Isolation of Clostridium difficile and molecular detection of binary and A/B toxins in faeces of dogs

Document Type: Short paper

Authors

1 Ph.D. Student in Bacteriology, Department of Microbiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

Abstract

The aim of this study was to isolate Clostridium difficile from dogs’ faeces, and to study the frequency of its virulence genes. A total of 151 samples of dogs’ faeces were collected. The isolation of C. difficile was performed by using the bacterial culture methods followed by DNA extraction using boiling method. Multiplex PCR method was performed for identification of tcdA, tcdB, cdtA and cdtB genes and single method was carried out for detection of tcdC. Twelvesamples (7.9%) were positive in bacteriological assay and based on molecular assay, 66.7% of the isolates (8 of 12 C. difficile isolated) had shown tcdA+, tcdB+ profile. This is the first investigation on molecular assay of C. difficilein Iran’s dog population.

Keywords


Aldous, WK; Pounder, JI; Cloud, JL and Woods, GL (2005). Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR. J. Clin. Microbiol., 43: 2471-2473.

Antikainen, J; Pasanen, T; Mero, S; Tarkka, E; Kirveskari, J; Kotila, S; Mentula, S; Könönen, E; Virolainen Julkunen, AR and Vaara, M (2009). Detection of virulence genes of Clostridium difficile by multiplex PCR. APMIS., 117: 607-613.

Clooten, J; Kruth, S; Arroyo, L and Weese, JS (2008). Prevalence and risk factors for Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit. Vet. Microbiol., 129: 209-214.

Dabard, J; Dubos, F; Martinet, L and Ducluzeau, R (1979). Experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with Clostridium difficile and other Clostridium strains. Infect. Immun., 24: 7-11.

Deneve, C; Janoir, C; Poilane, I; Fantinato, C and Collignon, A (2009). New trends in Clostridium difficile virulence and pathogenesis. Int. J. Antimicrob. Agents. 33: S24-S28.

Doosti, A and Mokhtari-Farsani, A (2014). Study of the frequency of Clostridium difficile tcdA, tcdB, cdtA and cdtB genes in feces of calves in south west of Iran. Ann. Clin. Microbiol. Antimicrob., 13: 1-6.

Fedorko, DP and Williams, EC (1997). Use of cycloserine-cefoxitinfructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile. J. Clinic. Microbiol., 35: 1258-1259.

Fooladi, AAI; Rahmati, S; Abadi, JFM; Halabian, R; Sedighian, H; Soltanpour, MJ and Rahimi, M (2014). Isolation of Clostridium difficile and detection of A and B toxins encoding genes. Int. J. Entric. Pathog., 2: e15238.

Frazier, KS; Herron, AJ; Hines, ME; Gaskin, JM and Altman, NH (1993). Diagnosis of enteritis and entero-toxemia due to Clostridium difficile in captive ostriches (Struthio camelus). J. Vet. Diagn. Invest., 5: 623-625.

Ghose, C (2013). Clostridium difficile infection in the twenty-first century. Emerg. Microbes. Infect., 2: 1-8.

Hasanzade, A and Rahimi, E (2013). Isolation of Clostridium difficile from turkey and ostrich meat sold in meat stores of Isfahan city. I.J.A.B.B.R., 1: 963-967.

Jalali, M; Khorvash, F; Warriner, K and Weese, JS (2012). Clostridium difficile infection in an Iranian hospital. BMC. Res. Notes. 5: 159.

Kevin, K; Brazier, JS; Post, KW; Weese, S and Songer, JG (2007). Prevalence of PCR ribotypes among Clostridium difficile isolates from pigs, calves, and other species. J. Clin. Microbiol., 45: 1963-1964.

Koene, MGJ; Mevius, D; Wagenaar, JA; Harmanus, C; Hensgens, MPM; Meetsma, AM; Putirulan, FF; Bergen, MAP and Kuijper, EJ (2011). Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates. Clin. Microbiol. Infect., 18: 778-784.

Marks, SL and Kather, EJ (2003). Antimicrobial sus-ceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs. Vet. Microbiol., 94: 39-45.

Marks, S; Kather, EJ; Kass, PH and Melli, AC (2002). Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J. Vet. Intern. Med., 16: 533-540.

Marks, S; Rankin, S; Byrne, B and Weese, J (2011). Enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control. J. Vet. Intern. Med., 25: 1195-1208.

McKee, R; Mangalea, M; Purcell, E; Borchardt, E and Tamayo, R (2013). The second messenger cyclic Di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD. J. Bacteriol., 195: 5174-5185.

O’Neill, G; Adams, JE; Bowman, RA and Riley, TV (1993). A molecular characterization of Clostridium difficile isolates from humans, animals and their environments. Epidemiol. Infect., 111: 257-264.

Ossiprandi, MC; Buttrini, M; Bottarelli, E and Zerbini, L (2012). A preliminary molecular typing by PCR assays of
Clostridium perfringens and Clostridium difficile isolates from dogs. AiM., 2: 87-92.

Persson, S; Torpdahl, M and Olsen, KEP (2008). New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtAcdtB) genes applied to a Danish strain collection. Clin. Microbiol. Infect., 14: 1057-1064.

Pituch, H; ObuchWoszczatyñski, P; Van Den Braak, N; Van Belkum, A; Kujawa, M; Luczak, M and Meisel Mikolajczyk, F (2002). Variable flagella expression among clonal toxin A–/B+ Clostridium difficile strains with highly homogeneous flagellin genes. Clin. Microbiol. Infect., 8: 187-188.

Rahimi, E; Jalali, M and Weese, JS (2014). Prevalence of Clostridium difficile in raw beef, cow, sheep, goat, camel and buffalo meat in Iran. BMC. Public Health. 14: 119.

Riley, T; Adams, J; O’Neill, G and Bowman, R (1991). Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics. Epidemiol. Infect., 107: 659-665.

Silva, ROS; Santos, RLR; Pires, PS; Pereira, LC; Duarte, MC; de Assis, RA and Lobato, FC (2013). Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil. Braz. J. Microbiol., 44: 133-137.

Weese, JS; Staempfli, HR; Prescott, JF; Kruth, SA; Greenwood, SJ and Weese, HE (2001). The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs. J. Vet. Intern. Med., 15: 374-378.

Wetterwik, K; Trowald-Wigh, G; Fernström, L and Krovacek, K (2013). Clostridium difficile in faeces from healthy dogs and dogs with diarrhea. Acta Vet. Scand., 55: 23.