indirect ELISAs (iELISAs) that use baculovirus-expressed RPV or PPRV nucleoprotein as coating antigens. A single larva contains enough viral antigen to test more than 10,000 serum samples, in duplicate. African scientists trained at the ILMB successfully transferred the iELISA kit technology to more than 30 countries in Africa, providing a model for technology transfer among developing countries. Vaccination with v2RVFH, in conjunction with the iELISA kits, greatly enhances the prospects for global eradication of rinderpest, as developing nations achieve independence in control efforts.

Induction of apoptotic cellular death in lymphatic tissues of cattle experimentally infected with different strains of rinderpest virus.

The presence, type, and extent of cellular death in lymphatic tissues of cattle experimentally infected with rinderpest virus strains of different virulence was investigated morphologically. Cells with DNA strand breaks were identified in histological sections of palatine tonsil, spleen, and mesenteric and mandibular lymph nodes by the TUNEL (terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. In addition, representative samples of lymphatic tissues were examined by transmission electron microscopy. The results indicated that cellular disassembly in lymphatic tissues was caused by both apoptosis and oncosis. Cells with DNA strand breaks were observed in follicular and parafollicular areas of lymphatic tissues and their numbers were determined. A significant correlation was found between the number of TUNEL-positive cells and viral virulence. These results suggest that, in addition to oncosis, apoptotic cellular death in lymphatic tissues contributes substantially to the pathogenesis of rinderpest.

Reaction of goats to infection with infectious bovine rhinotracheitis virus.

Intranasal exposure of goats to infectious bovine rhinotracheitis virus resulted in mild respiratory disease and virus reisolation from nasal secretions. No disease was produced in goats exposed to the same virus by the genital or ocular routes. There was serological evidence of contact transmission of infection from infected goats to cattle. Virus recrudescence was not detected in goats treated with dexamethasone two months after virus inoculation.

Improved isolation of rinderpest virus in transformed bovine T lymphoblast cell lines.

Bovine T lymphoblast cell lines transformed by the protozoan Theileria parva were compared with bovine kidney (BK) and Vero cells for their ability to isolate various strains of rinderpest virus from tissues and infected secretions. All of the strains of rinderpest virus that were tested, including attenuated cell-culture, caprinised and lapinised vaccines, and both mild and virulent pathogenic strains, readily induced syncytial cytopathic effect (cpe) in T lymphoblasts. The cpe could often be detected within one day of inoculation of lymphoblasts, whereas it took three to 14 days to appear in Vero and BK cells. Using lymphoblasts it was possible to reisolate rinderpest virus from nine of 42 swabs collected from three cattle experimentally infected with an isolate from a recent outbreak of mild disease whereas the same swabs yielded only one reisolate on BK cells. It was also possible using the lymphoblasts to detect infectious virus in the ocular, nasal and oral secretions of goats and rabbits infected with caprinised and lapinised virus, respectively. Peste des petits ruminants virus appeared to grow as rapidly as rinderpest virus in the lymphoblasts whereas canine distemper virus readily induced cpe on first passage but less readily on subsequent passage. Measles virus induced relatively little cpe when inoculated into lymphoblasts and did not appear to passage in these cells. The lymphoblasts are easy to maintain in culture and since they rapidly recovered 11 isolates from 37 diagnostic samples could prove useful in laboratories carrying out rinderpest diagnosis.

Observations on rinderpest in Kenya, 1986-1989.

Rinderpest was confirmed in Kenya in 1986, 1987, 1988 and 1989. Three epidemiologically distinct events appear to have occurred: repeated outbreaks in West Pokot district related to cross-border movement of stock, an outbreak in Marsabit district in 1987 (thought to have been caused by illegal movement of cattle, possibly in vehicles, from countries further north) and a series of related outbreaks in and near Nairobi between 1988 and 1989 due to the unauthorized movement from abattoirs and holding grounds of slaughter stock possibly introduced from West Pokot or Marsabit. In West Pokot the disease affected unvaccinated calves and yearlings. In Marsabit cattle of all ages were affected. In August 1988, a major outbreak was confirmed in Kiambu and Kajiado districts in central Kenya, near Nairobi. At the same time a provisional diagnosis of rinderpest was made in a herd of cattle at a slaughterhouse in Nairobi. Rinderpest virus was isolated from sick cattle in all the outbreaks. Experimental infection of susceptible cattle with the Kiambu isolate demonstrated this to be of low virulence. Emergency vaccination and quarantine measures instituted immediately after confirmation eliminated clinical disease within three to four weeks in West Pokot, Kiambu and Nairobi. In Kajiado, however, the disease persisted for at least nine months, during which time a series of virus isolates was recovered. There was no evidence of infection in susceptible wildlife. This increase in the incidence of rinderpest in Kenya in recent years serves to highlight the problems of control and the need for concerted efforts to eradicate the threat of the disease from East Africa.

Development and distribution of rinderpest virus antigen in experimentally infected goats.

Three goats, experimentally infected with rinderpest virus were examined for the development and distribution of precipitating antigens in various tissues and secretions using the agar gel immunodiffusion test. Virus antigens were detected in ocular secretions and lymph node biopsies from the second to the fourth and fifth days of pyrexia, respectively, but were not detected in nasal secretions. Precipitating antigens were demonstrated in various lymphoid organs, the lung and abomasum of a goat killed on the fourth day of pyrexia. These findings are discussed in relation to the epidemiology of rinderpest in goats in Africa.

Preliminary observations on rinderpest in pregnant cattle.

A Kabete 'O' strain of rinderpest virus enhanced in virulence was inoculated subcutaneously into four cows which were between six and eight months pregnant. All the cows developed clinical signs of rinderpest from the third day after inoculation and shed high titres of virus in their ocular and vaginal secretions during the course of the clinical disease. Three of the cows died of rinderpest on the third day after the onset of fever but no virus was isolated from their fetuses which were examined post mortem. The fourth cow showed complete clinical and virological recovery by the eighth day after the onset of fever and aborted an eight-and-a-half-month-old fetus on the 12th day after it recovered. Rinderpest virus was demonstrated in a wide range of the aborted fetal tissues. Virus was also detected in the maternal vaginal discharges up to 24 hours after abortion. The only gross pathological change observed was a severe necrotising placentitis.

Rinderpest epidemic in wild ruminants in Kenya 1993-97.

A severe epidemic of rinderpest, affecting mainly wild ruminants, occurred between 1993 and 1997 in East Africa. Buffalo (Syncerus caffer), eland (Taurotragus oryx) and lesser kudu (Tragelaphus imberbis) were highly susceptible. The histopathological changes, notably individual epithelial cell necrosis with syncytia formation, were consistent with an infection with an epitheliotrophic virus. Serology, the polymerase chain reaction, and virus isolation confirmed the diagnosis and provided epidemiological information. The virus was related to a strain which was prevalent in Kenya in the 1960s, of a second lineage (II), and distinct from isolations of rinderpest virus in the region since 1986. The source of the virus was presumed to be infected cattle from the Eastern region of Kenya and Somalia. The pathogenicity of the virus varied during the epidemic. The mortality in buffalo populations was estimated to be up to 80 per cent, and population data suggested that the virus had an adverse effect on a wide range of species. The virus caused only a mild disease in cattle, with minimal mortality. The results confirmed the importance of wildlife as sentinels of the disease, but although wildlife were important in the spread of the virus, they did not appear to act as reservoirs of infection.

Use of Vero cells for the isolation and propagation of malignant catarrhal fever virus.

Vero cells were compared with primary bovine thyroid (BTh) cultures for the isolation of malignant catarrhal fever virus from infected blood and tissues. Comparative titrations showed Vero cells detected only two-fold less infectivity in rabbit spleen suspensions than BTh cells. Twenty three of 32 bovine buffy coat cell preparations which were positive on BTh cells were also positive on Vero cells. The cytopathic effects (CPE) of virus isolates in Vero cells consisted of syncytia and refractile cytomegalic cells which were as easy to recognise and developed as rapidly as CPE in BTh cells. Two laboratory strains of malignant catarrhal fever virus were readily adapted to and maintained by passage in Vero cells.

Confirmation of rinderpest in experimentally and naturally infected cattle using microtitre techniques.

Virulent rinderpest virus was detected by immunoperoxidase staining of microtitre bovine kidney cell cultures within 24 to 48 hours of inoculation with prescapular lymph node and spleen homogenates from experimentally infected steers. Rinderpest virus specific cytopathic effects were evident from 48 hours in microtitre plates and from 72 hours in rolled tube cultures. Nasal and ocular secretions collected from cattle naturally infected with rinderpest and inoculated into bovine kidney cell cultures did not readily yield cytopathic virus in both tubes and microtitre plates, but immunoperoxidase staining of microtitre cultures on the fourth day of inoculation detected replication of virus in cultures inoculated with ocular and nasal secretions from seven of 17 cattle tested.

Immunohistological localisation of rinderpest virus in formalin-fixed, paraffin-embedded tissues from experimentally infected cattle.

Six Freesian steers were subcutaneously inoculated with the virulent rinderpest virus strain Kabete "0" and sacrificed at the height of fever. Sections of formalin-fixed, paraffin-embedded tissues were stained according to the peroxidase anti-peroxidase (PAP) technique. Labelling of viral antigen, both in the cytoplasm and in the nuclei of infected cells, was observed in the epithelial cells of the upper and lower alimentary tract and in lymphoid organs, i.e. spleen, lymph nodes, pharyngeal tonsils, Peyer's patches and thymus. Electron microscopy studies confirmed the results.