Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR

<p>Abstract</p> <p>Background</p> <p>Culture of <it>M. bovis </it>from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of <it>M. bovis </it>by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than <it>M. bovis </it>may be associated with the tissues.</p> <p>Results</p> <p>Previously published IS<it>6110 </it>based PCR diagnostic assays, along with one developed in house, were tested against environmental mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Services Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS<it>6110 </it>based assays. Of the 13 non-tuberculous mycobacteria tested with IS6110_T only <it>M. wolinskyi </it>was positive. Thirty <it>M. bovis </it>infected tissue homogenates and 18 control tissues were used to evaluate the potential for the assay as a diagnostic test. In this small sample, IS6110_T detected 20/30 samples from <it>M. bovis </it>infected animals and 0/18 control tissues.</p> <p>Conclusions</p> <p>The IS6110_T assay provides a PCR based assay system that is compatible with current diagnostic protocols for the detection of <it>M. bovis </it>in the USA and compliments current testing strategies.</p

Mycobacterium bovis: A model pathogen at the interface of livestock, wildlife, and humans

Complex and dynamic interactions involving domestic animals, wildlife, and humans create environments favorable to the emergence of new diseases, or reemergence of diseases in new host species. Today, reservoirs of Mycobacterium bovis, the causative agent of tuberculosis in animals, and sometimes humans, exist in a range of countries and wild animal populations. Free-ranging populations of white-tailed deer in the US, brushtail possum in New Zealand, badger in the Republic of Ireland and the United Kingdom, and wild boar in Spain exemplify established reservoirs of M. bovis. Establishment of these reservoirs is the result of factors such as spillover from livestock, translocation of wildlife, supplemental feeding of wildlife, and wildlife population densities beyond normal habitat carrying capacities. As many countries attempt to eradicate M. bovis from livestock, efforts are impeded by spillback from wildlife reservoirs. It will not be possible to eradicate this important zoonosis from livestock unless transmission between wildlife and domestic animals is halted. Such an endeavor will require a collaborative effort between agricultural, wildlife, environmental, and political interests.Peer Reviewe

Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection

Phylogeny, diversification, and biogeography of a hemiclonal hybrid system of native Australian freshwater fishes (Gobiiformes:Gobioidei: Eleotridae: Hypseleotris)

BACKGROUND: Carp gudgeons (genus Hypseleotris) are a prominent part of the Australian freshwater fish fauna, with species distributed around the western, northern, and eastern reaches of the continent. We infer a calibrated phylogeny of the genus based on nuclear ultraconserved element (UCE) sequences and using Bayesian estimation of divergence times, and use this phylogeny to investigate geographic patterns of diversification with GeoSSE. The southeastern species have hybridized to form hemiclonal lineages, and we also resolve relationships of hemiclones and compare their phylogenetic placement in the UCE phylogeny with a hypothesis based on complete mitochondrial genomes. We then use phased SNPs extracted from the UCE sequences for population structure analysis among the southeastern species and hemiclones. RESULTS: Hypseleotris cyprinoides, a widespread euryhaline species known from throughout the Indo-Pacific, is resolved outside the remainder of the species. Two Australian radiations comprise the bulk of Hypseleotris, one primarily in the northwestern coastal rivers and a second inhabiting the southeastern region including the Murray–Darling, Bulloo-Bancannia and Lake Eyre basins, plus coastal rivers east of the Great Dividing Range. Our phylogenetic results reveal cytonuclear discordance between the UCE and mitochondrial hypotheses, place hemiclone hybrids among their parental taxa, and indicate that the genus Kimberleyeleotris is nested within the northwestern Hypseleotris radiation along with three undescribed species. We infer a crown age for Hypseleotris of 17.3 Ma, date the radiation of Australian species at roughly 10.1 Ma, and recover the crown ages of the northwestern (excluding H. compressa) and southeastern radiations at 5.9 and 7.2 Ma, respectively. Range-dependent diversification analyses using GeoSSE indicate that speciation and extinction rates have been steady between the northwestern and southeastern Australian radiations and between smaller radiations of species in the Kimberley region and the Arnhem Plateau. Analysis of phased SNPs confirms inheritance patterns and reveals high levels of heterozygosity among the hemiclones. CONCLUSIONS: The northwestern species have restricted ranges and likely speciated in allopatry, while the southeastern species are known from much larger areas, consistent with peripatric speciation or allopatric speciation followed by secondary contact. Species in the northwestern Kimberley region differ in shape from those in the southeast, with the Kimberley species notably more elongate and slender than the stocky southeastern species, likely due to the different topographies and flow regimes of the rivers they inhabit

Whole-Genome SNP Analysis Identifies Putative Mycobacterium bovis Transmission Clusters in Livestock and Wildlife in Catalonia, Spain

The high-resolution WGS analyses of MTBC strains have provided useful insight for determining sources of infection for animal tuberculosis. In Spain, tuberculosis in livestock is caused by Mycobacterium bovis and Mycobacterium caprae, where wildlife reservoirs play an important role. We analyzed a set of 125 M. bovis isolates obtained from livestock and wildlife from Catalonia to investigate strain diversity and identify possible sources and/or causes of infection. Whole-genome SNP profiles were used for phylogenetic reconstruction and pairwise SNP distance analysis. Additionally, SNPs were investigated to identify virulence and antimicrobial resistance factors to investigate clade-specific associations. Putative transmission clusters (≤12 SNPs) were identified, and associated epidemiological metadata were used to determine possible explanatory factors for transmission. M. bovis distribution was heterogeneous, with 7 major clades and 21 putative transmission clusters. In order of importance, the explanatory factors associated were proximity and neighborhood, residual infection, livestock-wildlife interaction, shared pasture, and movement. Genes related to lipid transport and metabolism showed the highest number of SNPs. All isolates were pyrazinamide resistant, and five were additionally resistant to isoniazid, but no clade-specific associations could be determined. Our findings highlight the importance of high-resolution molecular surveillance to monitor bovine tuberculosis dynamics in a low-prevalence setting.info:eu-repo/semantics/publishedVersio

Screening of Microbial Volatile Organic Compounds for Detection of Disease in Cattle: Development of Lab-scale Method

The primary hurdle for diagnosis of some diseases is the long incubation required to culture and confirm the presence of bacteria. The concept of using microbial VOCs as “signature markers” could provide a faster and noninvasive diagnosis. Finding biomarkers is challenging due to the specificity required in complex matrices. The objectives of this study were to (1) build/test a lab-scale platform for screening of microbial VOCs and (2) apply it to Mycobacterium avium paratuberculosis; the vaccine strain of M. bovis Bacillus Calmette-Guérin; and M. kansasii to demonstrate detection times greater those typically required for culture. SPME-GC-MS was used for sampling, sample preparation, and analyses. For objective (1), a testing platform was built for headspace sampling of bacterial cultures grown in standard culture flasks via a biosecure closed-loop circulating airflow system. For (2), results show that the suites of VOCs produced by Mycobacteria ssp. change over time and that individual strains produce different VOCs. The developed method was successful in discriminating between strains using a pooled multi-group analysis, and in timepoint-specific multi- and pair-wise comparisons. The developed testing platform can be useful for minimally invasive and biosecure collection of biomarkers associated with human, wildlife and livestock diseases for development of diagnostic point-of-care and field surveillance

Effects of Inactivated \u3ci\u3eMycobacterium bovis\u3c/i\u3e Vaccination on Molokai-Origin Wild Pigs Experimentally Infected with Virulent \u3ci\u3eM. bovis\u3c/i\u3e

The wild pig population on Molokai, Hawaii, USA is a possible reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and has been implicated in decades past as the source of disease for the island’s domestic cattle. Heat-inactivated vaccines have been effective for reducing disease prevalence in wild boar in Spain and could prove useful for managing M. bovis in Molokai wild pigs. We designed an experiment to test this vaccine in wild pigs of Molokai genetics. Fifteen 3–4-month-old pigs were orally administered 106–107 colony forming units (cfu) of heat-inactivated M. bovis (Vaccinates; n = 8; 0.2 mL) or phosphate buffered saline (Controls; n = 7; 0.2 mL). Each dose was administered in a 0.5 mL tube embedded in a fruit candy/cracked corn mix. Boosters were given seven weeks post-prime in the same manner and dose. Nineteen weeks post-prime, pigs were orally challenged with 1 × 106 cfu of virulent M. bovis. Twelve weeks post-challenge, pigs were euthanized and necropsied, at which time 23 different tissues from the head, thorax, and abdomen were collected and examined. Each tissue was assigned a lesion score. Ordinal lesion score data were analyzed using non-zarametric Wilcoxon Signed Rank test. Effect size was calculated using Cohen’s d. Four of eight Vaccinates and four of seven Controls had gross and microscopic lesions, as well as culture-positive tissues. Vaccinates had statistically lower lesion scores than Controls in the following areas: gross thoracic lesion scores (p = 0.013 Cohen’s d = 0.33) and microscopic thoracic lesion scores (p = 0.002, Cohen’s d = 0.39). There were no differences in head lesion scores alone, both gross and microscopic, nor were there differences when comparing combined gross and microscopic head and thoracic lesion scores. These results are indicative that this vaccination protocol affords a modest degree of infection containment with this vaccine in Molokai wild pigs

Increased TNF-α/IFN-γ/IL-2 and Decreased TNF-α/IFN-γ Production by Central Memory T Cells Are Associated with Protective Responses against Bovine Tuberculosis Following BCG Vaccination

Central memory T cells (Tcm) and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector / memory populations from bacille Calmette Guerin (BCG) vaccinated and non-vaccinated calves prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves; only differing in magnitude (i.e., infected > vaccinated). BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (three weeks post-infection), non-vaccinates had greater antigen-specific IFN-γ/TNF-α and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains) were detected in memory subsets, as well as in effector cells, as early as three weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Acknowledgments Some of the figures (Figures 4–6 and Supplementary Material S1) were generated using BioRender and draw.io, respectively. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Wellcome Foundation (grant #222941/Z/21/Z), the South African Medical Research Council, American Association of Zoo Veterinarians Wild Animal Health Fund [S005651 and S007355], the National Research Foundation South African Research Chair Initiative [grant #86949], and MHM was supported by Wellcome Trust (grant #216634/Z/19/Z). AGL is supported by the EDCTP TESA III network (CSA2020NoE-3104).Peer reviewedPublisher PD