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. 1996 Sep;62(9):3446–3452. doi: 10.1128/aem.62.9.3446-3452.1996

IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales.

D Millar 1, J Ford 1, J Sanderson 1, S Withey 1, M Tizard 1, T Doran 1, J Hermon-Taylor 1
PMCID: PMC168142  PMID: 8795236

Abstract

IS900 PCR for Mycobacterium paratuberculosis was applied to cream, whey, and pellet fractions of centrifuged whole cows' milk. The test and simultaneous control reactions gave correct results for spiked milk and for native milk samples obtained directly from M. paratuberculosis-free, subclinically infected, and clinically infected cows. The test was then applied to units of whole pasteurized cows' milk widely obtained from retail outlets throughout central and southern England from September 1991 to March 1993. With peak periods in January to March and in September to November, when up to 25% of units were affected, an overall 22 of 312 samples (7%) tested positive for M. paratuberculosis. In 18 of the 22 positive samples (81%), the PCR signal segregated to the cream or pellet fractions or both, consistent with the presence of intact mycobacteria. Nine of 18 PCR-positive milk samples (50%) and 6 of 36 PCR-negative milk samples (16%) yielded long-term liquid cultures which tested positive for M. paratuberculosis after 13 to 40 months of incubation, despite overgrowth by other organisms. Taken together with data on the prevalence of M. paratuberculosis infection in herds in the United Kingdom, the known secretion of M. paratuberculosis in milk from subclinically infected animals, and the inability of laboratory conditions simulating pasteurization to ensure the killing of all these slowly growing or unculturable organisms, there is a high risk, particularly at peak times, that residual M. paratuberculosis will be present in retail pasteurized cows' milk in England.

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Selected References

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  1. Buergelt C. D., Hall C., McEntee K., Duncan J. R. Pathological evaluation of paratuberculosis in naturally infected cattle. Vet Pathol. 1978 Mar;15(2):196–207. doi: 10.1177/030098587801500206. [DOI] [PubMed] [Google Scholar]
  2. Carrigan M. J., Seaman J. T. The pathology of Johne's disease in sheep. Aust Vet J. 1990 Feb;67(2):47–50. doi: 10.1111/j.1751-0813.1990.tb07693.x. [DOI] [PubMed] [Google Scholar]
  3. Cetinkaya B., Egan K., Harbour D. A., Morgan K. L. An abattoir-based study of the prevalence of subclinical Johne's disease in adult cattle in south west England. Epidemiol Infect. 1996 Jun;116(3):373–379. doi: 10.1017/s0950268800052705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cetinkaya B., Egan K., Morgan K. L. A practice-based survey of the frequency of Johne's disease in south west England. Vet Rec. 1994 May 7;134(19):494–497. doi: 10.1136/vr.134.19.494. [DOI] [PubMed] [Google Scholar]
  5. Chiodini R. J. Crohn's disease and the mycobacterioses: a review and comparison of two disease entities. Clin Microbiol Rev. 1989 Jan;2(1):90–117. doi: 10.1128/cmr.2.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chiodini R. J., Hermon-Taylor J. The thermal resistance of Mycobacterium paratuberculosis in raw milk under conditions simulating pasteurization. J Vet Diagn Invest. 1993 Oct;5(4):629–631. doi: 10.1177/104063879300500424. [DOI] [PubMed] [Google Scholar]
  7. Chiodini R. J., Van Kruiningen H. J., Merkal R. S. Ruminant paratuberculosis (Johne's disease): the current status and future prospects. Cornell Vet. 1984 Jul;74(3):218–262. [PubMed] [Google Scholar]
  8. Chiodini R. J., Van Kruiningen H. J., Merkal R. S., Thayer W. R., Jr, Coutu J. A. Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease. J Clin Microbiol. 1984 Nov;20(5):966–971. doi: 10.1128/jcm.20.5.966-971.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chiodini R. J., Van Kruiningen H. J., Thayer W. R., Merkal R. S., Coutu J. A. Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified Mycobacterium species isolated from patients with Crohn's disease. Dig Dis Sci. 1984 Dec;29(12):1073–1079. doi: 10.1007/BF01317078. [DOI] [PubMed] [Google Scholar]
  10. Clarke C. J., Little D. The pathology of ovine paratuberculosis: gross and histological changes in the intestine and other tissues. J Comp Pathol. 1996 May;114(4):419–437. doi: 10.1016/s0021-9975(96)80017-x. [DOI] [PubMed] [Google Scholar]
  11. Dell'Isola B., Poyart C., Goulet O., Mougenot J. F., Sadoun-Journo E., Brousse N., Schmitz J., Ricour C., Berche P. Detection of Mycobacterium paratuberculosis by polymerase chain reaction in children with Crohn's disease. J Infect Dis. 1994 Feb;169(2):449–451. doi: 10.1093/infdis/169.2.449. [DOI] [PubMed] [Google Scholar]
  12. Erasmus D. L., Victor T. C., van Eeden P. J., Falck V., van Helden P. Mycobacterium paratuberculosis and Crohn's disease. Gut. 1995 Jun;36(6):942–942. doi: 10.1136/gut.36.6.942-b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fellows I. W., Freeman J. G., Holmes G. K. Crohn's disease in the city of Derby, 1951-85. Gut. 1990 Nov;31(11):1262–1265. doi: 10.1136/gut.31.11.1262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fidler H. M., Thurrell W., Johnson N. M., Rook G. A., McFadden J. J. Specific detection of Mycobacterium paratuberculosis DNA associated with granulomatous tissue in Crohn's disease. Gut. 1994 Apr;35(4):506–510. doi: 10.1136/gut.35.4.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gitnick G., Collins J., Beaman B., Brooks D., Arthur M., Imaeda T., Palieschesky M. Preliminary report on isolation of mycobacteria from patients with Crohn's disease. Dig Dis Sci. 1989 Jun;34(6):925–932. doi: 10.1007/BF01540280. [DOI] [PubMed] [Google Scholar]
  16. Grant I. R., Ball H. J., Neill S. D., Rowe M. T. Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures. Appl Environ Microbiol. 1996 Feb;62(2):631–636. doi: 10.1128/aem.62.2.631-636.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Green E. P., Tizard M. L., Moss M. T., Thompson J., Winterbourne D. J., McFadden J. J., Hermon-Taylor J. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 1989 Nov 25;17(22):9063–9073. doi: 10.1093/nar/17.22.9063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hermon-Taylor J., Moss M., Tizard M., Malik Z., Sanderson J. Molecular biology of Crohn's disease mycobacteria. Baillieres Clin Gastroenterol. 1990 Mar;4(1):23–42. doi: 10.1016/0950-3528(90)90037-h. [DOI] [PubMed] [Google Scholar]
  19. Kyle J. Crohn's disease in the northeastern and northern Isles of Scotland: an epidemiological review. Gastroenterology. 1992 Aug;103(2):392–399. doi: 10.1016/0016-5085(92)90826-k. [DOI] [PubMed] [Google Scholar]
  20. LEMCKE R. M., HURST A. Antibody content of colostrum and piglet serum following vaccination of the sow. J Comp Pathol. 1961 Jul;71:268–278. doi: 10.1016/s0368-1742(61)80033-7. [DOI] [PubMed] [Google Scholar]
  21. Lisby G., Andersen J., Engbaek K., Binder V. Mycobacterium paratuberculosis in intestinal tissue from patients with Crohn's disease demonstrated by a nested primer polymerase chain reaction. Scand J Gastroenterol. 1994 Oct;29(10):923–929. doi: 10.3109/00365529409094864. [DOI] [PubMed] [Google Scholar]
  22. McClure H. M., Chiodini R. J., Anderson D. C., Swenson R. B., Thayer W. R., Coutu J. A. Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides). J Infect Dis. 1987 May;155(5):1011–1019. doi: 10.1093/infdis/155.5.1011. [DOI] [PubMed] [Google Scholar]
  23. McFadden J. J., Butcher P. D., Chiodini R., Hermon-Taylor J. Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J Clin Microbiol. 1987 May;25(5):796–801. doi: 10.1128/jcm.25.5.796-801.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Millar D. S., Withey S. J., Tizard M. L., Ford J. G., Hermon-Taylor J. 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. Anal Biochem. 1995 Apr 10;226(2):325–330. doi: 10.1006/abio.1995.1232. [DOI] [PubMed] [Google Scholar]
  25. Moss M. T., Sanderson J. D., Tizard M. L., Hermon-Taylor J., el-Zaatari F. A., Markesich D. C., Graham D. Y. Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues. Gut. 1992 Sep;33(9):1209–1213. doi: 10.1136/gut.33.9.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ridley D. S. Histological classification and the immunological spectrum of leprosy. Bull World Health Organ. 1974;51(5):451–465. [PMC free article] [PubMed] [Google Scholar]
  27. Rose J. D., Roberts G. M., Williams G., Mayberry J. F., Rhodes J. Cardiff Crohn's disease jubilee: the incidence over 50 years. Gut. 1988 Mar;29(3):346–351. doi: 10.1136/gut.29.3.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanderson J. D., Moss M. T., Tizard M. L., Hermon-Taylor J. Mycobacterium paratuberculosis DNA in Crohn's disease tissue. Gut. 1992 Jul;33(7):890–896. doi: 10.1136/gut.33.7.890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Streeter R. N., Hoffsis G. F., Bech-Nielsen S., Shulaw W. P., Rings D. M. Isolation of Mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows. Am J Vet Res. 1995 Oct;56(10):1322–1324. [PubMed] [Google Scholar]
  30. Suenaga K., Yokoyama Y., Okazaki K., Yamamoto Y. Mycobacteria in the intestine of Japanese patients with inflammatory bowel disease. Am J Gastroenterol. 1995 Jan;90(1):76–80. [PubMed] [Google Scholar]
  31. Sweeney R. W., Whitlock R. H., Hamir A. N., Rosenberger A. E., Herr S. A. Isolation of Mycobacterium paratuberculosis after oral inoculation in uninfected cattle. Am J Vet Res. 1992 Aug;53(8):1312–1314. [PubMed] [Google Scholar]
  32. Sweeney R. W., Whitlock R. H., Rosenberger A. E. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J Clin Microbiol. 1992 Jan;30(1):166–171. doi: 10.1128/jcm.30.1.166-171.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taylor T. K., Wilks C. R., McQueen D. S. Isolation of Mycobacterium paratuberculosis from the milk of a cow with Johne's disease. Vet Rec. 1981 Dec 12;109(24):532–533. [PubMed] [Google Scholar]
  34. Thorel M. F. Relationship between Mycobacterium avium, M. paratuberculosis and mycobacteria associated with Crohn's disease. Ann Rech Vet. 1989;20(4):417–429. [PubMed] [Google Scholar]
  35. Wall S., Kunze Z. M., Saboor S., Soufleri I., Seechurn P., Chiodini R., McFadden J. J. Identification of spheroplast-like agents isolated from tissues of patients with Crohn's disease and control tissues by polymerase chain reaction. J Clin Microbiol. 1993 May;31(5):1241–1245. doi: 10.1128/jcm.31.5.1241-1245.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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