Alpha-toxin Gene Research Articles

Carriage of Clostridium perfringens in domestic and farm animals across the central highlands of Colombia: implications for gut health and zoonotic transmission.

Clostridium perfringens inhabits the guts of humans and animal species. C. perfringens can proliferate and express an arsenal of toxins, promoting the development of multiple gut illnesses. Healthy animals carrying C. perfringens represents a risk of transmission to other animals or humans through close contact and an increased likelihood of acquisition of toxin plasmids. The aim of this study was to evaluate the frequency of C. perfringens carriage in domestic and farm animals in the central highlands of Colombia. C. perfringens was detected in six animal species using PCR targeting alpha toxin (cpa) and 16S ribosomal RNA (16S-rRNA) genes from 347 fecal samples collected in two Departments: 177 from farm animals of Boyacá and 170 from domestic animals of both Cundinamarca and Boyacá. The overall frequency of C. perfringens detection was 22.1% (n = 77/347), with the highest frequency observed in cats 34.2% (n = 41/120), followed by dogs 30.0% (n = 15/50). The lowest frequency was detected in ruminants: goats 11.1% (n = 3/27), sheep 8.0% (n = 4/50) and cattle 6.0% (n = 6/50). Domestic animals showed a higher frequency of C. perfringens carriage than farm animals. This difference could be associated with dietary patterns, as domestic animals have diets rich in proteins and carbohydrates, while ruminants have low-carbohydrate diets, resulting in high production of endopeptidase-type enzymes and differences in pH due to the anatomy of gastrointestinal tract, which can influence bacterial proliferation. These findings indicate a potential risk of transmission of C. perfringens among animals and from animals to humans through close contact.

Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32–35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

The biology and pathogenicity of Clostridium perfringens type F: a common human enteropathogen with a new(ish) name.

SUMMARYIn the 2018-revised Clostridium perfringens typing classification system, isolates carrying the enterotoxin (cpe) and alpha toxin genes but no other typing toxin genes are now designated as type F. Type F isolates cause food poisoning and nonfoodborne human gastrointestinal (GI) diseases, which most commonly involve type F isolates carrying, respectivefooly, a chromosomal or plasmid-borne cpe gene. Compared to spores of other C. perfringens isolates, spores of type F chromosomal cpe isolates often exhibit greater resistance to food environment stresses, likely facilitating their survival in improperly prepared or stored foods. Multiple factors contribute to this spore resistance phenotype, including the production of a variant small acid-soluble protein-4. The pathogenicity of type F isolates involves sporulation-dependent C. perfringens enterotoxin (CPE) production. C. perfringens sporulation is initiated by orphan histidine kinases and sporulation-associated sigma factors that drive cpe transcription. CPE-induced cytotoxicity starts when CPE binds to claudin receptors to form a small complex (which also includes nonreceptor claudins). Approximately six small complexes oligomerize on the host cell plasma membrane surface to form a prepore. CPE molecules in that prepore apparently extend β-hairpin loops to form a β-barrel pore, allowing a Ca2+ influx that activates calpain. With low-dose CPE treatment, caspase-3-dependent apoptosis develops, while high-CPE dose treatment induces necroptosis. Those effects cause histologic damage along with fluid and electrolyte losses from the colon and small intestine. Sialidases likely contribute to type F disease by enhancing CPE action and, for NanI-producing nonfoodborne human GI disease isolates, increasing intestinal growth and colonization.

Development of real-time PCR assay for quantitative detection of Clostridium septicum

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.

Isolation and identification of Clostridium perfringens and its toxins from mutton in Lahore City, Punjab, Pakistan.

The present study was aimed to detect C. perfringens and identify its toxins in mutton samples collected from Lahore City in the Punjab Province of Pakistan. A total of 40 samples of minced and non‑minced mutton were collected from local butcher and retail shops representing four areas of the city. The samples were subjected to ELISA for the detection of C. perfringens alpha, beta and epsilon toxins. The samples were simultaneously processed for bacterial isolation. The isolates were confirmed both by biochemical testing and a multiplex PCR targeting alpha, beta and epsilon toxin genes of C. perfringens. While 10% (4/40) of the samples were positive for C. perfringens alpha toxins, 17.5% (7/40) of the samples were positive for the alpha toxin gene. The present study indicated that the samples collected from the local butcher shops were contaminated with C. perfringens and its toxins. Interestingly, no such contamination was detected in any of the samples collected from retail meat shops. In conclusion, improper hygienic conditions at butcher shops could lead to the contamination of mutton with C. perfringens and its toxins.

Impact of buffered sodium butyrate as a partial or total dietary alternative to lincomycin on performance, IGF-1 and TLR4 genes expression, serum indices, intestinal histomorphometry, Clostridia, and litter hygiene of broiler chickens

BackgroundSodium butyrate (SB) is a short-chain fatty acid and a safe antibiotic alternative. During 35 days, this study compared the impact of coated SB (Butirex C4) and lincomycin (Lincomix) on broiler growth, gut health, and litter hygiene in 1200 one-day-old Ross-308 broiler chicks that were randomly assigned into 5-dietary groups with 5-replications each. Groups divided as follows: T1: Basal diet (control), T2: Basal diet with buffered SB (1 kg/ton starter feed, 0.5 kg/ton grower-finisher feeds), T3: Basal diet with 100 g/ton lincomycin, T4: Basal diet with buffered SB (0.5 kg/ton starter feed, 0.25 kg/ton grower-finisher feeds) + 50 g/ton lincomycin, and T5: Basal diet with buffered SB (1 kg/ton starter feed, 0.5 kg/ton grower-finisher feeds) + 50 g/ton lincomycin. Birds were housed in a semi-closed deep litter house, where feed and water were available ad libitum. Results were statistically analyzed using ANOVA and Tukey’s post hoc tests.ResultsCombined dietary supplementation with SB and lincomycin (T4 and T5) significantly enhanced body weights, weight gains, feed conversion ratio, and profitability index. Also, carcasses in T4 and T5 exhibited the highest dressing, breast, thigh, and liver yields. T5 revealed the best blood biochemical indices, while T3 showed significantly elevated liver and kidney function indices. T4 and T5 exhibited the highest expression levels of IGF-1 and TLR4 genes, the greatest villi length of the intestinal mucosa, and the lowest levels of litter moisture and nitrogen. Clostridia perfringens type A alpha-toxin gene was confirmed in birds’ caeca, with the lowest clostridial counts defined in T4.ConclusionsReplacing half the dose of lincomycin (50 g/ton) with 0.5 or 1 kg/ton coated SB as a dietary supplement mixture showed the most efficient privileges concerning birds’ performance and health.

Gastrointestinal Impaction and Necrotic Enteritis in a Backyard Chicken.

The carcass of a 4-mo-old, female, mixed-breed backyard chicken was submitted for postmortem evaluation and diagnostic workup. The bird was previously presented to a veterinary clinic because of chronic weight loss and loose stool, and was euthanized before submission to the California Animal Health and Food Safety, Turlock lab. On gross examination, the proventriculus, gizzard, and duodenum were markedly distended and impacted with a mixture of fibrous plant material, cereal grain, and litter material. The koilin layer of the gizzard was eroded. There were multifocal to coalescing, 0.2-1-cm diameter white nodules on the serosal surface of the duodenal loop and lesions extended into the distal jejunum. The duodenum had multifocal, transmural, umbilicated, and ulcerated mucosal lesions, which were covered with a white pseudomembrane. Microscopically, there was segmental, transmural necrosis of the intestinal wall with diffuse sloughing of villi epithelium and accumulation of fibrino-hemorrhagic exudate with numerous bacterial colonies in the lumen. The gross and microscopic findings were indicative of gastrointestinal impaction and necrotic enteritis. Proliferation of Clostridium perfringens within the intestine was demonstrated by anaerobic bacterial culture, intestinal gram stains, and immunohistochemistry. The C. perfringens isolate was type F (encoding the gene for alpha toxin -cpa- and for enterotoxin -cpe) by PCR toxinotyping. Overgrowth of C. perfringens was likely exacerbated by the rough fibrous forage and highly fermentable grain diet. To our knowledge, gastrointestinal impaction concurrent with necrotic enteritis has not been described in backyard chickens. In addition, to our knowledge, C. perfringens type F has not been associated with necrotic enteritis in chickens.

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.

Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens

Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase + . These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-h period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.

Prevalence, molecular characterization, and antimicrobial resistance profile of Clostridium perfringens from India: A scoping review

Clostridium perfringens is one of the most important foodborne pathogens that causes histotoxic diseases and intestinal infections in both humans and animals. The present scoping review has been designed to analyze the literature published during 2000–2021 from India on the prevalence, molecular characterization, and antimicrobial resistance profile of C. perfringens isolates recovered from humans, animals, animal-based foods, and associated environmental samples. The peer-reviewed articles retrieved from four electronic databases (Google Scholar, PubMed, Science Direct, and Web of Science) were assessed using PRISMA-ScR guidelines. A total of 32 studies from India were selected on the basis of their relevance and inclusion criteria. The overall prevalence of C. perfringens among domestic animals having history of clinical symptoms and among healthy animals was found to be 65.8% (508/772) and 42.8% (493/1152), respectively. The pathogen was also detected in clinically affected wild animals (75%), healthy wild animals (35.4%), and captive birds (24.5%). The detection of C. perfringens among poultry having necrotic enteritis and among healthy birds was found to be 66.8% (321/480) and 25.6% (80/312), respectively. The detection of pathogen among animal-based foods (i.e., meat, milk, and fish and their products) and environmental samples depicted a prevalence of 20.8% (325/1562) and 30.2% (23/76), respectively. However, the prevalence of C. perfringens among humans having history of diarrhea and among healthy humans was found to be 25% (70/280) and 23.2% (36/155), respectively. The genotyping of C. perfringens isolates revealed that toxin type A was found to be the most prevalent genotype. Along with the alpha toxin gene (cpa), beta (cpb), epsilon (etx), iota (itx), enterotoxin (cpe), beta-2 toxin (cpb2), and NetB (netB) toxins were also detected in different combinations. Antimicrobial resistance profile of C. perfringens isolates recovered from different sources demonstrated that the highest resistance was detected against sulphonamides (76.8%) and tetracycline (41.3%) by phenotypic and genotypic detection methods, respectively. Comprehensive scientific studies covering different geographical areas at the human-animal-environment interface are crucial to generalize the real magnitude of C. perfringens-associated problem in India and for establishing a reliable database.

First molecular evidence of Clostridium perfringens in adult Fasciola spp. isolates in cattle hosts.

Fasciolosis is a parasitic disease caused by Fasciola spp. It is a prevalent helminth infection globally. Clostridial hepatitis is a general name refer to disorders caused by a few clostridial agents that most severely affect the liver. Migration of young parasite forms (mostly Fasciola hepatica) in the parenchymal tissue of the liver causes necrosis and anaerobic environment, stimulating the proliferation of C. novyi type B spores. This study investigated the occurrence of Clostridium spp in adult Fasciola spp isolates. Isolates (n = 100) were collected from the bile ducts of infected cattle after slaughter. Total genomic DNA was extracted from each sample. A multiplex-PCR based on the flagellin C (fliC) gene was used for quick identification of C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. In addition, a pair of primers Cpa (F) and Cpa (R) were used for detection of the C. perfringens alpha toxin gene. The products were sequenced. No band was obtained after multiplex-PCR of the fliC gene. A 247 bp band was detected in two isolates using the Cpa primers. BLAST analysis of these two isolates characterized both as C. perfringens alpha toxin. This is the first description of the molecular detection of C. perfringens in flukes. Further studies are needed to investigate whether Clostridum species is also carried by other developmental forms (egg and larval stages) of Fasciola spp.

Novel Lethal Clostridial Infection in Florida Manatees (Trichechus manatus latirostris): Cause of the 2013 Unusual Mortality Event in the Indian River Lagoon

The Indian River Lagoon (IRL) on Florida’s east coast is a biologically diverse estuary and an important habitat to the threatened Florida manatee (Trichechus manatus latirostris). An unusual mortality event (UME) was declared by the Working Group on Marine Mammal Unusual Mortality Events in 2013 after a marked increase in manatee deaths in the IRL of an unknown cause. This UME followed a dramatic reduction of seagrass coverage in the IRL due to chronic non-toxic phytoplankton blooms, with a resultant ecosystem shift to mixed macroalgal dominance. At least 199 manatee deaths fitting the UME case definition were documented in and adjacent to the IRL during 2012–2019; mortality was highest in 2013, when 111 of these deaths were documented. The case definition included carcasses in good nutritional condition, with multiorgan congestion or wet lungs consistent with drowning without trauma. The gastrointestinal compartments of manatee carcasses were filled with diverse macroalga species, and the contents were notably more fluid than usual. Gross intestinal findings included blebbing to segmental thickening of the wall. Microscopic lesions were primarily intestinal, including necrosis, edema, hemorrhage, mucosa-associated lymphoid changes, and inflammation, sometimes associated with Gram-positive bacterial rods. A multidisciplinary approach of environmental and carcass sampling found no causative evidence through tests for micro- and macroalgal biotoxins, trace metals, general toxin screening, or vitreum biochemistry. Microbiological, cytological, immunohistochemical, and molecular analyses of Clostridiales from intestinal samples identified Clostridioides difficile toxin A, toxins A/B and toxin A gene; Paeniclostridium sordellii lethal gene (and other potential virulence factors from a sequenced strain); and Clostridium perfringens alpha and epsilon toxin genes. The results from this 8 year-long investigation are indicative that the cause of death in this manatee UME was associated with clostridial infection, initiated by a shift to a predominantly macroalgal diet.

English

Necrotic enteritis (NE) is one of the important enteric disease in the poultry industry worldwide, caused by C. perfringens type A. This study describes the isolation, identification, and toxinotyping of C. perfringens in necrotic enteritis affected broiler chicken in Pakistan. A total of 430 intestinal samples from dead carcasses and birds suspected of NE outbreak, in and around Faisalabad, Pakistan were collected from 36 broiler farms which yielded 87 alpha toxin gene (cpa) positive C. perfringens type A isolates. The birds having 4-5 weeks of age, clinical signs, and reared in open (conventional) sheds showed higher C. perfringens isolation rate. The study concluded netB negative C. perfringens type A as a causative agent for NE outbreaks in broiler birds in Faisalabad, Pakistan.

Sequencing of Clostridium perfringens toxin genes (cpa, etx, iap) from Iraqi hospitals and detection by PCR of the genes encoding resistance to metronidazole, tetracycline, and clindamycin

PurposeThis cross sectional study was designed to investigate the molecular detection of C. perfringens toxins (alpha, epsilon, iota) and antibiotics resistance genes, as well as sequencing of their toxin genes. MethodsDifferent wound swabs were taken from 140 patients. PCR was applied for detection of clostridial toxins; alpha toxin (cpa) gene, epsilon toxin (etx) gene, and iota toxin (iap) gene. Metronidazole nim gene, tetracycline resistance genes; (tetQ, tetM, tetB, and tetW) and clindamycin resistance genes erm (A,B), were used for genotypic detection of antibiotic resistance. ResultsOut of 140 clinical samples collected, 7 isolates were detected using specific primer 16S–23S intergenic rRNA spacer gene of C. perfringens. Results showed presence of alpha toxin (cpa) genes in all clostridial isolates, Epsilon toxin (etx) genes in 2/7(28.4%), and iota toxin (iap) genes in 2/7 (28.4%). Results of antibiotic resistance genes showed that all isolates were not able to produce nim, tet W and Q, tet B, erm (A), erm (B) genes except for tetM. Gene sequence analysis for cpa gene showed that there were 3 mutations in the sample of this gene, the results of etx gene when had been sent 2 samples from this gene. The first sample showed that there was one mutation and the results of iap gene showed that there were no mutation in 2 samples of the iap gene. The samples had been showed identical 100%. ConclusionThe DNA sequence analysis of bacterial genome revealed several important feature including that may allow to confirm new the presence of toxin and to identify new or mutant toxins that may be missed by diagnostic PCR specifically target toxin encoding genes.

Phenotypic detection and genotyping of Clostridium perfringens associated with enterotoxemia in sheep in the Qassim Region of Saudi Arabia.

Background and Aim:Enterotoxemia caused by Clostridium perfringens toxinotypes is an often fatal disease of sheep of all ages, with a substantial economic loss to the sheep industry. This study was conducted to isolate C. perfringens from suspected cases of enterotoxemia in sheep in the central part of the Qassim Region, Saudi Arabia, and to determine the prevalent toxinotype by detecting alpha (cpA), beta (cpB), and epsilon (etX) toxin genes, which might help control this disease locally.Materials and Methods:A total of 93 rectal swabs and intestinal content samples were collected from diseased and animals suspected of having died of enterotoxemia in early 2020. Samples were subjected to bacteriological examination, biochemical analysis of isolates by VITEK 2, and molecular toxinotyping of isolates by LightCycler® real-time polymerase chain reaction (RT-PCR).Results:Our results revealed that only 14 isolates were confirmed by VITEK 2 as being C. perfringens, with excellent identification (probability of 95% and 97%). According to the toxinotyping of isolates by RT-PCR, all 14 isolates possessed both the cpA and etX toxin genes, while the cpB toxin gene was not detected in any of the isolates.Conclusion:Our findings demonstrated that C. perfringens type D was the only toxinotype found in the central part of the Qassim Region in 2020; moreover, according to the culture method, only 15% (14/93) of the suspected cases of enterotoxemia were confirmed to be caused by C. perfringens infection, which highlighted the importance of clinical and laboratory differential diagnosis of enterotoxemia in sheep.

Molecular Cloning of a New Bi-Functional Fusion Protein of Clostridium perfringens Type A Alpha and Clostridium septicum Alpha Toxin Genes in E. coli

Background: A synthetic construct bi-functional protein fusion includes two protein domains, or proteins bind by a fragment. The synthetic construct is designed to achieve better characterize and new functionality. Therefore, having proper cells is essential for cloning fusion genes. Clostridium perfringens type A produces the alpha-toxin and can cause gas gangrene and gastrointestinal diseases. C. septicum produces the alpha-toxin and can cause non-traumatic and traumatic gas gangrene. Objectives: The current study aimed to investigate molecular cloning of a new bi-functional fusion protein of C. perfringens alpha (cpa) and C. septicum alpha (csa) toxin genes in E. coli TOP10. In silico analysis was used for the chimeric fusion protein structural prediction. Methods: To produce chimeric fusion protein, the alpha-alpha (α-α) fusion gene was designed according to nucleotide sequences of cpa (KY584046.1) and csa (JN793989.2) genes. Tertiary structural prediction and validation of the fusion protein were determined by online software. In the new synthetic construction, α-α fusion protein genes are bind via the linker AEAAAKEAAAKA. The linker was introduced between the two domains by fusion PCR. The synthetic fusion gene was cloned into the pUC57cloning vector and then transferred into the host cell. Results: Analysis of the chimeric protein fusion is showed using the I-TASSER server as C-score equal to -2.68 as well as Rampage software in order to confirm the geometrical model as a natural like protein. Also, 1.0% agarose gel electrophoresis of fusion PCR product and sequencing analysis revealed a DNA fragment length of 2346 bp. Screening gel electrophoresis showed 996 bp length, which the designed linker was contained in it. Gel electrophoresis of extracted and purified recombinant plasmid (pUC57/αα) showed that a pUC57/αα of 5056 bp. The digested recombinant pUC57/αα showed one 2.3 kb (our fusion gene) band and one 2.7 kb (pUC57) band. Conclusions: This study presented a new approach for the fusion of cpa and csa genes based on the fusion PCR strategy. According to the latest information, this is the first time that α-α fusion gene is designed and cloned into a suitable cloning vector.

Identification of Clostridium septicum in Clinical Cases Suspected to Braxy and Malignant Edema by Traditional and Molecular Method and Evaluation of Toxigenic Isolates for Vaccine Production

Clostridium septicum is the causative agent of the acute fetal disease braxy and gas gangrene with major economic losses in the dairy industry. Accurate and rapid detection is great importance in this regard. Vaccination is an effective process for protection against C. septicum infection. It is necessary to identify and evaluate toxigenic Iranian isolates to produce a vaccine. This study aimed to detect Iranian isolates and evaluate toxigenic isolates as candidates for vaccine production. To this end, a total of 17 samples of animals, clinically suspected to braxy and malignant edema, were obtained. All samples were then cultured on media and microbiological and biochemical tests were performed on the colonies. The test results were confirmed by PCR amplification of the alpha-toxin gene. The toxigenic isolates were then evaluated using MLD. The experimental vaccine was produced and evaluated according to the British Pharmacopoeia Standard. According to the results, out of 17 samples, 15 samples were considered C. septicum. All samples were confirmed by PCR amplification. The most toxigenic isolate was used for experimental vaccine production. The result was successful. The Iranian isolate could be, therefore, used for vaccine production although further studies should be conducted in this regard.

Faecal PCR panel results and clinical findings in Western Australian dogs with diarrhoea.

To describe faecal PCR (fPCR) results and clinical findings of dogs seen at a university teaching hospital for diarrhoea. Retrospective case series (April 2015 to July 2018). Data were collected from the hospital electronic medical records. Data extracted included signalment, history, clinical signs, treatment, fPCR panel results, other faecal diagnostic test results and antimicrobial use. One hundred and sixty-eight dogs with diarrhoea had a fPCR panel submitted. Most dogs (115, 68.5%) had diarrhoea of 3 days or less duration. Clostridium perfringens alpha toxin gene was most frequently detected (156, 92.9%) by fPCR, followed by Campylobacter spp. (55, 32.7%), canine parvovirus (CPV) (29, 17.3%), Salmonella spp. (14, 8.3%) and Giardia spp. (9, 5.4%). For the 45 dogs that had a negative point-of-care CPV test, 13 were CPV fPCR positive; some of which were adult dogs with current vaccination status. A total of 94/168 (56%) dogs received antimicrobials at some time during the treatment of diarrhoea. Faecal PCR panels can identify dogs with enteric organisms in their faeces that traditional faecal diagnostics may miss, thus contributing additional information to the diagnostic process. Nonetheless, fPCR results should be interpreted in light of the clinical findings, and particular consideration given to avoiding inappropriate use of antimicrobials. This study highlights that testing for C. perfringens alpha toxin gene is not likely to be diagnostically helpful, and that adult dogs with diarrhoea might be identified as CPV positive with PCR testing, despite a negative point-of-care CPV test result and a current vaccination status.

In silico analysis of a chimeric fusion protein as a new vaccine candidate against Clostridium perfringens type A and Clostridium septicum alpha toxins

In silico analysis is the most important approach to understand protein structure and functions, and the most important problem for designing and producing a fusion construct is producing large amounts of functional protein. Clostridium perfringens type A and Clostridium septicum produce alpha (plc) and alpha toxins respectively. C. perfringens can cause gas gangrene and gastrointestinal diseases. C. septicum can cause traumatic and non-traumatic gas gangrene. The aim of current research was in silico analysis of a chimeric fusion protein against C. perfringens type A and C. septicum alpha toxins. Firstly, the chimeric fusion gene was designed according to nucleotide sequences of C. perfringens type A alpha (KY584046.1) and C. septicum alpha (JN793989.2) toxin genes and then its fusion protein is constructed by amino acid sequences of C. perfringens type A and C. septicum alpha toxins. Secondly, online software was used to determine prediction of secondary and tertiary structures and physicochemical characteristics of the fusion protein. Finally, the validation of the fusion protein was confirmed by Rampage and proSA program. The designed fusion protein has 777 amino acids in length. TASSER server and physicochemical parameters are showed: C-score = − 2.68 and molecular weight = 87.9 KD respectively. Rampage and proSA software revealed the fusion protein is valid. Deposited accession number for the sequence of the fusion gene in the GenBank is MK908396. The designed fusion protein is valid and functional. Thus, the fusion gene could be used for clone and expression in a proper prokaryotic cell and also as a recombinant vaccine candidate.

Isolation and Genotyping Study of Clostridium Perfringens From Broiler Farms Infected with Necrotic Enteritis in Sulaimania Province

The study is conducting to isolate and toxinotype the suspected cases of Clostridium perfringens infections of broiler farms in Sulaimania province. A total of 108 samples were collected from intestinal contents, mucosal scraping, and hemorrhagic lymphoid nodules from suspected cases of necrotic enteritis in broilers. The result of isolated and identified bacteria were revealed that 63 (58%) out of 108 samples were positive for C. perfringens. The results revealed that the isolates were only positive for alpha and beta2 toxin genes. Phylogenetic and DNA sequence analysis of cpa and cpb2 gene showed that cpa genes were highly identical to isolates from broiler in Iran, poultry stool and broiler in Brazil, and blue calves in Belgium. While cpb2 gene is closely related to the isolates of broiler in Iran, India and isolates of goat in Pakistan. The results indicated that the causative agent of necrotic enteritis in broiler farms in the region was mainly due to C. perfringens type A infection