Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer)

The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and non-pathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic Theileria parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterise the full-length 18S rRNA genes of Theileria mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridised only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria species-specific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.     

Infection of African buffalo (Syncerus caffer) and cattle with Theileria parva lawrencei after serial passage in cattle

The infectivity of a Theileria parva lawrencei stabilate, from a stock derived from an African buffalo (Syncerus caffer) in the Serengeti National Park, Tanzania, was investigated. In the first experiment a buffalo and three cattle were inoculated with a stabilate from a stock passaged three times in cattle. All cattle developed fatal theilerial infections. Isolations from the buffalo by tick feeding and cell culture isolation showed that it was infected with T p lawrencei at the time of inoculation, but the second isolation made 19 days after inoculation behaved like T p parva in cattle, developing a high parasitosis, while the third isolation made three months later behaved like T p lawrencei with low parasitosis. It was concluded that two biological types of T parva could exist in a buffalo at one time, but it was not shown that the buffalo had become a carrier of T p lawrencei adapted to cattle. In the second experiment two buffaloes and three cattle were inoculated with T p lawrencei (Serengeti) stabilate which had been passaged six times through cattle and ticks. The two buffaloes had mild theilerial infections and developed serological titres in the indirect fluorescent antibody test, but the cattle had fatal infections. Tick and cell culture isolations of T parva were possible during the clinical reactions of the buffaloes, but no carrier state was demonstrated. Theileria-infected cell lines were established from the buffaloes and the cattle and were examined using monoclonal antibodies against T parva schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haemoparasite prevalence and Theileria parva strain diversity in Cape buffalo (Syncerus caffer) in Uganda

Cape buffalo (Syncerus caffer) are considered to be an important reservoir for various tick-borne haemoparasites of veterinary importance. In this study we have compared the haemoparasite carrier prevalence in buffalo from four geographically isolated national parks in Uganda [Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP)]. Differences were seen in haemoparasite prevalence in buffalo from the four national parks. All the buffalo sampled in LMNP were carriers of Theileria parva however, buffalo from MFNP and KVNP, which are both located in the north of Uganda, were negative for T. parva. Interestingly, 95% of buffalo in the northern part of QENP were T. parva positive, however all buffalo sampled in the south of the park were negative. A high multiplicity of infection was recorded in all the buffalo found to be carrying T. parva, with evidence of at least nine parasite genotypes in some animals. Most of the buffalo sampled in all four national parks were carriers of T. mutans and T. velifera, however none were carriers of T. taurotragi, Babesia bovis, Babesia bigemina, Ehrlichia bovis or Ehrlichia ruminantium. All the buffalo sampled from LMNP were positive for T. buffeli and T. sp. (buffalo) however, buffalo from the parks in the north of the country (KVNP and MFNP) were negative for these haemoparasites. Anaplasma centrale and Anaplasma marginale were circulating in buffalo from all four national parks. T. parva gene pools from two geographically separated populations of buffalo in two of the national parks in Uganda (LMNP and QENP) were compared. The T. parva populations in the two national parks were distinct, indicating that there was limited gene flow between the populations. The results presented highlight the complexity of tick-borne pathogen infections in buffalo and the significant role that buffalo may play as reservoir hosts for veterinary haemoparasites that have the potential to cause severe disease in domestic cattle.     

Gousiekte in African buffalo (Syncerus caffer)

Three African buffalo (Syncerus caffer) that died after capture and translocation from Mutirikwe Recreational Park in southern Zimbabwe showed macroscopic and microscopic lesions of cardiomyopathy compatible with a diagnosis of gousiekte. The buffalo had had access to Pavetta schumanniana, a plant that is known to cause gousiekte. Death was attributed to cardiac failure as a result of previous consumption of the plant, exacerbated by the stress of translocation.     

Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva

Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo.     

Isolation of Theileria parva lawrencei-infected lymphoid cell lines from free-ranging African buffaloes (Syncerus caffer)

Lymphoid cells collected from the peripheral blood of 45 free-ranging buffaloes sampled in the Masai Mara Game Reserve in Kenya were cultured in vitro in an attempt to establish cell lines of intralymphocytic theilerial schizonts. Theileria parva lawrencei-infected lymphoblastoid cell lines were established with samples taken from 12 buffaloes, and 11 of these were maintained continuously in vitro. Sixteen of the buffalo samples were contaminated with either trypanosomes or viruses. The successful in vitro isolation of Theileria species from 27 per cent of the buffaloes sampled demonstrates the applicability of this technique for field isolation of T parva lawrencei.     

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.     

The occurrence of Theileria and Cowdria parasites in African buffalo (Syncerus caffer) and their associated Amblyomma hebraeum ticks

The polymerase chain reaction and oligonucleotide probing were used to detect Theileria and Cowdria species in DNA extracted from blood and ticks recovered from 24 African buffalo during a gamecapture operation in the Kruger National Park, South Africa. Species-specific probing indicated that all but one of the buffalo were carrying at least one Theileria species. Indirect fluorescent antibody (IFA) serology indicated that all animals had been exposed to Theileria parva infection but only 33% were positive for T. parva by probing. Twelve (50%) of the animals but only six of the 214 adult Amblyomma hebraeum ticks examined (2.8%) were probe-positive for Cowdria. Only one Cowdria 16S genotype was detected in the animals and ticks.     

Further evaluation of the use of buparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer).

Three experiments were undertaken to determine the efficacy of different doses of buparvaquone in the infection and treatment immunization of cattle against Theileria parva derived from African buffalo (Syncerus caffer). Two of these experiments also compared buparvaquone with standard doses of long- and short-acting formulations of oxytetracycline. In addition, different dilutions of stabilates were used in the experiments. In the first experiment, a 10(-1.0) dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-1 body weight (bwt) on Day 0 after infection. All control cattle developed severe theileriosis and none of the treatment regimes (including those utilizing long-acting oxytetracycline) prevented the development of theileriosis. Treatment with buparvaquone at 2.5 mg kg-1 bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10(-2.0) dilution, buparvaquone treatment at 5 and 2.5 mg kg-1 bwt and short- and long-acting formulations of oxytetracycline reduced reactions greatly. While all the oxytetracycline treated animals produced a serological response and were immune to a 50-fold higher challenge with the immunizing stabilate, several animals in the buparvaquone groups did not show a serological response and were not immune to challenge. In the third experiment, groups of cattle were infected with 10(-1.2), 10(-1.4) and 10(-1.6) dilutions of stabilate and were treated with 2.5 mg kg-1 bwt of buparvaquone. No animals developed severe theileriosis and all seroconverted. On homologous challenge, however, two out of 14 cattle showed severe reactions. It was concluded that further work on immunization using buparvaquone treatment at 2.5 mg kg-1 bwt and 10(-1.6) dilution of the stabilate would have to be carried out before such a system could be used in the field.     

羊泰勒虫吉林省分离株18S rRNA基因的克隆和序列分析

为分析羊泰勒虫吉林省分离株的18S rRNA基因序列,根据羊泰勒虫18S rRNA基因的保守区序列设计1对引物,对吉林省的绵羊血液基因组DNA进行PCR扩增,结果扩增出长度为459 bp的基因片段,并成功地将该基因克隆到pMD18-T载体。将经纯化、筛选及酶切鉴定和PCR鉴定为阳性的重组质粒进行测序,与已发表的羊泰勒虫基因序列进行比较。序列分析显示,羊泰勒虫吉林省分离株与羊泰勒虫China 1的同源性为100%。     

The epidemiology of tuberculosis in free-ranging African buffalo (Syncerus caffer) in the Kruger National Park, South Africa

The presence of bovine tuberculosis (Mycobacterium bovis) in the Kruger National Park (KNP) was determined for the first time in 1990. It was diagnosed in an African buffalo (Syncerus caffer) bull, which was found recumbent and in an emaciated and moribund state near the south-western boundary fence. This prompted an investigation into the bovine tuberculosis (BTB) status of the KNP, with emphasis on its epidemiological determinants and risk factors. This report documents the findings of surveys that were conducted from 1990 to 1996. It was found that BTB had entered the KNP ecosystem relatively recently (+/- 1960), and has found favourable circumstances for survival and propagation in a fully susceptible and immunologically naive buffalo population. Indications are that it entered the KNP from across the southern river boundary, where the presence of infected domestic cattle herds had been documented. From there the infection spread through the southern buffalo population and is currently spreading in a northward direction. It was estimated that this northward spread took place at a rate of about 6 km per year; the prospect being that, if this rate of spread is maintained, the entire KNP may be affected in less than 30 years from now. Spillover from buffalo had already occurred in species such as chacma baboon (Papio ursinus), lion (Panthera leo), cheetah (Acinonyx jubatus), kudu (Tragelaphus strepsiceros) and leopard (Panthera pardus). Although there is no indication yet that these species act as maintenance hosts, the possibility is raised that these, or an as yet overlooked species, might assume such a role in future. In the KNP, BTB manifests itself as a chronic and predominantly subclinical disease in buffalo. It may take years for clinical signs to develop, and then only at a terminal stage, when emaciation is a constant feature. It is suspected that the time from infection to death is variable and dependent on the animal's immune response, which can be weakened by such factors as stress, old age or droughts. It was found that, in the interim, buffalo have a normal reproductive life. On necropsy, buffalo show almost exclusively lung and upper respiratory tract involvement, pointing to an aerogenous mode of transmission. Histologically, little sign of encapsulation of lesions was detected, which suggests that they are exceptionally susceptible to BTB and that most lesions are open and infectious and progressive, leading ultimately to death of the individual. Evidence also indicates that BTB is progressive within the herd context (92% being the highest prevalence rate thus far determined in a buffalo herd) as well as progressive within the KNP buffalo population (the implication being that virtually all buffalo herds in the KNP will eventually be infected). Preliminary data suggest a positive correlation between disease prevalence and mortality, with potential mortality reaching up to 10% in buffalo herds having BTB prevalence rates of 50 % and higher. Only the future will tell what the effect of the disease on the population dynamics of buffalo will be.     

Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle

Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.     

Coccidian oocyst and nematode egg counts of free-ranging African buffalo (Syncerus caffer) in the Kruger National Park, South Africa

Faecal specimens collected in the Kruger National Park from 103 African buffaloes (Syncerus caffer) up to 1 year old and 283 buffaloes older than 1 year were examined for the presence of coccidian oocysts and nematode eggs. Most specimens from animals older than 1 year had negative coccidian oocyst counts. Positive specimens from younger animals had significantly higher coccidian oocyst counts than those from older animals. No such difference was found for nematode egg counts.     

Disease constraints for utilization of the African buffalo (Syncerus caffer) on game ranches in Zambia

Eco-tourism depending on wildlife is becoming increasingly profitable and landowners are beginning to favor game farming and ecotourism. In these areas, large-scale translocation of wildlife involves a diversity of species and large populations. The African buffalo (Syncerus caffer) is one of the major tourist attractions in Zambia. It accounts for 8.7% and 12.4% of the total animal species hunted in the Game Management Areas and the total hunting revenue earned in Zambia, respectively. It is ecologically an important animal species essential for the purpose of habitat control and facilitating the provision of suitable grazing pastures. However, the rearing of the African buffalo on game ranches has been hampered by its carrier state of the Southern Africa Terroritory (SAT) serotypes of foot and mouth disease virus (FMD). The African buffalo is also known to be a carrier of Theileria parva lawrencei, the causative agent of corridor disease (CD) that continues to have devastating effects on the livestock industry in Zambia. In addition, the importation of buffaloes from countries with populations endemic to bovine tuberculosis is highly restricted. Veterinary regulations in Zambia, strongly advocate against the translocation of buffaloes from protected areas to private ranches for disease control purposes thereby mounting a considerable constraint on the economic and ecological viability of the industry. It is hoped that this review will motivate the relevant government authorities in exploiting ways in which this animal species play a central role in eco-tourism.     

Potential for the transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle

Foot-and-mouth disease viruses of types SAT 1 and SAT 2 isolated from diseased cattle and carrier buffalo, either on the same farm or in the same ecological area within a short time of each other, were compared by T1 oligonucleotide mapping. No similarity was observed between the maps obtained, indicating that the different populations of virus were unique to each species and that no interspecies transmission had occurred.     

Phagocytosis of Trypanosoma (Nannomonas) congolense by circulating macrophages in the African buffalo (Syncerus caffer).

Nineteen African buffalo were short in the Mara region of Kenya. Leucocytes were separated from the buffalo samples by sedimentation and centrifugation. In leucocyte smears of six buffalo, trypanosomes were detected and in two of them, high levels of phagocytosis of Trypanosoma (Nannomonas) congolense by circulating macrophages were demonstrated.