Phenotypical Identification and Toxinotyping of Clostridium perfringens Isolates from Healthy and Enteric Disease-Affected Chickens

Clostridium perfringens is a ubiquitous spore-forming anaerobic pathogen that is frequently associated with enteric disease in chickens. Moreover, enterotoxin-producing C. perfringens has high zoonotic potential as well as serious public health concerns due to the emanation of food-borne intoxication. The present study was designed to isolate, identify, and toxinotype C. perfringens from both healthy and cases of necrotic or ulcerative enteritis chickens. A total of 110 samples were collected from July 2019 to February 2021. Among the samples, 38 (34.5%, 95% CI: 26.39–43.83) were positive for C. perfringens and were obtained from broiler 21 (33.3%, 95% CI: 22.91–45.67), Sonali 9 (34.6%, 95% CI: 19.31–53.88), and layer 8 (38%, 95% CI: 20.68–59.20). C. perfringens was highly prevalent (35.7%, 95% CI: 25.48–47.44) in enteritis chickens compared with healthy ones. In multiplex PCR toxinotyping, 34 (89.4%) isolates were identified as C. perfringens type A by the presence of the alpha toxin gene (cpa). Moreover, in addition to the cpa gene, 3 (14.3%, 95% CI: 4.14–35.48) broiler and 1 (11.1%, 95% CI: 0.01–45.67) Sonali isolates harbored the enterotoxin gene (cpe) and were classified as type F. However, none of the isolates carried genes encoding beta (cpb), epsilon (etx), iota (iap), or beta-2 (cpb2) toxins. Multivariable logistic regression analysis identified the following variables such as; “previously used litter materials” (OR 21.77, 95% CI 2.22–212.66, p ≤ 0.008); intestinal lesions, “presence of ulceration” (OR 30.01, 95% CI 3.02–297.91, p ≤ 0.004); “ballooned with gas” (OR 24.74, 95% CI 4.34–140.86, p ≤ 0.001) and “use of probiotics” (OR 5.24, 95% CI 0.74–36.75, p ≤ 0.095) act as risk factors for C. perfringens colonization in chicken gut. This is the first study of molecular toxinotyping of C. perfringens from healthy and enteric-diseased chickens in Bangladesh, which might have a potential food-borne zoonotic impact on human health.


Introduction
C. perfringens is an anaerobic, spore forming enteric pathogen that causes both clinical and subclinical enteric disease in chickens. Te most severe clinical form of enteric disease is necrotic enteritis, which is characterized by a ballooned, friable intestine with necrosis of the intestinal mucosa that is often covered by a tan-to-yellow pseudomembrane [1]. In addition, due to secondary bacterial infection and a roughened intestinal mucosal surface, it appears like a Turkish towel [1]. Te clinical form of the disease is associated with a huge economic burden [2], and the subclinical form of the disease signifcantly reduces the growth performance of chickens by causing extensive damage to the gut epithelial layer [3]. Te principal mechanism of disease manifestation by C. perfringens is associated with the release of six major extracellular toxins, which are described as alpha (α), beta (β), epsilon (ε), iota (ι), enterotoxin (cpe), and NetB. Based on the production of the abovementioned toxins, C. perfringens is classifed into seven toxinogenic types, A to G [4,5]. In the toxinogenic typing scheme, type A and all other types of C. perfringens produce alpha (a) toxin. In addition to alpha (α) toxin, type B produces beta (β) and epsilon (ε) toxins, type C produces beta (β) toxin, type D produces epsilon (ε) toxin, type E produces iota (ι) toxin, type F produces enterotoxin (cpe), and type G produces NetB toxin [4,5]. Among all the C. perfringens types, type F is most frequently associated with food poisoning in humans and causes food-borne illness by producing an enterotoxin (cpe) [4][5][6][7]. Still, to date, it is positioned as the third most common food poisoning agent in the industrialized world [2].
Although clinical necrotic enteritis cases are frequently found in poultry farms in Bangladesh, they are all diagnosed based only on clinical signs and postmortem fndings. To date, this is the frst study on the molecular identifcation of C. perfringens, which is circulating in chicken focks in Bangladesh. As a result, the purpose of this study was to determine the phenotypic and toxinogenic typing of C. perfringens isolates from healthy and necrotic enteritisinfected chickens in Bangladesh.

Samples.
We collected intestinal swabs and intestinal contents from broiler, Sonali (a crossbreed of Fayoumi and Rhode Island Red), and layer chickens from July 2019 to February 2021. Samples were collected from chickens that were brought to the poultry practitioners for postmortem examinations at the Department of Pathology and Parasitology and the Department of Physiology, Biochemistry, and Pharmacology at Chattogram Veterinary and Animal Sciences University (CVASU). Te samples only included chickens that showed enteric disease, including necrotic or ulcerative lesions during postmortem examination (Figures 1(a) and 1(b)). Additionally, specimens were collected from apparently healthy birds from live-broiler markets that were kept for selling. Intestinal swabs and intestinal contents (Figures 1(c) and 1(d)) were collected in 10 mL of cooked meat medium (Oxoid Limited, Hampshire, England) and immediately transported on ice to the Department of Microbiology and Veterinary Public Health laboratory for isolation and identifcation of C. perfringens.

Isolation and Identifcation of C. perfringens.
For primary enrichment of C. perfringens, cooked meat medium containing samples was incubated anaerobically at 37°C for 48 hrs in an anaerobic jar (Oxoid Limited, Termo Fisher Scientifc Inc., UK) with an anaerobic GasPak (Oxoid Limited, Hampshire, England). Te pre-enriched samples were then inoculated onto 5% bovine blood agar with added colistin sulfate (2 mg/litter) and incubated anaerobically for 24 hours. Te characteristic colonies with double zones of beta hemolysis (inner zone: complete hemolysis; outer zone: partial hemolysis) were presumptively identifed as C. perfringens. For further confrmation, the suspected bacterial colonies were subjected to Gram staining (Gram positive, large bacilli) and the catalase test (negative). Two to three subcultures were performed to obtain the pure culture, and fnally, the pure colonies were subcultured on brain heart infusion broth (BHI) (Oxoid Limited, Termo Fisher Scientifc Inc., UK). All the isolates were preserved at −80°C for further analysis.

Extraction of DNA and Identifcation of C. perfringens
Isolates by PCR. Te preparation of template DNA was performed according to previously published literature [14]. Briefy, three to fve pure bacterial colonies were suspended in 150 μl of ultrapure water in a 1.5 ml microcentrifuge tube, boiled at 100°C for 10 min, and immediately cooled at −20°C for fve minutes. Finally, the cell lysates were centrifuged at 12,000 g for 5 minutes, and 100 μl of the supernatant containing template DNA was transferred into a microcentrifuge tube and stored at −20°C for further analysis. A species-specifc primer (16S rRNA gene) was used for confrmation of C. perfringens [15]. A total of 25 µl of the PCR reaction was prepared with 12.5 µl of Taq PCR Mas-terMix (Qiagen, Merelbeke, Belgium), 1 µl (20 picomole/µl) of both forward and reverse primer, 4 µl (5 ng/µl) of template DNA, and 6.5 µl of nuclease-free water. Amplifcation was performed in the thermal cycler (DLAB Scientifc Inc., USA) with the PCR program consisting of initial denaturation at 95°C for 15 min, followed by 94°C for 30 sec, 53°C for 1.5 min, and 72°C for 1.5 min for 35 cycles, and fnal extension at 72°C for 10 min.

Toxinotyping by PCR.
After confrmation of the C. perfringens species, all positive isolates were selected for toxinotyping using fve diferent toxin genes α (cpa), β (cpb), ε(etx), ί(iap), and enterotoxin (cpe); their primer sequences are listed in Table 1. Multiplex PCR (m-PCR) was performed for specifc amplifcation of the toxinotyping gene, which was described in a previous study [11]. For m-PCR, a total volume of 50 µl reaction was prepared, which comprises 25 µl of Taq PCR Master Mix, 1 µl (20 pmol/µl) of each primer for six toxin genes, 4 µl of C. perfringens confrmed genomic DNA, and 9 µl of nuclease-free water for every sample. A uniplex PCR was performed for the detection of β2 (cpb-2) and enterotoxin (cpe) gene, comprising 25 µl of the reaction volume. Also, the similar PCR condition was followed to perform the uniplex PCR. Finally, all of the PCR products were separated on an ethidium bromide-stained 1.5% agarose gel (Oxoid Limited, Termo Fisher Scientifc Inc., UK) and visualized using UV lights in a gel documentation system (Alphalmager, Alpha Innotech, San Leandro, CA, USA). Previously confrmed C. perfringens isolates were used as positive controls, and nuclease-free water was used as a negative control.

Data Analysis.
All targeted demographic as well as postmortem data was recorded into a Microsoft Excel 2010 spread sheet. Te prevalence was calculated by considering the number of positive C. perfringens isolates as the numerator, divided by the number of chickens sampled as the denominator. Chi-square test was performed to fnd out the association between the binary result of C. perfringens and the farm and chicken factors. Firstly, univariable logistic regression analysis was performed to identify possible risk factors, and subsequently, any factor having a p-value of ≤0.20 was selected to build the further multivariable logistic regression model. Any variables with a p-value of 0.05 were considered signifcant and kept in the fnal model. All descriptive and analytical analyses were performed using STATA ® 13.0 software [16]. Finally, the representative heat map was constructed using Graphpad Prism (version 7.05).

Discussion
C. perfringens are spore-forming bacilli that commonly inhabit soil, poultry litter, and are also harbored as gut pathogens in chickens and other animals [17,18]. Te diferent toxinotypes cause a wide variety of signifcant systemic and enteric diseases in diferent species of animals, including chickens. Another signifcant aspect is that it is involved in food-borne intoxication (food-borne zoonosis), which evolved from consumption of diferent raw and canned foods, particularly chicken meat and meat products [19].
In the present study, the overall prevalence of C. perfringens in chicken is 34.5%, but in some countries, particularly Jordan and Egypt, it is higher than 40% [20,21]. Besides, the prevalence of C. perfringens in broiler chicken was 33.3% in our study, which is slightly higher than the recent fndings of Praveen Kumar et al. [22] and Zhang et al. [23]. Tey described the prevalence as 21.97% and 23.1% in broiler chicken in India and China, respectively. Tis variation may be due to geographical conditions as well as the earlier contaminated soil and unchanged bedding materials of the poultry farm. As C. perfringens is a spore-forming pathogen that can survive in soil for an extended period of time, chickens are frequently infected. On the other hand, chickens that harbor C. perfringens continuously shed it in the poultry litter and environment, and it acts as a potential source of infection for chickens. Cross-infection through droppings, litter materials, poultry waste products, feed, and water is a major route of infection for chickens and other animals [23,24]. In addition, the prevalence of C. perfringens may increase signifcantly when chickens have a concurrent clinical or subclinical coccidial infection in the gut [25].
Toxinotyping of C. perfringens by multiplex PCR assay displayed the presence of the alpha toxin gene in all isolates and the absence of other major lethal toxin genes except the enterotoxin (cpe) gene, which was expressed by type F isolates. Te current fndings demonstrate that 34 (89.4%) of the PCR-confrmed C. perfringens isolates are type A. Tis study revealed that C. perfringens type A is the most dominant toxin type circulating both in healthy and diseased chickens. Tese fndings are strongly supported by several previous studies [2,20,23,26]. However, none of the C. perfringens isolates carried the cpb, etx, or iap toxin genes, which indicate the absence of C. perfringens toxinotypes B, C, D, and E in this study. Similar fndings were also previously reported [26][27][28], showing that C. perfringens obtained from chickens encoded only the alpha toxin gene. In addition to the four major toxinotyping toxins, β2 toxin (cpb2) was also absent in all chicken isolates, which were considered minor or nonessential virulence factors for disease manifestation in chicken [29].
Although C. perfringens is a signifcant food-borne zoonotic pathogen, very limited microbiological and epidemiological studies have been reported in Bangladesh. In our study, 4 (10.5%) of the type F isolates harbored the enterotoxigenic gene (cpe), which is a potential virulence determinant of C. perfringens for food poisoning in humans [4,5,19,30]. Te low incidence of enterotoxin in chicken may be due to sample source variation as well as the genetic absence of an encoded gene. However, in addition to foodborne intoxication, the enterotoxin producing C. perfringens strains also frequently cause neonatal diarrhea [31]. Furthermore, the enterotoxin gene (cpe) encoded by C. perfringens type F isolates clearly poses the risk of human food-borne intoxication as well as food-borne zoonosis [4,5,32]. Finally, it is recognized that any C. perfringens isolates with alpha toxin and enterotoxin have a signifcant impact on the production of necrotic enteritis in chickens and also cause fatal food-borne enteric disease in humans. However, in the current study, we classifed C. perfringens from A to F, with the exception of type G, which harbored a net B toxin.
We identifed previously used litter materials as a potential risk factor for the colonization of C. perfringens in chickens. Besides, the presence of associated lesions, such as ulceration and ballooned gas in the chicken gut, is also asserted as a colonization factor. Long-term and repeated use of the same litter materials in poultry houses may harbor the spores of C. perfringens [23,33] and act as an initial trigger of exposure. Te presence of intestinal lesions may involve the initial intestinal damage caused by coccidial pathogens [34] and subsequent colonization of C. perfringens. Finally, C. perfringens releases various toxins and enzymes responsible for gut lesions in chicken. Moreover, further details on the sequencing of C. perfringens  Veterinary Medicine International isolates helped to fnd out the mechanisms of colonization and toxin production, as well as the control of necrotic enteritis in chickens and food-borne zoonosis in humans.

Conclusions
Tis study describes the prevalence and toxinotyping of C. perfringens isolated from chicken in Bangladesh. We found that 33.3% of broiler, 34.6% Sonali, and 38.0% of layer chickens tested positive for C. perfringens. It is interesting that, among the enteric-diseased and healthy chickens, the prevalence of C. perfringens was higher in the enteric-diseased birds (35.7%).
In the present investigation, alpha toxin-producing C. perfringens type A was the predominant genotype, and relatively considerable numbers of isolates were type F (10.5%) that carried the enterotoxin gene (cpe). Previously used litter materials were identifed as risk factors that were associated with a higher prevalence of C. perfringens in chickens.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Ethical approval was obtained from the institutional ethical approval committee of Chattogram Veterinary and Animal Sciences University (Approval no. CVASU/Dir (R&E) EC/ 2019/12 (5/9)) to implement the study.

Consent
Informed consent was obtained from all individual farm owners included in the study.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Conceptualization, funding acquisition, data curation, investigation, methodology, project administration, resources, validation, formal analysis, visualization, writing the original draft, and writing, reviewing and editing the manuscript were performed by EAR. TAN contributed to the investigation, methodology, reviewing, and editing the manuscript. MSI was involved in visualization, validation, and reviewing, and editing of the manuscript. HB was involved in supervision, visualization, validation, and reviewing, and editing the manuscript. MZI was involved in conceptualization, supervision, visualization, validation, and reviewing and editing of the manuscript. All authors read and approved the fnal manuscript.