Cardiac lesions in sheep and goats with spontaneous Clostridium perfringens type D enterotoxemia

Enterotoxemia caused by Clostridium perfringens type D is one of the most prevalent clostridial diseases of sheep. The lesions of the acute form of this disease, particularly the cerebral lesions, are well characterized; however, detailed descriptions of the cardiac and pulmonary lesions are lacking. Here we describe cardiopulmonary lesions in experimental acute type D enterotoxemia in sheep and determine the role of epsilon toxin (ETX) in the development of these lesions. Four groups of 6 sheep were intraduodenally inoculated with either a wild-type C. perfringens type D strain; its etx knockout mutant, which is unable to produce ETX; the etx mutant complemented with the wild-type etx gene, which regains the ETX toxigenic ability; or sterile culture medium as a control. All sheep were subjected to postmortem examination within 24 hours of inoculation. Lesion scores were compared between groups for pulmonary edema; hydrothorax; ascites; hydropericardium; endocardial, myocardial and epicardial hemorrhages; microscopic lesions of acute myocardial degeneration and necrosis; and myocardial, endocardial, and epicardial edema, hemorrhage, and inflammation. Only sheep inoculated with the wild-type and complemented ETX-toxigenic bacterial strains developed cardiopulmonary lesions, which were present in varying degrees of severity and proportions. These lesions were not present in sheep inoculated with the etx mutant or in the negative control. We conclude that severe acute cardiopulmonary lesions in sheep with experimental enterotoxemia are associated with the capacity of the strains to produce ETX. These changes are likely contributors to the clinical signs and even death of affected animals.

Analyses of miRNA in the ileum of diarrheic piglets caused by Clostridium perfringens type C

Simple SummaryClostridium perfringens (C. perfringens) type C is a spore-forming pathogenic bacterium characterized by the secretion of fatal toxins, which are absorbed into the body, causing diarrhea. Diarrhea has already brought about tremendous economic losses in pig farms worldwide. However, the understanding of lncRNAs’ regulatory mechanisms of the spleen in piglets challenged by C. perfringens type C is still limited. This paper aimed to identify antagonistic lncRNAs associated with the spleen in piglets challenged by C. perfringens type C. The study found that four lncRNAs are involved in immune-/inflammation-related pathways to regulate cytokine genes against C. perfringens type C infection.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.

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.

Evaluation of the efficacy of chlorogenic acid in reducing small intestine injury, oxidative stress, and inflammation in chickens challenged with Clostridium perfringens type A

Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa.

Enterotoxemia is a severe and peracute disease caused by Clostridium perfringens (C. perfringens) rendering high mortality leading to huge economic losses, especially in small ruminants. The bacterium induces peracute death in animals based on the rapid production of different lethal toxins. Mortality occurred three private herds of two breeds, i.e., Makhi Cheeni and Beetal, and one non-descriptive (Teddy) herds reared in the desert area of Bahawalpur, Pakistan. At necropsy, tissue samples for histopathology and intestinal contents for bacterial isolation and culture were collected. Following the standard procedure, tissue slides were prepared. Multiplex PCR was used to identify toxinotypes using specific primers. Morbidity, mortality, and case fatality in Makhi Cheeni, Beetal, and Teddy goats caused by enterotoxemia were 87.58, 75.81, and 76.11%, respectively. Based on toxinotypes in the present outbreaks, C. perfringens type A (cpα = 20.7%; cpα + cpβ2 = 11.2%) and C. perfringens type D (cpα + cpβ2 + etx = 47.7%; cpα + etx = 20.7%) were detected. Deaths due to C. perfringens type D (68.10%) were significantly higher (p < 0.001) compared with deaths by C. perfringens type A (34.90%). Petechiation of serosal surfaces, hemorrhage of intestines, lungs, and liver were seen. Kidneys were soft, and under the microscope, tubules were studded with erythrocytes. There was stunting and fusion in the intestinal villi. From this study, we concluded that endotoxemia can occur in any season; thus, a proper vaccination schedule must be followed for the protection of small ruminants' health.

Clinicopathological and immunological studies on toxoids vaccine as a successful alternative in controlling clostridial infection in broilers

Enterotoxaemia (pulpy kidney) is a severe condition that has a very brief clinical course in small ruminants (young sheep and goats) and results in unexpected deaths. Therefore, therapeutic interventions often do not work. It is brought on by the absorption of the Clostridium perfringens (C. perfringens) type D toxin from the intestinal tract. This bacterium has five toxin types, and type D, which produces epsilon toxin, is primarily linked to enterotoxaemia in caprine and ovine species. This study aimed to improve the efficacy of the immunization by investigating the toxigenicity of C. perfringens type D and the impact of each element of its production media on the potency of the epsilon toxin, three types of sugars (glucose, sucrose and Dextrin) were tested with four types of proteins (peptone, tryptone, protease peptone and N-Z casein) with different incubation periods (18, 24 and 48 hours) to improve the media to reach to maximum toxigenicity. Among all these trials, sucrose with N-Z casein with 24 hours’ incubation has been ascended the throne.

Background: Clostridium perfringens (C. perfringens) type A is a major cause of enteritis in farmed and wild deer populations, leading to significant economic losses in the deer industry. This bacterium produces toxins that damage the intestine. Methods: In this study, we performed transcriptome analysis by establishing an intestinal circulation model of the intestines of fallow deer (Dama Dama) inoculated with C. perfringens type A versus those not inoculated with C. perfringens type A. In a further step, we determined the protein content of immunoinflammation-related molecules by ELISA and the antioxidant capacity of the intestine to investigate the molecular mechanisms of C. perfringens type A-induced enteritis. Results: Transcriptome analysis revealed significant enrichment of pathways related to the haematopoietic system, oxidative stress, the immune system and intestinal tight junctions. Additionally, C. perfringens α-toxin enters the intestine and may be recognized by TLR6, activating the immune system, increasing the secretion of various cytokines and inflammasome components, inducing oxidative stress and damaging the intestine. Conclusions: This study provides a comprehensive transcriptomic basis for understanding the selective differential expression of genes in deer enteritis induced by C. perfringens type A and provides a broader guide for finding therapeutic approaches to deer enteritis.

BackgroundClostridium perfringens (C. perfringens) type C is the most common bacteria causing piglet diarrheal disease and it greatly affects the economy of the global pig industry. The spleen is an important immune organ in mammals; it plays an irreplaceable role in resisting and eradicating pathogenic microorganisms. Based on different immune capacity in piglets, individuals display the resistance and susceptibility to diarrhea caused by C. perfringens type C. Recently, long non-coding RNA (lncRNA) and mRNA have been found to be involved in host immune and inflammatory responses to pathogenic infections. However, little is known about spleen transcriptome information in piglet diarrhea caused by C. perfringens type C.MethodsHence, we infected 7-day-old piglets with C. perfringens type C to lead to diarrhea. Then, we investigated lncRNA and mRNA expression profiles in spleens of piglets, including control (SC), susceptible (SS), and resistant (SR) groups.ResultsAs a result, 2,056 novel lncRNAs and 2,417 differentially expressed genes were found. These lncRNAs shared the same characteristics of fewer exons and shorter length. Bioinformatics analysis identified that two lncRNAs (ALDBSSCT0000006918 and ALDBSSCT0000007366) may be involved in five immune/inflammation-related pathways (such as Toll-like receptor signaling pathway, MAPK signaling pathway, and Jak-STAT signaling pathway), which were associated with resistance and susceptibility to C. perfringens type C infection. This study contributes to the understanding of potential mechanisms involved in the immune response of piglets infected with C. perfringens type C.

This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.

To determine the percentage of broodmares and foals that shed Clostridium perfringens in their feces and classify the genotypes of those isolates. Prospective cross-sectional study. 128 broodmares and their foals on 6 equine premises. Anaerobic and aerobic bacteriologic cultures were performed on feces collected 3 times from broodmares and foals. All isolates of C. perfringens were genotyped. Clostridium perfringens was isolated from the feces of 90% of 3-day-old foals and 64% of foals at 8 to 12 hours of age. A lower percentage of broodmares and 1- to 2-month-old foals shed C. perfringens in their feces, compared with neonatal foals. Among samples with positive results, C. perfringens type A was the most common genotype identified (85%); C. perfringens type A with the beta2 toxin gene was identified in 12% of samples, C. perfringens type A with the enterotoxin gene was identified in 2.1% of samples, and C. perfringens type C was identified in < 1% of samples. Clostridium perfringens was identified from the feces of all but 6 foals by 3 days of age and is likely part of the normal microflora of neonatal foals. Most isolates from broodmares and foals are C. perfringens type A; thus, the clinical relevance of culture results alone is questionable. Clostridium perfringens type C, which has been associated with neonatal enterocolitis, is rarely found in the feces of horses.

This study was conducted to investigate the effects of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative statuses of broiler chickens under Clostridium perfringens (CP) type A challenge. Two organic acid products were tested: organic acid 1 (OA1), consisting of butyrate, medium-chain fatty acids, organic acids, and phenolics; and organic acid 2 (OA2), consisting of buffered short-chain fatty acids. Six hundred 1-day-old male Arbor Acres broiler chicks were randomly assigned to one of five treatments: Control 1, basal diet, nonchallenged birds; Control 2, basal diet, with CP challenge; antimicrobial growth promoters (AGP), basal diet supplemented with Aureomycin (chlortetracycline), with CP challenge; OA1, basal diet supplemented with OA1, with CP challenge; and OA1OA2, basal diet supplemented with OA1 and OA2, with CP challenge. Each treatment had eight replicate pens of 15 birds. The experiments lasted for 29 days. The disease challenge was performed on days 15-17, with an oral gavage of 0.5 mL of CP culture (2.0 × 108 colony-forming units [CFU]/mL) for each bird. Body weights (BWs), intestinal lesion scores, immune organ indices, and serum malondialdehyde (MDA) concentrations were measured on days 19, 22, and 29, respectively, in three birds per pen. Carcass characteristics were determined on day 29. No treatment-related differences in mortality were noted before (P = 0.28) or after (P = 0.64) challenge or over the whole study period (days 0-28; P = 0.66). On day 19, the BW of Control 2 was lower than other treatments (P < 0.0001). On day 22, AGP, OA1, and OA1OA2 had higher BW than Control 2 (P = 0.001). The breast muscle yield of OA1 and OA1OA2 was higher than AGP (P < 0.05). The abdominal fat yield of OA1OA2 was lower than AGP and Control 2 (P < 0.05). On day 22, the birds fed OA1OA2 showed lower intestinal lesion scores than OA1 (P < 0.05). No treatment-related differences in immune organ (spleen, thymus, and bursa) indices were noted (P > 0.05). On day 29, the MDA concentration of OA1 and OA1OA2 was lower than those of Control 1 and AGP (P < 0.05). In conclusion, the addition of organic acids may protect broiler chickens from severe intestinal lesions and oxidative stress and may help reduce abdominal fat mass deposition. There is potential for organic acid-based products as alternatives for AGP in preventing necrotic enteritis in broilers.

LncRNA play important roles in regulation of host immune and inflammation responses in defending bacterial infection. Clostridium perfringens (C. perfringens) type C is one of primary bacteria leading to piglet diarrhea and other intestinal inflammatory diseases. For the differences of host immune capacity, individuals usually show resistance and susceptibility to bacterial infection. However, whether and how lncRNAs involved in modulating host immune resistance have not been reported. We have investigated the expression patterns of ileum lncRNAs of 7-day-old piglets infected by C. perfringens type C through RNA sequencing. A total of 16 lncRNAs and 126 mRNAs were significantly differentially expressed in resistance (IR) and susceptibility (IS) groups. Many lncRNAs and mRNAs were identified to regulate resistance and susceptibility of piglets through immune related pathways. Five lncRNAs may have potential function on regulating the expressions of cytokines, these lncRNAs and cytokines work together to co-regulated piglet immune response to C. perfringens, affecting host resistance and susceptibility. These results provide valuable information for understanding the functions of lncRNA and mRNA in affecting piglet diarrhea resistance of defensing to C. perfringens type C, these lncRNAs and mRNAs may be used as the important biomarkers for decreasing C. perfringens spread and diseases in human and piglets.