Pathogen detection and disease diagnosis in wildlife: challenges and opportunities.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

Retrospective study of bacterial and fungal causes of abortion in domestic ruminants in northern regions of South Africa (2006-2016).

This initial retrospective study presents information on bacterial and fungal causes of abortion in domestic ruminants in South Africa over 10 years (2006-2016). A complete set of data was collected at the Faculty of Veterinary Science through a keyword search of pathology and bacteriology laboratory registers. Additional electronic data were received from an external laboratory. A total of 288 cases were recorded from six provinces. Overall diagnostic rate was 35.1%. In 14.6% of cases, histological evidence of an infectious process was found, but no aetiological agent was detected. Several cases did not include aetiological diagnoses because applicable diagnostic techniques were not available or not applied when necessary. Increased submission of placenta, as well as additional conventional and molecular diagnostic methods, can contribute to an improved diagnostic rate. In addition, the study highlights the superior significance of Brucella abortus as a major bovine pathogen in South Africa.

Prevalence of bovine tuberculosis in cattle, goats, and camels of traditional livestock raising communities in Eritrea.

BACKGROUND: The aim of the current study was to assess the prevalence of bovine tuberculosis (BTB) in cattle, goats, and camels, and its zoonotic potential within the traditional livestock raising communities in four regions of Eritrea. The Single Intradermal Comparative Tuberculin Test (SICTT) as indicator of M. bovis infection was conducted on 1077 cattle, 876 goats, and 195 camels. To elucidate possible risk factors for BTB transmission between animals and its potential zoonotic implication, questionnaire based face-to-face interviews were conducted in households of which 232 raised cattle, 128 goats, and 29 camels. RESULTS: The results of the SCITT were interpreted using the OIE standard (> 4 mm cut-off) for positive responses. In cattle, individual animal (n = 1077) and herd (n = 413) prevalences were 1.2% (n = 13) [Confidence Interval (CI) 95% CI, 1.0-1.3%] and 3.2% (n = 13) (95% CI, 3.0-3.4%), respectively. In goats (n = 876), none of the animals was positive. In camels, individual animal (n = 195) and herd (n = 70), BTB prevalences were 1.5% (n = 3) (95% CI,1.4-1.6%) and 2.9(n = 2) (95% CI, 0.9-4.6%), respectively. Overall, male animals were more at risk (OR = 2.6; 95% CI:1.0-8.7) when compared to females. Sharing of water points, introduction of new animals into herds and migration of animals over large distances were common events that may contribute to intra and inter-species transmission of BTB. Consumption of raw milk, lack of BTB transmission awareness, and low levels of education were common in the farming communities. CONCLUSION: The current study highlighted a low prevalence of M. bovis in cattle, goats and camels in extensive traditional livestock in Eritrea. Despite this, the spatial distribution of affected animals across most of the sampled regions and consumption of unpasteurized milk warrants surveillance, cautious and timely control measures for the disease.

Cross reactive immune responses in cattle arising from exposure to Mycobacterium bovis and non-tuberculous mycobacteria.

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-É£). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-É£) responses were measured in whole blood cultures using the IFN-É£ assay and the IFN-É£ elispot assay on purified peripheral blood mononuclear cells (PBMC). We observed the expected strong IFN-É£ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-É£ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-É£ immune responses to the 10 R-Mag measured by the IFN-É£ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests.

Farm-level risk factors associated with bovine tuberculosis in the dairy sector in Eritrea.

The aim of our study was to determine the association of selected potential risk factors with the presence of bovine tuberculosis (BTB) in dairy herds in Eritrea. A case-control study was conducted in the three major milk-producing regions of the country by stratified random sampling of 61 case and 65 control herds combined with completion of a standardized pretested questionnaire pertaining 36 relevant risk factors (variables). The variables were divided into two clusters, based on potential association with either "introduction" or "establishment" of BTB on the farms to elucidate association with incident or prevalent cases separately. Subsequent to univariable analysis of the 36 risk factors at herd level, 14 of these were offered to multivariable logistic regression models. Farms with higher numbers of cows, and those with concrete floors, were 3.6, and 7.5 times more at risk for presence of BTB, respectively, compared with their references. These findings will be useful as entry points for future informed decision-making towards BTB control and eradication programme in the country.

Tuberculosis serosurveillance and management practices of captive African elephants (Loxodonta africana) in the Kavango-Zambezi Transfrontier Conservation Area.

Transfrontier conservation areas represent an international effort to encourage conservation and sustainable development. Their success faces a number of challenges, including disease management in wildlife, livestock and humans. Tuberculosis (TB) affects humans and a multitude of non-human animal species and is of particular concern in sub-Saharan Africa. The Kavango-Zambezi Transfrontier Conservation Area encompasses five countries, including Zimbabwe, and is home to the largest contiguous population of free-ranging elephants in Africa. Elephants are known to be susceptible to TB; thus, understanding TB status, exposure and transmission risks to and from elephants in this area is of interest for both conservation and human health. To assess risk factors for TB seroprevalence, a questionnaire was used to collect data regarding elephant management at four ecotourism facilities offering elephant-back tourist rides in the Victoria Falls area of Zimbabwe. Thirty-five working African elephants were screened for Mycobacterium tuberculosis complex antibodies using the ElephantTB Stat-Pak and the DPP VetTB Assay for elephants. Six of 35 elephants (17.1%) were seropositive. The risk factor most important for seropositive status was time in captivity. This is the first study to assess TB seroprevalence and risk factors in working African elephants in their home range. Our findings will provide a foundation to develop guidelines to protect the health of captive and free-ranging elephants in the southern African context, as well as elephant handlers through simple interventions. Minimizing exposure through shared feed with other wildlife, routine TB testing of elephant handlers and regular serological screening of elephants are recommended as preventive measures.

Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates.

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.

Isolation and Potential for Transmission of Mycobacterium bovis at Human-livestock-wildlife Interface of the Serengeti Ecosystem, Northern Tanzania.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a multihost pathogen of public health and veterinary importance. We characterized the M. bovis isolated at the human-livestock-wildlife interface of the Serengeti ecosystem to determine the epidemiology and risk of cross-species transmission between interacting hosts species. DNA was extracted from mycobacterial cultures obtained from sputum samples of 472 tuberculosis (TB) suspected patients and tissue samples from 606 livestock and wild animal species. M. bovis isolates were characterized using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) on 24 loci. Only 5 M. bovis were isolated from the cultured samples. Spoligotyping results revealed that three M. bovis isolates from two buffaloes (Syncerus caffer) and 1 African civet (Civettictis civetta) belonged to SB0133 spoligotype. The two novel strains (AR1 and AR2) assigned as spoligotype SB2290 and SB2289, respectively, were identified from indigenous cattle (Bos indicus). No M. bovis was detected from patients with clinical signs consistent with TB. Of the 606 animal tissue specimens and sputa of 472 TB-suspected patients 43 (7.09%) and 12 (2.9%), respectively, yielded non-tuberculous mycobacteria (NTM), of which 20 isolates were M. intracellulare. No M. avium was identified. M. bovis isolates from wildlife had 45.2% and 96.8% spoligotype pattern agreement with AR1 and AR2 strains, respectively. This finding indicates that bTB infections in wild animals and cattle were epidemiologically related. Of the 24 MIRU-VNTR loci, QUB 11b showed the highest discrimination among the M. bovis strains. The novel strains obtained in this study have not been previously reported in the area, but no clear evidence for recent cross-species transmission of M. bovis was found between human, livestock and wild animals.

Prevalence and risk factors of bovine tuberculosis in dairy cattle in Eritrea.

BACKGROUND: The prevalence of bovine tuberculosis (BTB) in dairy cattle in the three major milk producing regions of Eritrea was assessed by subjecting 15,354 dairy cattle, 50 % of Eritrea's dairy cattle population, to the single intradermal comparative tuberculin test (SICTT). Skin test results were interpreted according to guidelines of the World Organization for Animal Health (OIE) with >4 mm as cutoff in skin thickness increase. In addition, we studied the relation between 'physiological' variables related to pregnancy and lactation, and the variable 'region' on the probability to be skin test positive. RESULTS: The BTB prevalences at animal and herd levels were: 21.5% and 40.9% in Maekel, 7.3% and 10% in Debub, and 0.2% and 1.6% in the Anseba region, respectively. Overall, in the regions included, prevalence was 11.3% (confidence interval (CI) 95% CI, 11.29 - 11.31%) and 17.3% (95% CI, 17.27-17.33%), at animal and herd level, respectively. Considering positive herds only, the animal BTB prevalence was 36.8%, 30.1%, and 1.8%, in Maekel, Debub and Anseba, respectively, and the overall animal prevalence within these herds was 32%. In adult dairy cattle the probability of positive reactivity in the SICTT test was highest in pregnant animals as compared to the other categories. CONCLUSION: This study reports persistent prevalence of BTB as defined by positive SICTT in the dairy sector of Eritrea, especially in the regions of Maekel and Debub that are located in the central highlands of the country. To our understanding this is the first report that has encompassed all the major dairy farms in Eritrea and it will be instrumental in advocating future BTB control programs in the dairy sector.

Field application of immunoassays for the detection of Mycobacterium bovis infection in the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is considered the most important maintenance host of bovine tuberculosis (BTB) in wildlife in Southern Africa. The diagnosis of Mycobacterium bovis infection in this species mostly relies on the single intradermal comparative tuberculin test (SICTT). As an alternative, the BOVIGAM® 1G, an interferon-gamma (IFN-γ) release assay, is frequently used. The test performance of cell-mediated immunity (CMI-) and humoral immunity (HI-) based assays for the detection of M. bovis infections in buffaloes was compared to identify the test or test combination that provided the highest sensitivity in the study. Buffaloes were sampled during the annual BTB SICTT testing in the Hluhluwe-iMfolozi-Park (KwaZulu-Natal, South Africa) during June 2013. A total of 35 animals were subjected to the SICTT, 13 of these tested positive and one showed an inconclusive reaction. CMI-based assays (BOVIGAM® 1G (B1G) and BOVIGAM® 2G (B2G)) as well as a serological assay (IDEXX TB ELISA) were used to further investigate and compare immune responsiveness. Thirteen SICTT positive buffaloes and one inconclusive reactor were slaughtered and a post-mortem (PM) examination was conducted to confirm BTB. Lesions characteristic of BTB were found in 8/14 animals (57.1%). Test results of individual assays were compared with serial and parallel test interpretation and the sensitivity was calculated as a percentage of test positives out of the number of SICTT positive animals with granulomatous lesions (relative sensitivity). The B1G assay showed the highest individual sensitivity (100%; 8/8) followed by the B2G assay (75%; 6/8) and the IDEXX TB ELISA (37.5%; 3/8). Therefore, using in parallel interpretation, any combination with the B1G showed a sensitivity of 100% (8/8), whereas combinations with the B2G showed a 75% sensitivity (6/8). Out of the 21 SICTT negative animals, 7 animals showed responsiveness in the B2G or IDEXX TB ELISA. In conclusion, this study has shown that the BOVIGAM® IFN-γ assay had the highest test performance.

Evidence of increasing intra and inter-species transmission of Mycobacterium bovis in South Africa: are we losing the battle?

Tuberculosis caused by Mycobacterium bovis is recognized worldwide as a significant health risk in domestic cattle, farmed and wild animal species as well as in humans. We carried out spoligotyping and variable number of tandem repeat (VNTR) typing methods to characterize 490 M. bovis isolates from livestock (cattle, n=230; pig n=1) and wildlife species (n=259) originating from different farms and regions in South Africa, with the aim to further establish the genetic diversity of the isolates, study the population structure of M. bovis and elucidate the extent of interspecies transmission of bovine tuberculosis. A total of ten spoligotype patterns were identified, two of which were novel (SB2199 and SB2200) and reported for the first time in the literature, while VNTR typing revealed a total of 97 VNTR profiles. Our results showed evidence of clonal expansion for some ancestral strains as well as co-infections with two or three M. bovis strains on some of the cattle and game farms, which suggested independent introductions of infected animals from epidemiologically unrelated sources. Five spoligotypes and nine VNTR profiles were shared between cattle and wildlife. Our findings showed that besides cattle, at least 16 different animal species in South Africa are infected with bovine tuberculosis, and highlight a strong evidence of inter and intra-species transmission of M. bovis. Infection of the blue wildebeest (Connochaetes taurinus) with M. bovis is described for the first time in this report. This update in epidemiological information raises concerns that bovine tuberculosis has increased its spatial distribution in South Africa and is also affecting an increasing number of wildlife species compared to ten years ago.

An assessment of zoonotic and production limiting pathogens in rusa deer (Cervus timorensis rusa) from Mauritius.

A population of approximately 70,000 rusa deer (Cervus timorensis russa) represents the most important mammal species reared for food on the island of Mauritius, being the main source of red meat for the local population. However, very limited information is available on the circulation of pathogens affecting the productivity and health of this species. To produce baseline data on the circulation of infectious pathogens in rusa deer under production, a serological survey and/or direct pathogen detection for six selected infectious diseases was undertaken in 2007 in a sample of 53% of the herds reared in semi-free-ranging conditions in hunting estates. Seropositive results were recorded for Johne's disease with an indirect ELISA test (1.7%, n = 351), heartwater with an immunofluorescence antibody test (IFAT) (95.5%, n = 178) and leptospirosis with a Microscopic Agglutination Test (MAT) (25.9%, n = 363). Significant associations were found between seroprevalence to some of the leptospiral serogroups detected (Tarassovi, Pomona, Sejroe and Mini) and age of the animals, animal density or location of the estates (being more prevalent in hotter and more humid areas). In addition, Mycobacterium bovis and M. avium subspecies paratuberculosis were confirmed in two deer carcasses by culture and PCR, respectively. No antibodies against Brucella spp. nor Rift Valley Fever virus were detected with the use of respective indirect ELISA's. The results obtained suggest that the population of rusa deer from Mauritius is exposed to a wide range of pathogens which may affect their productivity. In addition, the results highlight the potential public health risks incurred by deer industry workers and consumers. This survey fills an important gap in knowledge regarding the health of tropical deer meat in Mauritius and justifies the need to implement more regular surveys of selected pathogens in the deer population.

Improving specific disease outcomes through a One Health approach--tuberculosis.

Early discoveries in the field of tuberculosis more than a century ago indicated that the success of disease control in human populations would depend on the success of control measures in animals, and vice versa. Recognising the zoonotic importance of a cattle-derived pathogen was the beginning of the eradication of bovine tuberculosis from most of the cattle population in Europe. It was a costly and resource-intensive process, but a successful one. The resulting near disappearance of zoonotic tuberculosis from the human population exemplifies probably one of the largest One Health successes in medical history. Since that time many advances in tuberculosis diagnosis, vaccinology, molecular epidemiology and immunopathogenetic studies have been made within the disciplinary divides of human and animal health research. More recently, the discovery of similarities in the interactions between the natural hosts and the causative agents of tuberculosis, as well as similarities in the resulting disease consequences, have led to a renewed appraisal of the benefits of collaborative approaches. It is to be hoped that, in the future, the combined body of scientific knowledge will also provide the basis for practical One Health initiatives at community level.

Mycobacterium tuberculosis at the human/wildlife interface in a high TB burden country.

This study reports on an investigation of Mycobacterium tuberculosis cases in mostly captive wild animals using molecular typing tools [Variable Number of Tandem Repeat (VNTR) typing and Restriction Fragment Length Polymorphism typing]. The investigation included cases from (i) the National Zoological Gardens of South Africa (NZG) recorded between 2002 and 2011; (ii) Johannesburg Zoo, where tuberculosis was first diagnosed in 2007 and has since been detected in three antelope species; (iii) a rehabilitation centre for vervet monkeys (Chlorocebus pygerythrus) in which M. tuberculosis was diagnosed in 2008; and (iv) incidental cases in other facilities including a sable antelope (Hippotragus niger), two unrelated cases in chacma baboons (Papio ursinus) (one of which was from a free-ranging troop) and a colony of capuchin monkeys (Cebus capucinus). Identical genetic profiles of the latter three isolates indicate the persistence of a single M. tuberculosis strain in this population since at least 2006. Results of the outbreak investigation in the captive vervet monkey colony indicate that it was caused by two unrelated strains, while all 13 M. tuberculosis isolates from 11 animal species in the NZG showed different VNTR patterns. A substantial increase in tuberculosis cases of 60% was recorded in the NZG, compared with the previous reporting period 1991-2001, and may indicate a countrywide trend of increasing spillover of human tuberculosis to wild animals. South Africa ranks among the countries with the highest-tuberculosis burden worldwide, complicated by an increasing rate of multidrug-resistant strains. Exposure and infection of captive wildlife in this high prevalence setting is therefore a growing concern for wildlife conservation but also for human health through potential spillback.

Towards establishing a rhinoceros-specific interferon-gamma (IFN-γ) assay for diagnosis of tuberculosis.

Mycobacterium bovis is the causal agent of bovine tuberculosis (BTB), with a diverse host range, extending from livestock to domestic and captive wild animals as well as free-ranging wildlife species. In South Africa, BTB is endemic in the Kruger National Park (KNP) and the Hluluwe iMfolozi National Park (HiP), where the high prevalence of M. bovis infections in buffalo herds has led to infection of a number of wildlife species. This has raised concerns about the spillover into the rhinoceros population, a species known to be susceptible to both M. bovis and Mycobacterium tuberculosis, jeopardizing breeding and relocation projects that serve to conserve and protect this species. In view of the advantages of the interferon-gamma (IFN-γ) assay in the diagnosis of BTB in a variety of species worldwide, such an assay has been developed for rhinoceroses by Morar and co-workers in 2007. In this study, this assay was optimized using recombinant eukaryotic rhinoceros IFN-γ and the lower detection limit was calculated to be 0.5 ng/ml. Subsequently, assessing the detection of native rhinoceros IFN-γ protein in whole-blood samples revealed stimulation with each of the mitogens: pokeweed (PWM), phytohaemagglutinin (PHA) & phorbol 12-myristate 13-acetate and calcium ionophore (PMA/CaI), though most prominently with the latter two. In addition, samples collected from 52 clinically healthy rhinoceroses, of presumed negative BTB status, from two different areas in South Africa were used to determine the cut-off value for a negative test result. This was calculated to be 0.10 (OD490 nm ) and as determined in this study is a preliminary recommendation based on IFN-γ responses observed in samples from BTB-free rhinoceroses only.

Facts and dilemmas in diagnosis of tuberculosis in wildlife.

Mycobacterium bovis, causing bovine tuberculosis (BTB), has been recognized as a global threat at the wildlife-livestock-human interface, a clear "One Health" issue. Several wildlife species have been identified as maintenance hosts. Spillover of infection from these species to livestock or other wildlife species may have economic and conservation implications and infection of humans causes public health concerns, especially in developing countries. Most BTB management strategies rely on BTB testing, which can be performed for a range of purposes, from disease surveillance to diagnosing individual infected animals. New diagnostic assays are being developed for selected wildlife species. This review investigates the most frequent objectives and associated requirements for testing wildlife for tuberculosis at the level of individual animals as well as small and large populations. By aligning those with the available (immunological) ante mortem diagnostic assays, the practical challenges and limitations wildlife managers and researchers are currently faced with are highlighted.

Development of a lion-specific interferon-gamma assay.

The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species.

Economics of Helicobacter pylori eradication therapy.

The annual incidence of peptic ulcer disease in developed countries is around one to three per thousand inhabitants. Since the introduction of acid-secretion inhibitors, the indirect costs of this disease, which has a high rate of relapse, have steadily decreased, although direct costs have been increasing. The possibility of healing the patient through Helicobacter pylori eradication has the potential for a huge economic impact considering the long-term cost: benefit ratio. A recent study has shown that H. pylori eradication therapy can save between US$750,000 and US$1,000,000 per year per million inhabitants in western Europe compared to maintenance or episodic therapy. This paper reviews the cost implications of various management strategies for peptic ulcer disease, comparing the cost: benefit ratios of five different treatment regimens.