Pulsed-field gel electrophoresis profile homogeneity of Mycobacterium avium subsp. paratuberculosis isolates from cattle and heterogeneity of those from sheep and goats

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied. RESULTS: All bovines, 4 and 26% of Spanish sheep and goats, respectively, and the deer and wild boar studied, carried IS1311-Cattle type strains. IS1311-Sheep type encompassed 96% and 74% of Spanish sheep and goats, and all three Portuguese sheep. Thirty-seven distinct multiplex PFGE profiles were found, giving 32 novel profiles. Profiles 2-1 and 1-1 accounted for the 85% of cattle isolates. Ten distinct profiles were detected in Spanish sheep, none of them with an incidence higher than 25%. Profile 16-11 (43%) and another three profiles were identified in Spanish caprine cultures. The hierarchical analysis, clustered all profiles found in cattle, "wild" hosts and some small ruminants within the same group. The other group included 11 profiles only found in Spanish sheep and goats, including Spanish pigmented profiles. Differences in growth requirements associated with isolate genotype were observed. CONCLUSION: Cattle in Spain are infected with cattle type strains, while sheep and goats are mainly infected with sheep type strains. Although 7H9 broth based culture media seem to broadly cover the growth requirements of most Map strains, the use of various solid media is recommended to reduce any recovery biases. High genetic homogeneity of isolates from cattle, and heterogeneity of those from sheep and goats have been detected

Oral vaccination of cattle with heat inactivated Mycobacterium bovis does not compromise bovine TB diagnostic tests

AbstractIn this study we investigated whether oral uptake of a heat inactivated M. bovis wildlife vaccine by domestic cattle induced systemic immune responses that compromised the use of tuberculin or defined antigens in diagnostic tests for bovine TB. Positive skin test and blood-based IFN-γ release assay (IGRA) results were observed in all calves vaccinated via the parenteral route (i.e. intramuscular). In contrast, no positive responses to tuberculin or defined antigens were observed in either the skin test or IGRA test when performed in calves vaccinated via the oral route. In conclusion, our results suggest that the heat inactivated M. bovis vaccine could be used to vaccinate wildlife in a baited form in conjunction with the following in cattle: (i) continuation of existing tuberculin skin testing or novel skin test formats based on defined antigens; and (ii) the use of IGRA tests utilizing tuberculin or defined antigens

Comparative analysis of Mycobacterium avium subsp. paratuberculosis isolates from cattle, sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and <it>Sna</it>BI-<it>Spe</it>I pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar).</p> <p>Results</p> <p>A total of nineteen different multi-locus SSR (SSR1_SSR8) types were identified amongst the 268 isolates compared to the 37 multiplex profiles differentiated by the <it>Sna</it>BI-<it>Spe</it>I PFGE. SSR type 7_4 was the predominant genotype (51.2% of all isolates and 54.3% of cattle isolates), but combined with PFGE results the abundance of the most prevalent genotype (7_4&{2-1}) dropped down to 37.7%. SSR types 7_3 and 14_3 were significantly spread amongst isolates recovered from small ruminants. The comparison of SSR1_SSR8 and <it>Sna</it>BI-<it>Spe</it>I PFGE typing of these isolates has shown that both methods perform at similar discriminatory level. These were 0.691 and 0.693, respectively for SSR and PFGE as indicated Simpson's Index of Diversity, and 0.82 when calculated for combined SSR and PFGE genotypes. Overall, SSR1_SSR8 analysis seemed to detect higher levels of within-farm strain diversity and seemed to give higher year-related information. Combination of both typing methods revealed 20 multi-type farms out of the 33 bovine farms studied with more than one isolate.</p> <p>Conclusion</p> <p>The particular SSR and PFGE typing approaches described here are in general agreement but they showed some discrepancies that might reflect differing evolutionary processes of Map strains. Both methods are able to reciprocally complement their results and neither should be replaced with the other if sufficient material and time is available. Overall, the results of our comparative analyses suggest that, based on current methodologies available, a combined approach that includes SSR and PFGE seems to provide the highest level of discrimination for Map strain typing with meaningful epidemiological information.</p

Wild Boar and Red Deer Display High Prevalences of Tuberculosis-Like Lesions in Spain

We describe the distribution of tuberculosis-like lesions (TBL) in wild boar (Sus scrofa) and red deer (Cervus elaphus) in Spain. Animals with TBL were confirmed in 84.21% of mixed populations (n = 57) of red deer and wild boar and in 75% of populations of wild boar alone (n = 8) in central and southern Spain (core area). The prevalence of TBL declined towards the periphery of this region. In the core area, the prevalence ranged up to 100% in local populations of wild boar (mean estate prevalence 42.51%) and up to 50% in red deer (mean estate prevalence 13.70%). We carried out exploratory statistical analyses to describe the epidemiology of TBL in both species throughout the core area. Prevalence of TBL increased with age in both species. Wild boar and red deer mean TBL prevalence at the estate level were positively associated, and lesion scores were consistently higher in wild boars than in red deer. The wild boar prevalence of TBL in wild boar did not differ between populations that were or were not cohabiting with red deer. Amongst the wild boars with TBL, 61.19% presented generalized lesions, and the proportion of generalized cases was similar between sex and age classes. In red deer, 57.14% of TBL-positive individuals presented generalized lesions, and the percentage of generalized cases increased with age class, but did not differ between the sexes. These results highlight the potential importance of wild boar and red deer in the maintenance of tuberculosis in south central Spain

Progress in the control of bovine tuberculosis in Spanish wildlife

Special issue: 5th International Conference on Mycobacterium bovis.-- HAL Id: hal-00701898Despite the compulsory test and slaughter campaigns in cattle, bovine tuberculosis (bTB) is still present in Spain, and the role of wildlife reservoirs is increasingly recognized. We provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination.At the high densities and in the particular circumstances of Mediterranean environments, wild ungulates, mainly Eurasian wild boar and red deer, are able to maintain Mycobacterium bovis circulation even in absence of domestic livestock. Infection is widespread among wild ungulates in the south of the country, local infection prevalence being as high as 52% in wild boar and 27% in red deer. Risk factors identified include host genetic susceptibility, abundance, spatial aggregation at feeders and waterholes, scavenging, and social behaviour. An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Southwestern Spain. Sporadic cases of badger TB have been detected, further complicating the picture.Gene expression profiles were characterized in European wild boar and Iberian red deer naturally infected with M. bovis. The comparative analysis of gene expression profiles in wildlife hosts in response to infection advanced our understanding of the molecular mechanisms of infection and pathogenesis, revealed common and distinctive host responses to infection and identified candidate genes associated with resistance to bTB and for the characterization of host response to infection and vaccination.Ongoing research is producing valuable knowledge on vaccine delivery, safety and efficacy issues. Baits for the oral delivery of BCG vaccine preparations to wild boar piglets were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy. Safety experiments yielded no isolation of M. bovis BCG from faeces, internal organs at necropsy and the environment, even after oral delivery of very high doses. Finally, preliminary vaccination and challenge experiments suggested that a single oral BCG vaccination may protect wild boar from infection by a virulent M. bovis field strain.Studies on TB at IREC are supported by Grupo Santander – Fundacion Marcelino Botin, and by TB-STEP EU FP7 212414, INIA-MICINN FAU 06-017, and PAI07-0062-6611 Castilla – La Mancha.Peer Reviewe

Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa)

Boadella et al.New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or crossreacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.The current study is a contribution to MCINN Plan Nacional I+D+i research grant AGL2008-03875 and FEDER and to FP7 EU grant TB-STEP (212414). Studies on diseases shared between domestic animals and wildlife are also supported by Grupo Santander Fundacion Marcelino Botin. Tania Carta acknowledges a grant from Regione Sardegna.Peer Reviewe

Identification of loci associated with pathological outcomes in Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis using whole-genome sequence data

[EN]Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis that affects cattle worldwide. According to their severity and extension, PTB-associated histological lesions have been classified into the following groups; focal, multifocal, and diffuse. It is unknown whether these lesions represent sequential stages or divergent outcomes. In the current study, the associations between host genetic and pathology were explored by genotyping 813 Spanish Holstein cows with no visible lesions (N = 373) and with focal (N = 371), multifocal (N = 33), and diffuse (N = 33) lesions in gut tissues and regional lymph nodes. DNA from peripheral blood samples of these animals was genotyped with the bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data using the 1000 Bull genomes reference population. A genome-wide association study (GWAS) was performed using the WGS data and the presence or absence of each type of histological lesion in a case-control approach. A total of 192 and 92 single nucleotide polymorphisms (SNPs) defining 13 and 9 distinct quantitative trait loci (QTLs) were highly-associated (P <= 5 x 10(-7)) with the multifocal (heritability = 0.075) and the diffuse (heritability = 0.189) lesions, respectively. No overlap was seen in the SNPs controlling these distinct pathological outcomes. The identified QTLs overlapped with some QTLs previously associated with PTB susceptibility, bovine tuberculosis susceptibility, clinical mastitis, somatic cell score, bovine respiratory disease susceptibility, tick resistance, IgG level, and length of productive life. Pathway analysis with candidate genes overlapping the identified QTLs revealed a significant enrichment of the keratinization pathway and cholesterol metabolism in the animals with multifocal and diffuse lesions, respectively. To test whether the enrichment of SNP variants in candidate genes involved in the cholesterol metabolism was associated with the diffuse lesions; the levels of total cholesterol were measured in plasma samples of cattle with focal, multifocal, or diffuse lesions or with no visible lesions. Our results showed reduced levels of plasma cholesterol in cattle with diffuse lesions. Taken together, our findings suggested that the variation in MAP-associated pathological outcomes might be, in part, genetically determined and indicative of distinct host responses.Financial support for this study was provided by a Grant from the Spanish Ministry of Science, Innovation, and Universities (RTI2018-094192-R-C21) and by European Regional Development Funds (FEDER) to MAH. MC and GBB have been awarded fellowships from the National Institute for Agricultural Research (INIA) and the Spanish Ministry of Science, Innovation and Universities programs, respectively. This work has been possible thanks to the support of the computing infrastructure of the i2BASQUE Research and Academic Network. We gratefully acknowledge the Bull Genomes Consortium for providing accessibility to the WGS data that was used in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Detection of Mycobacteria by Culture and DNA-Based Methods in Animal-Derived Food Products Purchased at Spanish Supermarkets

Mycobacteria include obligate and opportunistic pathogens that cause significant human and animal disease. The burden of tuberculosis has been largely reduced in developed territories but remains a huge problem worldwide. The significance of nontuberculous mycobacteria is growing considerably, especially in developed regions with higher life expectancy and more therapy-related immunosuppressed individuals. Due to their robustness mycobacteria can contaminate animal products by direct transmission from infected individuals or by environmental contamination during processing. The situation at market level is poorly known. Most studies analyzing commercially available foods are limited to a small or local scale and mainly focused on a particular mycobacterial species. There is a need to investigate if animal products that have passed the established controls to be for sale at main supermarkets could represent a route of contact with any mycobacteria. Thus, our goal was to study the prevalence of mycobacteria in these foods to assess if this could represent a source of human exposure. Five stores from the main supermarket chains in Spain were selected. 138 dairy and 119 meat products were purchased. All were processed using culture and multiplex real-time PCR methods. Additional molecular methods were used to specifically identify any positive result. Mycobacterium avium subsp. hominissuis (2), M. avium subsp. avium (1), and M. fortuitum (1) were isolated from powdered infant formula and ground beef, chicken sausage, and mortadella cold cut, respectively. Mycobacterial DNA (M. avium, M. tuberculosis complex and other nontuberculous mycobacteria) was detected in 15% of dairy products and 2% of meat products. These results show that the prevalence of viable mycobacteria in foods of animal origin obtained at the supermarket was not substantial although a considerable proportion of them contained mycobacterial DNA. Contact with mycobacteria through this route could be ensured over time. Further investigation is necessary to determine the real impact of foodborne mycobacterial exposure on human health and identify critical points in the food production system to enable setting up more stringent control measures