Perfringens Type Research Articles

Detection of Enterotoxigenic Escherichia coli and Clostridia in the Aetiology of Neonatal Piglet Diarrhoea: Important Factors for Their Prevention.

This study aimed to research the involvement of enterotoxigenic E. coli (ETEC) and C. difficile or C. perfringens type C in the aetiology of neonatal piglet diarrhoea in Greece and to identify preventive factors for them. A total of 78 pooled faecal samples were collected randomly from 234 suckling piglets (1-4 days of age) with diarrhoea from 26 pig farms (3 piglets × 3 litters × 26 farms = 234 piglets = 78 faecal pool samples). The collected samples were initially screened for the presence of E. coli and C. difficile or C. perfringens via cultivation on MacConkey and anaerobic blood agar, respectively. Subsequently, the samples were pooled on ELUTE cards. From samples tested, 69.23% of those in the farms were ETEC F4-positive, 30.77% were ETEC F5-positive, 61.54% ETEC were F6-positive, 42.31% were ETEC F4- and E. coli enterotoxin LT-positive, 19.23% were ETEC F5- and LT-positive, 42.31% were ETEC F6- and LT-positive, while LT was found in 57.69% of those in the farms. C. difficile was involved in many cases and identified as an emerging neonatal diarrhoea etiological agent. Specifically, Toxin A of C. difficile was found in 84.62% and Toxin B in 88.46% of those in the farms. Antibiotic administration to sows in combination with probiotics or acidifiers was revealed to reduce the detection of antigens of ETEC and the enterotoxin LT of E. coli.

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.

Microbiological Findings and Associated Histopathological Lesions in Neonatal Diarrhoea Cases between 2020 and 2022 in a French Veterinary Pig Practice

This retrospective study described the aetiologies of neonatal diarrhoea cases and their associations with histological findings. A total of 106 diarrhoeic neonatal piglets were selected. Cultures, MALDI typings, PCRs and evaluation of intestinal lesions were performed. A total of 51 cases (48.1%) were positive for only one pathogen and 54 (50.9%) were positive for more than one pathogen. Clostridium perfringens type A was the most frequently detected pathogen (61.3%), followed by Enterococcus hirae (43.4%), rotavirus type A (38.7%), rotavirus type C (11.3%) and enterotoxigenic Escherichia coli (3.8%). Only lesions in the small intestine were correlated with detected pathogens. The detection of rotavirus was associated with an increased probability of observing villous atrophy (p < 0.001), crypt hyperplasia (p = 0.01) and leucocyte necrosis in the lamina propria (p = 0.05). The detection of Clostridium perfringens type A was associated with an increased probability of observing bacilli in close proximity to the mucosa (p < 0.001) and a decreased probability of observing epithelial necrosis (p = 0.04). Detection of Enterococcus hirae was associated with an increased probability of observing enteroadherent cocci (p < 0.001). Multivariate regression logistic models revealed that epithelial necrosis was more likely to occur in Enterococcus hirae-positive piglets (p < 0.02) and neutrophilic infiltrate was more likely to occur in Clostridium perfringens type A- and Enterococcus hirae-positive piglets (p = 0.04 and p = 0.02, respectively).

Effects of three different probiotics of Tibetan sheep origin and their complex probiotics on intestinal damage, immunity, and immune signaling pathways of mice infected with Clostridium perfringens type C.

Tibetan sheep have unique intestinal microorganisms in their intestines that are adapted to the highland alpine and anoxic environment. To further clarify the probiotic properties of Tibetan sheep-derived probiotics, we selected three Tibetan sheep-derived probiotic isolates (Enterococcus faecalis EF1-mh, Bacillus subtilis BS1-ql, and Lactobacillus sakei LS-ql) to investigate the protective mechanisms of monocultures and their complex strains against Clostridium perfringens type C infection in mice. We established a model of C. perfringens type C infection and used histology and molecular biology to analyze the effects and mechanisms of different probiotic treatments on mice after C. perfringens type C infection. After supplementation with either probiotics or complex probiotics, mice were improved in terms of weight reduction and reduced the levels of cytokines in serum and increased the levels of intestinal sIgA, and supplementation with complex probiotics was effective. In addition, both probiotic and complex probiotic supplementation effectively improved the damage of intestinal mucosa and spleen tissue. The relative expressions of Muc 2, Claudin-1, and Occludin genes were increased in the ileum. The three probiotics and the compound probiotics treatment significantly reduced the relative mRNA expression of toll-like/MyD88/NF-κB/MAPK. The effect of probiotic treatment was similar to the results of engramycin treatment, but the effect of engramycin treatment on intestinal sIgA was not significant. Our results clarify the immunomodulatory effects of the three probiotic isolates and the complex probiotics on C. perfringens infection, and the repair of the intestinal mucosal barrier.

In silico mutation of aromatic with aliphatic amino acid residues in Clostridium perfringens epsilon toxin (ETX) reduces its binding efficiency to Caprine Myelin and lymphocyte (MAL) protein receptors

Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between ‘ETX (wild)-MAL protein’ and ‘ETX (mutated)-MAL protein complex’ interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats. Communicated by Ramaswamy H. Sarma

Infectious Rotavirus and Coronavirus of Calves

Recently, some progress has been made in the study of bacterial and viral infectious diseases of calves. However, as effective systems and methods are put in place to control these diseases, other new emerging diseases will also become important. Among diseases of the neonatal period in calves, diarrhea, which causes significant economic damage, is one of the main problems in cattle breeding. Calves diarrhea is divided into two groups: infectious and non-infectious. Diarrhea of infectious origin is mainly caused by Escherichia coli, Salmonella sp., Campylobacter sp., Clostridium perfringens type C, Rotavirus, Coronavirus, Parvovirus, Astrovirus, Calicivirus, Breda virus, Cryptosporidium, Coccidia, Giardia and Neoascaris vitulorum. During field studies, it was found that 75-95% of infectious diarrhea in calves is caused by enterotoxic Escherichia coli, rotaviruses, coronaviruses, cryptosporidium, including 27-36% rotaviruses, 20-26% coronaviruses. These diseases are often registered in livestock farms of the republic of Azerbaijan and cause the death of newborn calves.

Identification of MicroRNAs Regulating Clostridium perfringens Type C Infection in the Spleen of Diarrheic Piglets

Clostridium perfringens (C. perfringens) type C is one of the common bacteria in piglet diarrhea, which seriously affects the swine industry's development. The spleen plays crucial roles in the resistance and elimination of pathogenic microorganisms, and miRNAs play important roles in regulating piglet diarrhea caused by pathogens. However, the mechanism by which miRNAs in the spleen are involved in regulating C. perfringens type C causing diarrhea in piglets remains unclear. The expression profiles of the spleen miRNAs of 7-day-old piglets challenged by C. perfringens type C were studied using small RNA-sequencing in control (SC), susceptible (SS), and resistant (SR) groups. Eight-eight differentially expressed miRNAs were screened. The KEGG pathway analysis of target genes revealed that the miRNAs were involved in the MAPK, p53, and ECM-receptor interaction signaling pathways. NFATC4 was determined to be a direct target of miR-532-3p and miR-133b using a dual-luciferase reporter assay. Thus, miR-133b and miR-532-3p targeted to NFATC4 were likely involved to piglet resistance to C. perfringens type C. This paper provides the valuable resources to deeply understand the genetic basis of C. perfringens type C resistance in piglets and a solid foundation to identify novel markers of C. perfringens type C resistance.

Drug-independent control strategy of clostridial infection in broiler chickens using anti-toxin environmentally friendly multienzymes

The study investigated the effect of enzymes as a toxin detoxifier (DETOXIZYME) dietary supplementation on performance during growth, blood chemistry, and immunity under clostridia infection in chickens. A total of 480, day-old male chicks were randomly distributed to four groups, with six replicates of 20 birds each. The first control negative treatment (A) fed the basal formula as commercial feed prepared following the strain's needs, the second control positive group (B) fed the basal formula challenged with Clostridium perfringens (C. perfringens) type A, the third group (C) fed the basal formula with 100 g DETOXIZYME/ton of feed and challenged with clostridia, and the fourth group (D) fed the control basal formula with 100 g DETOXIZYME/ton of feed. DETOXIZYME dietary supplementation significantly boosted body weight (BW), body weight gain (BWG), feed intake (FI), and European production efficiency factor (EPEF) and improved the feed conversion rate (FCR) of the broilers. The dietary supplementation of DETOXIZYME significantly increased carcass trait and spleen. However, liver and abdominal fat weight significantly decreased compared with clostridia-challenged groups. The values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid, creatinine, and Malondialdehyde (MDA) were decreased. While calcium, phosphate, zinc, and glutathione peroxidase (GPx) levels were improved in birds that took basal formulas fortified with DETOXIZYME contrary to the other treatment groups during 35 days of age. Plasma total cholesterol, triglyceride, and low-density lipoprotein (LDL) values were reduced versus the other treatment groups. Dietary supplementation of DETOXIZYME increased total protein, albumin, globulin, and Newcastle Disease (ND) immunity titer levels in the overall period compared to other groups. Dietary DETOXIZYME supplementation decreased clostridia and E. coli bacteria counts and improved gut morphometry. In conclusion, dietary supplementation of DETOXIZYME had a positive impact on performance, blood biochemistry, immunity, and bacterial counts and improved the gut morphology in broilers under clostridia infection.

Lnc001776 Affects CPB2 Toxin-Induced Excessive Injury of Porcine Intestinal Epithelial Cells via Activating JNK/NF-kB Pathway through ssc-let-7i-5p/IL-6 Axis

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.

Clostridium perfringens in Broiler Chickens: Isolation, Identification, Typing, and Antimicrobial Susceptibility

Necrotic enteritis (NE) is a common worldwide poultry disease caused by the bacterium of Clostridium perfringens (C. perfringens) which has significant economic losses in the poultry industry as well as the cost of treatment and preventive measures. The current study was conducted to evaluate the incidence of NetB toxin positive in C. perfringens on different farms in Egypt. In the years 2020 and 2021, on industrial broiler farms (15-45 days- old), 100 intestinal samples were collected consisting of 30 healthy Ross broiler chickens and 70 unhealthy Ross broiler chickens. Culture and biochemical characterization (Catalase, urease, sugar fermentation, gelatin liquefaction, nitrate reduction, and lecithinase reaction tests) confirmed that C. perfringens was isolated at a rate of 10% (3/30) from apparently healthy broiler chickens and 40% from unhealthy broiler chickens. Thirty-one isolates were tested for toxigenicity and typing by ELISA kits and the results showed that 80% of the isolates from unhealthy broiler chickens were C perfringens type A alpha-toxin (toxigenic), 20% were non-toxigenic, and 66.7% isolates from apparently normal broiler chickens were toxigenic. The same thirty-one (44%) C. perfringens isolates were detected by PCR to investigate the presence of the NetB toxin gene in apparently healthy and unhealthy broilers and subsequently detect the role of NetB toxin in inducing NE. Of the samples, 82% of the isolates from unhealthy chicks were found to incode NetB gene, while none of the isolates from healthy broiler chickens had NetB. Clostridium perfringens showed sensitivity to amoxicillin, amoxicillin with clavulanic acid and ampiclox, intermediate for ofloxacin, and high resistance to cephalexin, streptomycin, colistin sulfate, erythromycin, sulfa trimethoprim, gentamycin, and oxytetracycline. The present study revealed the importance of NetB gen in the appearance of clinical signs of NE in broiler chickens. Keywords: Alpha toxin, Antibiotic sensitivity test, Clostridium perfringens, Necrotic enteritis, NetB

METTL3-Mediated LncRNA EN_42575 m6A Modification Alleviates CPB2 Toxin-Induced Damage in IPEC-J2 Cells.

Long non-coding RNAs (lncRNAs) modified by n6-methyladenosine (m6A) have been implicated in the development and progression of several diseases. However, the mechanism responsible for the role of m6A-modified lncRNAs in Clostridium perfringens type C piglet diarrhea has remained largely unknown. We previously developed an in vitro model of CPB2 toxin-induced piglet diarrhea in IPEC-J2 cells. In addition, we previously performed RNA immunoprecipitation sequencing (MeRIP-seq), which demonstrated lncRNA EN_42575 as one of the most regulated m6A-modified lncRNAs in CPB2 toxin-exposed IPEC-J2 cells. In this study, we used MeRIP-qPCR, FISH, EdU, and RNA pull-down assays to determine the function of lncRNA EN_42575 in CPB2 toxin-exposed IPEC-J2 cells. LncRNA EN_42575 was significantly downregulated at different time points in CPB2 toxin-treated cells. Functionally, lncRNA EN_42575 overexpression reduced cytotoxicity, promoted cell proliferation, and inhibited apoptosis and oxidative damage, whereas the knockdown of lncRNA EN_42575 reversed these results. Furthermore, the dual-luciferase analysis revealed that METTL3 regulated lncRNA EN_42575 expression in an m6A-dependent manner. In conclusion, METTL3-mediated lncRNA EN_42575 exerted a regulatory effect on IPEC-J2 cells exposed to CPB2 toxins. These findings offer novel perspectives to further investigate the function of m6A-modified lncRNAs in piglet diarrhea.

Change in Long Non-Coding RNA Expression Profile Related to the Antagonistic Effect of Clostridium perfringens Type C on Piglet Spleen.

LncRNAs play important roles in resisting bacterial infection via host immune and inflammation responses. Clostridium perfringens (C. perfringens) type C is one of the main bacteria causing piglet diarrhea diseases, leading to major economic losses in the pig industry worldwide. In our previous studies, piglets resistant (SR) and susceptible (SS) to C. perfringens type C were identified based on differences in host immune capacity and total diarrhea scores. In this paper, the RNA-Seq data of the spleen were comprehensively reanalyzed to investigate antagonistic lncRNAs. Thus, 14 lncRNAs and 89 mRNAs were differentially expressed (DE) between the SR and SS groups compared to the control (SC) group. GO term enrichment, KEGG pathway enrichment and lncRNA-mRNA interactions were analyzed to identify four key lncRNA targeted genes via MAPK and NF-κB pathways to regulate cytokine genes (such as TNF-α and IL-6) against C. perfringens type C infection. The RT-qPCR results for six selected DE lncRNAs and mRNAs are consistent with the RNA-Seq data. This study analyzed the expression profiling of lncRNAs in the spleen of antagonistic and sensitive piglets and found four key lncRNAs against C. perfringens type C infection. The identification of antagonistic lncRNAs can facilitate investigations into the molecular mechanisms underlying resistance to diarrhea in piglets.

Animal Model Studies, Antibiotic Resistance and Toxin Gene Profile of NE Reproducing Clostridium perfringens Type A and Type G Strains Isolated from Commercial Poultry Farms in China

Poultry necrotic enteritis (NE) is a complex and multifactorial disease caused by Clostridium perfringens types. Earlier, the disease was prevented and/or controlled through the addition of in-feed antibiotics and antimicrobial growth promoters (AGPs). The ban on the use of these agents as feed additives has been a major reason for re-emergence of this disease leading to huge economic losses to the world poultry industry. Understanding the pathogenesis of NE by developing an effective experimental model remains challenging and lacks consistency owing to the involvement of several critical factors involved in causing lesions of disease in the field. In this study, locally characterized C. perfringens types, i.e., ACP (toxinotype A), and GCP (toxinotype G), obtained from NE outbreaks on commercial farms in China (2020-2022), were used to experimentally induce NE in Specific-Pathogen-Free (SPF) chicks. The lesion scores observed on day 20 were 1.9 ± 1.10 (GCP strain) and 1.5 ± 1.08 (ACP strain), and both had significant difference as compared to the control group. The inclusion of fishmeal in addition to oral clostridial dose, i.e., fishmeal (day 7 onward) + Clostridia (7.5 × 108 cfu/mL consecutively for 04 days) induced a lesion score of 2.0 ± 1.15 in respective groups. Use of coccidia (Eimeria necatrix) on day 9 followed by clostridia challenge enhanced the lesion scores to 2.5 ± 1.08 and 2.2 ± 1.23 for type G and type A strains, respectively. When both predisposing factors (coccidia + fish meal) were given together, i.e., fishmeal (day 7 onward) and coccidia (day 9) along with clostridia, the lesion scores were 3.2 ± 1.22 (GCP + coccidia + fish meal) and 3.0 ± 1.15 (ACP + coccidia + fish meal). These results were significantly different from group 1 (ACP) and 2 (GCP), in which only C. perfringens was used to induce NE. The clinical signs as well as histopathological lesions in experimentally induced groups were found similar as reported in the literature. The two type G strains identified in this study were also used for susceptibility testing against various drugs. Both strains were found to be resistant to amikacin, doxycycline, metronidazole, neomycin, nystatin, polymyxin B, streptomycin, and tetracycline. Variable susceptibility was seen against ceftriaxone, florfenicol, gentamicin, and kanamycin drugs. Amoxicillin, ampicillin, cefotaxime, ciprofloxacin, enrofloxacin, ofloxacin, and penicillin were effective drugs based upon their low level of resistance and therefore they might be preferred over other antimicrobial agents for proper treatment/prophylaxis of NE infections. Further studies are needed to study the pathogenesis of NE in detail in experimentally induced models along with continuous monitoring of the resistance pattern of C. perfringens strains in the field.

Necrotic enteritis in a commercial turkey flock coinfected with hemorrhagic enteritis virus.

Four turkeys from a commercial flock with acutely elevated mortality levels were submitted for postmortem examination and diagnostic workup. No clinical signs had been observed before death. On gross examination, hemorrhage and necrosis were present throughout the intestinal tracts, and the spleens were markedly enlarged and speckled. Microscopically, numerous, large basophilic-to-amphophilic intranuclear inclusion bodies were observed in mononuclear cells of the spleen and the lamina propria of the small intestine. In addition, there were lesions of diffuse villus blunting and necrosis of the small intestine, with large numbers of rod-shaped bacteria adhered to the epithelium and in the intestinal lumen. Hemorrhagic enteritis virus (HEV) infection was confirmed via PCR on the spleen. Clostridium perfringens was demonstrated in the small intestine by anaerobic culture and immunohistochemistry. The C. perfringens isolate was type F by PCR and, to our knowledge, necrotic enteritis in turkeys has not been described in association with C. perfringens type F infection.

Nano selenium-enriched probiotic Lactobacillus enhances alum adjuvanticity and promotes antigen-specific systemic and mucosal immunity.

Nano selenium-enriched probiotics have been identified to improve immune responses, such as alleviating inflammation, antioxidant function, treatment of tumors, anticancer activity, and regulating intestinal flora. However, so far, there is little information on improving the immune effect of the vaccine. Here, we prepared nano selenium-enriched Levilactobacillus brevis 23017 (SeL) and heat-inactivated nano selenium-enriched L. brevis 23017 (HiSeL) and evaluated their immune enhancing functions on the alum-adjuvanted, inactivated Clostridium perfringens type A vaccine in mouse and rabbit models, respectively. We found that SeL enhanced immune responses of the vaccine by inducing a more rapid antibody production, eliciting higher immunoglobulin G (IgG) antibody titers, improving secretory immunoglobulin A (SIgA) antibody level and cellular immune response, and regulating Th1/Th2 immune response, thus helping to induce better protective efficacy after challenge. Moreover, we confirmed that the immunoenhancement effects are related to regulating oxidative stress, cytokine secretion, and selenoprotein expression. Meanwhile, similar effects were observed in HiSeL. In addition, they show enhanced humoral immune response at 1/2 and 1/4 standard vaccine doses, which confirms their prominent immune enhancement effect. Finally, the effect of improving vaccine immune responses was further confirmed in rabbits, which shows that SeL stimulates the production of IgG antibodies, generates α toxin-neutralizing antibodies rapidly, and reduces the pathological damage to intestine tissue. Our study demonstrates that nano selenium-enriched probiotics improve the immune effect of the alum adjuvants vaccine and highlight its potential usage in remedying the disadvantages of alum adjuvants.

Decreased S100A9 expression alleviates Clostridium perfringens beta2 toxin-induced inflammatory injury in IPEC-J2 cells.

S100 calcium-binding protein A9 (S100A9) is a commonly known pro-inflammatory factor involved in various inflammatory responses. Clostridium perfringens (C. perfringens ) type C is known to cause diarrhea in piglets. However, the role of S100A9 in C. perfringens type C-induced infectious diarrhea is unclear. Here, the S100A9 gene was overexpressed and knocked down in the IPEC-J2 cells, which were treated with C. perfringens beta2 (CPB2) toxin. The role of S100A9 in CPB2 toxin-induced injury in IPEC-J2 cells was assessed by measuring the levels of inflammatory cytokines, reactive oxygen species (ROS), lactate dehydrogenase (LDH), cell proliferation, and tight junction-related proteins. The results showed elevated expression of S100A9 in diarrhea-affected piglet tissues, and the elevation of S100A9 expression after CPB2 toxin treatment of IPEC-J2 was time-dependent. In CPB2 toxin-induced IPEC-J2 cells, overexpression of S100A9 had the following effects: the relative expression of inflammatory factors IL-6, IL8, TNF-α, and IL-1β was increased; the ROS levels and LDH viability were significantly increased; cell viability and proliferation were inhibited; the G0/G1 phase cell ratio was significantly increased. Furthermore, overexpression of S100A9 reduced the expression of tight junction proteins in CPB2-induced IPEC-J2 cells. The knockdown of S100A9 had an inverse effect. In conclusion, our results confirmed that S100A9 exacerbated inflammatory injury in CPB2 toxin-induced IPEC-J2 cells, inhibited cell viability and cell proliferation, and disrupted the tight junctions between cells. Thus, decreased S100A9 expression alleviates CPB2 toxin-induced inflammatory injury in IPEC-J2 cells.

Concurrent infection by Clostridium perfringens type A, Clostridium septicum, and Clostridium sordelli in mouflon (Ovis aries musimon) - A case report

A female mouflon, weighing 40 kilograms, was submitted to the diagnostic laboratory of the Institute of Veterinary Medicine of Serbia for determining the potential cause of death. Necropsy revealed massive hemorrhages in subcutaneous and intermuscular tissue and on papillary muscle. Petechiae and ecchymoses were found on the omentum, mesentery and adipose tissue of heart, kidney and costal pleura. Haemorrhagic-necrotic enteritis in duodenum and jejunum was characterized by catarrhal hemorrhagic inflammation with the presence of mucous and bloody content, whereas gas bubbles in the submucosa have also been confirmed. Bacterial cultures from sampled organs were identified as Clostridium perfringens type A, Clostridium septicum, and Clostridium sordelli. Based on the established pathological and histological changes and the results of the bacteriological, biochemical, and molecular examination, the state of septic shock and toxemia with disseminated massive bleeding was the immediate cause of mouflon death. The septic condition is a consequence of enterotoxemia caused by Clostridium perfringens type A, Clostridium septicum, and Clostridium sordelli infection.

Detection and molecular characterization of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum from lambs and goat kids with hemorrhagic abomasitis in Turkey

BackgroundThe pathogenic Clostridia cause neurotoxic, histotoxic and enterotoxic infections in humans and animals. Several Clostridium species have been associated with abomasitis in ruminants. The present study aimed to investigate the frequency, and the presence of virulence genes, of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum in lambs and goat kids with hemorrhagic abomasitis.ResultsA total of 38 abomasum samples, collected from lambs and goat kids of 1 week to 1 month of age in different farms located in eastern Turkey between 2021 and 2022, were evaluated by histopathology, culture and PCR. At necropsy, the abomasum of the animals was excessively filled with caseinized content and gas, and the abomasum mucosa was hemorrhagic in varying degrees. In histopathological evaluation, acute necrotizing hemorrhagic inflammation was noted in abomasum samples. The examination of swab samples by culture and PCR revealed that C. perfringens type A was the most frequently detected species (86.84%) either alone or in combination with other Clostridium species. P. sordellii, C. perfringens type F and C. septicum were also harboured in the samples, albeit at low rates. Beta2 toxin gene (cpb2) was found in three of C. perfringens type A positive samples.ConclusionIt was suggested that vaccination of pregnant animals with toxoid vaccines would be beneficial in terms of protecting newborn animals against Clostridial infections. This study investigated the presence of clostridial toxin genes in abomasal samples for the first time in Turkey.

S100A9 plays a key role in Clostridium perfringens beta2 toxin-induced inflammatory damage in porcine IPEC-J2 intestinal epithelial cells

BackgroundAs an important regulator of autoimmune responses and inflammation, S100A9 may serve as a therapeutic target in inflammatory diseases. However, the role of S100A9 in Clostridium perfringens type C infectious diarrhea is poorly studied. The aim of our study was to screen downstream target genes regulated by S100A9 in Clostridium perfringens beta2 (CPB2) toxin-induced IPEC-J2 cell injury. We constructed IPEC-J2 cells with S100A9 knockdown and a CPB2-induced cell injury model, screened downstream genes regulated by S100A9 using RNA-Seq technique, and performed functional enrichment analysis. The function of S100A9 was verified using molecular biology techniques.ResultsWe identified 316 differentially expressed genes (DEGs), of which 221 were upregulated and 95 were downregulated. Functional enrichment analysis revealed that the DEGs were significantly enriched in cilium movement, negative regulation of cell differentiation, immune response, protein digestion and absorption, and complement and coagulation cascades. The key genes of immune response were TNF, CCL1, CCR7, CSF2, and CXCL9. When CPB2 toxin-induced IPEC-J2 cells overexpressed S100A9, Bax expression increased, Bcl-2 expression and mitochondrial membrane potential decreased, and SOD activity was inhibited.ConclusionIn conclusion, S100A9 was involved in CPB2-induced inflammatory response in IPEC-J2 cells by regulating the expression of downstream target genes, namely, TNF, CCL1, CCR7, CSF2, and CXCL9; promoting apoptosis; and aggravating oxidative cell damage. This study laid the foundation for further study on the regulatory mechanism underlying piglet diarrhea.

LncRNA EN-90756 promotes CPB2-induced proliferation and inhibits apoptosis in IPEC-J2 cells by affecting the JAK-STAT signaling pathway activation.

Long non-coding RNAs (lncRNAs), as key regulators, are closely associated with the development of a variety of disease. However, the mechanisms by which lncRNAs regulate Clostridium perfringens type C induced piglet diarrhea are unclear. In the present study, we explored the expression and characterization of lncRNAs in a C. perfringens beta2 (CPB2) toxin-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) using RNA-sequencing (RNA-seq). A total of 6,558 lncRNAs were identified, of which 49 lncRNAs were significantly differentially expressed between the control and CPB2 groups. Functional enrichment analysis showed that the target genes of differentially expressed lncRNA EN-90756 were mainly associated with defense response to virus, and negative regulation of apoptotic process. LncRNA EN-90756 was significantly up-regulated in IPEC-J2 cells at different time points after CPB2 treatment. Functionally, knockdown of lncRNA EN-90756 might regulate the proliferation and apoptosis of IPEC-J2 cells by affecting the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. LncRNA EN-90756 may be involved in CPB2 toxin-induced piglet diarrhea by regulating the expression of its target gene MX1 (encoding MX dynamin like GTPase 1). Long non-coding RNA EN-90756 affected the antiviral ability of IPEC-J2 cells by regulating the expression of MX1. Meanwhile, lncRNA EN-90756 might regulate cell proliferation and apoptosis by affecting JAK-STAT signaling pathway activation. These findings provide novel perspectives and directions for further exploration of the regulatory mechanisms of lncRNAs on CPB2 toxin-induced diarrhea in piglets.