Effects of freezing on ability to detect Mycobacterium avium subsp. paratuberculosis from bovine tissues following culture

Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium that causes Johne’s disease in cattle. Although infected cattle can be identified by examining fecal, blood, or milk samples, the gold standard is identification of MAP in tissue samples postmortem. Although tissue samples are commonly frozen, the ability to detect MAP in frozen–thawed tissue samples has apparently not been reported. We therefore determined the ability to detect MAP in tissue samples following freezing. Tissue samples were collected from calves that were either inoculated (IN) 3 mo prior, or contact-exposed (CE) for 3 mo. Following autopsy, tissues were immediately processed for culture, followed by DNA extraction and detection by qPCR. Samples were categorized as positive or negative based on the cycle threshold (Ct) value. The remaining unprocessed tissue samples were frozen at −80°C. After 18 mo, 50 tissue samples designated MAP-positive were thawed and processed for detection of MAP. Four (8%) samples were qPCR-negative, and Ct values of the remaining 46 samples were higher after freezing. Given the small numerical change in Ct values for MAP-positive samples after 18 mo of frozen storage, freezing and thawing may have had some deleterious effects on MAP detection in tissues. Although the decrease in ability to detect MAP-positive samples was minor for IN calves, there may be a greater effect for CE calves that should be considered when freezing tissue samples.

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Elucidating Transmission Patterns of Endemic Mycobacterium avium subsp. paratuberculosis Using Molecular Epidemiology

Veterinary Sciences ◽

10.3390/vetsci6010032 ◽

2019 ◽

Vol 6 (1) ◽

pp. 32 ◽

Cited By ~ 1

Author(s):

Rebecca Mitchell ◽

Annabelle Beaver ◽

Elena Knupfer ◽

Abani Pradhan ◽

Terry Fyock ◽

...

Keyword(s):

New York ◽

Mycobacterium Avium ◽

Dairy Herd ◽

Mycobacterium Avium Subsp ◽

Minor Role ◽

Tissue Samples ◽

Control Programs ◽

Infection Dynamics ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

A Minor

Mycobacterial diseases are persistent and characterized by lengthy latent periods. Thus, epidemiological models require careful delineation of transmission routes. Understanding transmission routes will improve the quality and success of control programs. We aimed to study the infection dynamics of Mycobacterium avium subsp. paratuberculosis (MAP), the causal agent of ruminant Johne’s disease, and to distinguish within-host mutation from individual transmission events in a longitudinally MAP-defined dairy herd in upstate New York. To this end, semi-annual fecal samples were obtained from a single dairy herd over the course of seven years, in addition to tissue samples from a selection of culled animals. All samples were cultured for MAP, and multi-locus short-sequence repeat (MLSSR) typing was used to determine MAP SSR types. We concluded from these precise MAP infection data that, when the tissue burden remains low, the majority of MAP infections are not detectable by routine fecal culture but will be identified when tissue culture is performed after slaughter. Additionally, we determined that in this herd vertical infection played only a minor role in MAP transmission. By means of extensive and precise longitudinal data from a single dairy herd, we have come to new insights regarding MAP co-infections and within-host evolution.

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Mycobacterium avium subsp. paratuberculosis ELISA Responses in Milk Samples from Vaccinated and Nonvaccinated Dairy Goat Herds in The Netherlands

Veterinary Sciences ◽

10.3390/vetsci6020058 ◽

2019 ◽

Vol 6 (2) ◽

pp. 58 ◽

Cited By ~ 2

Author(s):

Saskia Luttikholt ◽

Karianne Lievaart-Peterson ◽

Maaike Gonggrijp ◽

Marian Aalberts ◽

Gerdien van Schaik ◽

...

Keyword(s):

The Netherlands ◽

Mycobacterium Avium ◽

Relative Sensitivity ◽

Herd Size ◽

Mycobacterium Avium Subsp ◽

Dairy Goat ◽

Dairy Goats ◽

Milk Samples ◽

Goat Population ◽

Mycobacterium Avium Subsp Paratuberculosis

The aims of our study were to calculate the most appropriate cut-off value for milk samples in a serum-validated Mycobacterium avium subsp. paratuberculosis (MAP) ELISA and to analyze MAP ELISA responses in milk samples from vaccinated and nonvaccinated dairy goats in the Netherlands. Analyzed herds were representative for location and herd size of dairy goat herds in the Netherlands. A significantly higher proportion of the analyzed 49 herds were organic as compared with the total Dutch dairy goat population. First, the MAP ELISA was optimized using 992 paired serum and milk samples. At a cut-off of 25 S/P%, the relative sensitivity (Se) was 58.4% (n = 992, 95% CI: 48.8%−67.6%) and relative specificity (Sp) was 98.5% (n = 992, 95% CI: 97.5%−99.2%), as compared to serum ELISA results. The percentage of positively tested herds was 78.2% (n = 49, 95% CI: 63.4%−88.1%). The percentage of positive milk samples per herd (n = 22) was on average 4.6% (median, min, and max of 4.7%, 0.0%, and 10.7%, respectively). Average age of ELISA-positive (3.2 years) and -negative goats (3.2 years) was not different. Significantly more vaccinated goats tested positive (6.7%) as compared with nonvaccinated goats (1.1%). This study shows that a high number of vaccinated and nonvaccinated commercial dairy goat herds in the Netherlands have MAP-ELISA-positive goats.

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Identification of Mycobacterium avium subsp. paratuberculosis (MAP) in Sheep Milk, a Zoonotic Problem

Microorganisms ◽

10.3390/microorganisms8091264 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1264

Author(s):

Sepideh Hosseiniporgham ◽

Tiziana Cubeddu ◽

Stefano Rocca ◽

Leonardo A. Sechi

Keyword(s):

Mycobacterium Avium ◽

Gold Standard ◽

Dairy Product ◽

Mycobacterium Avium Subsp ◽

Individual Level ◽

Reference Models ◽

Sheep Milk ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

The Individual ◽

Serum Elisa

Johne’s disease (JD) is a life-threatening gastrointestinal disease affecting ruminants, which causes crucial economical losses globally. This ailment is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious intracellular pathogen that belongs to the Mycobacteriaceae family. This acid-fast, hard-to-detect bacterium can resist milk pasteurization and be conveyed to dairy product consumers. Many studies have emphasized the zoonotic nature of MAP, suggesting an association between MAP and some gastroenteric conditions such as Crohn’s disease in humans. This underlines the importance of utilizing efficient pasteurization alongside a state-of-the-art diagnostic system in order to minimize the possible ways this pathogen can be conveyed to humans. Until now, no confirmatory MAP screening technique has been developed that can reveal the stages of JD in infected animals. This is partially due to the lack of an efficient gold-standard reference method that can properly evaluate the performance of diagnostic assays. Therefore, the following research aimed to compare the merits of qPCR and ELISA assessments of milk for the detection of MAP in a total of 201 Sardinian unpasteurized sheep milk samples including 73 bulk tank milk (BTM) and 128 individual samples from a MAP-infected flock (MIF) applying various reference models. Accordingly, milk qPCR and ELISA assessments, together and individually, were used as reference models in the herd-level study, while serum ELISA and fecal PCR were similarly (together and in isolation) considered as the gold standards in the individual-level diagnosis. This study showed that the type of gold-standard test affects the sensitivity and specificity of milk qPCR and ELISA significantly. At the individual level in the MAP-infected flock, serum ELISA in isolation and together with fecal PCR were recognized as the best references; however, the best correlation was seen between milk and serum ELISA (p < 0.0001). Regarding the detection of MAP in BTM, qPCR IS900 was recognized as the most sensitive and specific diagnostic test (p < 0.0001) for monitoring the MAP shedders and animals with clinically developed symptoms within herds, under the condition that both milk qPCR and milk ELISA tests formed a binary reference model. The BTM analyses (qPCR and ELISA) revealed that MAP positivity has a seasonal pattern. This hypothesis was proven through a longitudinal study on 14 sheep herds.

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Comparison between two in situ methods for Mycobacterium avium subsp. paratuberculosis detection in tissue samples from infected cattle

Veterinary Microbiology ◽

10.1016/j.vetmic.2008.08.023 ◽

2009 ◽

Vol 134 (3-4) ◽

pp. 383-387 ◽

Cited By ~ 12

Author(s):

F DELGADO ◽

D ETCHECHOURY ◽

A GIOFFRE ◽

F PAOLICCHI ◽

F BLANCOVIERA ◽

...

Keyword(s):

Mycobacterium Avium ◽

Mycobacterium Avium Subsp ◽

Infected Cattle ◽

Tissue Samples ◽

In Situ Methods ◽

Mycobacterium Avium Subsp Paratuberculosis

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Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene

Veterinary Microbiology ◽

10.1016/s0378-1135(01)00348-0 ◽

2001 ◽

Vol 81 (3) ◽

pp. 257-271 ◽

Cited By ~ 13

Author(s):

S. Englund ◽

G. Bölske ◽

A. Ballagi-Pordány ◽

K.-E. Johansson

Keyword(s):

Mycobacterium Avium ◽

Nested Pcr ◽

Mycobacterium Avium Subsp ◽

Tissue Samples ◽

Mycobacterium Avium Subsp Paratuberculosis

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A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test

Vaccines ◽

10.3390/vaccines9090997 ◽

2021 ◽

Vol 9 (9) ◽

pp. 997

Author(s):

Sepideh Hosseiniporgham ◽

Franck Biet ◽

Christelle Ganneau ◽

John P. Bannantine ◽

Sylvie Bay ◽

...

Keyword(s):

Mycobacterium Avium ◽

Elisa Test ◽

Cross Reactivity ◽

Mycobacterium Avium Subsp ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

Individual Level ◽

Milk Samples ◽

Milk Elisa ◽

Mycobacterium Avium Subsp Paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.

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A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

Scientific Reports ◽

10.1038/s41598-021-04451-w ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Sepideh Hosseiniporgham ◽

Lucio Rebechesu ◽

Pierangela Pintore ◽

Stefano Lollai ◽

Maria Dattena ◽

...

Keyword(s):

Magnetic Separation ◽

Mycobacterium Avium ◽

Limit Of Detection ◽

Goat Milk ◽

Mycobacterium Avium Subsp ◽

Milk Samples ◽

Sheep Milk ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Higher Sensitivity

AbstractParatuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

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Modeling the Occurrence of Mycobacterium avium subsp. paratuberculosis in Bulk Raw Milk and the Impact of Management Options for Exposure Mitigation

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-005 ◽

2011 ◽

Vol 74 (7) ◽

pp. 1126-1136 ◽

Cited By ~ 10

Author(s):

CHRISTOPHE BOULAIS ◽

RON WACKER ◽

JEAN-CHRISTOPHE AUGUSTIN ◽

MOHAMED HEDI BEN CHEIKH ◽

FABRICE PELADAN

Keyword(s):

Mycobacterium Avium ◽

Raw Milk ◽

Mycobacterium Avium Subsp ◽

Dairy Herds ◽

Management Options ◽

Scientific Debate ◽

Milk Samples ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Exposure Mitigation ◽

The Impact

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (Johne's disease) in cattle and other farm ruminants. The potential role of MAP in Crohn's disease in humans and the contribution of dairy products to human exposure to MAP continue to be the subject of scientific debate. The occurrence of MAP in bulk raw milk from dairy herds was assessed using a stochastic modeling approach. Raw milk samples were collected from bulk tanks in dairy plants and tested for the presence of MAP. Results from this analytical screening were used in a Bayesian network to update the model prediction. Of the 83 raw milk samples tested, 4 were positive for MAP by culture and PCR. We estimated that the level of MAP in bulk tanks ranged from 0 CFU/ml for the 2.5th percentile to 65 CFU/ml for the 97.5th percentile, with 95% credibility intervals of [0, 0] and [16, 326], respectively. The model was used to evaluate the effect of measures aimed at reducing the occurrence of MAP in raw milk. Reducing the prevalence of paratuberculosis has less of an effect on the occurrence of MAP in bulk raw milk than does managing clinically infected animals through good farming practices.

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