Comparison of 2 PCR assays on environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Environmental samples are often used to classify the paratuberculosis status of cattle herds. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), predominantly through oral ingestion during infancy. In this explorative study, the presence of MAP was determined in the barn environment of a paratuberculosis-infected vaccinated dairy goat herd. A total of 256 bedding, dust, feed, and water samples were collected at eight time points and examined using culture and qPCR. Detection rates of both methods were compared, and factors determining MAP confirmation were identified. MAP was cultured from 28 bedding and one dust sample, while MAP DNA was detected in all materials (117/256). Samples from high animal traffic areas and those collected during the indoor season were more likely to yield positive culture and qPCR results. Cultivation of MAP from kidding pens indicated this area as a possible infection site. Dust proved to be the most suitable material for detecting MAP DNA, as bedding was for MAP culture. Environmental sampling was demonstrated to be an effective way to detect MAP in a dairy goat herd. qPCR results could confirm herd infection, while culture results provided insight into crucial areas for MAP transmission. These findings should be considered when designing farm-specific paratuberculosis control plans.

Application of cattle slurry containing Mycobacterium avium subsp. paratuberculosis (MAP) to grassland soil and its effect on the relationship between MAP and free-living amoeba

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

Global increase in diabetes (DM) prevalence necessitated the need to establish the association between DM and environmental triggers including MAP (Mycobacterium avium subsp. paratuberculosis) that have been postulated to play a role in DM etiopathology for effective management. The present investigation aimed to assess the odds ratio (OR) presenting the association between MAP and DM. MAP-related DM studies were systematically retrieved from 6 databases until 31 September 2021 according to PRISMA principles for data abstraction. The abstracted dataset was fitted to the fixed-effects (FE) and random-effects (RE) models using the Mantel–Haenszel approach. Sixteen studies involving 2072 participants (1152 DM patients (957 type 1 diabetes mellitus (T1DM) & 195 type 2 diabetes mellitus (T2DM)) and 920 healthy controls) met the inclusion criteria. Results revealed a significant association between anti-MAP antibodies (abs) seroprevalence and T1DM (FE: OR 7.47, 95% CI 5.50–10.14, p value < 0.0001; RE: OR 7.92, 95% CI 4.39–14.31, p < 0.0001) and MAP DNA with T1DM (FE: OR 4.70 (95% CI 3.10–7.13, p value < 0.0001), RE: OR 3.90 (95% CI 0.93–16.38, p value = 0.06)). Both anti-MAP abs and MAP DNA based meta-analyses had medium heterogeneity (I2 = 47.2–61.0%). Meanwhile, no significant association between MAP and T2DM (FE: OR 1.13, 95% CI 0.54–2.37, p value = 0.74; RE: OR 1.19; 95% CI 0.34–4.12, p value = 0.69), its OR magnitude exceeded 1 and prediction interval (0.09–15.29) suggest possibility of association between the duo in the future. The leave-one-out sensitivity analysis depicts a robust meta-analysis in all cases. In conclusion, the study manifests a positive association between MAP and T1DM, highlighting that MAP prevention and environmental control would indubitably revolutionize T1DM management. Also, its projects possible link between MAP and T2DM as more data becomes available. However, it remains elusive whether MAP triggers T1/T2DM or a mere comorbidity in T1/T2DM. Epidemiological activities to fill the global/regional data gaps on MAP-related T1DM and T2DM are advocated in order to assess the burden of MAP-related DM and improve their clinical management.

Risk factors associated with Mycobacterium avium subsp. paratuberculosis herd status in Québec dairy herds

Evaluation of three commercial PCR kits for the direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine faeces

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease, a chronic progressive enteritis. It is generally assumed that calves rarely shed MAP bacteria and that calf-to-calf transmission is of minor importance. The objectives were 1) to estimate the prevalence of MAP-shedding young stock in MAP-infected dairy herds, and identify predictors for test-positive young stock; and 2) to estimate proportions of MAP-contaminated young stock group housing pens and air spaces, and furthermore, identify predictors for test-positive pens. Fecal samples were collected from 2606 young stock on 18 MAP-infected dairy farms. Environmental fecal samples were collected from all group-housing pens and dust samples were collected from all barns. All individual samples were analysed using IS900 and F57 qPCR; fecal samples positive by either PCR and all environmental and dust samples were cultured. Overall, 8.1, 1.2 and 2.0% of cattle were positive on IS900 qPCR, F57 qPCR and bacterial culture, respectively. Young stock housed on farms with culture-positive environmental samples collected from adult cow housing and manure storage had higher odds of testing IS900 qPCR-positive than young stock housed on farms with only negative environmental samples. Furthermore, 14% of collected environmental samples, but no dust samples, were test-positive. Age of cattle in the pen was a significant predictor for environmental sample results. Young stock excreted MAP bacteria in their feces which provided strong evidence for calves as sources of within-herd transmission of MAP on dairy farms known to be infected with this organism.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0192-1) contains supplementary material, which is available to authorized users.

Detection of Viable Mycobacterium avium subsp. paratuberculosis in Retail Pasteurized Whole Milk by Two Culture Methods and PCR

Copper causes significant damage to the integrity of many bacteria, mainly at the DNA level, through its redox states, as well as its reactive oxygen species (ROS) generating capacity at the cellular level. But whether these mechanisms also apply to Mycobacterium avium subsp. paratuberculosis (MAP) is unknown. In the present study, we have evaluated whether copper ions produce damage at the DNA level of MAP, either through their redox states or through ROS production. MAP-spiked PBS was first supplemented with different copper chelators (2) and ROS antioxidants (3), followed by treatment with copper ions at 942ppm. MAP DNA integrity (qPCR, magnetic phage separation) was then evaluated. We found that bathocuproine (BCS), as a chelator, and D-mannitol, as an antioxidant of hydroxyl radicals, had a significant protective effect (P < 0.05) on DNA molecules, and that EDTA, as a chelator, and D-mannitol, as an antioxidant had a significant positive effect (P < 0.05) on the viability of this pathogen in contrast to the control and other chelators and anti-oxidants used. In light of the reported findings, it may be concluded that copper ions within MAP cells are directly related to MAP DNA damage.

Environmental samples are considered to be a cost-effective method of identifying Mycobacterium avium subsp. paratuberculosis (MAP)-positive dairy herds, but evidence for beef cow-calf herds is weak. This study aims at evaluating this approach in a total of 20 German herds that were characterized by individual faecal samples (n = 2545) of all cows. For 14 MAP-positive herds having at least one MAP-positive animal, the within-herd prevalence was calculated from concurrent individual faecal culture-based testing. Six herds certified as 'MAP free' based on the negative results of previous years served as MAP-negative controls. On average, six environmental samples were taken at the end of winter from areas with high cow traffic and tested for MAP by faecal culture. According to the environmental samples, nine (64·3%) out of the 14 MAP-positive cow-calf herds were infected. The percentage of positive environmental samples and the apparent within-herd prevalence (Spearman's P = 0·73, P < 0·001) as well as the herd-level test results (positive and negative) and the herd's status based on individual testing (Fisher's exact test, P = 0·014) showed a positive association. Considering limitations in low-prevalence herds, MAP-positive beef cow-calf herds are detectable by environmental samples in temperate climate zones.

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.

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Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), a chronic infectious disease that causes intractablediarrhea in ruminants. To control the occurrence of JD in cattle, a national surveillance is conducted in Japan. Since 2013, real-time quantitative PCR has been used for definite diagnosis.In this study, we compared the amount of fecal MAP DNA with histopathological classification of ileocecal lesions. Multinomial logistic regression models enabled us to predict theprobability of finding the histopathological classification from the amount of fecal MAP DNA. These results suggest that shedding level of MAP DNA could act as an indicator of JDprogression.

Johne’s disease (JD), also referred to as paratuberculosis, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is a chronic disease of ruminants that primarily affects the enteric system and is seen worldwide. The study aimed towards molecular detection of MAP in bovine samples. A total of 220 bovine samples (intestinal tissue-60, faecal-60 and milk-100) were collected from cattle herds and slaughter point and were examined for detection of MAP targeting the IS900 insertion sequence through conventional PCR. MAP DNA was detected in 7 intestinal tissue samples (11.66%) and 1 faecal sample (1.66%). Only 3/60 (5.0%) intestinal tissue samples could be detected as acid fast bacilli by Z-N staining. The results were further re-confirmed by nBLAST analysis following Sanger dideoxynucleotide sequencing. The results indicate the presence of MAP-infections in dairy herds of the study area, Assam. This may be the first report of molecular detection of MAP in bovine intestinal tissue and faecal samples in Assam, Northeast India as per our literature search. Additional research is required across all Northeastern regions of India to determine the precise prevalence of the pathogen and to mitigate the spread of the disease.