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.

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.

Caracterización de las vías de señalización asociadas a la activación celular en macrófagos infectados con Mycobacterium bovis.

Tesis (Doctor en Ciencias con Especialidad en Microbiología) UANL, 2013.

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)

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Animal-side serologic assay for rapid detection of Mycobacterium Bovis infection in multiple species of free-ranging wildlife

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n = 1532), white-tailed deer (n = 463), brushtail possums (n = 129), and wild boar (n = 177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.

Estimating the extent of spatial association of Mycobacterium bovis infection in badgers in Ireland

Mycobacterium bovis infects the wildlife species badgers Meles meles who are linked with the spread of the associated disease tuberculosis (TB) in cattle. Control of livestock infections depends in part on the spatial and social structure of the wildlife host. Here we describe spatial association of M. bovis infection in a badger population using data from the first year of the Four Area Project in Ireland. Using second-order intensity functions, we show there is strong evidence of clustering of TB cases in each the four areas, i.e. a global tendency for infected cases to occur near other infected cases. Using estimated intensity functions, we identify locations where particular strains of TB cluster. Generalized linear geostatistical models are used to assess the practical range at which spatial correlation occurs and is found to exceed 6 in all areas. The study is of relevance concerning the scale of localized badger culling in the control of the disease in cattle.

Development of a Model of Natural Infection with Mycobacterium bovis in White-Tailed Deer

The objective of this study was to develop a suitable experimental model of natural Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus), describe the distribution and character of tuberculous lesions, and to examine possible routes of disease transmission. In October 1997, 10 mature female white-tailed deer were inoculated by intratonsilar instillation of 2 3 103 (low dose) or 2 3 105 (high dose) colony forming units (CFU) of M. bovis. In January 1998, deer were euthanatized, examined, and tissues were collected 84 to 87 days post inoculation. Possible routes of disease transmission were evaluated by culture of nasal, oral, tonsilar, and rectal swabs at various times during the study. Gross and microscopic lesions consistent with tuberculosis were most commonly seen in medial retropharyngeal lymph nodes and lung in both dosage groups. Other tissues containing tuberculous lesions included tonsil, trachea, liver, and kidney as well as lateral retropharyngeal, mandibular, parotid, tracheobronchial, mediastinal, hepatic, mesenteric, superficial cervical, and iliac lymph nodes. Mycobacterium bovis was isolated from tonsilar swabs from 8 of 9 deer from both dosage groups at least once 14 to 87 days after inoculation. Mycobacterium bovis was isolated from oral swabs 63 and 80 days after inoculation from one of three deer in the low dose group and none of four deer in the high dose group. Similarly, M. bovis was isolated from nasal swabs 80 and 85 days after inoculation in one of three deer from the low dose group and 63 and 80 days after inoculation from two of four deer in the high dose group. Intratonsilar inoculation with M. bovis results in lesions similar to those seen in naturally infected white-tailed deer; therefore, it represents a suitable model of natural infection. These results also indicate that M. bovis persists in tonsilar crypts for prolonged periods and can be shed in saliva and nasal secretions. These infected fluids represent a likely route of disease transmission to other animals or humans.

Evaluation of Blood Assays for Detection of Mycobacterium bovis in White-Tailed Deer (Odocoileus virginianus) in Michigan

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all .92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Shared Feed as a Means of Deer-to-Deer Transmission of Mycobacterium bovis

To determine the ability of experimentally inoculated white-tailed deer (Odocoileus virginianus) to transmit Mycobacterium bovis to naive deer through the sharing of feed, four deer were intratonsillarly inoculated with 4x105 colony-forming units of M. bovis. On a daily basis, feed not consumed by inoculated deer after approximately 8 hr was offered to four naıve deer maintained in a separate pen, where direct contact, aerosol transmission, or transmission through personnel were prevented. After 150 days, naıve deer were euthanized and examined. All naıve deer had lesions consistent with tuberculosis and M. bovis was isolated from various tissues. The most commonly affected tissues were lung, tracheobronchial lymph nodes, and mediastinal lymph nodes. This study demonstrates the potential for indirect transmission of M. bovis through the sharing of feed. Intentional or unintentional feeding of deer by wildlife or agricultural interests in regions where M. bovis infection is endemic should be avoided because both direct and indirect transmission through sharing of feed are enhanced.

Investigation of the Transmission of Mycobacterium bovis from Deer to Cattle Through Indirect Contact

Objective—To investigate the infection of calves with Mycobacterium bovis through oral exposure and transmission of M bovis from experimentally infected white-tailed deer to uninfected cattle through indirect contact. Animals—24 11-month-old, white-tailed deer and 28 6-month-old, crossbred calves. Procedure—In the oral exposure experiment, doses of 4.3 X 106 CFUs (high dose) or 5 X 103 CFUs (low dose) of M bovis were each administered orally to 4 calves; as positive controls, 2 calves received M bovis (1.7 X 105 CFUs) via tonsillar instillation. Calves were euthanatized and examined 133 days after exposure. Deer-to-cattle transmission was assessed in 2 phases (involving 9 uninfected calves and 12 deer each); deer were inoculated with 4 X 105 CFUs (phase I) or 7 X 105 CFUs (phase II) of M Bovis. Calves and deer exchanged pens (phase I; 90 days’ duration) or calves received uneaten feed from deer pens (phase II; 140 days’ duration) daily. At completion, animals were euthanatized and tissues were collected for bacteriologic culture and histologic examination. Results—In the low- and high-dose groups, 3 of 4 calves and 1 of 4 calves developed tuberculosis, respectively. In phases I and II, 9 of 9 calves and 4 of 9 calves developed tuberculosis, respectively. Conclusions and Clinical Relevance—Results indicated that experimentally infected deer can transmit M bovis to cattle through sharing of feed. In areas where tuberculosis is endemic in free-ranging white-tailed deer, management practices to prevent access of wildlife to feed intended for livestock should be implemented.

Aerosol Exposure of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium Bovis

Tuberculosis due to Mycobacterium bovis affects both captive and free-ranging Cervidae in the United States. Various animal models have been developed to study tuberculosis of both humans and animals. Generally, tuberculosis is transmitted by aerosol and oral routes. Models of aerosol exposure of large animals to M. bovis are uncommon. In order to develop a reliable method of aerosol exposure of white-tailed deer (Odocoileus virginianus) to M. bovis, 12 healthy white-tailed deer, aged 8–10 mo, were infected by aerosol exposure to 2x105 to 1x106 colony forming units (CFU) (high dose, n=4) of M. bovis or 6x102 to 1.6 x 103 CFU (low dose, n=8) of M. bovis. Tuberculous lesions were more widely disseminated in deer receiving the high dose, while lesions in deer receiving the low dose were more focused on the lungs and associated lymph nodes (tracheobronchial and mediastinal). Aerosol delivery of M. bovis to white-tailed deer results in a reliable manner of experimental infection that may be useful for studies of disease pathogenesis, immune response, mycobacterial shedding, and vaccine efficacy.