Clostridium perfringens: A review of enteric diseases in dogs, cats and wild animals

 Rodrigo Otavio Silveira Silva, Francisco Carlos Faria Lobato Veterinary School, Universidade Federal de Minas Gerais (UFMG), Antonio Carlos Avenue, 6627, 31 270-901 Belo Horizonte, MG, Brazil article info:
Clostridium perfringens is a gram-positive anaerobic bacillus that is commonly part of the microbiota of humans and animals. It is considered a common enteric pathogen, but the pathogenesis and the pre-disposing factors of the disease commonly differ between host species. Thus, specific research is necessary to understand the role of this pathogen, how to diagnose it, and which control measures are applicable. The aim of this paper is to review the current knowledge of C. perfringens infections in dogs, cats and wild animals.

1. Introduction
Clostridia produce the highest number of toxins of any type of bacteria. Among Clostridium species, Clostridium perfringens is the
largest toxin producer and also the most widespread, being found as part of the microbiota of animals and humans and in the soil. This bacterium is a gram-positive anaerobic bacillus that is classified into five types according to the production of four major toxins:alpha (a), beta (b), epsilon (ε) and iota (i). In addition to the majortoxins, it can produce more than 15 other known toxins. Some of these additional virulence factors, including enterotoxin, necrotic enteritis B-like toxin (NetB) and beta-2 toxin, have received more attention than others due to their role in the pathogenesis of C. perfringens-associated disease (CPAD) in humans and animals [1,2]. C. perfringens is recognized as one of the most important causes of foodborne disease in humans, and it is also commonly involved in human and animal cases of gas gangrene [3,4]. In veterinary medicine, C. perfringens is responsible for several, mostly enteric, diseases. In the last few years, significant progress in the understanding of C. perfringens in animals has been achieved [5,6]. Here, we review the most recent advances in the understanding of C. perfringens enteric disease in dogs, cats and wild species.

2. Dogs and cats
The role of C. perfringens type A as an enteropathogen in dogs and cats is not fully understood, but it is suspected to be associated with anything from mild, self-limiting diarrhea to rapidly fatal necrohemorrhagic enteritis [7e14]. Most studies indicate that C. perfringens is a primary enteropathogen, but some authors also suggest that it could act as an opportunistic agent in dogs and cats. Disruption of the normal microbiota, such as a sudden change to a high protein diet, or enteric infection by other pathogens, such as parvovirus, are considered predisposing factors [15,16]. C. perfringens type A is the most common genotype in dogs and cats, and 10e30% of the isolated strains are also positive for beta-2 toxin gene (cpb2). The enterotoxin gene (cpe) is also common in canine and feline C. perfringens strains. No reports of strains positive for necrotic enteritis-like B toxin gene (netB) from cats and dogs have been published [17]. In addition to type A, it is believed that C. perfringens type C can also be responsible for acute fatal hemorrhagic enteritis in dogs and cats; however, there have been no case reports in the literature until now [10].

Diagnosis of C. perfringens type A-associated diarrhea in dogs is very difficult because the clinical signs cannot be differentiated from enteritis caused by other enteropathogens. Additionally, C. perfringens type A is a normal inhabitant of the canine intestine, so isolation of this microorganism is not diagnostic [17]. A corre- lation between the presence of CPE in feces and diarrhea in recent studies suggests that the detection of this toxin in stool samples could be useful to diagnose CPAD in dogs [10,11,18]. However, the specificity and sensitivity of most enzyme immunoassays (EIA) for the detection of CPE in dog stool samples is unknown [10]. Isolation of the bacteria followed by detection of the enterotoxin gene (cpe) by PCR was also suggested and supported by studies that reported that detection of cpe is correlated with the occurrence of diarrhea in dogs [8,11,19]. In summary, (1) despite this commonly reported association, there is no confirmatory study about the role of the enterotoxin of C. perfringens in dogs; (2) both enterotoxin and isolates positive for the cpe gene can be found in healthy dogs, so these methods could suggest that C. perfringens is involved, but they are not confirmatory. A more robust diagnosis could be ach- ieved by the exclusion of other potential enteropathogens and the detection of enterotoxin followed by the isolation of a cpe-positive C. perfringens strain [10]. In addition, if death occurs, visualization of a large number of clostridia-like bacilli on the mucosal surfaces and associated with the lesions could add more confidence to the final diagnosis of CPAD [14,20,21]. It is important to note that, in contrast with dogs, studies have failed to show an association between diarrhea and the detection of enterotoxin in stool samples or detection of cpe in C. perfringens strains isolated from cats [16,22]. Thus, the diagnosis should be based only on the isolation of C. perfringens (positive or negative for cpe) in conjunction with the absence of other enteropathogens.

A presumptive diagnosis of C. perfringens type C diarrhea can be determined by the association of clinical signs (bloody diarrhea, commonly in young animals) with isolation of the agent, which is not commonly part of the feline microbiota. A confirmatory diagnosis would require the detection of beta toxin in stool samples or in intestinal contents [10]. The most commonly  recommended drugs for the treatment of C. perfringens infection in dogs are metronidazole and tylosin, although amoxicillin, tetracycline, erythromycin and cephalexin are also used. Studies regarding the susceptibility of C. perfringens isolates from dogs showed that most C. perfringens isolates are strongly susceptible to metronidazole, but resistance to macrolides and tetracyclines is common [23,24].

Future studies might focus on the pathogenesis and diagnosis of C. perfringens infection in dogs. Despite the known association between diarrheic dogs and detection of enterotoxin or cpe in C. perfringens isolates, few studies, such as those employing animal models or examining molecular differences among canine C. perfringens cpe positive strains, have been conducted to understand the true role of this toxin in canine diarrhea.

3. Wild animals
Knowledge about C. perfringens as a cause of enteric disease in wild animals is extremely limited mainly because of the difficulty in
accessing fecal samples from these species, which makes studies of diseased and healthy animals rare. Another complicating factor is that most accessible animals are in captivity, which might interfere directly with the microbiota composition. Thus, for most wild species, knowledge is limited to a few case reports, and the role of this microorganism as a pathogen or even as part of the normal microbiota is still poorly understood. There are reports of single cases or outbreaks of necrotic enteritis (NE) caused by C. perfringens type A with elevated mortality in several wild avian species such as swans, capercaillies, white storks, crows, geese, western bluebirds, lories, lorikeets, and bobwhite quail [25e33]. C. perfringens type C was also reported but seems to be uncommon [34]. The lesions are similar to those described for NE in poultry, including confluent mucosal necrosis restricted to the small intestine along with hepatitis and cholangiohepatitis. In addition to the common macroscopic lesions, the diagnosis is made by an association between histology, isolation ofC. perfringens type A and the absence of other enteropathogens [26,30,35].

The main predisposing factors are not well understood for wild avian species and, in contrast with broiler chicken, coccidian infection is not commonly associated with NE in most reports [25e34]. It is also important to note that the netB gene, which has been associated with NE in broiler chickens, has not been detected in C. perfringens isolates from wild avian species to date. Similar to broiler chickens, the beta-2 toxin gene (cpb2) and enterotoxin (cpe) can be found, but these genes do not seem important for the pathogenesis of NE in wild avian species [36]. In contrast with domestic avian species, it seems that C. perfringens is not commonly isolated from healthy wild birds. Silva et al. [36] reported an isolation rate of 5.6% in a study of Ramphastidae, Tinamidae and Cracidae species in Brazil, raising the hypothesis that C. perfringens might not be part of the normal microbiota in these species. However, there are marked anatomical differences in the intestinal tract and also in the habits of each avian species, and therefore, specific studies are needed to better understand the role of this bacterium in wild avian species. Regarding wild carnivores, C. perfringens type A is commonly isolated from healthy animals, and the strains can also be positive for cpb2 and cpe genes [37]. However, as there was no balance between diarrheic and healthy animals, no conclusions could be made regarding the importance of these additional virulence factors in C. perfringens isolated from wild carnivore species. Recently, Zhang et al. [38] reported the death of a Siberian tiger (Pantheratigris altaica) and a lion (Panthera leo) due to hemorrhagic enterocolitis associated with C. perfringens type A. Both developed depression, anorexia and bloody diarrhea, which quickly led to death. Neither the cpe nor cbp2 genes were found in the C. perfringens isolates from the animals. A similar case was also reported in an Amur tiger (Panthera pardus orientalis) by Neiffer [39]; however, in addition to the isolation of the microorganism, the authors also detected the enterotoxin in its feces. In both re- ports, the source of infection and the predisposing factors were not confirmed, but the authors suggested that the supply of partially thawed chicken meat could have contributed to the development of disease in these three animals [38,39]. Another interesting recent report in wild felines is a suspected case of neurotoxicity due to C. perfringens type B in a tiger (P. tigris) [40]. In this case, the tiger was referred with acute onset of severe abnormal consciousness with a suspected diffuse forebrain lesion.

C. perfringens type B was found in high numbers in the feces in the absence of other infections, vascular lesions or metabolic abnor-
malities. The animal recovered after antibiotic therapy against the C. perfringens infection. C. perfringens type A cpb2-positive strains were also reported to be associated with cases of necrotic and hemorrhagic enteritis in Asiatic black bears (Selenarctos thibetanus) and pygmy hogs (Sussalvanius) in Italy and India, respectively [41,42]. In both cases, the animals developed acute hemorrhagic enteritis, which led to death. Cpb2-positive C. perfringens type A was obtained from the intestine in the absence of other enteropathogens. C. perfringens type A infection was also reported in ferrets suggested as one of the most common causes of death in free-living seals (Phoca vitulina)in Germany, but there is limited data about the predisposing factors or laboratory diagnosis in these two reports [43,44]. In simians, in R.O.S. Silva, F.C.F. Lobato / Anaerobe 33 (2015) 14e17 15 addition to case reports [46], there are some studies showing that this microorganism could be responsible for foodborne diarrhea and acute gastric dilatation in these animals [46,47]. Anyway, similar to almost all wild species, there is no recognized method to differentiate C perfringens strains that are causing infection from strains that are only part of the microbiota. In light of this, the diagnostic is still based in an association between histological lesions, isolation of C. perfringens and absence of other enteropathogens [45,48].

There are a few case reports of C. perfringens type C infection in wild species. Carvalho et al. [49] reported an outbreak in four young captive collared (Pecari tajacu) and three white-lipped (Tayassupecari) peccaries in Brazil. The animals showed lethargy and inappetence followed by sudden death within 24 h of the beginning of clinical signs. In addition to typical post mortem findings and isolation of C. perfringens type C, the diagnosis was confirmed by detection of beta-toxin from the intestinal contents. The authors suggested that the ingestion of a feed containing sweet potato was an important predisposing factor in this outbreak [49]. Sweet potato is a potent trypsin inhibitor, and it is well known as a predisposing factor of type C infection in humans [50]. There are few reports about the antimicrobial susceptibility of C. perfringens isolates from wild animals. In studies with isolates from wild carnivores and wild avian species, Silva et al. [36,37] reported that these isolates were widely susceptible to florfenicol, metronidazole, penicillin and vancomycin, but some strains were resistant to erythromycin, tylosin and oxytetracycline.

Conflict of interest
The authors declare that they have no conflicts of interest.

Acknowledgments
This work was supported by funds from Fapemig, Capes, CNPq,
INCT and PRPq-UFMG.
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