Identification, Isolation, and Phylogenetic Analysis of Clostridium perfringens Type A and Type C from Wild Boar (Sus scrofa) in the People's Republic of China

Clostridium perfringens is one the most common causes of foodborne or nonfoodborne diarrhea in human beings; an estimated one million individuals are affected by C. perfringens in the US each year (Grass et al. 2013). Clostridium perfringens is a large, Gram-positive, spore-forming, anaerobic, rod-shaped pathogenic bacterium of the genus Clostridium that is ubiquitously present in the environment, and that can be a normal component of gut microbiota (Freedman et al. 2015). When the dynamic balance of the intestinal microbiome is altered by sudden diet change, antibiotic treatment, or parasite infection, C. perfringens can induce enteritis in a wide range of animals (Uzal 2004), including ruminant and nonruminant mammals and poultry, as well as human beings (Qiu et al. 2014; Prescott 2016).

Clostridium perfringens encodes for up to 17 exotoxins (Freedman et al. 2015). Based on the production of four major toxins (α, β, ɛ, and ι), C. perfringens strains are further classified into five subtypes (A, B, C, D, and E). Clostridium perfringens type A and type C usually cause diseases in either humans or swine (Freedman et al. 2015).

Wild boar (Sus scrofa) are widely distributed over the world, and are listed as one of the Class II Protected Species in the People's Republic of China (PRC). Wild boars are known reservoirs and vectors for many infectious agents (Ruiz-Fons et al. 2008) that are transmissible to livestock or human beings in several ways: 1) direct contact with wild pigs, 2) through insect vectors (for example, ticks), and 3) indirect contact with contaminated environmental factors (such as soil, water, or vegetation). Thus, surveillance, prevention, and control of wild boar infectious diseases have great significance on public health and economics. Here, we report an outbreak of C. perfringens in wild boar in Zhaosu County, Xinjiang Province, PRC, near the border with Kazakhstan.

From 10–21 February 2016, 10 dead wild boars were found in the mountains in Zhaosu County, Xinjiang Province, PRC. Body surface inspection showed no obviously lesions (Fig. 1A). We conducted postmortem examinations on two fresh corpses (Fig. 1A). The predominant lesions were observed in the digestive, respiratory, and immune systems. In detail, the small intestine, lung, and inguinal lymph nodes showed severe hemorrhage (Fig. 1B–D). Emphysema was seen in the large intestine (Fig. 1B). Additionally, bloody pleural effusion was found in the chest. The other organs showed no significant impairments. Based on the appearance of the carcasses and the presence of considerable chyme in the stomach, we speculated that the dead wild pigs were well-nourished.

Based on the postmortem examination, we made a presumptive diagnosis that the wild boars were killed by C. perfringens, with a lesser probable diagnosis porcine epidemic diarrhea virus (PEGV) or transmissible gastroenteritis virus (TGEV). To confirm our diagnosis, we performed PCRs specific for C. perfringens, PEGV, or TGEV. Details of the PCR analyses are given in Supplementary Materials. The PCR was positive for C. perfringens in intestine and lymph node samples (Fig. 2A), whereas PCRs for PEGV and TGEV were negative.

As mentioned, wild pigs are important reservoirs and vectors of many infectious diseases; therefore, the carcasses were potential sources of infection. Gross postmortem changes did not support the speculation that the wild boars were infected by African swine fever virus, classical swine fever virus, porcine pseudorabies virus, or other fulminating infectious agents; however, those concerns cannot be excluded. Thus, we conducted specific tests for animal diseases that can cause serious damage to humans and animals. All specimens were negative for infectious agents tested except for C. perfringens.

All subtypes of C. perfringens can be normal inhabitants of the intestine of most animals, so the mere detection of this microorganism from intestinal contents is not diagnostic for enteritis (Uzal 2004; Baker et al. 2010). We next conducted several auxiliary procedures to test our preliminary diagnosis. First, Gram-stained smears of intestinal mucosa were used to observe the bacteria in the gut. A predominant number of large Gram-positive, rod-shaped bacteria were seen, which is a positive indicator of C. perfringens infection (Fig. 2B; Dworkin et al. 2006). Second, one of the most accepted criteria on which to establish a definitive diagnosis of enteritis by C. perfringens is the detection of its toxins in intestinal contents (Songer and Uzal 2005). We inoculated five BALB/C mice with small intestine contents (Uzal et al. 2010). As a result, three mice died within 12 h postchallenge, while the other two showed mental depression and cowered together. Necropsy of one dead mouse revealed severe and extensive hemorrhage in the lung, as well as edema in the stomach and small intestine (Fig. 2C). Third, we conducted an enzyme-linked immunosorbent assay to detect alpha-toxin in intestine contents of the dead wild boar (Goldstein et al. 2012). This experiment gave a positive result for alpha-toxin, as well as for beta-toxin. These results support our presumption that the wild boar died of C. perfringens intoxication.

Histopathological examination of the intestine mucosa is of equal importance in a final diagnosis (Uzal 2004); however, the boars died in the wild and were frozen due to the cold weather, so we did not make histopathological sections from the intestines.

Clostridium perfringens was successfully isolated from the collected intestine samples. After 18 h of anaerobic incubation at 37 C in inoculated tryptose sulfite cyclosporine sandwich plates (Sigma Chemical, St. Louis, Missouri, USA), this microorganism produced typical black colonies (Fig. 3A), and yielded foam in fluid thioglycollate broth (Sigma Chemical) after overnight incubation at 37 C (Fig. 3B). Further details of culture are given in the Supplementary Material. Gram smears of the isolated strains revealed large amount of Gram-positive, rod-shaped bacteria (Fig. 3C). A C. perfringens-specific PCR was conducted for further identification. The results showed that C. perfringens was successfully isolated. Genotyping of isolated colonies showed that two strains of C. perfringens, type A (Fig. 3D) and type C (Fig. 3E), were isolated (details in Supplementary Materials). The C. perfringens enterotoxin gene (cpe), an important virulence factor of C. perfringens types A and C, was not detected in either of the isolated strains (Fig. 3D, E). The C. perfringens toxin netF gene is another widely reported virulence factor potentially related to poultry infection; the PCR was negative for netF. In addition, the cpb2 gene is an important virulence factor related to enteritis. In our analyses, both of the isolated C. perfringens strains were negative for cpb2 (Fig. 3D, E).

We performed phylogenetic analyses (details in Supplementary Material) based on the sequence of the 16S rDNA (Fig. 4). The specific primers used are shown in Supplementary Table S1. The results showed that the 16S rDNA sequences of C. perfringens were closely related, forming a monophyletic group. Estimates of evolutionary divergence between sequences range from 0 to 0.018. The type-A strain XJWB01 showed highest homology with strain W16-2a, whereas type-C strain XJWB02 showed highest homology with strain E020. The phylogeny of C. perfringens 16S rDNA neither displayed relationships to toxin typing, nor revealed characteristics of population structures, which is consistent with previous studies. The 16s rDNA sequences from our study were submitted to GenBank (KX094441–KX094442).

Here, we reported an outbreak with cointoxication of wild boars by C. perfringens type-A and type-C in Xinjiang Province, China. Clostridium perfringens is considered as one the most common causes for food-borne poisoning (Grass et al. 2013). Our research highlighted the importance that wild boars might serve as vectors for foodborne pathogens (Wacheck et al. 2010). Considering the expanding range of wild boar, it is reasonable to speculate that wild boar pose an increasing threat to public health (Miller et al. 2013).

Wild boar are numerous and widespread across PRC (IUCN 2016). Due to their high reproductive capacity, low natural mortality, and excellent adaptive capacity, wild pigs continuously expand their habitats and range (Bevins et al. 2014). For these reasons, contacts between humans or livestock with wild boar are becoming more frequent. Diseases of wild boars pose increasing concerns for several reasons: 1) wild boars harbor important zoonotic disease pathogens such as Brucella spp., Mycobacterium tuberculosis, and foot-and-mouth disease virus (Meng et al. 2009); 2) wild boars are sources for livestock infectious diseases such as African swine fever (Meng et al. 2009); and 3) wild pigs are potential carriers of alien invasive diseases (Ruiz-Fons et al. 2008). We should pay more attention on prevention and control of wild boar diseases.

All types of C. perfringens are considered commensal microbes in the intestinal tract of most healthy animals. Therefore, diagnosis of the diseases caused by C. perfringens is complicated and difficult. A presumptive diagnosis can be made based on the integrative factors including history, clinical, and pathologic results. Definitive diagnosis should take more specific evidence into consideration. Type C enteritis has been well-studied and controlled by vaccination in swine herds in recent years, whereas type A enteritis is poorly understood (Baker et al. 2010). Diagnosis of type A enteritis is often equivocal; detection of type A C. perfringens and alpha toxin are supportive but does not have diagnostic relevance in itself (Uzal 2004; Songer and Uzal 2005). The role of type A strains in natural diseases of these species remains controversial and poorly documented. Therefore, which strain of C. perfringens plays a leading role in the pathogenesis remains unclear.

This work was funded by grants from the Wildlife-Borne Disease Surveillance Program from the State Forestry Administration of the People's Republic of China (CZBZX-1), the National Key Research and Development Program of the People's Republic of China (2016YFC1201600), and the External Cooperation Program of Bureau of International Corporation, Chinese Academy of Sciences (grant 152111KYSB20150023).

Supplementary material for this article is online at http://dx.doi.org/10.7589/2016-08-199.