Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil

Abstract

The objective of this study was to detect C. difficile A/B toxins and to isolate strains of C. perfringens and C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficile A/B toxins were detected by ELISA, and C. perfringens and C. difficile were identified by multiplex PCR. C. difficile A/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+ and two were A−B−. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtB was found in one strain, which was A+B+ and was derived from a non-diarrheic dog. C. perfringens strains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringens type A and there was an association between the detection of the cpe gene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2 gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficile and C. perfringens in dogs and to better our understanding of C. difficile as a zoonotic agent. This is the first study to report the binary toxin gene in C. difficile strains isolated from dogs in Brazil.

An outbreak of Clostridium difficile-associated diarrhea in piglets in Brazil

“…This result corroborates previous studies with piglets and other domestic species (BÅVERUD et al, 2003;CLOOTEN et al, 2008;SILVA et al, 2011) and may be due to the susceptibility of some C. difficile strains to one or both of the antibiotics used in the medium (SONGER; UZAL, 2005). The detection of A/B toxins in non-diarrheic piglets is also a previously reported event.…”

“…On the other hand, little is known about the prevalence of this disease in all of Latin America. Until now, only two reports were conducted in Brazil and both showed a prevalence of approximately 16% in piglets (LIPPKE et al, 2011;SILVA et al, 2011). It is also interesting to note that, according to Silva et al (2011), 53% of the sampled farms had at least one animal positive for A/B toxins, suggesting a high dissemination of the disease in Brazilian farms.…”

“…Stool samples were submitted to coccidian oocyst detection by the flotation method (HOFFMANN, 1987), rotavirus detection by polyacrylamide gel electrophoresis followed by silver staining (HERRING et al, 1982), isolation of C. perfringens and C. difficile (SILVA et al, 2011;VIEIRA et al, 2008) and routine bacteriologic culture for aerobic bacteria in MacConkey agar (Biobrás®, Prodimol Biotechnology) and MullerHinton agar supplemented with 5% of equine blood (Difco Laboratories, Detroit, USA). For A/B toxin detection, a cytotoxicity assay method (CTA) was used with African green monkey kidney cells (Vero-ATCC CCL 81) ( VAN DER BERG et al, 2005).…”

“…Despite this high production, little is known about the occurrence of C. difficile infection in piglets. Recently, the occurrence of the disease was confirmed by survey studies (LIPPKE et al, 2011;SILVA et al, 2011); additionally, a prevalence study about the most important enteric pathogens in swine suggested that C. difficile is one of the main causes of neonatal diarrhea in piglets in Brazil (SILVA; GUEDES; LOBATO, 2013). Despite the increased importance of C. difficile as a swine pathogen, until now, there has been no description of an outbreak of the disease in piglets in Brazil.…”

Clostridium difficile in Food and Animals: A Comprehensive Review

“…Different other studies have associated the presence of C. difficile in faeces with diarrhoea in dogs and cats (Weese et al 2001a;2001b;Weese and Armstrong 2003;Koene et al 2012;Wetterwik et al 2013). However, dogs can also be healthy carriers of C. difficile strains belonging to human epidemic PCR-ribotypes (Schneeberg et al 2012;Silva et al 2013b;Spigaglia et al 2015), with a high colonisation in the first period of live (Perrin et al 1993;Á lvarez-Pérez et al 2015).…”