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 significanthealth risk in domestic cattle, farmed and wild animal species as well as in humans. Wecarried out spoligotyping and variable number of tandem repeat (VNTR) typing methodsto characterize 490 M. bovis isolates from livestock (cattle, n = 230; pig n = 1) and wildlifespecies (n = 259) originating from different farms and regions in South Africa, with the aimto further establish the genetic diversity of the isolates, study the population structureof 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 (SB2199and SB2200) and reported for the first time in the literature, while VNTR typing revealeda total of 97 VNTR profiles. Our results showed evidence of clonal expansion for someancestral strains as well as co-infections with two or three M. bovis strains on some of thecattle and game farms, which suggested independent introductions of infected animalsfrom epidemiologically unrelated sources. Five spoligotypes and nine VNTR profiles wereshared between cattle and wildlife. Our findings showed that besides cattle, at least16 different animal species in South Africa are infected with bovine tuberculosis, andhighlight a strong evidence of inter and intra-species transmission of M. bovis. Infectionof the blue wildebeest (Connochaetes taurinus) with M. bovis is described for the first timein this report. This update in epidemiological information raises concerns that bovinetuberculosis has increased its spatial distribution in South Africa and is also affecting anincreasing number of wildlife species compared to ten years ago.Department of Scienceand Technology and the National Research Foundation of South Africa.http://www.elsevier.com/locate/prevetmedhb201

Evaluation of the discriminatory power of variable number of tandem repeat typing of Mycobacterium bovis isolates from southern Africa

The usefulness of variable number tandem repeat (VNTR) typing based on limited numbers of loci has previously proven inferior compared to IS6110-RFLP typing when applied to the study of the molecular epidemiology of bovine tuberculosis (BTB) in both livestock and wildlife in southern Africa. In this study, the discriminatory power of 29 published VNTR loci in the characterization of 131 Mycobacterium bovis strains isolated predominantly from wildlife and a smaller number from livestock in southern Africa was assessed. Allelic diversities calculated when loci were evaluated on a selected panel of 23 M. bovis isolates with identified varying degrees of genetic relatedness from different geographic origins as well as M. bovis BCG ranged from 0.00 to 0.63. Of the 29 loci tested, 13 were polymorphic (QUB 11a, QUB 11b, QUB 18, ETR-B and -C, Mtub 21, MIRU 16 and 26, ETR-E, QUB 26, MIRU 23, ETR-A, and Mtub 12). In addition, a comparative evaluation of the 13 loci on a panel of 65 isolates previously characterized by IS6110 restriction fragment length polymorphism (RFLP) typing and further evaluation on 41 isolates with no typing history from Kruger National Park (KNP) highlighted that M. bovis from epidemiologically unrelated cases of BTB in different geographic regions can be adequately distinguished. However, there is a need for improvement of the method to fully discriminate between the parental KNP strain and its clones to allow the detection of evolutionary events causing transmission between and within wildlife species.Department of Agriculture and the Department of Science and Technologyhttp://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1865-1682hb2014ab201

Wildlife on the move : a hidden tuberculosis threat to conservation areas and game farms through introduction of untested animals

In South Africa, African buffaloes (Syncerus caffer) are one of the wildlife maintenance hosts for bovine tuberculosis (BTB) and play a key role in the spread of the disease to other wildlife species and potentially back to cattle. We report a trace-back investigation following the diagnosis of BTB in a previously BTB-free provincial game reserve, founded in the early 1990s in the North West Province of South Africa (SA). Using the intradermal tuberculin and interferon gamma tests, we detected Mycobacterium bovis infection in captured African buffaloes intended for sale. Detection of M. bovis was confirmed by culture and PCR. Molecular typing of M. bovis isolates from three African buffaloes revealed spoligotype SB0140 and a variable number of tandem repeat genotypes which had been previously isolated from wildlife in the KwaZulu-Natal Province of SA. Diagnosis of BTB in a previously uninfected buffalo population provides evidence that the disease can be introduced into an ecosystem through the translocation of untested plains game species. We further illustrate how BTB can remain unnoticed for considerable periods of time in free-ranging wildlife populations and emphasize the need for validated diagnostic tests for application in suitable and practical monitoring programs. This is especially important for species with maintenance host potential and those in high demand at game auctions.Department of Science and Technology and the National Research Foundation of South Africa.http://www.jwildlifedis.org/hb2016Veterinary Tropical Disease

Some South African Rubiaceae tree leaf extracts have antimycobacterial activity against pathogenic and non-pathogenic Mycobacterium species

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic M. smegmatis, M. aurum and M. bovis BCG using a two-fold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. MIC values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied and Oxyanthus speciosus had the most promising selectivity index values.The University of Pretoria Institutional Research Theme for Animal and Zoonotic Diseases (IRT-AZD), National Research Foundation (NRF, Grant No 81010) and Medical Research Council (MRC).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-15732016-07-30hb201

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.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1865-1682hb201

Isolation and molecular characterization of Mycobacterium bovis causing pulmonary tuberculosis and epistaxis in a Thoroughbred horse

Background: Tuberculosis caused by Mycobacterium bovis (M. bovis) is very uncommon in horses worldwide. Case presentation: In the current study, an eight-year-old male Thoroughbred in good body condition was admitted to the Equine Clinic at the Onderstepoort Veterinary Academic Hospital in 2005 due to bilateral epistaxis accompanied by coughing. Routine examinations were conducted to determine the cause of the condition. Endoscopic examination revealed the major source of the epistaxis as the trachea, whereas thoracic radiography indicated the presence of a primary pulmonary mass. M. bovis was isolated from a broncho-alveolar lavage (BAL) sample collected. The pulmonary mass reduced in size three months later following an oral administration of enrofloxacin (7.5 mg/kg PO SID). Genetic fingerprinting by spoligotyping identified the M. bovis isolate as spoligotype SB0868 strain. This M. bovis strain type was never described previously in South Africa (SA). This is the first case of M. bovis infection in a horse in SA which has been fully documented including clinical findings, isolation and genetic characterisation of the causative pathogen. Conclusions: This report indicates that horses may contract and harbour M. bovis despite their lower susceptibility compared to other domestic animals. It also suggests that the infection may be more easily contained and eliminated from the host

Molecular characterization of Mycobacterium bovis from livestock and wildlife in South Africa : genetic marker optimization and identification using whole genome sequence data

Bovine tuberculosis (BTB) is a disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, which are bacteria that infect both domestic and wildlife host species. The zoonotic nature of the disease is of great concern globally. Over 50 million animals are infected all over the world, resulting in economic losses of approximately three billion United States dollars per year. Molecular typing techniques have greatly improved knowledge regarding the sources and modes of BTB transmission in many countries including South Africa. The current study was aimed at filling the knowledge gap regarding the epidemiology of the disease in livestock and wildlife species in the country for the purpose of effective control and management (in wildlife) as well as ultimate eradication (in livestock). Identification of a suitable typing technique for potential application in South Africa was the core purpose of the study. In an attempt to determine the manner of transmission within and between animal populations and map the geographical spread of this zoonotic disease, samples were collected from a variety of animal species throughout the country. In Chapter 2, molecular techniques (i.e. Restriction Fragment Length Polymorphism, spoligotyping and variable number tandem repeat) were used to identify and characterize Mycobacterium bovis infecting buffaloes in the Hluhluwe-iMfolozi Park (HiP) and three epidemiologically related game reserves over a 15-year time frame. The findings demonstrated that the majority of the M. bovis infections in buffaloes were as a result of a single strain which has persisted throughout the years with spill-over to other wildlife species. Evidence of further M. bovis infections due to strains previously undetected in the park was also shown. With a view to providing a simple and reliable method suitable for studying the epidemiology of BTB in the country, we describe, in Chapter 3, how we evaluated the utility of known VNTR sequences as epidemiological markers to describe the molecular epidemiology of BTB in South Africa, with special emphasis on the Kruger National Park (KNP) where there is evidence of clonal expansion of the C8 parental cattle strain. A set of VNTR loci that provided sufficient discriminatory power for application in epidemiological studies was identified. Potential use of VNTR typing in exploring evolutionary changes was also highlighted. To further establish the genetic diversity of the isolates and study the population structure of M. bovis, the loci were used to study a larger panel of M. bovis isolates from different regions (Chapter 4). The findings showed that besides cattle, at least 16 animal species contracted the infection, and highlighted a strong evidence of intra and inter-species transmission of M. bovis. Furthermore, the study also showed that despite the fact that a national control programme for BTB is applied in commercial cattle, there is a high diversity of M. bovis persisting in the country, highlighting the importance and need for intensified diagnostic testing and consideration of alternative control measures such as vaccination. Together with results obtained in Chapter 5, the study further indicated the presence of M. bovis infection in previously uninfected (or unknown status) game parks or reserves. Overall, three novel M. bovis strains (i.e. SB1474, SB2199 and SB2200) were identified and their patterns are now available in the international M. bovis database. We also describe for the first time in South Africa, M. bovis infection in a blue wildebeest (Comochaetes taurinus), nyala (Tragelaphus angasii) and giraffe (Giraffa camelopardalis); and a rare case of M. bovis infection causing pulmonary tuberculosis in a horse (Equus ferus caballus) is described in Chapter 6. To improve VNTR typing method for clonally linked isolates from the KNP, we sequenced whole genomes of local M. bovis isolates in Chapter 7. The tandem repeat loci identified were exploited for polymorphisms in M. tuberculosis complex isolates including M. bovis, with two of the five polymorphic loci identified showing potential discriminatory capabilities for clonally linked isolates. The epidemiological information gained in this investigation is particularly important in the context of the establishment of the Greater Limpopo Transfrontier Park (GLTFP) where the potential spread of BTB from Kruger National Park to Limpopo National Park (Mozambique) and Gonarezhou National Park (Zimbabwe) (or vice versa) is of great concern. The availability of the local M. bovis genomes provides an opportunity for future comprehensive sequence comparisons which may assist in further deducing the phylogenetic and evolutionary relationships of M. bovis isolates and efforts towards vaccine developments.Thesis (PhD)--University of Pretoria, 2014.National Research Foundation (NRF)Department of Science and TechnologyDepartment of Agriculture, Forestry and FisheriesUniversity of PretoriaARC-Onderstepoort Veterinary Institute and the Joy Liebenberg TrustVeterinary Tropical DiseasesPhDUnrestricte

Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa

Abstract Background Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa. Results In the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the ‘One Health’ concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match. Conclusions This is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country

The prevalence of bovine tuberculosis in cattle at the wildlife/livestock interface in the Mnisi community, South Africa

Bovine tuberculosis (BTB) is caused by Mycobacterium bovis (M. bovis), of the Mycobacterium tuberculosis complex. The pathogen has a wide host range including humans, wildlife, livestock as well as domestic animals. The disease has great implications in livestock productivity, conservation of endangered wildlife and public health due to its zoonotic potential (Firdessa et al., 2012). Reports have mentioned the endemic levels and potential spread of BTB in South African game reserves, particularly the Kruger National Park (Michel et al., 2006). However, limited to no research has been conducted to investigate the potential spill-back of BTB into livestock, especially in communities' bordering the game reserves at the wildlife/livestock/human interface.Includes bibliographical referencesThis study was funded by the Institute of Tropical Medicine in Antwerp, Belgium (ITM) and University of Pretoria (UP)ab202

Non-tuberculous Mycobacterium species causing mycobacteriosis in farmed aquatic animals of South Africa

Abstract Background Mycobacteriosis caused by non-tuberculous mycobacteria (NTM), is among the most chronic diseases of aquatic animals. In addition, fish mycobacteriosis has substantial economic consequences especially in the aquaculture and fisheries industry as infections may significantly decrease production and trade. Some fish NTM pathogens are highly virulent and zoonotic; as such, infection of aquaria with these pathogens is a public health concern. In this study, we report isolation of nine different NTM species from sixteen aquatic animals including different fish species, frogs and a crocodile. Given the clinical significance of Mycobacterium marinum and its close relation to Mycobacterium tuberculosis, as well as the significance of ESAT 6 and CFP-10 secretion in mycobacterial virulence, we analysed the esxA and esxB nucleotide sequences of M. marinum isolates identified in this study as well as other mycobacteria in the public databases. Results Mycobacterium shimoidei, Mycobacterium marinum, Mycobacterium chelonae, Mycobacterium septicum /M. peregrinum and Mycobacterium porcinum were isolated from gold fish, Guppy, exotic fish species in South Africa, koi and undefined fish, Knysna seahorse, as well Natal ghost frogs respectively, presenting tuberculosis like granuloma. Other NTM species were isolated from the studied aquatic animals without any visible lesions, and these include Mycobacterium sp. N845 T, Mycobacterium fortuitum, a member of the Mycobacterium avium complex, and Mycobacterium szulgai. Phylogenetic analysis of mycobacteria, based on esxA and esxB genes, separated slow growing from rapidly growing mycobacteria as well as pathogenic from non-pathogenic mycobacteria in some cases. Conclusions Isolation of the different NTM species from samples presenting granuloma suggests the significance of these NTM species in causing mycobacteriosis in these aquatic animals. The study also revealed the potential of esxA and esxB sequences as markers for phylogenetic classification of mycobacteria. Observations regarding use of esxA and esxB sequences for prediction of potential pathogenicity of mycobacteria warrants further investigation of these two genes in a study employing NTM species with well-defined pathogenicity