Vaccines to control tuberculosis in cattle.

The age-old cattle disease has resisted rigorous control, but the BCG vaccine may do better.

Optimization and Application of Real-Time qPCR Assays in Detection and Identification of Chlamydiales in Products of Domestic Ruminant Abortion.

Domestic ruminant abortions due to infectious agents represent an important cause of economic losses in the agricultural industry. This study aimed to optimise and apply qPCR assays for detection of Chlamydiales in domestic ruminant abortion cases. Primers and probes for detection of the order Chlamydiales, Chlamydia abortus, Chlamydia pecorum, Parachlamydia acanthamoeba and Waddlia chondrophila were taken from the literature to create one singleplex and two duplex assays and the assays were optimised. Placentitis and pneumonia are pathological lesions associated with Chlamydiales infection. In a previous study, twenty-five clinical cases had pathological lesions of placentitis or pneumonia. These cases were investigated further by application of the qPCR assays in this study. Chlamydiales were detected in 16 cases. C. abortus, P. acanthamoeba and W. chondrophila were detected in bovine; and C. pecorum and W. chondrophila in ovine and caprine cases. Chlamydiales were detected in three previously inconclusive cases. Identification was improved from genus to species level (C. pecorum). Four cases remained inconclusive. In conclusion, detection of Chlamydiales and differentiation to species level was improved. This study reports the first detection of P. acanthamoeba and W. chondrophila in abortion cases in South Africa, indicating a potentially significant role in abortions in this country.

Approaches to increase recovery of bacterial and fungal abortion agents in domestic ruminants.

Abortions in domestic ruminants cause significant economic losses to farmers. Determining the cause of an abortion is important for control efforts, but it can be challenging. All available diagnostic methods in the bacteriology laboratory should be employed in every case due to the many limiting factors (autolysis, lack of history, range of samples) that complicate the investigation process. The purpose of this study was to determine whether the recovery of diagnostically significant isolates from domestic ruminant abortion cases could be increased through the use of a combination of the existing aerobic culture and Brucella selective method with methods that are commonly recommended in the literature reporting abortion investigations. These methods are examination of wet preparations and impression smears stained by the modified Ziehl-Neelsen method, anaerobic, microaerophilic, Leptospira, Mycoplasma and fungal culture. Samples of placenta and aborted foetuses from 135 routine clinical abortion cases of cattle (n = 88), sheep (n = 25) and goats (n = 22) were analysed by the new combination of methods. In 46 cases, bacteria were identified as aetiological agents and in one case a fungus. Isolation of Brucella species increased to 7.4% over two years compared with the previous 10 years (7.3%), as well as Campylobacter jejuni (n = 2) and Rhizopus species (n = 1). Salmonella species (5.9%) and Trueperella pyogenes (4.4%) were also isolated more often. In conclusion, the approach was effective in removing test selection bias in the bacteriology laboratory. The importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by increased isolation of Brucella abortus and isolation of C. jejuni. The combination of methods that yielded the most clinically relevant isolates was aerobic, microaerophilic, Brucella and fungal cultures.

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda.

BACKGROUND: Bovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health. MATERIALS AND METHODS: A cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence. FINDINGS: Of the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle. CONCLUSIONS: This study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.

Zoonotic Tuberculosis - The Changing Landscape.

Despite slow reductions in the annual burden of active human tuberculosis (TB) cases, zoonotic TB (zTB) remains a poorly monitored and an important unaddressed global problem. There is a higher incidence in some regions and countries, especially where close association exists between growing numbers of cattle (the major source of Mycobacterium bovis) and people, many suffering from poverty, and where dairy products are consumed unpasteurised. More attention needs to be focused on possible increased zTB incidence resulting from growth in dairy production globally and increased demand in low income countries in particular. Evidence of new zoonotic mycobacterial strains in South Asia and Africa (e.g. M. orygis), warrants urgent assessment of prevalence, potential drivers and risk in order to develop appropriate interventions. Control of M. bovis infection in cattle through detect and cull policies remain the mainstay of reducing zTB risk, whilst in certain circumstances animal vaccination is proving beneficial. New point of care diagnostics will help to detect animal infections and human cases. Given the high burden of human tuberculosis (caused by M. tuberculosis) in endemic areas, animals are affected by reverse zoonosis, including multi-drug resistant strains. This, may create drug resistant reservoirs of infection in animals. Like COVID-19, zTB is evolving in an ever-changing global landscape.

Tracing cross species transmission of Mycobacterium bovis at the wildlife/livestock interface in South Africa.

BACKGROUND: Bovine tuberculosis (bTB) affects cattle and wildlife in South Africa with the African buffalo (Syncerus caffer) as the principal maintenance host. The presence of a wildlife maintenance host at the wildlife/livestock interface acting as spill-over host makes it much more challenging to control and eradicate bTB in cattle. Spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) genotyping methods were performed to investigate the genetic diversity of Mycobacterium bovis (M. bovis) isolates from cattle and wildlife, their distribution and transmission at the wildlife/livestock interface in northern Kwa-Zulu Natal (KZN), South Africa. RESULTS: SB0130 was identified as the dominant spoligotype pattern at this wildlife/livestock interface, while VNTR typing revealed a total of 29 VNTR profiles (strains) in the KZN province signifying high genetic variability. The detection of 5 VNTR profiles shared between cattle and buffalo suggests M. bovis transmission between species. MIRU-VNTR confirmed co-infection in one cow with three strains of M. bovis that differed at a single locus, with 2 being shared with buffalo, implying pathogen introduction from most probably unrelated wildlife sources. CONCLUSION: Our findings highlight inter and intra species transmission of bTB at the wildlife/livestock interface and the need for the implementation of adequate bTB control measures to mitigate the spread of the pathogen responsible for economic losses and a public health threat.

Wildlife-cattle interactions emerge as drivers of bovine tuberculosis in traditionally farmed cattle.

Bovine tuberculosis (bTB) in cattle is recognized to be associated with several risk factors that include herd size, cattle movement, ownership of other domestic animals, confinement of cattle in enclosures at night, water sources, communal grazing area and proximity to wildlife, especially bTB maintenance hosts. A questionnaire survey was used to investigate the risk factors associated with Mycobacterium bovis (M. bovis) infection and transmission in traditionally farmed cattle at the wildlife/livestock interface in uMkhanyakude district, northern Kwa-Zulu Natal (KZN), South Africa. The questionnaire comprised of semi-structured questions that were used to gather data on livestock management practices and knowledge about bTB from 71 respondents from households that owned either bTB infected cattle herds or uninfected herds. Multiple correspondence analysis (MCA) was used to explore the association between the risk factors for M. bovis transmission and the bTB herd status. Bovine TB positive herds were associated with a herd size of n > 15, movement of cattle to areas adjacent to the game parks for grazing, cattle grazing inside the game parks as well as cattle sharing water and pasture with wildlife. The multivariable logistic regression model identified movement of animals to areas adjacent to the game parks and cattle sharing water with wildlife as highly significant risk factors for bTB infection in cattle. The findings of this study emphasized the need for the implementation of bTB control strategies in both cattle and wildlife populations for the successful control of the disease.

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.

Detection of native interferon-γ in nyala (Tragelaphus angasii): Towards diagnosing tuberculosis.

Mycobacterium bovis is the main cause of tuberculosis in wildlife. In South Africa, African buffaloes (Syncerus caffer) are a wildlife maintenance host while a number of other species are considered spillover hosts. Nyala (Tragelaphus angasii), a large antelope species from Southern Africa, is frequently traded and can be infected with M. bovis. Interferon gamma (IFN-γ) release assays that detect cell-mediated immune (CMI) responses to M. bovis infection have shown promise in elephants, rhinoceroses and buffaloes. The BOVIGAM® assay is a commercial IFN-γ release assay designed to detect tuberculosis in cattle and has been validated in buffaloes. We tested the suitability of the BOVIGAM® assay to detect native IFN-γ release in nyala. Blood samples collected from 17 nyalas were stimulated with different mitogens and IFN-γ release measured. We found that incubating whole blood with phorbol 12-myristate 13-acetate and calcium ionophore (PMA/CaI) resulted in the highest levels of IFN-y release. Samples stimulated with tuberculin purified protein derivatives of M. bovis (PPDb) and M. avium (PPDa) did not show significant IFN-γ production. An intradermal tuberculin test (IDT) and culture of tissues from 15 of the 17 culled nyala were also performed, which supported the findings of the BOVIGAM® assay, suggesting the potential value of this assay for the diagnosis of tuberculosis in nyala.

Risk Factors for Zoonotic Tuberculosis at the Wildlife-Livestock-Human Interface in South Africa.

A cross-sectional study was conducted to investigate the risk factors associated with zoonotic tuberculosis in humans and its transmission to people living at the wildlife-livestock-human interface. A questionnaire was administered to collect information on food consumption habits, food handling practices, and knowledge of zoonotic TB. Sputum samples were also collected from 150 individuals that belonged to households of cattle farmers with or without a bTB infected herd. In addition, 30 milk samples and 99 nasal swabs were randomly collected from cattle in bTB infected herds for isolation of Mycobacterium bovis (M. bovis). The sputum samples were screened for TB using the GeneXpert test and this was followed by mycobacterial culture and speciation using molecular techniques. No M. bovis was isolated from TB positive sputum samples and only one sample was confirmed as Mycobacterium tuberculosis (M. tuberculosis). M. bovis was isolated from 6.6% (n = 2/30) milk samples and 9% (n = 9/99) of nasal swabs. Ownership of a bTB infected herd and consumption of milk were recognized as highly significant risk factors associated with a history of TB in the household using multiple correspondence analysis (MCA) and logistic regression. The findings from this study have confirmed the potential for zoonotic TB transmission via both unpasteurized milk and aerosol thus, the role of M. bovis in human TB remains a concern for vulnerable communities.

First detection of Mycobacterium bovis infection in Giraffe (Giraffa camelopardalis) in the Greater Kruger National Park Complex: Role and implications.

Bovine tuberculosis (bovine TB) caused by Mycobacterium bovis has become endemic in some wildlife populations in South Africa. The disease has been reported in 21 wildlife species in the country. In this study, we report M. bovis infection in two female giraffes (Giraffa camelopardalis) from two different nature reserves within the Greater Kruger National Park Complex (GKNPC). Mycobacterium bovis was isolated from tissue lesions consistent with macroscopic appearance of tuberculosis (TB) and confirmed by polymerase chain reactions (PCRs), targeting the RD4 region of difference on the genome of the isolates. Spoligotyping and variable number of tandem repeat (VNTR) typing revealed infection of one giraffe with a strain (SB0294) previously not detected in South Africa, while a resident M. bovis strain (SB0121) was detected from the other giraffe. Our work is first to report M. bovis infection in free-ranging giraffes in South Africa. We have further demonstrated the existence of at least three genetically unrelated strains currently infecting wildlife species within the GKNPC. This finding suggests that the epidemiological situation of M. bovis within the GKNPC is not only driven by internal sources from its established endemic presence, but can be additionally fuelled by strains introduced from external sources. It further emphasizes that regular wildlife disease surveillance is an essential prerequisite for the timely identification of new pathogens or strains in ecospheres of high conservation value.

Genetic profiling of Mycobacterium bovis strains from slaughtered cattle in Eritrea.

Mycobacterium bovis (M.bovis) is the main causative agent for bovine tuberculosis (BTB) and can also be the cause of zoonotic tuberculosis in humans. In view of its zoonotic nature, slaughterhouse surveillance, potentially resulting in total or partial condemnation of the carcasses and organs, is conducted routinely. Spoligotyping, VNTR profiling, and whole genome sequencing (WGS) of M. bovis isolated from tissues with tuberculosis-like lesions collected from 14 cattle at Eritrea's largest slaughterhouse in the capital Asmara, were conducted.The 14 M. bovis isolates were classified into three different spoligotype patterns (SB0120, SB0134 and SB0948) and six VNTR profiles. WGS results matched those of the conventional genotyping methods and further discriminated the six VNTR profiles into 14 strains. Furthermore, phylogenetic analysis of the M. bovis isolates suggests two independent introductions of BTB into Eritrea possibly evolving from a common ancestral strain in Europe.This molecular study revealed the most important strains of M. bovis in Eritrea and their (dis)similarities with the strains generally present in East Africa and Europe, as well as potential routes of introduction of M. bovis. Though the sample size is small, the current study provides important information as well as platform for future in-depth molecular studies on isolates from both the dairy and the traditional livestock sectors in Eritrea and the region. This study provides information onthe origin of some of the M. bovis strains in Eritrea, its genetic diversity, evolution and patterns of spread between dairy herds. Such information is essential in the development and implementation of future BTB control strategy for Eritrea.

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

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.

Mycobacterium komaniense sp. nov., a rapidly growing non-tuberculous Mycobacterium species detected in South Africa.

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020T (=CIP 110823T=ATCC BAA-2758).

Experimental Mycobacterium bovis infection in three white rhinoceroses (Ceratotherium simum): Susceptibility, clinical and anatomical pathology.

Tuberculosis caused by Mycobacterium bovis is endemic in the African buffalo (Syncerus caffer) population in the Kruger National Park and other conservation areas in South Africa. The disease has been diagnosed in a total of 21 free ranging or semi-free ranging wildlife species in the country with highly variable presentations in terms of clinical signs as well as severity and distribution of tuberculous lesions. Most species are spillover or dead-end hosts without significant role in the epidemiology of the disease. White rhinoceroses (Ceratotherium simum) are translocated from the Kruger National Park in substantial numbers every year and a clear understanding of their risk to manifest overt tuberculosis disease and to serve as source of infection to other species is required. We report the findings of experimental infection of three white rhinoceroses with a moderately low dose of a virulent field isolate of Mycobacterium bovis. None of the animals developed clinical signs or disseminated disease. The susceptibility of the white rhinoceros to bovine tuberculosis was confirmed by successful experimental infection based on the ante mortem isolation of M. bovis from the respiratory tract of one rhinoceros, the presence of acid-fast organisms and necrotizing granulomatous lesions in the tracheobronchial lymph nodes and the detection of M. bovis genetic material by PCR in the lungs of two animals.

Tuberculosis Infection: Occurrence and Risk Factors in Presumptive Tuberculosis Patients of the Serengeti Ecosystem in Tanzania.

BACKGROUND: Cross-species tuberculosis (TB) transmission between humans and animals has been reported for quite a long time in sub-Saharan Africa. Because humans and animals coexist in the same ecosystem, exploring their potential for cross-species transmission and the impact the disease may have on the health of humans, animals, and their products is critical. OBJECTIVES: This study aimed to identify risk factors for transmission of TB (Mycobacterium tuberculosis) and to assess the potential for zoonotic TB (Mycobacterium bovis) transmission in the Serengeti ecosystem where humans and animals are in intense contact. Our aim is to create a base for future implementation of appropriate control strategies to limit infection in both humans and animals. METHODOLOGY: We administered a semi-structured questionnaire to 421 self-reporting patients to gather information on risk factors and TB occurrence. In a parallel study, researchers screened sputum smears using Ziehl-Neelsen staining and confirmed by mycobacterial culture. We then performed descriptive statistics (Pearson's chi-square test) and logistic regression analysis to establish frequencies, association, and quantification of the risk factors associated with TB cases. RESULTS: Our findings showed 44% (95% confidence interval [CI], 0.40-0.49) of the results were positive from sputum samples collected over a 1-year duration in areas with a high TB burden, particularly the Bunda district, followed by the Serengeti and Ngorongoro districts. Of the culture-positive patients who also had infections other than TB (43/187 patients), 21 (49%) were HIV positive. Contact with livestock products (odds ratio [OR] 6.0; 95% CI, 1.81-19.9), infrequent milk consumption (OR 2.5; 95% CI, 1.42-4.23), cigarette smoking (OR 2.9; 95% CI, 1.19-7.1.2), and alcohol consumption (OR 2.3; 95% CI, 1.22-4.23) were associated with a higher likelihood of TB infection. CONCLUSION: There was no evidence of direct cross-species transmission of either M tuberculosis or M bovis between humans and animals using the study methods. The absence of cross-species TB transmission could be due to limited chances of contact rather than an inability of cross-species disease transmission. In addition, not all people with presumptive TB are infected with TB, and therefore control strategies should emphasise confirming TB status before administering anti-TB drugs.

DIAGNOSIS AND IMPLICATIONS OF MYCOBACTERIUM BOVIS INFECTION IN BANDED MONGOOSES (MUNGOS MUNGO) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

Bovine tuberculosis (bTB) was first diagnosed in the Kruger National Park (KNP) in 1990. Research has since focused on the maintenance host, the African buffalo ( Syncerus caffer ) and clinically affected lion ( Panthera leo ). However, little is known about the role of small predators in tuberculosis epidemiology. During 2011-12, we screened banded mongooses ( Mungos mungo ) in the bTB high-prevalence zone of the KNP for Mycobacterium tuberculosis complex members. Fecal swabs, tracheal swabs, and tracheal lavages of 76 banded mongooses caught in cage traps within a 2-km radius of Skukuza Rest Camp were submitted for Mycobacterium culture, isolation, and species identification. Lesions and lymph node samples collected from 12 animals at postmortem examination were submitted for culture and histopathology. In lung and lymph nodes of two banded mongooses, well demarcated, irregularly margined, gray-yellow nodules of up to 5 mm diameter were identified with either central necrosis or calcification, characterized on histopathology as caseating necrosis with epithelioid macrophages or necrogranuloma with calcified centre. No acid fast bacteria were identified with Ziehl-Neelsen stain. We isolated Mycobacterium bovis from lung, lymph node, and liver samples, as well as from tracheal lavages and tracheal swab from the same two banded mongooses. Blood samples were positive by ElephantTB STAT-PAK® Assay for 12 and Enferplex™ TB Assay for five animals. Only the two banded mongooses positive on pathology and M. bovis culture were positive on both serologic assays. We provide evidence of bTB infection in banded mongooses in the KNP, demonstrate their ability to shed M. bovis , and propose a possible antemortem diagnostic algorithm. Our findings open the discussion around possible sources of infection and their significance at the human/wildlife interface in and around Skukuza.

Mapping of Mycobacterium tuberculosis Complex Genetic Diversity Profiles in Tanzania and Other African Countries.

The aim of this study was to assess and characterize Mycobacterium tuberculosis complex (MTBC) genotypic diversity in Tanzania, as well as in neighbouring East and other several African countries. We used spoligotyping to identify a total of 293 M. tuberculosis clinical isolates (one isolate per patient) collected in the Bunda, Dar es Salaam, Ngorongoro and Serengeti areas in Tanzania. The results were compared with results in the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Genotyping and phylogeographical analyses highlighted the predominance of the CAS, T, EAI, and LAM MTBC lineages in Tanzania. The three most frequent Spoligotype International Types (SITs) were: SIT21/CAS1-Kili (n = 76; 25.94%), SIT59/LAM11-ZWE (n = 22; 7.51%), and SIT126/EAI5 tentatively reclassified as EAI3-TZA (n = 18; 6.14%). Furthermore, three SITs were newly created in this study (SIT4056/EAI5 n = 2, SIT4057/T1 n = 1, and SIT4058/EAI5 n = 1). We noted that the East-African-Indian (EAI) lineage was more predominant in Bunda, the Manu lineage was more common among strains isolated in Ngorongoro, and the Central-Asian (CAS) lineage was more predominant in Dar es Salaam (p-value<0.0001). No statistically significant differences were noted when comparing HIV status of patients vs. major lineages (p-value = 0.103). However, when grouping lineages as Principal Genetic Groups (PGG), we noticed that PGG2/3 group (Haarlem, LAM, S, T, and X) was more associated with HIV-positive patients as compared to PGG1 group (Beijing, CAS, EAI, and Manu) (p-value = 0.03). This study provided mapping of MTBC genetic diversity in Tanzania (containing information on isolates from different cities) and neighbouring East African and other several African countries highlighting differences as regards to MTBC genotypic distribution between Tanzania and other African countries. This work also allowed underlining of spoligotyping patterns tentatively grouped within the newly designated EAI3-TZA lineage (remarkable by absence of spacers 2 and 3, and represented by SIT126) which seems to be specific to Tanzania. However, further genotyping information would be needed to confirm this specificity.

Longevity of Mycobacterium bovis in Raw and Traditional Souring Milk as a Function of Storage Temperature and Dose.

BACKGROUND: Unpasteurised fresh and souring dairy products form an essential component of household diets throughout many rural communities in southern Africa. The presence of milk-borne zoonotic pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis and zoonotic tuberculosis in humans, constitute a public health threat, especially in remote areas with poor disease surveillance in livestock and highly compromised human health due to HIV/AIDS. METHODS: In this study we used culture to determine the longevity of M. bovis in experimentally inoculated fresh and naturally souring milk obtained from communal cattle in the KwaZulu-Natal province of South Africa. The effect of bacterial load and storage temperature on the survival of M. bovis was evaluated by spiking mixtures of fresh milk and starter soured milk (aMasi) culture with three concentrations of bacteria (10(2), 10(4), 10(7) colony forming units/ml), followed by incubation under controlled laboratory conditions that mimicked ambient indoor (20°C) and outdoor (33°C) temperatures and periodic sampling and testing over time (0-56 days). RESULTS: M. bovis cultured from samples of the fresh and souring milk was identified by PCR analysis. At the highest spiking concentration (10(7) cfu/ml), M. bovis survived for at least 2 weeks at 20°C; but, at all concentrations in the 33°C treatment, M. bovis was absent by three days after inoculation. Logistic regression analysis was used to assess the effects of bacterial concentration and time since inoculation, as well as determine the potential half-life of M. bovis in raw souring milk. Given the most favourable tested conditions for bacterial survival (20°C), approximately 25% of mycobacteria were alive after one day of storage (95% CI: 9-53%), giving an estimated half-life of M. bovis in raw souring milk of approximately 12 hours (95% CI: 7-27 hours). CONCLUSIONS: This study demonstrates that M. bovis may survive in fresh and souring milk for periods of time that represent a risk of exposure to people consuming these products, as well as domestic or wild animal populations that have reported opportunities to consume homemade unpasteurised dairy products. The temperature at which the milk is soured and stored substantially affects the survival time of M. bovis.