A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis causes Johne's disease in ruminants, whereas the antigenically and genetically similar subspecies Mycobacterium avium ssp. avium is less virulent. In this study, we compared one strain of each subspecies for its ability to survive, induce cytokines, suppress MHC class I and II expression and induce apoptosis or necrosis in ovine monocyte-derived macrophages. Both subspecies survived intracellularly and induced the secretion of IL-10. Low levels of TNF-alpha were detected after infection with both subspecies at 4 h. IL-12 was not upregulated after infection. Downregulation of MHC class I and II was evident in response to infection with both M. avium ssp. avium and M. avium ssp. paratuberculosis. No significant cytotoxicity was detectable in ovine macrophages after the addition of bacteria. M. avium ssp. paratuberculosis induced slightly more apoptosis than M. avium ssp. avium. Still the overall rate of apoptosis was very low and both subspecies suppressed LPS-induced macrophage apoptosis.     

Mycobacterium avium ssp. paratuberculosis and its potential survival tactics

Mycobacterium avium ssp. paratuberculosis (Map) is an important animal pathogen with a potential, but as yet unproven, role in human disease. This review briefly describes the characteristics of Map that distinguish it from other Mycobacterium spp., presenting new information arising from completion of the sequencing of the Map genome. It then focuses on the potential mechanisms Map might employ to survive and disseminate in the environment, including interaction with protozoa and insects, dormancy, biofilm formation and aerosolization.     

Inactivation of Mycobacterium avium ssp. paratuberculosis in milk by UV treatment

Aims:  To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk.
Methods and Results:  Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV-C radiation from 0 to 1836 mJ ml⁻¹ using a pilot-scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold's egg yolk medium, Middlebrook 7H10 medium and the FASTPlaque TB™ phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml⁻¹ a Map kill rate of 0·1–0·6 log₁₀ was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count.
Conclusions:  The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk.
Significance and Impact of the Study:  To the authors' knowledge the work here represents the first pilot-scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory-scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments.     

Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal–oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-κB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.     

Identification of a QTL on BTA20 affecting susceptibility to Mycobacterium avium ssp. paratuberculosis infection in US Holsteins

The objective of this study was to identify QTL affecting susceptibility to Mycobacterium paratuberculosis infection in US Holsteins. Twelve paternal half-sib families were selected for the study based on large numbers of daughters in production and limited relationships among sires. Serum and faecal samples from 4350 daughters of these 12 sires were obtained for disease testing. Case definition for an infected cow was an ELISA sample-to-positive ratio >/=0.25, a positive faecal culture or both. Three families were selected for genotyping based on a high apparent prevalence (6.8-10.4% infected cows), high faecal culture prevalence (46.2-52.9% positive faecal cultures) and large numbers of daughters tested for disease (264-585). DNA pooling was used to genotype cows, with an average of 159 microsatellites within each sire family. Infected cows (the positive pool) were matched with two of their non-infected herdmates in the same lactation (the negative pool) to control for herd and age effects. Eight chromosomal regions putatively linked with susceptibility to M. paratuberculosis infection were identified using a Z-test (P < 0.01). Significant results were more rigorously tested by individually genotyping cows with three to five informative microsatellites within 15 cM of the significant markers identified with the DNA pools. Probability of infection based on both diagnostic tests was estimated for each individual and used as the dependent variable for interval mapping. Based on this analysis, evidence for the presence of a QTL segregating within families on BTA20 was found (chromosome-wide P-value = 0.0319).     

Effect of high pressure and pasteurization on Mycobacterium avium ssp. paratuberculosis in milk

AIMS: To determine the effect of high pressures alone and in conjunction with pasteurization on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map). METHODS AND RESULTS: Map in a milk matrix was subjected to 400, 500 and 600 MPa with and without pasteurization (72 degrees C for 15 s) and plated onto Herrold's egg yolk medium (HEYM) and Middlebrook 7H10 (7H10) agar, both containing antibiotic supplements. Medium 7H10 was found to give a significantly (P < 0.001) better recovery than HEYM. A significantly greater (P < 0.001) reduction in viable numbers was observed using 500 MPa (mean log reduction of 6.52) compared with 400 MPa (mean log reduction of 2.56) and between 400 MPa and control (no applied pressure) for 10 min treatments. A treatment time of 10 min resulted in significantly (P < 0.001) fewer survivors than 5 min. Low numbers of survivors were still detected when pressure treatment at 400 and 600 MPa was combined with subsequent pasteurization. CONCLUSIONS: The use of high-pressure was effective in reducing viable numbers of Map but even when combined with pasteurization there were still survivors, albeit when high inoculum levels of Map were used. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge the work reported here represents the first study of the efficacy of high-pressure treatments alone and in combination with pasteurization to kill Map. The results indicate that further research is warranted before more commercial-scale studies are commissioned.     

Genetic Loci Involved in Antibody Response to Mycobacterium avium ssp. paratuberculosis in Cattle

Background

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic enteritis in a wide range of animal species. In cattle, MAP causes a chronic disease called Johne''s disease, or paratuberculosis, that is not treatable and the efficacy of vaccine control is controversial. The clinical phase of the disease is characterised by diarrhoea, weight loss, drop in milk production and eventually death. Susceptibility to MAP infection is heritable with heritability estimates ranging from 0.06 to 0.10. There have been several studies over the last few years that have identified genetic loci putatively associated with MAP susceptibility, however, with the availability of genome-wide high density SNP maker panels it is now possible to carry out association studies that have higher precision.

Methodology/Principal Findings

The objective of the current study was to localize genes having an impact on Johne''s disease susceptibility using the latest bovine genome information and a high density SNP panel (Illumina BovineSNP50 BeadChip) to perform a case/control, genome-wide association analysis. Samples from MAP case and negative controls were selected from field samples collected in 2007 and 2008 in the province of Lombardy, Italy. Cases were defined as animals serologically positive for MAP by ELISA. In total 966 samples were genotyped: 483 MAP ELISA positive and 483 ELISA negative. Samples were selected randomly among those collected from 119 farms which had at least one positive animal.

Conclusion/Significance

The analysis of the genotype data identified several chromosomal regions associated with disease status: a region on chromosome 12 with high significance (P<5×10⁻⁶), while regions on chromosome 9, 11, and 12 had moderate significance (P<5×10⁻⁵). These results provide evidence for genetic loci involved in the humoral response to MAP. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information into breeding programmes for the improvement of health status.     

Systemic and Mucosal Immune Reactivity upon Mycobacterium avium ssp. paratuberculosis Infection in Mice

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne''s disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn''s disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2 ^−/− mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4⁺ and CD8⁺ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4⁺ T cells. Whereby inflammation in infected and CD4⁺CD45RB^hi T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8⁺ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4⁺CD45RB^lo/int T cells. We propose, the usual non-colitogenic CD4⁺CD45RB^lo/int T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne''s diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.     

Development of an IMS-PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water

AIMS: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique. METHODS AND RESULTS: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 microm pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR. CONCLUSIONS: This method will be used to determine the incidence of Map in water destined for domestic use in future studies. SIGNIFICANCE AND IMPACT OF THE STUDY: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.     

Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) causes a chronic, granulomatous inflammatory condition of the intestines in ruminants and wild-type species. It causes significant economic losses to the dairy and beef industries owing to reduced productivity, premature culling and mortality. Bovine peptidoglycan recognition protein 1 is an important pattern recognition molecule that is capable of directly killing microorganisms. The goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine peptidoglycan recognition protein 1 and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples were collected from Holsteins in Southwestern and Eastern Ontario and tested for MAP infection using blood and milk ELISAs. A resource population consisting of 197 infected (S/P > 0.25) and 242 healthy (S/P < 0.10) cattle was constructed. Sequencing of pooled DNA was used to identify three SNPs (c.102G>C, c.480G>A and c.625C>A) that were genotyped in the resource population. Statistical analysis was performed using a logistic regression model fitting the additive and dominance effects of each SNP in the model. SNP c.480G>A (P = 0.054) was found to be associated with susceptibility to MAP infection. Cows with a copy of the major allele 'G' at this locus had an odds ratio of 1.51 (95% CI: 0.99-2.31) for being infected with MAP.     

Identification of a repetitive DNA sequence specific to Mycobacterium paratuberculosis

Abstract A 0.2-kb DNA sequence specific to Mycobacterium paratuberculosis , the causative organism of Johne's disease, was isolated from a partial genomic library. The sequence was part of a larger repetitive DNA element and was present in strains of M. paratuberculosis from cattle, sheep, goat, deer and also a woman with Crohn's disease but not in M. paratuberculosis strain 18. The sequence was not present in strains of 19 other mycobacterial species including 31 reference serotype strains of the M. avium-M. intracellular-M. scrofulaceum (MAIS) complex, some strains of which are closely related to M. paratuberculosis . The sequence may be useful for developing a diagnostic test for Johne's disease.     

Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.     

Survival of Mycobacterium avium ssp. paratuberculosis in yoghurt and in commercial fermented milk products containing probiotic cultures

Aims: To assess the survival of Mycobacterium avium ssp. paratuberculosis (MAP) in yoghurt and commercial fermented milk products containing probiotic strains. Methods and Results: Whole and skimmed UHT milk artificially inoculated with MAP were used to manufacture yoghurt, using two different yoghurt starter cultures. Five commercial fermented milk products were inoculated with MAP. Two different MAP strains were studied. The survival of MAP in all products was monitored by culture over a 6‐week storage period at 6°C. In yoghurt, MAP counts did not change appreciably during the storage period. Fat content and type of yoghurt starter culture had no consistent effect on the survival of MAP. In the fermented milk products, survival patterns varied but resulted in a 1·5 to ≥3·8 log reduction for the Niebüll strain and a 1·2–2·2 log reduction for the NIZO strain after 6 weeks, depending on the probiotic starters present in the product. Conclusions: MAP easily survived in yoghurt but MAP numbers decreased in fermented milk products containing probiotic cultures. Significance and Impact of the Study: The results contribute to the lack of knowledge on the behaviour of MAP in yoghurt and fermented milk products containing probiotic cultures. This knowledge is valuable in the context of the risk of MAP transmission to humans via yoghurt and the possible contribution of probiotic fermented milk products to the elimination of MAP.     

Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA ( P <0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.     

Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subsp. avium infections in a tule elk (Cervus elaphus nannodes) herd

Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P) > or = 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/P > or = 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC.     

Heat inactivation of Mycobacterium avium subsp. paratuberculosis in milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10(3) to 10(4) organisms/ml were processed with combinations of three temperatures of 72, 75, and 78 degrees C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented > 6-log10 reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72 degrees C for 15 s, 75 degrees C for 25 s, and 78 degrees C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of > 6 log10 in 85% of runs and > 4 log10 in 14% of runs.     

Heat Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10³ to 10⁴ organisms/ml were processed with combinations of three temperatures of 72, 75, and 78°C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented >6-log₁₀ reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72°C for 15 s, 75°C for 25 s, and 78°C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of >6 log₁₀ in 85% of runs and >4 log₁₀ in 14% of runs.