Approaches towards optimising the gamma interferon assay for diagnosing Mycobacterium bovis infection in African buffalo (Syncerus caffer)

The application of diagnostic tests for bovine tuberculosis in wildlife poses formidable technical difficulties and the use of the gamma interferon assay offers a simplified approach to testing wild animal species. We compared the performance of the gamma interferon assay in African buffalo (Syncerus caffer) under the recommended guidelines for interpretation of test results and found a high sensitivity (92.1%) at the cost of a greatly reduced specificity (68.3%). The optimised cut-off value for positive test results under local conditions was identified at an optical density of 0.385 at wavelength 450nm as the preferred compromise between sensitivity and specificity. Additional optimisation approaches to improve test performance were examined and showed that the application of 'a priori exclusions' of test results on the basis of reactivity to fortuitum PPD (sensitin produced from Mycobacterium fortuitum) and to a lesser degree, avian PPD, increased specificity without losing sensitivity. The implications of these findings on a modified testing protocol adjusted to include measurement of immune responsiveness to fortuitum PPD and other interpretation schemes are discussed.     

Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer)

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.     

An experimental intratonsilar infection model for bovine tuberculosis in African buffaloes, Syncerus caffer

An infection model for Mycobacterium bovis in African buffaloes, Syncerus caffer, was developed, using the intratonsilar route of inoculation. Two groups of 11 buffaloes each, aged approximately 18 months, were infected with either 3.2 x 10(2) cfu (low dose) or 3 x 10(4) cfu (high dose) of M. bovis strain isolated from a buffalo. A control group of six buffaloes received saline via the same route. The infection status was monitored in vivo using the comparative intradermal tuberculin test, and in vitro by the modified interferon-gamma assay. All buffaloes were euthanazed 22 weeks post infection and lesion development was assessed by macroscopic examination, culture and histopathology. It was found that the high dose caused macroscopic lesions in nine out of 11 buffaloes. Mycobacterium bovis was isolated from all buffaloes in the high-dose group and from six out of 11 in the low-dose group.     

Comparative field evaluation of two rapid immunochromatographic tests for the diagnosis of bovine tuberculosis in African buffaloes (Syncerus caffer)

Panels of sera from African buffalo with confirmed bovine tuberculosis and from known uninfected controls were used to evaluate the performance of two commercial rapid chromatographic immunoassays (A and B) for the detection of antibodies to Mycobacterium bovis. The sensitivity was 33% and 23%, respectively, while the specificity was determined at 90% and 94%, respectively. Overall the performance of both diagnostic tests under field conditions was not found sufficiently high to support their use in bovine tuberculosis management and control strategies in South African game reserves.     

The host status of African buffaloes, Syncerus caffer, for Rhipicephalus (Boophilus) decoloratus

The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for the one-host tick Rhipicephalus (Boophilus) decoloratus. To this end the R. (B.) decoloratus burdens of ten buffaloes examined in three north-eastern KwaZulu-Natal Province (KZN) nature reserves were compared with those of medium-sized to large antelope species in these reserves and in the southern Kruger National Park (KNP), Mpumalanga Province. The R. (B.) decoloratus burdens of the buffaloes were considerably smaller than those of the antelopes in the KNP, but not those in the KZN reserves. The life-stage structure of the R. (B.) decoloratus populations on the buffaloes, in which larvae predominated, was closer to that of this tick on blue wildebeest, Connochaetes taurinus, a tick-resistant animal, than to that on other antelopes. A single buffalo examined in the KNP was not infested with R. (B.) decoloratus, whereas a giraffe, Giraffa camelopardalis, examined at the same locality and time, harboured a small number of ticks. In a nature reserve in Mpumalanga Province adjacent to the KNP, two immobilized buffaloes, from which only adult ticks were collected, were not infested with R. (B.) decoloratus, whereas greater kudus, Tragelaphus strepsiceros, examined during the same time of year in the KNP harboured large numbers of adult ticks of this species. African buffaloes would thus appear to be resistant to infestation with R. (B.) decoloratus, and this resistance is expressed as the prevention of the majority of tick larvae from developing to nymphs.     

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.     

Development of a diagnostic gene expression assay for tuberculosis and its use under field conditions in African buffaloes (Syncerus caffer)

The development of diagnostic tests for tuberculosis (TB) in exotic species is constrained by host biology and the limited availability of suitable assay reagents. As such, we evaluated a gene expression assay (GEA) which is easily modified for novel species and allows for initial sample processing under field conditions. African buffaloes (Syncerus caffer) were categorized using the single comparative intradermal tuberculin test, and blood from test-positive and test-negative animals was incubated for 20h in "Nil" tubes (containing saline) and "TB Antigen" tubes (containing Mycobacterium tuberculosis complex (MTC)-specific antigens) of a commercial human TB test, the QuantiFERON(?)-TB Gold (In-Tube) (QFT) assay. Blood samples were then stabilized in RNAlater(?) and transported to the laboratory for RNA extraction. A Custom TaqMan GEA was used to calculate the relative abundance of interferon-gamma (IFN-γ) mRNA in the TB Antigen tube compared to that in the Nil tube as a marker of immune activation in response to MTC antigen recognition. The GEA results from the two buffalo groups were compared and a cutoff value of 2.85 was calculated to differentiate between animals from these groups with a sensitivity of 80% (95% C.I.: 56-94%) and a specificity of 95% (95% C.I.: 75-100%). Further optimization of this assay could provide a highly useful tool for the diagnosis of MTC infection in exotic species.     

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.     

Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattle-specific serological assay for BTB (TB ELISA). Buffaloes (n = 997) were tested with the tuberculin skin test and TB ELISA; a subset (n = 119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes.     

The gamma-interferon test: its usefulness in a bovine tuberculosis survey in African buffaloes (Syncerus caffer) in the Kruger National Park

A survey to determine the bovine tuberculosis status of buffalo herds north of the Olifants River in the Kruger National Park was conducted, using a new diagnostic approach. Diagnosis of Mycobacterium bovis infection was accomplished using the gamma-interferon assay technique in 608 adult buffaloes out of a total of 29 discreet herds. The animals were immobilized in groups of 10-15, bled, individually marked and then revived and released on site. As soon as test results were available (after 26-36 h), the same buffalo herd was relocated by tracking the frequency of a radio-collar previously fitted to one adult cow per group during the initial operation. Bovine reactors were identified, darted and euthanased from the helicopter. Necropsy and culture findings of all culled buffaloes showed excellent correlation with the results of the ante-mortem gamma-interferon test. The survey revealed that over and above the two positive herds that had been identified during a previous survey carried out in 1996, there were three additional, but previously unidentified, infected herds in the region north of the Olifants River.     

Hypothyroidism in an African forest buffalo (Syncerus caffer nanus).

An adult female African forest buffalo (Syncerus caffer nanus) of unknown age was presented with signs of recurrent hoof overgrowth, persistent anestrous, obesity, dull hair coat, and decreased activity level. Complete blood counts and serum biochemistry values were unremarkable. Decreased concentrations of total triiodothyronine and total thyroxine were noted compared with values for normal domestic cattle and a healthy African forest buffalo. Treatment with oral levothyroxine increased blood concentrations of total triiodothyronine and total thyroxine, and subsequent improvement in clinical signs included weight loss, hair regrowth, and reproductive cycling.     

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.     

Survey of ixodid ticks and two tick-borne pathogens in African buffaloes, Syncerus caffer, from the Caprivi Strip, Namibia

A capture operation to ascertain health status in free-ranging buffaloes from six different areas in the Caprivi Strip in the northeast corner of Namibia was conducted in October 2009. Basic information on the ticks and tick-borne pathogens normally found in wildlife from this area are scarce. The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for ixodid ticks and two selected tick-borne pathogens in the Caprivi Strip, a key area bordering Angola, Zambia, Botswana, and Zimbabwe. Four different tick species have been identified among the 233 collected specimens, and, of 95 tested buffaloes, 54 (57%) were positive for Theileria parva, whereas only 3 (3%) showed evidence of being infected with Ehrlichia ruminantium.     

Foot-and-mouth disease and the African buffalo (Syncerus caffer). 1. Carriers as a source of infection for cattle

Ten pregnant buffalo cows, six of which were subsequently shown to be carriers of SAT 1, 2 and 3 viruses, were captured in the Kruger National Park (KNP) and allowed to calve in captivity. The buffalo cows and calves were separated by a fence from 6 FMD susceptible cattle but the buffalo and cattle were obliged to use common drinking troughs and hay racks. Over a period of 15 months, during which the buffalo calves lost their maternally-derived immunity, neither the buffalo calves nor the susceptible cattle became infected with FMD virus. By the end of the observation period, however, only 1 buffalo cow still had detectable virus in its oesophageal/pharyngeal specimens.