SERPİL KAHYA DEMİRBİLEK, Burcu TUĞ KESİN, SERAN TEMELLİ, K. Tayfun ÇARLI, Ayşegül EYİGÖR

Yumurtacı tavuklarda Salmonella izolatlarının tanısı ve tiplendirilmesi

Çalışmada, farklı yetiştirme dönemlerindeki yumurtacı tavuklarda Salmonella aranmasında farklı metotların değerlendirilmesi, elde edilen izolatların serotiplendirilmesi, ribotiplendirilmesi ve antibiyotiplendirilmesi ile Salmonella yaygınlığının belirlenmesi amaçlandı. Bu amaçla, 58 adet ticari yumurtacı tavuk kümesinden yetiştirmenin 15., 25. ve 40. haftalık dönemlerinde alınan toplam 174 adet drag svap örneği, Salmonella varlığı açısından standart kültür metodu olan ISO (ISO 6579:2002/Amd 1:2007 - ISO) ve 2 farklı gerçek zamanlı polimeraz zincir reaksiyonu (rPCR) sistemi (Light Cycler SYBR Green I rPCR - LCSGIrPCR ve DuPont BAX Q7 rPCR - BAXrPCR) kullanılarak analiz edildi. İncelenen 58 kümesin 17 adedi (%29.31), 174 adet drag svap örneğinin 15. ve 25. haftalık dönemlerde 6şar (%3.44 ve %3.44), 40. haftada ise 10 adedi (%5.74) olmak üzere toplam 22si (%12.62) her 3 metotla Salmonella yönünden pozitif olarak bulundu. Elde edilen 22 adet Salmonella izolatının 20sinin (%90.90) Salmonella enterica subsp. enterica olup 9unun Enteritidis (SE) (%40.9), 7sinin Infantis (SI) (%31.8), 1inin Hadar (%4.5), 1inin Montevideo (%4.5), 1inin Colombo (%4.5) ve 1inin de Spartel (%4.5) serovarı olduğu, 2sinin (%9.10) ise Salmonella enterica subsp. arizonae ile Salmonella enterica subsp. houtenae alttürlerine ait olduğu belirlendi. Ayrıca izolatların test edilen 24 antibiyotiğin 23üne karşı direnç oluşturduğu, en yüksek direnç oranının Ampicillin (%100), Neomycin (%100), Penicillin G (%100) ve Erythromycine (%95.45) karşı olduğu saptandı. Çalışmanın sonucunda, yumurtacı tavuklarda Salmonella nın hızlı deteksiyonunda LCSGIrPCR ve BAXrPCR sistemlerinin ISO metodunu destekleyici olarak kullanılabileceği, incelenen tüm yetiştirme dönemlerinde Salmonella enterica subsp. enterica varlığının yüksek olduğu belirlendi.

Detection of Salmonella from layer flocks and typing of the isolates

This study aimed to evaluate diferent detection methods in determining Salmonella prevalence in layers of various rearing periods, and to serotype, ribotype and antibiotype the isolates. For this, 174 drag swab samples taken from 58 commercial layer focks of 15, 25, and 40 week age were analyzed for Salmonella by ISO standard culture method (ISO 6579:2002/Amd 1:2007 - ISO) and by 2 real time polymerase chain reaction (rPCR) systems (Light Cycler SYBR Green I rPCR - LCSGIrPCR and DuPont BAX Q7 rPCR - BAXrPCR). Seventeen out of 58 (29.31%) fock samples, and 6 of the 15 th and 25 th weeks (3.44% and 3.44%), and 10 (5.74%) of the 40 th weeks drag swab samples were Salmonella positive by all 3 methods. Twenty out of 22 Salmonella isolates (90.90%) were Salmonella enterica subsp. enterica , including serovars of 9 Enteritidis (SE) (40.9%), 7 Infantis (SI) (31.8%), 1 Hadar (4.5%), 1 Montevideo (4.5%), 1 Colombo (4.5%), 1 Spartel (4.5%), and 2 subspecies (9.10%) as Salmonella enterica subsp. arizonae and Salmonella enterica subsp. houtenae. Also isolates showed resistance to 23 of 24 antibiotics, with the highest resistances to Ampicillin (100%), Neomycin (100%), Penicillin G (100%) and Erythromycin (95.45%). Results indicate that both LCSGIrPCR and BAXrPCR systems can be used to complement ISO method in rapid detection of Salmonella from layer chickens, and that there is high prevalence of Salmonella enterica subsp. enterica in all rearing periods examined.

PDF

___

1. CDC (Center of Diseases Control): Multistate outbreak of human Salmonella Enteritidis infections associated with shell eggs (Final Update). http://www.cdc.gov/salmonella/enteritidis/index.html, Accessed: 05.04.2014.
2. Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE: Salmonella infection. In, Gast RK (Ed): Diseases of Poultry. 12 th ed., 619-665, Blackwell Publishing, Iowae, 2008.
3. Eriksson E, Aspan A: Comparision of culture, ELISA and PCR techniques for Salmonella detection in faecal samples for cattle, pig and poultry. BMC Vet Res, 3, 1-19, 2007.
4. Temelli S, Kahya S, Eyigor A, Carli KT: Incidence of Salmonella Enteritidis in chicken layer focks in Turkey: Results by real-time polymerase chain reaction and International Organization for Standardization culture methods . Poultry Sci, 89, 1406-1410, 2010.
5. Medici DD, Croci L, Delibato E, Pasquale SD, Filetici E, Toti L: Evaluation of DNA extraction methods for use in combination with SYBR Green I Real-Time PCR to detect Salmonella enterica serotype Enteritidis in poultry. Appl Environ Microbiol, 69, 3456-3461, 2003.
6. Eyigor A, Goncagul G, Gunaydin E, Carli KT: Salmonella profile in chickens determined by real-time polymerase chain reaction and bacteriology from years 2000-2003 in Turkey. Avian Pathol, 34, 101-105, 2005.
7. Tomazelli IB, Freitas JB, Fabbi LM, Filipini TA, Silva CM, Bedin JM, Duarte DAM, Santos A, Baccarin A, Higa LRH, Yano DMY, Baccarin A, Higa LRG, Yano DMY, Killner M, Frezza ALC, Abecia ECG, Tronco VM, Junior OT, Junior WB: Comparison of the BAX system PCR method to Brazils official method for the detection of Salmonella in food, water and environmental samples. J Food Prot, 71, 2442-2447, 2008.
8. Tice G, Andaloro B, Fallon D, Wallace FM: Dupont qualicon BAX system polymerase chain reaction assay performance tested method 100201. J AOAC Int, 92, 1902-1905, 2009.
9. Tice G, Andaloro B, White HK, Bolton L, Wang S, Davis E, Wallace M: In-house validation study of the Dupont qualicon BAX system Q7 instrument with the BAX System PCR assay for Salmonella (Modification of AOAC Official Method 2003.09 and AOAC Research Institute Performance- Tested method 100201. Vet Drug Res, 92, 989-994, 2009.
10. Löfström C, Kravse M, Josefsen MH, Hansen F, Hoorfar J: Validation of a same-day real-time PCR method for screening of meat and carcases swabs for Salmonella. BMC Microbiol, 9, 1-9, 2009.
11. Franchin P, Ogliari PJ, Andrade DF, Chiapinoto M, Silva IGD, Batista CRV: Comparision of the BAX system with an in house MRSV method for the detection of Salmonella in chicken carcasses and pork meat. Braz J Microbiol, 37, 521-526, 2006.
12. Sommer D, Enderlein D, Antakli A, Schönenbrücher H, Slaghuis J, Redmann T, Lier M: Salmonella detection in poultry samples. Comparision of two commercial real-time PCR systems with culture methods for the detection of Salmonella spp. in environmental and fecal samples of poultry. Tierartl Prax, 40, 383-389, 2012.
13. Hollis RJ, Bruce L, Fritschel SJ, Pfaller MA: Comparative evaluation of an automated riboprinting instrument versus Pulsed-field gel electrophoresis for epidemiological investigation of clinical isolates of bacteria. Diagn Microbiol Infect Dis, 34, 263-268, 1999.
14. De Cesare A, Manfreda G, Dambaugh TR, Guerzoni ME, Franchini A: Automated ribotyping and random amplified poly- morphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy. J Appl Microbiol, 91, 780-785, 2001.
15. Li X, Payne JB, Santos FB, Levine JF, Anderson KE, Sheldon BW: Salmonella populations and prevalence in layer feces from commercial high-rise houses and characterization of the Salmonella isolates by serotyping, antibiotic resistance analysis and pulsed field gel electrophoresis. Poult Sci, 86, 591-597, 2007.
16. Temelli S, Kahya S, Eyigor A, Carli KT: Antibiotic resistance phenotypes of Salmonella isolates of chicken meat and chicken origin. Ankara Univ Vet Fak Derg, 59, 107-114, 2012.
17. Mahmud MS, Bari ML, Hossain MA: Prevalence of Salmonella serovars and antimicrobial resistance profiles in poultry of Savar Area, Bangladesh. Foodborne Path Dis, 8, 1111-1118, 2011.
18. Com mission Regulation (EC) No 1168/2006: Implementing Regulation No 2160/2003 as regards a community target for the reduction of the prevalence of certain Salmonella serotypes in laying hens of Gallus gallus and Amending regulation (EC) No 1003/2005.
19. ISO: Microbiology of food and animal feeding stufs-horizontal method for the detection of Salmonella (EN ISO 6579:2002/Amd 1:2007). Intenational Organization for Standardization, Geneva, Switerland, 2007.
20. Grimont PAD, Weill F, 2007: Antigenic formulae of the Salmonella serovars: White-Kaufmann-Le Minor Scheme. 9 th ed. WHO Collaborating Centre for Reference and Research on Salmonella.http://www.pasteur.fr/ ip/portal/action/WebdriveActionEvent/oid/01s-000036-089. Accessed: 05.04.2014.
21. Guibourdenche M, Roggent P, Mikoleit M, Fields PI, Bockemühl J, Grimont PA, Weill FX: Supplement 2003-2007 (No.47) to the White- Kaufmann-Le Minor scheme. Res Microbiol, 161, 26-29, 2010.
22. Riboprinter Kullanım Kılavuzu: http://www2.dupont.com/ Qualicon/en_US/products/RiboPrinter_System/ Erişim tarihi: 05.04.2014.
23. CLSI: Performance standards for antimicrobial susceptibility testing; twentieth informational supplement, CLSI document M100-S20, Clinical and Laboratory Standards Institute, Wayne, PA, USA, 2010.
24. Rahn K, De Grandis A, Clarke RC, McEwen SA, Galan JE, Ginocchio C, Curtiss R, Gyles CL: Amplification of an invA gene sequence of Salmonella Typhimurium by polymerase chain reaction as a specific method of detection of Salmonella . Mol Cell Probe, 6, 271-279, 1992.
25. SPSS: Windows (SPSS Inc. USA) Version 20.0.
26. Eyigor A, Carli KT, Unal CB: Implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. Lett Appl Microbiol, 34, 37-41, 2002.
27. Ata Z, Aydın N: Ankara bölgesindeki tavukçuluk işletmelerinden Salmonella spp. izolasyonu. Ankara Univ Vet Fak Derg, 55, 161-166, 2008.
28. Pitesky M, Charlton B, Bland M, Rolfe D: Surveillance of Salmonella enteritidis in layer houses: A retrospective comparison of the food and drug administrations egg safety rule (2010-2011) and the California egg quality assurance program (2007-2011). Avian Dis, 57, 51-56, 2013.
29. Zhang L, Yan Z, Ryser ET: Comparison of the reveal test, the U. S. Food and Drug Administration culture method, and selective media for recovery of Salmonella enteridis from commercial egg layer fock environments. J Food Prot, 69, 2766-2769, 2006.
30. Van Hoorebeke S, Van Immerseel F, Schulz J, Hartung J, Harisberger M, Barco L, Ricci A, Theodoropoulos G, Xylouri E, De Vylder J, Ducatelle R, Haesebrouck F, Pasmans F, De Kruif A, Dewulf J: Determination of the within and between and fock prevalence and identification of risk factors for Salmonella infections in laying hen focks housed in conventional and alternative systems. Prev Vet Med , 94, 94-100, 2010.
31. Lapuz RR, Umali DV, Suzuki T, Shirota K, Katoh H: Comparison of the prevalence of Salmonella infection in layer hens from commercial layer farms with high and low rodent densities. Avian Dis, 56, 29-34, 2012.
32. Iwabuchi E, Noriko M, Ayumi H, Masaaki N, Masatake M, Tameichi O, Katsuya H: Nationwide survey of Salmonella prevalence in Environmental dust from layer farms in Japan. J Food Prot, 11, 1962- 2140, 2010 .