HARUN HIZLISOY, Meral ÖZTÜRK KALIN, K. Semih GÜMÜŞSOY

Kayseri İlinde Satışa Sunulan Sokak Sütlerinde Mycobacterium paratuberculosis Varlığının Konvansiyonel ve Serolojik Yöntemlerle Araştırılması

Bu çalışmada Kayseri ilinde satışa sunulan sokak sütlerinde Mycobacterium avium subsp. paratuberculosis (MAP)'in varlığı konvansiyonel ve serolojik yöntemlerle araştırılması amaçlandı. Araştırmada Mayıs 2010 – Ekim 2011 yılları arasında Kayseri’nin ilçelerinden farklı satıcılardan 200 çiğ inek süt örneği toplandı. Her bir süt numunesi direkt mikroskopi, kültür ve serolojik yöntem olmak üzere üç aşamada değerlendirdi. Direkt mikroskopide süt örneklerinden hazırlanan preparatlar Ziehl-Neelsen boyama metodu ile boyandı. Ayrıca besiyerlerinden izole edilen şüpheli kolonilerin boyamasında da aynı yöntem kullanıldı. Etken izolasyonu amacıyla süt numunelerinin bakteriyolojik ekimleri gliserinli ve gliserinsiz Löwenstein-Jensen ve Watson-Reid agara yapıldı. İdentifikasyon için katalaz, niasin, nitrat redüksiyon, pirazinamidaz ve üreaz testleri kullanıldı. Sütteki antikorların araştırılmasında Mycobacterium paratuberculosis Enzyme-Linked ImmunoSorbent Assay (ELISA) kitinden yararlanıldı. İncelenen örneklerde üç yönteminin kullanılması sonucu sadece 1 numunede (% 0.5) MAP tespit edildi. Sonuç olarak, Kayseri ilinde satışa sunulan sokak sütlerinde paratüberkülozun yüksek oranda bulunmadığı belirlendi. Ancak bir örnekte etkenin tespiti infeksiyonun önemi göz önünde bulundurulduğunda halk sağlığı açısından önemli risk oluşturabileceği kanaatini ortaya koymuştur.

Anahtar Kelimeler:

ELISA, identifikasyon, izolasyon, Mycobacterium paratuberculosis, sokak sütü

The Investigation of Mycobacterium paratuberculosis by Serological and Cultural Methods In Raw Milks Retailed in Kayseri

In this study, it was aimed to investigate the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk sold in Kayseri province by conventional and serological methods. Between May 2010 and October 2011, 200 raw cow milk samples were collected from various sellers of the Kayseri provinces. Each milk sample was evaluated in three steps including direct microscopy, culture and serological method. Preparations from milk samples were stained with Ziehl-Neelsen staining method in direct microscopy method. In addition, the same method was used for the staining of suspicious colonies isolated from the media. For the purpose of isolation, the bacteriological inoculation of milk samples was carried out onto Watson-Reid agar and Löwenstein-Jensen supplemented with and without glycerin. Catalase, niacin, nitrate reduction, pyrazinamidase and urease tests were performed for the identification. Mycobacterium paratuberculosis Enzyme-Linked ImmunoSorbent Assay (ELISA) kit was used for the detection of antibodies in milk samples. MAP was detected in only one sample (0.5%) with the using of three identification methods. As a result, it has been understood that paratuberculosis was not found at high rate in raw milk sold in Kayseri province. However, it has been concluded that the presence of an agent in one sample may pose a significant risk for public health when considering the importance of infection.

Keywords:

ELISA, identification, isolation, Mycobacterium paratuberculosis, raw milk,

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1. Anzabi Y, Hanifian S. Detection of Mycobacterium avium subspecies paratuberculosis in pasteurized milk by IS900 PCR and culture method. African J Microbiol Res 2012; 6(7): 1453–56.
2. Ayele WY, Svastova P, Roubal P, Bartos M, Pavlik I. Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow’s milk in the Czech Republic. Appl Environ Microbiol 2005; 71(3): 1210-14.
3. Carvalho IA, Pietralonga PAG, Schwarz DGG, Faria ACS, Moreira MAS. Short communication: recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil. J Dairy Sci 2012; 95(12): 6946-48.
4. Çetinkaya B, Muz A, Ertaş HB, Güngör H, Sezen İY, Gülcü HB. Süt ineklerinde paratüberkülozun prevalansının polimeraz zincir reaksiyonu (PZR) ile saptanması. Tr J Vet Anim Sci 2000; 24(4): 371-9.
5. Djonne B, Pavlik I, Svastova P, Bartos M, Holstad G. IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway. Acta Vet Scand 2005; 46(1-2): 13-8.
6. Drobniewski FA, Uttley AHC. Mycobacterial speciation. Parish T, Stoker NG eds. In: Mycobacteria Protocols. Towata: Humana Press Inc, 1998; pp. 323-47.
7. Dundee L, Grant IR, Ball HJ, Rowe MT. Comparative evaluation of four decontamination protocols for the isolation of Mycobacterium avium subsp. paratuberculosis from milk. Lett Appl Microbiol 2001; 33(3): 173-7.
8. Eda S, Elliott B, Scott MC, Waters WR, Bannantine JP, Whitlock RH, Speer CA. New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne’s Disease) by flow cytometry. Foodborne Pathog Dis 2005; 2(3): 250-62.
9. Florou M, Leontides L, Kostoulas P, Billinis C, Sofia M, Kyriazakis I, Lykotrafitis F. Isolation of Mycobacterium avium subspecies paratuberculosis from non-ruminant wildlife living in the sheds and on the pastures of Greek sheep and goats. Epidemiol Infect 2008; 136(5): 644-52.
10. Grant IR, Kirk RB, Hitchings E, Rowe MT. Comparative evaluation of the MGITTM and BACTEC culture systems for the recovery of Mycobacterium avium subsp. paratuberculosis from milk. J Appl Microbiol 2003; 95(1): 196-201.
11. Gümüşsoy KS, İça T, Abay S, Aydın F, Hızlısoy H. Serological and molecular diagnosis of paratuberculosis in dairy cattle. Turkish J Vet Anim Sci 2015; 39(2): 147-53.
12. Hendrick SH, Duffield TF, Kelton DF, Leslie KE, Lissemore KD, Archambault M. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows. JAVMA 2005; 226(3): 424-8.
13. Hendrick SH, Kelton DF, Leslie KE, Lissemore KD, Archambault M, Duffield TF. Effect of paratuberculosis on culling, milk production, and milk quality in dairy herds. JAVMA 2005; 227(8): 1302-8.
14. Ikız S, Bagcıgıl AF, Ak S, Ozgur NY, Ilgaz A. Paratuberculosis in cattle in Turkey detected by PCR. Medycna Wet 2005; 61(8): 881-3.
15. Koenig GJ, Hoffsis GF, Shulaw WP, Bech-Nielsen S, Rings DM, St-Jean G. Isolation of Mycobacterium paratuberculosis from mononuclear cells in tissues, blood, and mammary glands of cows with advanced paratuberculosis. Am J Vet Res 1993; 54(9): 1441-5.
16. Makav V, Gökçe E. Kars yöresi sığırlarında subklinik paratüberkülozun seroprevalansı. Kafkas Unıv Vet Fak Derg 2013; 19(5): 913-6.
17. Merkal RS, Curran BJ. Growth and metabolic characteristics of Mycobacterium paratuberculosis. Appl Microbiol 1974; 28(2): 276-9.
18. Oken HA, Saleeb PG, Redfield RR, Schimpff SC. Is Mycobacterium avium paratuberculosis the trigger in the Crohn’s Disease spectrum. Open Forum Infect Dis 2017; 4(3): 1-3.
19. Ozturk D, Pehlivanoglu F, Tok AA, Gunlu S, Guldali Y, Turutoglu H. Seroprevalence of paratuberculosis in the Burdur province (Turkey), in dairy cattle using the enzyme linked immunosorbent assay (ELISA). Israel J Vet Med 2010; 65(2): 53-7.
20. Özpınar H, Tekiner İH, Karaman O, Kurt Y. Türkiye Trakya bölgesindeki süt işletmelerinden toplanan fekal ve çiğ süt örneklerinde Mycobacterium avium subsp. paratuberculosis (MAP) incelemesi. Kafkas Univ Vet Fak Derg 2015; 21(2): 247-52.
21. Patel A, Shah N. Mycobacterium avium subsp paratuberculosis--incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health. J Microbiol Immun Infect 2011; 44(6): 473-9.
22 Pierce ES. Could Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease, ulcerative colitis and colorectal cancer? Infect Agent Cancer 2018; 13(1): 1.
23. Robertson RE, Cerf O, Condron RJ, Donaghy JA, Heggume C, Jordan K. Review of the controversy over whether or not Mycobacterium avium subsp. paratuberculosis poses a food safety risk with pasteurised dairy products. Int Dairy J 2017; 73: 10-8.
24. Rossi G, Grohn YT, Schukken YH, Smith RL. The effect of Mycobacterium avium subspecies paratuberculosis infection on clinical mastitis occurrence in dairy cows. J Dairy Sci 2017; 100(9): 1-9.
25. Stabel JR, Steadham EM, Bolin CA. Heat inactivation of Mycobacterium paratuberculosis in raw milk: are current pasteurization conditions effective? Appl Environ Microbiol 1997; 63(12): 4975-7.
26. Stratmann J, Strommenger B, Stevenson K, Gerlach GF. Development of a peptide-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk. J Clin Microbiol 2002; 40(11): 4244-50.
27. Sweeney RW, Whitlock RH, Hamir AN, Rosenberger AE, Herr SA. Isolation of Mycobacterium paratuberculosis after oral inoculation of uninfected cattle. Am J Vet Res 1992; 53(8): 1312-4.
28. Uzun M. Tüberküloz tanı yöntemleri LJ besiyeri ile antitüberküloz duyarlılık testi. 21 Yüzyılda Tüberküloz Sempozyumu, 286-289 2003; Samsun-Türkiye.
29. Whitlocka RH, Wellsb SJ, Sweeneya RW, Van Tiem J. ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method. Vet Microbiol 2000; 77(3-4): 387-98.
30. Yıldırım D, Civelek T. Prevalence of subclinical paratuberculosis in dairy cattle in Uşak region. Kafkas Unıv Vet Fak Derg 2013; 19(1): 121-6.