Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia‐Romagna Region (Italy) by qPCR

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows’ milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia‐Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900‐qPCR. Seven hundred and eighty‐nine samples tested MAP‐positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL−1 (and maximum load of 1424 MAP cells mL−1). This study has shown that a small proportion (3.1%) of BTM samples from Emilia‐Romagna region contained MAP in excess of the limit of detection (1.5 × 101 MAP cells mL−1), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production.

[1]  Jaai Kim,et al.  Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. , 2006, Journal of biotechnology.

[2]  P. Bonilauri,et al.  Quantitative risk assessment of Mycobacterium avium subsp. paratuberculosis survival in pasteurized milk in three dairy plants in Italy , 2014 .

[3]  N. Toft,et al.  Occurrence of Mycobacterium avium subsp. paratuberculosis in milk at dairy cattle farms: a systematic review and meta-analysis. , 2012, Veterinary microbiology.

[4]  Ian A Gardner,et al.  An epidemiologic critique of current microbial risk assessment practices: the importance of prevalence and test accuracy data. , 2004, Journal of food protection.

[5]  M. Hornef,et al.  Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis. , 2014, International journal of medical microbiology : IJMM.

[6]  J. Sanderson,et al.  Mycobacterium avium Subspecies paratuberculosis Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities , 2007, Journal of Clinical Microbiology.

[7]  G. Chávez-Gris,et al.  Mycobacterium avium subsp. paratuberculosis detection in individual and bulk tank milk samples from bovine herds and caprine flocks. , 2010, Foodborne pathogens and disease.

[8]  E. Momotani,et al.  Molecular pathogenesis of bovine paratuberculosis and human inflammatory bowel diseases. , 2012, Veterinary immunology and immunopathology.

[9]  P. Ahrens,et al.  Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. , 2000, Veterinary microbiology.

[10]  C. Elliott,et al.  Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells , 2010, Applied and Environmental Microbiology.

[11]  C. Gill,et al.  Mycobacterium avium subsp. paratuberculosis in dairy products, meat, and drinking water. , 2011, Journal of food protection.

[12]  R. Whittington,et al.  PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311. , 1999, Molecular and cellular probes.

[13]  T. Bull,et al.  Crohn's disease caused by Mycobacterium avium subspecies paratuberculosis: a public health tragedy whose resolution is long overdue. , 2002, Journal of medical microbiology.

[14]  R. Sweeney,et al.  Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows , 1992, Journal of clinical microbiology.

[15]  D. Millar,et al.  Solid-phase hybridization capture of low-abundance target DNA sequences: application to the polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum. , 1995, Analytical biochemistry.

[16]  M. Rowe,et al.  Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57‐based qPCR assays , 2011, Journal of applied microbiology.

[17]  P. Vannuffel,et al.  Paratuberculosis , 1994, Clinical Microbiology Reviews.

[18]  M. Sisto,et al.  Proposing a relationship between Mycobacterium avium subspecies paratuberculosis infection and Hashimoto's thyroiditis , 2010, Scandinavian journal of infectious diseases.

[19]  F. J. Guitián,et al.  Contamination of food products with Mycobacterium avium paratuberculosis: a systematic review , 2009, Journal of applied microbiology.

[20]  J. Grange Mycobacterium avium. , 2020, European journal of respiratory diseases.

[21]  M. Burns,et al.  Modelling the limit of detection in real-time quantitative PCR , 2008 .

[22]  R. Stephan,et al.  Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland , 2002, BMC Microbiology.

[23]  M. Nielen,et al.  Milk quality assurance for paratuberculosis: simulation of within-herd infection dynamics and economics. , 2008, Veterinary research.

[24]  A. Lapanje,et al.  Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA , 2012, BMC Veterinary Research.

[25]  T. Schlederer,et al.  Automated high-throughput immunomagnetic separation-PCR for detection of Mycobacterium avium subsp. paratuberculosis in bovine milk. , 2006, International journal of food microbiology.

[26]  C. Dow M. paratuberculosis Heat Shock Protein 65 and Human Diseases: Bridging Infection and Autoimmunity , 2012, Autoimmune diseases.

[27]  N. Arrigoni,et al.  Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and Propidium Monoazide qPCR. , 2014, FEMS microbiology letters.

[28]  M. Moreira,et al.  Short communication: detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil. , 2009, Journal of dairy science.

[29]  R. Zadoks,et al.  Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants , 2009, BMC Microbiology.

[30]  L. Hutchinson,et al.  Evaluation of IS900-PCR assay for detection of Mycobacterium avium subspecies Paratuberculosis infection in cattle using quarter milk and bulk tank milk samples. , 2004, Foodborne pathogens and disease.

[31]  R. Juste,et al.  Estimation of Mycobacterium avium subsp. paratuberculosis Growth Parameters: Strain Characterization and Comparison of Methods , 2011, Applied and Environmental Microbiology.

[32]  F. Díaz,et al.  PRINCIPLES AND METHODS OF VALIDATION OF DIAGNOSTIC ASSAYS FOR INFECTIOUS DISEASES , 2009 .

[33]  A. Lindberg,et al.  A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk. , 2008, Journal of microbiological methods.

[34]  I. Pavlik,et al.  Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry , 2012, BMC Research Notes.

[35]  R. McCallum,et al.  Crohn’s disease and the mycobacterioses: A quarter century later. Causation or simple association? , 2012, Critical reviews in microbiology.

[36]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[37]  C. O’Reilly,et al.  Surveillance of Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Irish Liquid-Milk Pasteurization Plants To Determine the Incidence of Mycobacterium paratuberculosis , 2004, Applied and Environmental Microbiology.

[38]  N. Ahmed,et al.  Association of Mycobacterium avium subsp. paratuberculosis with Multiple Sclerosis in Sardinian Patients , 2011, PloS one.

[39]  M. Collins Food safety concerns regarding paratuberculosis. , 2011, The Veterinary clinics of North America. Food animal practice.

[40]  I. Pavlik,et al.  On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. , 2008, International journal of food microbiology.

[41]  M. Vissers,et al.  Short communication: Quantification of the transmission of microorganisms to milk via dirt attached to the exterior of teats. , 2007, Journal of dairy science.

[42]  M. Haghkhah,et al.  Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds. , 2008, Preventive veterinary medicine.

[43]  I. Grant,et al.  Incidence of Mycobacterium paratuberculosis in Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom , 2002, Applied and Environmental Microbiology.

[44]  I. Pavlik,et al.  Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR. , 2009, Preventive veterinary medicine.