J Vet Sci.  2012 Dec;13(4):371-376.

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

• Iraola G
• Hernandez
• Calleros L
• Paolicchi
• Silveyra S
• Velilla A
• Carretto L
• Rodriguez
• Perez R

Abstract

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.

Keyword

bovine aborted fetuses; Campylobacter fetus subspecies venerealis; multiplex PCR

MeSH Terms

Animals
Campylobacter
Campylobacter fetus
Cattle
Communicable Diseases
Fetus
Multiplex Polymerase Chain Reaction
Pathology, Molecular
Sheep

Figure

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  Fig. 1 Multiplex PCR results for C. fetus subsp. fetus (Cff), C. fetus subsp. venerealis (Cfv), C. fetus subsp. venerealis biovar intermedius (Cfvi), and the negative control strains. Lane 1: negative control (no template), Lane 2: 200-bp molecular weight ladder, Lane 3: Cff063, Lane 4: CffA28, Lane 5: Cfv3598, Lane 6: CfvD78, Lane 7: Cfvi470, Lane 8: C. sputorum subsp. bubulus, Lane 9: C. jejuni, Lane 10: C. hyointestinalis, Lane 11: C. Coli, and Lane 12: E. coli.

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  Fig. 2 Multiplex PCR results of the four field samples. Lane 1: negative control (no template), Lane 2: 200-bp molecular weight ladder, Lane 3: Cfv positive control, Lane 4: 6600, Lane 5: 3726, Lane 6: 3837, and Lane 7: 2733. The four field samples were identified as Cfv.

Reference

1. Abril C, Vilei EM, Brodard I, Burnens A, Frey J, Miserez R. Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation. Clin Microbiol Infect. 2007. 13:993–1000.

2. Campero CM, Anderson ML, Walker RL, Blanchard PC, Barbano L, Chiu P, Martínez A, Combessies G, Bardon JC, Cordeviola J. Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions. J Vet Med B Infect Dis Vet Public Health. 2005. 52:138–141.

3. Debruyne L, Gevers D, Vandamme P. Nachamkin I, Szymanski CM, Blaser MJ, editors. Taxonomy of the family Campylobacteraceae. Campylobacter. 2008. 3rd ed. Washington: ASM Press;3–25.

4. Dekeyser J. Butzler JP, editor. Bovine genital campylobacteriosis. Campylobacter Infection in Man and Animals. 1984. Boca Raton: CRC Press;181–191.

5. Elazhary M. An assay of isolation and identification of some animal vibrios and of elucidation of their pathological significance. Meded Veeartsschool Rijksuniv Gent. 1968. 12:1–80.

6. Garcia MM, Eaglesome MD, Rigby C. Campylobacters important in veterinary medicine. Vet Bull. 1983. 53:793–818.

7. Hum S, Quinn K, Brunner J, On SLW. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. Aust Vet J. 1997. 75:827–831.

8. Lappin MR. Infectious disease diagnostic assays. Top Companion Anim Med. 2009. 24:199–208.

9. Lecce JG. Some biochemical characteristics of Vibrio fetus and other related Vibrios isolated from animals. J Bacteriol. 1958. 76:312–316.

10. Linton D, Owen RJ, Stanley J. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Res Microbiol. 1996. 147:707–718.

11. Malorny B, Tassios PT, Rådström P, Cook N, Wagner M, Hoorfar J. Standardization of diagnostic PCR for the detection of foodborne pathogens. Int J Food Microbiol. 2003. 83:39–48.

12. Meier PA, Dooley DP, Jorgensen JH, Sanders CC, Huang WM, Patterson JE. Development of quinolone-resistant Campylobacter fetus bacteremia in human immunodeficiency virus-infected patients. J Infect Dis. 1998. 177:951–954.

13. Moolhuijzen PM, Lew-Tabor AE, Wlodek BM, Agüero FG, Comerci DJ, Ugalde RA, Sanchez DO, Appels R, Bellgard M. Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets. BMC Microbiol. 2009. 9:86.

14. On SLW, Harrington CS. Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods. J Appl Microbiol. 2001. 90:285–293.

15. Repisso MV, Gil A, Bañales P, D'Anatro N, Fernandez L, Guarino H, Herrera B, Núñez A, Olivera M, Osawa T, Silva M. Prevalencias de las principales enfermedades infecciosas que afectan el comportamiento reproductivo en la ganadería de carne y caracterización de los establecimientos de cría del Uruguay. Veterinaria. 2005. 40:5–28.

16. Schmidt T, Venter EH, Picard JA. Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates. J S Afr Vet Assoc. 2010. 81:87–92.

17. Schulze F, Bagon A, Müller W, Hotzel H. Identification of Campylobacter fetus subspecies by phenotypic differentiation and PCR. J Clin Microbiol. 2006. 44:2019–2024.

18. Skirrow MB, Blaser MJ. Nachamkin I, Blaser MJ, editors. Clinical aspects of Campylobacter infection. Campylobacter. 2000. 2nd ed. Washington: ASM Press;69–88.

19. Spence RP, Bruce IR, McFadden AMJ, Hill FI, Tisdall D, Humphrey S, van der Graaf L, van Bergen MAP, Wagenaar JA. Cross-reaction of a Campylobacter fetus subspecies venerealis real-time PCR. Vet Rec. 2011. 168:131.

20. Thibier M, Guerin B. Hygienic aspects of storage and use of semen for artificial insemination. Anim Reprod Sci. 2000. 62:233–251.

21. Thompson SA, Blaser MJ. Nachamkin I, Blaser MJ, editors. Pathogenesis of Campylobacter fetus infections. Campylobacter. 2000. 2nd ed. Washington: ASM Press;321–347.

22. Tramuta C, Lacerenza D, Zoppi S, Goria M, Dondo A, Ferroglio E, Nebbia P, Rosati S. Development of a set of multiplex standard polymerase chain reaction assays for the identification of infectious agents from aborted bovine clinical samples. J Vet Diagn Invest. 2011. 23:657–664.

23. van Bergen MAP, Simons G, van der Graaf-van Bloois L, van Putten JPM, Rombout J, Wesley I, Wagenaar JA. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. J Med Microbiol. 2005. 54:1217–1224.

24. Véron M, Chatelain R. Taxonomic study of the genus Campylobacter Sebald and Véron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) Sebald and Véron. Int J Syst Bacteriol. 1973. 23:122–134.

25. Wang G, Clark CG, Taylor TM, Pucknell C, Barton C, Price L, Woodward DL, Rodgers FG. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J Clin Microbiol. 2002. 40:4744–4747.

26. Willoughby K, Nettleton PF, Quirie M, Maley MA, Foster G, Toszeghy M, Newell DG. A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus-species venerealis: use on UK isolates of C. fetus and other Campylobacter spp. J Appl Microbiol. 2005. 99:758–766.

27. Yang S, Rothman RE. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis. 2004. 4:337–348.