Molecular detection and characterization of cpb2 gene in Clostridium perfringens isolates from healthy and diseased chickens

Clostridium perfringens is an important pathogen in both human and veterinary medicine. Necrotic enteritis (NE) is the most clinically dramatic bacterial enteric disease of poultry induced by C. perfringens. The pathogenicity of this bacterium is associated with the production of extracellular toxins produced by some of its strains, such as beta2 toxin. The exact role of beta2 toxin in NE pathogenesis is still controversial. In the present study, C. perfringens isolates from healthy and diseased poultry flocks from different parts of Iran were analyzed by PCR assay to determine the presence of all variants of the beta2 toxin gene (cpb2). The products of two positive cpb2 PCR reactions were sequenced, compared to each other and to the cpb2 sequences published in GenBank (by multiple alignment and phylogenetic analysis). The current work represents the first study of cpb2 in poultry C. perfringens isolates in Asia, and reports the highest percentage of cpb2-positive isolates in both apparently healthy chickens (97.7%) and those afflicted with NE (94.4 %). The sequenced isolates were classified as atypical. This study did not show a direct correlation between NE occurrence and cpb2 presence

Effect of treatment with Lactococcus lactis NZ9000 on intestinal microbiota and mucosal immune responses against Clostridium perfringens in broiler chickens

Alterations in intestinal microbiota can modulate the developing avian intestinal immune system and, subsequently, may impact on resistance to enteric pathogens. The aim was to demonstrate that early life exposure to Lactococcus lactis, could affect either susceptibility or resistance of broilers to necrotic enteritis (NE). L. lactis NZ9000 (rL. lactis) pre-treatment at 1, 7, 14 and 21 days of age (DOA) led to a significant decrease in NE lesion scores in Clostridium perfringens infected chickens. C. perfringens Infection was associated with spatial and temporal decreases in mononuclear phagocytes and CD4+ αβ T cells. However, rL. Lactis pre-treatment and subsequent C. perfringens infection led to a significant increase in mononuclear phagocytes, CD8α + γδ T, αβ T cells (CD4+ and CD8α+) and B cells (IgM+, IgA+ and IgY+), as well as IL-12p40, IFN-γ and CD40. Differential expression of interleukin (IL)-6, IL-8, IL-10, IL-13, IL-18, IL-22, and transforming growth factor (TGF)-β were observed in L. lactis treated chickens when compared to C. perfringens infected chickens. Microbiota analysis in C. perfringens infected chickens demonstrated an increase in abundance of Bacillota, Bacteroidota, Pseudomonadota and Actinomycetota. These findings suggests that modulation of the chicken intestinal immune system by L. lactis confers partial protection 30 against NE

Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+ FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 +/- 0.016) compared to the control (0.14 +/- 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 +/- 0.30 versus 6.3 +/- 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 +/- 2.22% versus 29.8 +/- 5.46%)

Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids

NetB (necrotic enteritis toxin B) is a recently identified β-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease

Determining drug resistance patterns of Clostridium perfringens isolates from acute necrotic enteritis (NE) outbreaks

In this study, after separating 40 Clostridium perfringens isolates from affected broiler flocks, the antibacterial susceptibility test revealed 39 drug resistance patterns in which 95% of the isolates were distributed into 38 different patterns (one isolate in each pattern) and only 5% of the isolates were placed in a single pattern (two isolates in a pattern). All 40 Clostridium perfringens isolates demonstrated resistance to chloramphenicol, vancomycin and sulfamethoxazole+trimethoprime, ranging from 0 to 17.5% while tetracycline, lincomycin and neomycin sulfate had a high level of resistance from 80 to 87.5%. Also an isolate demonstrated multiple resistance to more than 14 antibacterial compounds