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Background: Mycobacterium bovis (M. bovis) is the causative agent of animal tuberculosis (TB) which poses a threat to many of South Africa's most iconic wildlife species, including leopards (Panthera pardus). Due to limited tests for wildlife, the development of accurate ante-mortem tests for TB diagnosis in African big cat populations is urgently required. The aim of this study was to evaluate currently available immunological assays for their ability to detect M. bovis infection in leopards. Methods: Leopard whole blood (n=19) was stimulated using the QuantiFERON Gold Plus In-Tube System (QFT) to evaluate cytokine gene expression and protein production, along with serological assays. The GeneXpert® MTB/RIF ® Ultra (GXU ) qPCR assay, mycobacterial culture, and speciation by genomic regions of difference PCR, was used to confirm M. bovis infection in leopards. Results: Mycobacterium bovis infection was confirmed in six leopards and individuals that were tuberculin skin test (TST) negative were used for ® comparison. The GXU assay was positive using all available tissue homogenates (n=5) from M. bovis culture positive animals. Mycobacterium bovis culture-confirmed leopards had greater antigen-specific responses, in the QFT interferon gamma release assay, CXCL9 and CXCL10 gene expression assays, compared to TST-negative individuals. One M. bovis culture-confirmed leopard had detectable antibodies using the DPP® Vet TB assay. Conclusion: Preliminary results demonstrated that immunoassays and TST may be ® potential tools to identify M. bovis-infected leopards. The GXU assay provided rapid direct detection of infected leopards. Further studies should aim to improve TB diagnosis in wild felids, which will facilitate disease surveillance and screening. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis (M. bovis) infection has been identified in both domestic and wild animals and may threaten the conservation of vulnerable species including African lions (Panthera leo). There is a need to develop accurate antemortem tools for detection of M. bovis infection in African big cat populations for wildlife management and disease surveillance. The aim of this study was to compare the performances of two immunological assays, the QuantiFERON®-TB Gold Plus (QFT) Mabtech Cat interferon gamma release assay (IGRA) and QFT CXCL9 gene expression assay (GEA), which have both shown diagnostic potential for M. bovis detection in African lions. Lion whole blood (n=47), stimulated using the QFT platform, was used for measuring antigen-specific CXCL9 expression and IFN-g production and to assign M. bovis infection status. A subset (n=12) of mycobacterial culture-confirmed M. bovis infected and uninfected African lions was used to compare the agreement between the immunological diagnostic assays. There was no statistical difference between the proportions of test positive African lions tested by the QFT Mabtech Cat IGRA compared to the QFT CXCL9 GEA. There was also a moderate association between immunological diagnostic assays when numerical results were compared. The majority of lions had the same diagnostic outcome using the paired assays. Although the QFT Mabtech Cat IGRA provides a more standardized, commercially available, and cost-effective test compared to QFT CXCL9 GEA, using both assays to categorize M. bovis infection status in lions will increase confidence in results. [ABSTRACT FROM AUTHOR]

African wild dogs (Lycaon pictus) have undergone severe population reductions and are listed as endangered on the International Union for Conservation of Nature Red List. Small, isolated populations have the potential to suffer from threats to their genetic diversity that may impact species viability and future survival. This study provides the first set of population-wide genomic data to address conservation concerns for this endangered species. Whole genome sequencing data were generated for 71 free-ranging African wild dogs from the Kruger National Park (KNP), South Africa, and used to estimate important population genomic parameters. Genomic diversity metrics revealed that variation levels were low; however, this African wild dog population showed low levels of inbreeding. Very few first- and second-order relationships were observed in this cohort, with most relationships falling into the third-order or distant category. Patterns of homozygosity could have resulted from historical inbreeding or a loss in genome variation due to a population bottleneck. Although the results suggest that this stronghold African wild dog population maintains low levels of inbreeding, likely due to their cooperative breeding system, it may lead to a continuous population decline when a reduced number of suitable mates are available. Consequently, the low genomic variation may influence species viability over time. This study highlights the importance of assessing population genomic parameters to set conservation priorities. Future studies should include the investigation of the potential of this endangered species to adapt to environmental changes considering the low genomic diversity in this population. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis (M. bovis) infection in wildlife, including lions (Panthera leo), has implications for individual and population health. Tools for the detection of infected lions are needed for diagnosis and disease surveillance. This study aimed to evaluate the Mabtech Cat interferon gamma (IFN-γ) ELISABasic kit for detection of native lion IFN-γ in whole blood samples stimulated using the QuantiFERON® TB Gold Plus (QFT) platform as a potential diagnostic assay. The ELISA was able to detect lion IFN-γ in mitogen-stimulated samples, with good parallelism, linearity, and a working range of 15.6–500 pg/mL. Minimal matrix interference was observed in the recovery of domestic cat rIFN-γ in lion plasma. Both intra- and inter-assay reproducibility had a coefficient of variation less than 10%, while the limit of detection and quantification were 7.8 pg/mL and 31.2 pg/mL, respectively. The diagnostic performance of the QFT Mabtech Cat interferon gamma release assay (IGRA) was determined using mycobacterial antigen-stimulated samples from M. bovis culture-confirmed infected (n = 8) and uninfected (n = 4) lions. A lion-specific cut-off value (33 pg/mL) was calculated, and the sensitivity and specificity were determined to be 87.5% and 100%, respectively. Although additional samples should be tested, the QFT Mabtech Cat IGRA could identify M. bovis-infected African lions. [ABSTRACT FROM AUTHOR]

Tuberculous meningitis (TBM) is the most severe form of extrapulmonary tuberculosis (TB) that arises when a caseating meningeal granuloma discharges its contents into the subarachnoid space. It accounts for ~1% of all disease caused by Mycobacterium tuberculosis and the age of peak incidence is from 2–4 years. The exact pathogenesis of TBM is still not fully understood and the mechanism(s) by which the bacilli initially invade the blood-brain-barrier are still to be elucidated. This study investigated the involvement of the host genome in TBM susceptibility, by considering common variants (minor allele frequency (MAF) >5%) using microarray genotyping and rare variants (MAF <1%) via exome sequencing. A total of 123 TBM cases, 400 pulmonary TB (pTB) cases and 477 healthy controls were genotyped on the MEGA array. A genome-wide association study (GWAS) comparing 114 TBM cases to 395 healthy controls showed no association with TBM susceptibility. A second analysis comparing 114 TBM cases to 382 pTB cases was conducted to investigate variants associated with different TB phenotypes. No significant associations were found with progression from pTB to TBM. Ten TBM cases and 10 healthy controls were exome sequenced. Gene set association tests SKAT-O and SKAT Common Rare were used to assess the association of rare SNPs and the cumulative effect of both common and rare SNPs with susceptibility to TBM, respectively. Ingenuity Pathway Analysis (IPA) of the top-hits of the SKAT-O analysis showed that NOD2 and CYP4F2 are both important in TBM pathogenesis and highlighted these as targets for future study. For the SKAT Common Rare analysis Centriolar Coiled-Coil Protein 110 (CCP110) was nominally associated (p = 5.89x10−6) with TBM susceptibility. In addition, several top-hit genes ascribed to the development of the central nervous system (CNS) and innate immune system regulation were identified. Exome sequencing and GWAS of our TBM cohort has identified a single previously undescribed association of CCP110 with TBM susceptibility. These results advance our understanding of TBM in terms of both variants and genes that influence susceptibility. In addition, several candidate genes involved in innate immunity have been identified for further genotypic and functional investigation. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi‐host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species‐specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB‐endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi‐species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free‐ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis‐infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB‐endemic ecosystems and evaluate serological and other diagnostic tools in this species. [ABSTRACT FROM AUTHOR]

In South Africa, mycobacterial culture is regarded as the gold standard for the detection of Mycobacterium tuberculosis complex (MTBC) infection in wildlife even though it is regarded as "imperfect." We compared a novel decontamination and mycobacterial culture technique (TiKa) to the conventional mycobacterium growth indicator tube (MGIT) system using known amounts of bacilli and clinical samples from MTBC-infected African buffaloes (Syncerus caffer), white rhinoceros (Ceratotherium simum), and African elephants (Loxodonta africana). Use of the TiKa-KiC decontamination agent on samples spiked with 10,000 to 10 colony forming units (cfu) of M. bovis (SB0121) and M. tuberculosis (H37Rv) had no effect on isolate recovery in culture. In contrast, decontamination with MGIT MycoPrep resulted in no growth of M. bovis samples at concentrations < 1,000 cfu and M. tuberculosis samples < 100 cfu. Subsequently, we used the TiKa system with stored clinical samples (various lymphatic tissues) collected from wildlife and paucibacillary bronchoalveolar lavage fluid, trunk washes, and endotracheal tube washes from 3 species with known MTBC infections. Overall, MTBC recovery by culture was improved significantly (p < 0.01) by using TiKa compared to conventional MGIT, with 54 of 57 positive specimens versus 25 of 57 positive specimens, respectively. The TiKa mycobacterial growth system appears to significantly enhance the recovery of MTBC members from tissue and paucibacillary respiratory samples collected from African buffaloes, African elephants, and white rhinoceros. Moreover, the TiKa system may improve success of MTBC culture from various sample types previously deemed unculturable from other species. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis (M. bovis) infects a wide range of wildlife species and has recently been discovered in the endangered African wild dog (Lycaon pictus). This study aimed to characterize the epidemiology of tuberculosis (TB) in wild dogs in endemic areas of South Africa. We describe 12 TB cases in wild dogs from Kruger National Park (KNP), Hluhluwe–iMfolozi Park (HiP) and a private facility in Hoedspruit from 2015 to 2017. Spoligotyping was used to identify the disease‐causing M. bovis strain in these cases, and whole‐genome sequencing was performed on 5 M. bovis isolates (KNP = 2 and HiP = 3) to investigate genomic diversity as well as the relationship to other isolates found in these geographical areas. Three distinct strain types were responsible for the M. bovis infections in this species. The SB0121 strain was observed in wild dogs from KNP, whereas SB0130 was responsible for infection in wild dogs from HiP. A novel strain, SB2681, was also identified in the HiP wild dogs. Whole‐genome sequence analysis suggests that different infection sources exist among these wild dogs and that inter‐species transmission most likely occurred between wildlife predators and prey located within shared geographical areas. This study highlights the importance of regular disease surveillance to identify and characterize potential threats for successful control of infection and protection of endangered species. [ABSTRACT FROM AUTHOR]

Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis , is a complex disease. The risk of developing active TB is in part determined by host genetic factors. Most genetic studies investigating TB susceptibility fail to replicate association signals particularly across diverse populations. South African populations arose because of multi-wave genetic admixture from the indigenous KhoeSan, Bantu-speaking Africans, Europeans, Southeast Asian-and East Asian populations. This has led to complex genetic admixture with heterogenous patterns of linkage disequilibrium and associated traits. As a result, precise estimation of both global and local ancestry is required to prevent both false positive and false-negative associations. Here, 820 individuals from South Africa were genotyped on the SNP-dense Illumina Multi-Ethnic Genotyping Array (∼1.7M SNPs) followed by local and global ancestry inference using RFMix. Local ancestry adjusted allelic association (LAAA) models were utilized owing to the extensive genetic heterogeneity present in this population. Hence, an interaction term, comprising the identification of the minor allele that corresponds to the ancestry present at the specific locus under investigation, was included as a covariate. One SNP (rs28647531) located on chromosome 4q22 was significantly associated with TB susceptibility and displayed a SNP minor allelic effect (G allele, frequency = 0.204) whilst correcting for local ancestry for Bantu-speaking African ancestry (p -value = 5.518 × 10−7; OR = 3.065; SE = 0.224). Although no other variants passed the significant threshold, clear differences were observed between the lead variants identified for each ancestry. Furthermore, the LAAA model robustly captured the source of association signals in multi-way admixed individuals from South Africa and allowed the identification of ancestry-specific disease risk alleles associated with TB susceptibility that have previously been missed. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes. [ABSTRACT FROM AUTHOR]

Genomes retain records of demographic changes and evolutionary forces that shape species and populations. Remnant populations of African buffalo (Syncerus caffer) in South Africa, with varied histories, provide an opportunity to investigate signatures left in their genomes by past events, both recent and ancient. Here, we produce 40 low coverage (7.14×) genome sequences of Cape buffalo (S. c. caffer) from four protected areas in South Africa. Genome-wide heterozygosity was the highest for any mammal for which these data are available, while differences in individual inbreeding coefficients reflected the severity of historical bottlenecks and current census sizes in each population. PSMC analysis revealed multiple changes in Ne between approximately one million and 20 thousand years ago, corresponding to paleoclimatic changes and Cape buffalo colonisation of southern Africa. The results of this study have implications for buffalo management and conservation, particularly in the context of the predicted increase in aridity and temperature in southern Africa over the next century as a result of climate change. [ABSTRACT FROM AUTHOR]

Several genetic studies have implicated genes that encode for components of the innate immune response in tuberculosis (TB) susceptibility. The complement system is an early player in the innate immune response and provides the host with initial protection by promoting phagocytosis of apoptotic or necrotic cells. The C1q molecule is the first component of the classical pathway that leads to the activation of complement by binding to immune complexes and is encoded by the C1Q gene cluster. We investigated variants in this region to determine its association with TB susceptibility. Five single nucleotide polymorphisms (SNPs) (rs12033074, rs631090, rs172378, rs587585, and rs665691) were genotyped using TaqMan® SNP assays in 456 TB cases and 448 healthy controls and analysed by logistic regression models. The rs587585 variant showed a significant additive allelic association where the minor G allele was found more frequently in TB cases than in controls in both the discovery (p = 0.023; OR = 1.30; 95% CI, 1.04–1.64) and validation cohort (p = 0.038; OR = 1.31; 95% CI, 1.22–1.40). In addition, we detected increased C1qA expression when comparing cases and controls (p = 0.037) and linked this to a dosage effect of the G allele, which increased C1qA expression in TB cases. This is the first study to report the association of C1Q gene polymorphisms with progression to tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Bovine tuberculosis and tuberculosis are chronic infectious diseases caused by the Mycobacterium tuberculosis complex members, Mycobacterium bovis and Mycobacterium tuberculosis, respectively. Infection with M. bovis and M. tuberculosis have significant implications for wildlife species management, public health, veterinary disease control, and conservation endeavours. Results: Here we describe the first use of the VetMAX™ Mycobacterium tuberculosis complex (MTBC) DNA quantitative real-time polymerase chain reaction (qPCR) detection kit for African wildlife samples. DNA was extracted from tissues harvested from 48 African buffaloes and MTBC DNA was detected (test-positive) in all 26 M. bovis culture-confirmed animals with an additional 12 PCR-positive results in culture-negative buffaloes (originating from an exposed population). Of six MTBC-infected African rhinoceros tested, MTBC DNA was detected in antemortem and postmortem samples from five animals. The PCR was also able to detect MTBC DNA in samples from two African elephants confirmed to have M. bovis and M. tuberculosis infections (one each). Culture-confirmed uninfected rhinoceros and elephants' samples tested negative in the PCR assay. Conclusions: These results suggest this new detection kit is a sensitive screening test for the detection of MTBC-infected African buffaloes, African elephants and white rhinoceros. [ABSTRACT FROM AUTHOR]

Background: The X-linked recessive primary immunodeficiency disease (PIDD) Wiskott-Aldrich syndrome (WAS) is identified by an extreme susceptibility to infections, eczema and thrombocytopenia with microplatelets. The syndrome, the result of mutations in the WAS gene which encodes the Wiskott-Aldrich protein (WASp), has wide clinical phenotype variation, ranging from classical WAS to X-linked thrombocytopaenia and X-linked neutropaenia. In many cases, the diagnosis of WAS in first affected males is delayed, because patients may not present with the classic signs and symptoms, which may intersect with other thrombocytopenia causes. Case presentation: Here, we describe a three-year-old HIV negative boy presenting with recurrent infections, skin rashes, features of autoimmunity and atopy. However, platelets were initially reported as normal in numbers and morphology as were baseline immune investigations. An older male sibling had died in infancy from suspected immunodeficiency. Uncertainty of diagnosis and suspected severe PIDD prompted urgent further molecular investigation. Whole exome sequencing identified c. 397 G > A as a novel hemizygous missense mutation located in exon 4 of WAS. Conclusion: With definitive molecular diagnosis, we could target treatment and offer genetic counselling and prenatal diagnostic testing to the family. The identification of novel variants is important to confirm phenotype variations of a syndrome. [ABSTRACT FROM AUTHOR]

Integrating biological processes across scales remains a central challenge in disease ecology. Genetic variation drives differences in host immune responses, which, along with environmental factors, generates temporal and spatial infection patterns in natural populations that epidemiologists seek to predict and control. However, genetics and immunology are typically studied in model systems, whereas population-level patterns of infection status and susceptibility are uniquely observable in nature. Despite obvious causal connections, organizational scales from genes to host outcomes to population patterns are rarely linked explicitly. Here we identify two loci near genes involved in macrophage (phagocyte) activation and pathogen degradation that additively increase risk of bovine tuberculosis infection by up to ninefold in wild African buffalo. Furthermore, we observe genotype-specific variation in IL-12 production indicative of variation in macrophage activation. Here, we provide measurable differences in infection resistance at multiple scales by characterizing the genetic and inflammatory variation driving patterns of infection in a wild mammal. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is endemic in Kruger National Park, South Africa, home to the largest population of white rhinoceros (Ceratotherium simum) in the world. In 2016, the first cases of naturally occurring bTB were reported in white rhinoceros; however, there is a lack of understanding of infection and disease process in this species. Prevention and control of transmission depends on the availability of accurate tools to detect M. bovis infection. Interferon gamma (IFN-γ) assays are a reliable detection method for TB in other animal species, and studies have indicated that these tests can be used in white rhinoceros. We sought to screen and optimize a commercial IFN-γ enzyme-linked immunosorbent assay (ELISA) to detect endogenous white rhinoceros IFN-γ in mitogen-stimulated whole blood as a basis for developing a test for M. bovis infection. Optimizations included identifying ELISA antibodies and determining the effect of sample matrix, ELISA plate incubation temperature, ELISA linearity, assay reproducibility, and the assay's limit of quantification. The optimized assay employed an equine IFN-γ antibody pair that was used to create a commercial ELISA kit. This ELISA had a linear response to recombinant equine and endogenous rhinoceros IFN-γ (range: 7.8–125 pg/mL). When incubated at 37°C, the ELISA was highly reproducible, with an optimal recovery and a low limit of quantification, indicating that the Mabtech equine IFN-γ ELISAPRO kit is a robust assay for measuring white rhinoceros IFN-γ. [ABSTRACT FROM AUTHOR]

Molecular typing techniques are useful in understanding tuberculosis epidemiology; yet, they have been under-utilised at the human-animal interface in Nigeria. Sixty-four Mycobacterium tuberculosis complex (MTBC) isolates including 42 M. tuberculosis, 13 M. bovis and nine M. africanum obtained from livestock workers (LW, n = 47) and their cattle (n = 17) in three geographical zones of Nigeria were genotyped to identify and evaluate the genetic diversity of the circulating MTBC using spoligotyping. Distribution into clades of M. tuberculosis revealed; 45.3% Uganda I- [SIT46- cattle: 1; LW: 28], 14.1% Latin American Mediterranean- [SIT61, cattle: 1; LW: 8], and 1.6% T- [SIT53—LW: 1]. The M. bovis strains were 6.3% SB0944 [cattle: 4] and 1.6% each of SB0300, SB1026, SB1027 and SB1439 [cattle: 4]. Seventeen MTBC isolates [cattle: 7; LW: 10] yielded 14 new spoligotype patterns including three M. tuberculosis strains (three isolates), five M. bovis strains (five isolates) and six M. africanum strains (nine isolates), two of which belonged to MAF1. Only few families namely, the not previously described Uganda I-, LAM and SB0944 are predominant among the LW and cattle, with other types in lower prevalences. The strain population structure indicates an intriguing diversity and possible zoonotic linkage with consequences for TB control in the country. The need to employ newer molecular techniques such as Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats and whole genome sequence to decipher circulating MTBC strains in Nigeria is advocated. [ABSTRACT FROM AUTHOR]

Genotype imputation is a powerful tool for increasing statistical power in an association analysis. Meta-analysis of multiple study datasets also requires a substantial overlap of SNPs for a successful association analysis, which can be achieved by imputation. Quality of imputed datasets is largely dependent on the software used, as well as the reference populations chosen. The accuracy of imputation of available reference populations has not been tested for the five-way admixed South African Colored (SAC) population. In this study, imputation results obtained using three freely-accessible methods were evaluated for accuracy and quality. We show that the African Genome Resource is the best reference panel for imputation of missing genotypes in samples from the SAC population, implemented via the freely accessible Sanger Imputation Server. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), caused by Mycobacterium tuberculosis , is a complex disease with a known human genetic component. Males seem to be more affected than females and in most countries the TB notification rate is twice as high in males than in females. While socio-economic status, behavior and sex hormones influence the male bias they do not fully account for it. Males have only one copy of the X chromosome, while diploid females are subject to X chromosome inactivation. In addition, the X chromosome codes for many immune-related genes, supporting the hypothesis that X-linked genes could contribute to TB susceptibility in a sex-biased manner. We report the first TB susceptibility genome-wide association study (GWAS) with a specific focus on sex-stratified autosomal analysis and the X chromosome. A total of 810 individuals (410 cases and 405 controls) from an admixed South African population were genotyped using the Illumina Multi Ethnic Genotyping Array, specifically designed as a suitable platform for diverse and admixed populations. Association testing was done on the autosome (8,27,386 variants) and X chromosome (20,939 variants) in a sex stratified and combined manner. SNP association testing was not statistically significant using a stringent cut-off for significance but revealed likely candidate genes that warrant further investigation. A genome wide interaction analysis detected 16 significant interactions. Finally, the results highlight the importance of sex-stratified analysis as strong sex-specific effects were identified on both the autosome and X chromosome. [ABSTRACT FROM AUTHOR]

Background: The immune response against tuberculosis in lions is still poorly defined and our understanding is hampered by the lack of lion specific reagents. The process for producing antibodies against a specific antigen is laborious and not available to many research laboratories. As the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine, we have investigated the use of commercially available antibodies to characterize T cell subsets in African lions (Panthera leo). Results: Commercially available antibodies were screened and investigated the influence of two different sample processing methods, as well as the effect of time delay on cell surface marker expression on lion lymphocytes. Using commercially available antibodies, we were able to identify CD4+, CD5+, CD8+, CD14+, CD25+, CD44+ and CD45+ T lymphocytes in samples obtained by density gradient centrifugation as well as red cell lysis of lion whole blood. Two distinct lymphocyte populations, which differed in size and phenotype, were observed in the samples processed by density gradient centrifugation. Conclusion: Commercially available antibodies are able to differentiate between T lymphocyte subsets including immune effector cells in African lion whole blood, and possibly give insight into unique specie phenotypes. [ABSTRACT FROM AUTHOR]

Disease acts as a powerful driver of evolution in natural host populations, yet individuals in a population often vary in their susceptibility to infection. Energetic trade‐offs between immune and reproductive investment lead to the evolution of distinct life history strategies, driven by the relative fitness costs and benefits of resisting infection. However, examples quantifying the cost of resistance outside of the laboratory are rare. Here, we observe two distinct forms of resistance to bovine tuberculosis (bTB), an important zoonotic pathogen, in a free‐ranging African buffalo (Syncerus caffer) population. We characterize these phenotypes as "infection resistance," in which hosts delay or prevent infection, and "proliferation resistance," in which the host limits the spread of lesions caused by the pathogen after infection has occurred. We found weak evidence that infection resistance to bTB may be heritable in this buffalo population (h2 = 0.10) and comes at the cost of reduced body condition and marginally reduced survival once infected, but also associates with an overall higher reproductive rate. Infection‐resistant animals thus appear to follow a "fast" pace‐of‐life syndrome, in that they reproduce more quickly but die upon infection. In contrast, proliferation resistance had no apparent costs and was associated with measures of positive host health—such as having a higher body condition and reproductive rate. This study quantifies striking phenotypic variation in pathogen resistance and provides evidence for a link between life history variation and a disease resistance trait in a wild mammalian host population. Here, we identify two distinct forms of tuberculosis resistance that come at distinct fitness costs in a wild population of African buffalo. This empirical evidence directly supports previous theoretical work in evolutionary trade‐offs and provides a novel example of differences in pace‐of‐life life history evolution in a wild mammal. Furthermore, we present a novel method for the estimation of heritability using marker‐based relatedness estimates. [ABSTRACT FROM AUTHOR]

Background: Bovine tuberculosis (bTB) caused by Mycobacterium bovis has previously been diagnosed in warthogs and infection can be highly prevalent (> 30%) in endemic areas. Thus, warthogs could potentially be an important species to consider as sentinels for disease surveillance. However, disease surveillance is dependent on availability of accurate diagnostic assays and only a few diagnostic tests have been investigated for warthogs. Furthermore, the tests that have been used in this species require laboratory equipment and trained personnel to obtain results. Therefore, this study investigated the use of the intradermal tuberculin test (ITT) to screen warthogs for bTB, which can be done with minimal equipment and under field conditions by most veterinarians and other qualified professionals. Changes in skin fold thickness measurements at the bovine purified protein derivative (PPD) administration site, between 0 and 72 h, were compared with differential changes between the bovine and avian PPD sites, for 34 warthogs, to evaluate the performance when different interpretation criteria for the ITT was used. Results: Using an increase of 1.8 mm or more at the bovine PPD site as a cut-off for positive responders, 69% of 16 M. bovis culture-positive warthogs had a positive test result, with 100% of the 18 culture-negative warthogs considered as test negative. When a differential of 1.2 mm or more in skin fold thickness at the bovine PPD compared to the avian PPD site was used as a cut-off for the comparative ITT, 81% of culture-positive warthogs were considered as test positive, with 100% of culture-negative warthogs considered as test negative. Conclusion: The findings in this study suggest that the ITT is a promising tool to use when screening warthogs for M. bovis infection. [ABSTRACT FROM AUTHOR]

Rationale: Acquired resistance is an important driver of multidrug-resistant tuberculosis (TB), even with good treatment adherence. However, exactly what initiates the resistance and how it arises remain poorly understood.Objectives: To identify the relationship between drug concentrations and drug susceptibility readouts (minimum inhibitory concentrations [MICs]) in the TB cavity.Methods: We recruited patients with medically incurable TB who were undergoing therapeutic lung resection while on treatment with a cocktail of second-line anti-TB drugs. On the day of surgery, antibiotic concentrations were measured in the blood and at seven prespecified biopsy sites within each cavity. Mycobacterium tuberculosis was grown from each biopsy site, MICs of each drug identified, and whole-genome sequencing performed. Spearman correlation coefficients between drug concentration and MIC were calculated.Measurements and Main Results: Fourteen patients treated for a median of 13 months (range, 5-31 mo) were recruited. MICs and drug resistance-associated single-nucleotide variants differed between the different geospatial locations within each cavity, and with pretreatment and serial sputum isolates, consistent with ongoing acquisition of resistance. However, pretreatment sputum MIC had an accuracy of only 49.48% in predicting cavitary MICs. There were large concentration-distance gradients for each antibiotic. The location-specific concentrations inversely correlated with MICs (P < 0.05) and therefore acquired resistance. Moreover, pharmacokinetic/pharmacodynamic exposures known to amplify drug-resistant subpopulations were encountered in all positions.Conclusions: These data inform interventional strategies relevant to drug delivery, dosing, and diagnostics to prevent the development of acquired resistance. The role of high intracavitary penetration as a biomarker of antibiotic efficacy, when assessing new regimens, requires clarification. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis infection in wildlife species occurs worldwide. However, few cases of M. bovis infection in captive elephants have been reported. We describe 2 incidental cases of bovine tuberculosis in free-ranging African elephants (Loxodonta africana) from a tuberculosis-endemic national park in South Africa and the epidemiologic implications of these infections. [ABSTRACT FROM AUTHOR]

Natural history studies of tuberculosis (TB) have revealed a spectrum of clinical outcomes after exposure to Mycobacterium tuberculosis, the cause of TB. Not all individuals exposed to the bacteriumwill become diseased and depending on the infection pressure, many will remain infection-free. Intriguingly, complete resistance to infection is observed in some individuals (termed resisters) after intense, continuing M. tuberculosis exposure. After successful infection, the majority of individuals will develop latent TB infection (LTBI). This infection state is currently (and perhaps imperfectly) defined by the presence of a positive tuberculin skin test (TST) and/or interferon gamma release assay (IGRA), but no detectable clinical disease symptoms. The majority of healthy individuals with LTBI are resistant to clinical TB, indicating that infection is remarkably well-contained in these non-progressors. The remaining 5-15% of LTBI positive individuals will progress to active TB. Epidemiological investigations have indicated that the host genetic component contributes to these infection and disease phenotypes, influencing both susceptibility and resistance. Elucidating these genetic correlates is therefore a priority as it may translate to new interventions to prevent, diagnose or treat TB. The most successful approaches in resistance/susceptibility investigation have focused on specific infection and disease phenotypes and the resister phenotype may hold the key to the discovery of actionable genetic variants in TB infection and disease. This review will not only discuss lessons from epidemiological studies, but will also focus on the contribution of epidemiology and functional genetics to human genetic resistance to M. tuberculosis infection and disease. [ABSTRACT FROM AUTHOR]

Background: South Africa has the highest tuberculosis incidence globally (781/100,000), with an estimated 4.3% of cases being rifampicin resistant (RR). Control and elimination strategies will require detailed spatial information to understand where drug-resistant tuberculosis exists and why it persists in those communities. We demonstrate a method to enable drug-resistant tuberculosis monitoring by identifying high-burden communities in the Western Cape Province using routinely collected laboratory data.Methods and Findings: We retrospectively identified cases of microbiologically confirmed tuberculosis and RR-tuberculosis from all biological samples submitted for tuberculosis testing (n = 2,219,891) to the Western Cape National Health Laboratory Services (NHLS) between January 1, 2008, and June 30, 2013. Because the NHLS database lacks unique patient identifiers, we performed a series of record-linking processes to match specimen records to individual patients. We counted an individual as having a single disease episode if their positive samples came from within two years of each other. Cases were aggregated by clinic location (n = 302) to estimate the percentage of tuberculosis cases with rifampicin resistance per clinic. We used inverse distance weighting (IDW) to produce heatmaps of the RR-tuberculosis percentage across the province. Regression was used to estimate annual changes in the RR-tuberculosis percentage by clinic, and estimated average size and direction of change was mapped. We identified 799,779 individuals who had specimens submitted from mappable clinics for testing, of whom 222,735 (27.8%) had microbiologically confirmed tuberculosis. The study population was 43% female, the median age was 36 years (IQR 27-44), and 10,255 (4.6%, 95% CI: 4.6-4.7) cases had documented rifampicin resistance. Among individuals with microbiologically confirmed tuberculosis, 8,947 (4.0%) had more than one disease episode during the study period. The percentage of tuberculosis cases with rifampicin resistance documented among these individuals was 11.4% (95% CI: 10.7-12.0). Overall, the percentage of tuberculosis cases that were RR-tuberculosis was spatially heterogeneous, ranging from 0% to 25% across the province. Our maps reveal significant yearly fluctuations in RR-tuberculosis percentages at several locations. Additionally, the directions of change over time in RR-tuberculosis percentage were not uniform. The main limitation of this study is the lack of unique patient identifiers in the NHLS database, rendering findings to be estimates reliant on the accuracy of the person-matching algorithm.Conclusions: Our maps reveal striking spatial and temporal heterogeneity in RR-tuberculosis percentages across this province. We demonstrate the potential to monitor RR-tuberculosis spatially and temporally with routinely collected laboratory data, enabling improved resource targeting and more rapid locally appropriate interventions. [ABSTRACT FROM AUTHOR]

Objective: To investigate the distribution of Mycobacterium tuberculosis genotypes across Africa. Methods: The SITVIT2 global repository and PUBMED were searched for spoligotype and published genotype data respectively, of M. tuberculosis from Africa. M. tuberculosis lineages in Africa were described and compared across regions and with those from 7 European and 6 South-Asian countries. Further analysis of the major lineages and sub-lineages using Principal Component analysis (PCA) and hierarchical cluster analysis were done to describe clustering by geographical regions. Evolutionary relationships were assessed using phylogenetic tree analysis. Results: A total of 14727 isolates from 35 African countries were included in the analysis and of these 13607 were assigned to one of 10 major lineages, whilst 1120 were unknown. There were differences in geographical distribution of major lineages and their sub-lineages with regional clustering. Southern African countries were grouped based on high prevalence of LAM11-ZWE strains; strains which have an origin in Portugal. The grouping of North African countries was due to the high percentage of LAM9 strains, which have an origin in the Eastern Mediterranean region. East African countries were grouped based on Central Asian (CAS) and East-African Indian (EAI) strain lineage possibly reflecting historic sea trade with Asia, while West African Countries were grouped based on Cameroon lineage of unknown origin. A high percentage of the Haarlem lineage isolates were observed in the Central African Republic, Guinea, Gambia and Tunisia, however, a mixed distribution prevented close clustering. Conclusions: This study highlighted that the TB epidemic in Africa is driven by regional epidemics characterized by genetically distinct lineages of M. tuberculosis. M. tuberculosis in these regions may have been introduced from either Europe or Asia and has spread through pastoralism, mining and war. The vast array of genotypes and their associated phenotypes should be considered when designing future vaccines, diagnostics and anti-TB drugs. [ABSTRACT FROM AUTHOR]

A majority of emerging infectious diseases in humans are zoonoses. Understanding factors that influence the emergence and transmission of zoonoses is pivotal for their prevention and control. Toxoplasma gondii is one of the most widespread zoonotic pathogens known today. Whereas only a few genotypes of T. gondii dominate in the Northern Hemisphere, many genotypes coexist in South America. Furthermore, T. gondii strains from South America are more likely to be virulent than those from the Northern Hemisphere. However, it is not clear what factor(s) shaped modern-day genetic diversity and virulence of T. gondii. Here, our analysis suggests that the rise and expansion of farming in the past 11,000 years established the domestic cat/mouse transmission cycle for T. gondii, which has undoubtedly played a significant role in the selection of certain linages of T. gondii. Our mathematical simulations showed that within the domestic transmission cycle, intermediately mouse-virulent T. gondii genotypes have an adaptive advantage and eventually become dominant due to a balance between lower host mortality and the ability to superinfect mice previously infected with a less virulent T. gondii strain. Our analysis of the global type II lineage of T. gondii suggests its Old World origin but recent expansion in North America, which is likely the consequence of global human migration and trading. These results have significant implications concerning transmission and evolution of zoonotic pathogens in the rapidly expanding anthropized environment demanded by rapid growth of the human population and intensive international trading at present and in the future. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis is a zoonotic disease with largely unknown impact in Africa, with risk factors such as HIV and direct contact with animals or consumption of Mycobacterium bovis infected animal products. In order to understand and quantify this risk and design intervention strategies, good epidemiological studies are needed. Such studies can include molecular typing of M. bovis isolates. The aim of this study was to apply these tools to provide novel information concerning the distribution of bovine tuberculosis in cattle in Mozambique and thereby provide relevant information to guide policy development and strategies to contain the disease in livestock, and reduce the risk associated with transmission to humans. A collection of 178 M. bovis isolates was obtained from cattle in Mozambique. Using spoligotyping and regions of difference analysis, we classified the isolates into clonal complexes, thus reporting the first characterisation of M. bovis strains in this region. Data from MIRU-VNTR typing was used to compare isolates from a number of African countries, revealing a deeply geographically structured diversity of M. bovis. Eastern Africa appears to show high diversity, suggesting deep evolution in that region. The diversity of M. bovis in Africa does not seem to be a function of recent importation of animals, but is probably maintained within each particular region by constant reinfection from reservoir animals. Understanding the transmission routes of M. bovis in Mozambique and elsewhere is essential in order to focus public health and veterinary resources to contain bovine tuberculosis. [ABSTRACT FROM AUTHOR]

Primary immunodeficiency disorders (PIDs) render patients vulnerable to infection with a wide range of microorganisms and thus provide good in vivo models for the assessment of immune responses during infectious challenges. Priming of the immune system, especially in infancy, depends on different environmental exposures and medical practices. This may determine the timing and phenotype of clinical appearance of immune deficits as exemplified with early exposure to Bacillus Calmette-Guérin (BCG) vaccination and dissemination in combined immunodeficiencies. Varied phenotype expression poses a challenge to identification of the putative immune deficit. Without the availability of genomic diagnosis and data analysis resources and with limited capacity for functional definition of immune pathways, it is difficult to establish a definitive diagnosis and to decide on appropriate treatment. This study describes the use of exome sequencing to identify a homozygous recessive variant in MAP3K14, NIKVal345Met, in a patient with combined immunodeficiency, disseminated BCG-osis, and paradoxically elevated lymphocytes. Laboratory testing confirmed hypogammaglobulinemia with normal CD19, but failed to confirm a definitive diagnosis for targeted treatment decisions. NIKVal345Met is predicted to be deleterious and pathogenic by two in silico prediction tools and is situated in a gene crucial for effective functioning of the non-canonical nuclear factor-kappa B signaling pathway. Functional analysis of NIKVal345Met - versus NIKWT-transfected human embryonic kidney-293T cells showed that this mutation significantly affects the kinase activity of NIK leading to decreased levels of phosphorylated IkappaB kinase-alpha (IKKα), the target of NIK. BCG-stimulated RAW264.7 cells transfected with NIKVal345Met also presented with reduced levels of phosphorylated IKKα, significantly increased p100 levels and significantly decreased p52 levels compared to cells transfected with NIKWT. Ideally, these experiments would have been conducted in patient-derived immune cells, but we were unable to source these cells from the patient. The functional analysis described in this paper supports previous illustrations of the importance of NIK in human immune responses and demonstrates the involvement of function-altering mutations in MAP3K14 in PIDs. The genomic approach used for this patient demonstrates its value in the diagnosis of an unusual PID and as a tool for detecting rarer mutations to help guide treatment approaches. [ABSTRACT FROM AUTHOR]

The distinguishing factors that characterize the host response to infection with virulentMycobacterium tuberculosis(M.tb) are largely confounding. We present an infection study with 2 genetically closely relatedM.tbstrains that have vastly different pathogenic characteristics. The early host response to infection with these detergent-free cultured strains was analyzed through RNAseq in an attempt to provide information on the subtleties which may ultimately contribute to the virulent phenotype. Murine bone marrow derived macrophages (BMDMs) were infected with either a hyper- (R5527) or hypovirulent (R1507) BeijingM. tuberculosisclinical isolate. RNAseq revealed 69 differentially expressed host genes in BMDMs during comparison of these 2 transcriptomes. Pathway analysis revealed activation of the stress-induced and growth inhibitory Gadd45 signaling pathway in hypervirulent infected BMDMs. Upstream regulators of interferon activation such as and IRF3 and IRF7 were predicted to be upregulated in hypovirulent-infected BMDMs. Additional analysis of the host immune response through ELISA and qPCR included the use of human THP-1 macrophages where a robust proinflammatory response was observed after infection with the hypervirulent strain. RNAseq revealed 2 early-response genes (ier3andsaa3) and 2 host-defense genes (oasl1andslpi) that were significantly upregulated by the hypervirulent strain. The role of these genes underM.tbinfection conditions are largely unknown but here we provide validation of their presence with use of qPCR and Western blot. Further analysis into their biological role during infection with virulentM.tbis required. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

As scavengers, spotted hyenas (Crocuta crocuta) are exposed to a wide array of pathogens but exhibit low mortality rates due to infectious disease. This suggests that this species exhibits a unique and robust immune response to pathogens. However, few tools exist to measure cell-mediated immunity (CMI) in hyenas and we aimed to develop a gene expression assay to quantify antigen-specific responses. Whole blood from fiveMycobacterium bovis- sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genesACTB,GAPDH,YWHAZandTBPin these samples was determined as well as the mean fold change in the expression ofIFNG,CXCL8,CXCL9,CXCL10andCXCL11inM. bovis-antigen stimulated blood. The expression ofYWHAZandTBPshowed greatest stability, andYWHAZwas selected as a reference for further analysis. The expression ofCXCL9andCXCL11showed greatest upregulation in antigen-stimulated blood and the assay results for these genes were strongly correlated. The measurement of antigen-inducedCXCL9andCXCL11expression, relative to that ofYWHAZ, can be used to measureCMIresponses to infectious diseases in spotted hyenas. [ABSTRACT FROM PUBLISHER]

Background. Xpert MTB/RIF (Xpert) detects rifampicin-resistant tuberculosis (RR-tuberculosis), enabling physicians to rapidly initiate a World Health Organization-recommended 5-drug regimen while awaiting second-line drug-susceptibility test (DST) results. We quantified the second-line DST results time and proportion of patients potentially placed on suboptimal therapy. Methods. We included RR-tuberculosis patients detected using Xpert at the South African National Health Laboratory Services (NHLS) of the Western Cape between November 2011 and June 2013 and at Eastern Cape, Free State, and Gauteng NHLS between November 2012 and December 2013. We calculated time from specimen collection to phenotypic second-line DST results. We identified isoniazid and ethionamide resistance mutations on line probe assay and performed pyrazinamide sequencing. Results. Among 1332 RR-tuberculosis patients, only 44.7% (596) had second-line DST for both fluoroquinolones and second-line injectable: 55.8% (466 of 835) in the Western Cape and 26.2% (130 of 497) in the other provinces. Patients with smear negative disease and age ≤10 years were less likely to have a result (risk ratio [RR] = 0.72; 95% CI, 0.64-0.81 and RR = 0.49; 95% CI, 0.26-0.79). Median time to second-line DST was 53 days (range, 8-259). Of the 252 patients with complete second-line DST, 101 (40.1%) potentially initiated a suboptimal regimen: 46.8% in the Western Cape and 25.3% in the other provinces. Conclusions. Many South Africans diagnosed with RR-tuberculosis by Xpert initiate a suboptimal regimen, with information to adjust therapy available in half of all patients after a median 7 weeks. Algorithm completion and time delays remain challenging. [ABSTRACT FROM AUTHOR]

Utilizing data from published tuberculosis (TB) genome-wide association studies (GWAS), we use a bioinformatics pipeline to detect all polymorphisms in linkage disequilibrium (LD) with variants previously implicated in TB disease susceptibility. The probability that these variants had a predicted regulatory function was estimated using RegulomeDB and Ensembl’s Variant Effect Predictor. Subsequent genotyping of these 133 predicted regulatory polymorphisms was performed in 400 admixed South African TB cases and 366 healthy controls in a population-based case-control association study to fine-map the causal variant. We detected associations between tuberculosis susceptibility and six intronic polymorphisms located in MARCO, IFNGR2, ASHAS2, ACACA, NISCH and TLR10. Our post-GWAS approach demonstrates the feasibility of combining multiple TB GWAS datasets with linkage information to identify regulatory variants associated with this infectious disease. [ABSTRACT FROM AUTHOR]

Background: Trichohepatoenteric syndrome (THE-S) or phenotypic diarrhoea of infancy is a rare autosomal recessive disorder characterised by severe infantile diarrhoea, facial dysmorphism, immunodeficiency and woolly hair. It was first described in 1982 in two infants with intractable diarrhoea, liver cirrhosis and abnormal hair structure on microscopy. We report on two siblings from a consanguineous family of Somali descent who, despite extensive clinical investigation, remained undiagnosed until their demise. The index patient died of fulminant cytomegalovirus pneumonitis at 3 months of age. Methods: Whole exome sequencing (WES) was performed on a premortem DNA sample from the index case. Variants in a homozygous recessive state or compound heterozygous state were prioritized as potential candidate variants using TAPER™. Sanger sequencing was done to genotype the parents, unaffected sibling and a deceased sibling for the variant of interest. Results: Exome sequencing identified a novel homozygous mutation (c.4507C > T, rs200067423) in TTC37 which was confirmed by Sanger sequencing in the index case. The identification of this mutation led to the diagnosis of THE-S in the proband and the same homozygous variant was confirmed in a male sibling who died 4 years earlier with severe chronic diarrhoea of infancy. The unaffected parents and sister were heterozygous for the identified variant. Conclusions: WES permitted definitive genetic diagnosis despite an atypical presentation in the index case and suggests that severe infection, likely secondary to immunodeficiency, may be a presenting feature. In addition definitive molecular diagnosis allows for genetic counseling and future prenatal diagnosis, and demonstrates the value of WES for post-mortem diagnosis of disorders with a non-specific clinical presentation in which a Mendelian cause is suspected. [ABSTRACT FROM AUTHOR]

Background: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. Results: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. Conclusion: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals. [ABSTRACT FROM AUTHOR]

Background: The African buffalo (Syncerus caffer) is an important role player in the savannah ecosystem. It has become a species of relevance because of its role as a wildlife maintenance host for an array of infectious and zoonotic diseases some of which include corridor disease, foot-and-mouth disease and bovine tuberculosis. To date, no complete genome sequence for S. caffer had been available for study and the genomes of other species such as the domestic cow (Bos taurus) had been used as a proxy for any genetics analysis conducted on this species. Here, the high coverage genome sequence of the African buffalo (S. caffer) is presented. Results: A total of 19,765 genes were predicted and 19,296 genes could be successfully annotated to S. caffer while 469 genes remained unannotated. Moreover, in order to extend a detailed annotation of S. caffer, gene clusters were constructed using twelve additional mammalian genomes. The S. caffer genome contains 10,988 gene clusters, of which 62 are shared exclusively between B. taurus and S. caffer. Conclusions: This study provides a unique genomic perspective for the S. caffer, allowing for the identification of novel variants that may play a role in the natural history and physiological adaptations. [ABSTRACT FROM AUTHOR]

An accurate knowledge of anatomy, especially natural variation within individuals, is of vital clinical importance. Cadaver dissection during anatomical training may be a valuable introduction to pathology for undergraduate students, which can contribute greatly to a successful medical career. The purpose of this study was to determine the extent and type of pathology lesions in a cadaver population ( n = 127) used for medical dissection. This was done to gauge whether sufficient pathology lesions representative of all the organ systems were present in the cadaver population to warrant the use of cadavers as an additional pathology learning resource. This study demonstrated a wide variety of pathology lesions in different organ systems. The respiratory system was most affected with pulmonary tuberculosis (TB) lesions being the most common finding (seen in 76% of cadavers) followed by bronchopneumonia and emphysema. Other common pathology findings included atherosclerosis, colonic diverticula, and chronic pyelonephritis. Skeletal fractures and degenerative joint disease were also noted. This study shows that cadaveric dissection offers a chance to alert and expose students to a wide variety of gross pathology and histopathology. It has been suggested that most medical students will practice in primary health care and as such more attention should be given to the pathology of commonly encountered diseases. This is particularly true for developing countries, where diseases such as TB are commonly encountered. The integration of pathology into the dissection hall may therefore be beneficial to student learning while simultaneously optimizing the use of cadaver material. Anat Sci Educ 9: 575-582. © 2016 American Association of Anatomists. [ABSTRACT FROM AUTHOR]

We screened African wild dogs (Lycaon pictus) in Kruger National Park, South Africa, for Mycobacterium bovis infection using an interferon-gamma release assay. We detected M. bovis sensitization in 20 of 21 packs; overall apparent infection prevalence was 83%. These animals experience high infection pressure, which may affect long-term survival and conservation strategies. [ABSTRACT FROM AUTHOR]

During 2016-2017, when Kruger National Park, South Africa, was under quarantine to limit bovine tuberculosis spread, we examined 35 white and 5 black rhinoceroses for infection. We found 6 infected white rhinoceroses during times of nutritional stress. Further research on Mycobacterium bovis pathogenesis in white rhinoceroses is needed. [ABSTRACT FROM AUTHOR]

Background: User-friendly, rapid, inexpensive yet accurate TB diagnostic tools are urgently needed at points of care in resource-limited settings. We investigated host biomarkers detected in serum samples obtained from adults with signs and symptoms suggestive of TB at primary healthcare clinics in five African countries (Malawi, Namibia, South Africa, The Gambia and Uganda), for the diagnosis of TB disease.Methods: We prospectively enrolled individuals presenting with symptoms warranting investigation for pulmonary TB, prior to assessment for TB disease. We evaluated 22 host protein biomarkers in stored serum samples using a multiplex cytokine platform. Using a pre-established diagnostic algorithm comprising of laboratory, clinical and radiological findings, participants were classified as either definite TB, probable TB, questionable TB status or non-pulmonary TB.Results: Of the 716 participants enrolled, 185 were definite and 29 were probable TB cases, 6 had questionable TB disease status, whereas 487 had no evidence of TB. A seven-marker biosignature of C reactive protein, transthyretin, IFN-γ, complement factor H, apolipoprotein-A1, inducible protein 10 and serum amyloid A identified on a training sample set (n=491), diagnosed TB disease in the test set (n=210) with sensitivity of 93.8% (95% CI 84.0% to 98.0%), specificity of 73.3% (95% CI 65.2% to 80.1%), and positive and negative predictive values of 60.6% (95% CI 50.3% to 70.1%) and 96.4% (95% CI 90.5% to 98.8%), respectively, regardless of HIV infection status or study site.Conclusions: We have identified a seven-marker host serum protein biosignature for the diagnosis of TB disease irrespective of HIV infection status or ethnicity in Africa. These results hold promise for the development of a field-friendly point-of-care screening test for pulmonary TB. [ABSTRACT FROM AUTHOR]

A series of 2( 5H)-furanone-based compounds were synthesized from commercially available mucohalic acids. From the first-generation compounds, three showed inhibitory activity (10 µg/mL) of at least 35% against Mycobacterium smegmatis mc2 155 growth (Bioscreen C system). In screening the active first-generation compounds for growth inhibition against Mycobacterium tuberculosis H37Rv, the most active compound was identified with a minimum inhibitory concentration (MIC99) of 8.07 µg/mL (15.8 µM) using BACTEC 460 system. No cross-resistance was observed with some current first-line anti-TB drugs, since it similarly inhibited the growth of multidrug resistant (MDR) clinical isolates. The compound showed a good selectivity for mycobacteria since it did not inhibit the growth of selected Gram-positive and Gram-negative bacteria. It also showed synergistic activity with rifampicin (RIF) and additive activity with isoniazid (INH) and ethambutol (EMB). Additional time-kill studies showed that the compound is bacteriostatic to mycobacteria, but cytotoxic to the Chinese Hamster Ovarian (CHO) cell line. From a second generation library, two compounds showed improved anti-TB activity against M. tuberculosis H37Rv and decreased CHO cell cytotoxicity. The compounds exhibited MIC values of 2.62 µg/mL (5.6 µM) and 3.07 µg/mL (5.6 µM) respectively. The improved cytotoxicity against CHO cell line of the two compounds ranged from IC50 = 38.24 µg/mL to IC50 = 45.58 µg/mL when compared to the most active first-generation compound (IC50 = 1.82 µg/mL). The two second generation leads with selectivity indices (SI) of 14.64 and 14.85 respectively, warrant further development as anti-TB drug candidates. © 2016 IUBMB Life, 68(8):612-620, 2016 [ABSTRACT FROM AUTHOR]

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%–100%) and a specificity of 97% (95% CI, 85%–100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]