Improved Detection of Mycobacterium bovis in Bovine Tissues Using Immunomagnetic Separation Approaches

Immunomagnetic separation (IMS) represents a simple but effective method of selectively capturing and concentrating Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), from tissue samples. It is a physical cell separation technique that does not impact cell viability, unlike traditional chemical decontamination prior to culture. IMS is performed with paramagnetic beads coated with M. bovis-specific antibody and peptide binders. Once captured by IMS, M. bovis cells can be detected by either PCR or cultural detection methods. Increased detection rates of M. bovis, particularly from non-visibly lesioned lymph node tissues from bTB reactor animals, have recently been reported when IMS-based methods were employed.

Evaluation of the diagnostic potential of a novel field immunomagnetic separation-immunochromatographic lateral flow assay to detect Mycobacterium bovis cells in European badger (Meles meles) faeces

The European badger (Meles meles) is a natural reservoir for Mycobacterium bovis, the causative agent of Bovine Tuberculosis, and has consequently been implicated in transmission of the disease to cattle. This study describes application of a novel M. bovis-specific immunochromatographic (lateral flow) assay in combination with immunomagnetic separation (IMS-LFD), to test badger faeces samples. In total, 441 faeces samples from badgers of unknown disease status collected from latrines at 110 badger setts throughout Northern Ireland (NI) and 100 faeces samples from badgers of known infection status from Great Britain (GB) were tested. Faeces (approx. 1g) was homogenised in 9 ml phosphate buffered saline, filtered (70 µm), and then 6-8 ml subjected to the IMS-LFD test. Residual clarified faecal homogenates were subjected to automated IMS followed by MGIT™ liquid culture (AIMS-MGIT™ culture) and qPCR (AIMS-qPCR). Evidence for the presence of M. bovis was obtained for 78 (18%), 61 (14%) and 140 (32%) of 441 NI badger faeces samples, and 10 (10%), 41 (41%) and 56 (56%) of 100 GB badger faeces samples, by IMS-LFD, AIMS-MGIT culture and AIMS-qPCR tests, respectively. The IMS-LFD test was less sensitive than AIMS-qPCR for detection of M. bovis and was, therefore, detecting badgers shedding high numbers of M. bovis in their faeces only. However, these ‘super shedders’ may be primarily responsible for the spread of Bovine Tuberculosis so are, therefore, an important target. This non-invasive test could form the basis of a field surveillance tool to indicate infected badger groups which are actively spreading M. bovis.

Whole Genome Sequencing Reveals Local Transmission Patterns of Mycobacterium bovis in Sympatric Cattle and Badger Populations

Whole genome sequencing (WGS) technology holds great promise as a tool for the forensic epidemiology of bacterial pathogens. It is likely to be particularly useful for studying the transmission dynamics of an observed epidemic involving a largely unsampled 'reservoir' host, as for bovine tuberculosis (bTB) in British and Irish cattle and badgers. BTB is caused by Mycobacterium bovis, a member of the M. tuberculosis complex that also includes the aetiological agent for human TB. In this study, we identified a spatio-temporally linked group of 26 cattle and 4 badgers infected with the same Variable Number Tandem Repeat (VNTR) type of M. bovis. Single-nucleotide polymorphisms (SNPs) between sequences identified differences that were consistent with bacterial lineages being persistent on or near farms for several years, despite multiple clear whole herd tests in the interim. Comparing WGS data to mathematical models showed good correlations between genetic divergence and spatial distance, but poor correspondence to the network of cattle movements or within-herd contacts. Badger isolates showed between zero and four SNP differences from the nearest cattle isolate, providing evidence for recent transmissions between the two hosts. This is the first direct genetic evidence of M. bovis persistence on farms over multiple outbreaks with a continued, ongoing interaction with local badgers. However, despite unprecedented resolution, directionality of transmission cannot be inferred at this stage. Despite the often notoriously long timescales between time of infection and time of sampling for TB, our results suggest that WGS data alone can provide insights into TB epidemiology even where detailed contact data are not available, and that more extensive sampling and analysis will allow for quantification of the extent and direction of transmission between cattle and badgers. © 2012 Biek et al.

Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS) in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5 km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2 km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.

Infecção de mycobacterium bovis em células fagocíticas: estratégia aplicada ao diagnóstico da tuberculose bovina

Neste trabalho foram estudadas as interacções entre Mycobacterium bovis e células hospedeiras, na perspectiva de aplicar os conhecimentos resultantes desse estudo ao melhoramento do diagnóstico da tuberculose bovina. Foi estudada a dinâmica da infecção de células fagocíticas com estirpes de M. bovis, com ênfase para a invasão e multiplicação intracelular das micobactérias. Avaliações efectuadas por citometria de fluxo, microscopia de fluorescência e contagem de colónias demonstraram que as micobactérias invadiram e replicaram em todos os modelos celulares, sendo que as células epiteliais de pulmão de bovino foram as mais permissivas ao seu crescimento. Foi nas células macrofágicas J774 e THP-1 que se verificaram as maiores concentrações micobacterianas, pelo que foram utilizadas para a detecção e identificação de M. bovis por um método molecular. A optimização da extracção de DNA, por um processo mecânico, e o desenvolvimento do método de PCR-RFLP baseado no gene gyrB, com controlo interno, permitiram a identificação de M. bovis. A sensibilidade deste método foi de 100% quando aplicado a estirpes isoladas e apenas de 40% quando utilizado directamente em amostras de macerados de tecidos de bovinos com tuberculose. Uma pré-incubação (3 dias) das amostras nas culturas celulares contribuiu para melhorar significativamente a sensibilidade (77%) do PCR-RFLP gyrB. A cultura celular, como matriz a ser utilizada para aumentar a quantidade de M. bovis presente em amostras biológicas, revela-se um método promissor para o diagnóstico laboratorial rápido, específico e sensível da tuberculose bovina. ### - Summary - Interactions between Mycobacterium bovis and host cells were studied with the purpose to apply the outcome knowledge in the improvement of bovine tuberculosis diagnosis. The dynamic of infection of four cell models with three strains of M. bovis was evaluated, with emphasis given to the invasion and intracellular multiplication of mycobacteria. Assessments by flow cytometry, fluorescence microscopy and colony counting showed that every cell models permitted the replication of mycobacteria, although bovine lung epithelial cells had been the most permissive one. The highest mycobacteria) load was found, however, in J774 and THP-1 macrophages, and hence these cells were used for the optimization of a molecular method for detection and identification of M. bovis. The optimisation of a DNA extraction step, by a mechanical process, and the development of PCR-RFLP based on gyrB gene, with an internal control, allowed the identification of M. bovis. The sensitivity of this method was 100% when applied to isolated strains and only 40% when directly used on samples of macerated of tissues from cattle with tuberculosis. Assays in which a pre-incubation step (three days) of biological samples in cell cultures was introduced significantly improved the sensitivity (77%) of the gyrB PCR-RFLP. Cell cultures as a support for growth and rapid isolation of M. bovis is a promising method for the specific, sensitive and rapid laboratorial diagnosis of bovine tuberculosis.

Costs to farmers of a tuberculosis breakdown

An on-farm survey of 151 cattle farmers who had experienced a bovine tuberculosis (Mycobacterium bovis) breakdown on their farms was undertaken in 2003 to assess the costs associated with the breakdown. In 90 per cent of cases the cost was estimated to be less than 18,513 pound for dairy herds and less than El 1,462 for beef herds, but with a range from 229 pound to 103,817 pound. The main cost was the slaughter of reactor cattle. For the majority of the farmers, the compensation payments seemed to meet most of the costs of their breakdowns, although a majority was still left with net losses.

Sequence heterogeneity of an mpb70 gene analogue in Mycobacterium kansasii

The protein antigen MPB70 is a major component of culture supernatants of Mycobacterium bovis and Is an active ingredient of bovine PPD used for skin-testing cattle for tuberculosis. we have shown that Mycobacterium kansasii possesses a similar gene that cross-reacts in a PCR test for M. bovis. Single strand conformational polymorphism analysis, and the DNA sequence of the PCR product, shows differences between M. kansasii strains, supporting the suggestion that M. kansasii is not a homogeneous species.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes. (C) 2011 Elsevier Ltd. All rights reserved.

Disseminated avian tuberculosis in captive Ara macao

Avian mycobacteriosis was diagnosed in a captive scarlet macaw (Ara macao) that presented multifocal granulomas on subcutaneous tissue, sciatic nerves, infraorbital sinus, trachea, air sacs, muscles, spleen and liver. Microscopically, central areas of caseous necrosis surrounded by epithelioid macrophage, multinucleated giant cells, and lymphocytes were observed. Acid-fast bacilli were demonstrated by Ziehl-Neelsen stain. Inoculation into Lowenstein-Jensen, Stonebrink and Petragnani media, yielded Mycobacterium spp, which was identified as Mycobacterium avium by polymerase chain reaction technique (PCR).

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modulatory effects of tuberculosis vaccines on experimental autoimmune encephalomyelitis

Although BCG is the only accepted vaccine against tuberculosis (TB), its protective ability is very limited. Therefore, many new vaccines are being evaluated. Our group has been working on DNAhsp65 - a genetic construction containing the hsp65 gene from Mycobacterium leprae. In previous experimental works, we demonstrated that both DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with three doses of DNAhsp65 or primed with BCG followed by two DNAhsp65 boosters. The animals were, then, immunized with myelin associated with complete Freund's adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, central nervous system (CNS) inflammation and anti-myelin immune response. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and CNS inflammation did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN-gamma and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate multiple sclerosis, being therefore, safe in this aspect. In addition, they indicate that these vaccination regimens have a potential anti-inflammatory activity that could be better explored in the future.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

NO EVIDENCE of PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.