Lactobacillus casei displaying Clostridium perfringens NetB antigen protects chickens against necrotic enteritis.

Necrotic enteritis is a serious economical disease of poultry caused by Clostridium perfringens. NetB toxin of Clostridium perfringens is considered the causative agent of necrotic enteritis. Following the withdrawal of in-feed antibiotic growth promoters, there has been an urgent need to develop alternative approaches such as vaccination. Currently, there are no commercially available vaccines to control necrotic enteritis especially in broiler chickens as the target population. In the present study, we constructed a recombinant Lactobacillus casei strain expressing NetB protein of C. perfringens on the cell surface and used this probiotic-based vaccine strain to immunize broiler chickens orally against experimental induction of necrotic enteritis. The birds immunized with the oral vaccine strain were significantly protected against necrotic enteritis challenge and developed strong serum anti-NetB antibody responses to NetB protein. Furthermore, the immunized birds showed higher body weight gains during the challenge experiment compared with control birds. This study showed, for the first time, that a probiotic-based vector vaccine could be a promising vaccine candidate to provide protection against necrotic enteritis in broiler chickens. KEYPOINTS: • The probiotic L. casei carrying pT1NX-netB plasmid displayed NetB antigen on the cell surface. • The LC-NetB vaccine strain induced high anti-toxin antibody response in broiler chickens. • The LC-NetB vector vaccine provided significant protection against experimental NE challenge.

Clostridium perfringens-Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus).

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets (P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

NetB and necrotic enteritis: the hole movable story.

Clostridium perfringens is the primary causative agent of avian necrotic enteritis. Our understanding of the pathogenesis of this economically important disease has been enhanced by the discovery of C. perfringens NetB toxin, which belongs to the α-haemolysin family of ß-pore-forming toxins. In a chicken disease model, the analysis of an isogenic set of strains comprising the wild type, a netB mutant, and its complemented derivative, fulfilled molecular Koch's postulates and revealed that NetB was essential for disease. These results were consistent with epidemiological surveys, which generally found that there was a higher prevalence of netB carriage in C. perfringens isolates from diseased poultry compared to healthy birds. The netB gene has been shown to be located on large conjugative plasmids that are closely related to other toxin plasmids from C. perfringens, which has potential implications for the epidemiology of necrotic enteritis infections. The crystal structures of both monomeric NetB and the heptameric NetB pore have been determined, the latter revealed a central pore diameter of approximately 26 Å. Finally, it has been shown that vaccine preparations that include NetB can protect chickens against disease and a series of single amino acid substitution derivatives of NetB that have potential value for vaccine formulations have been isolated and analysed. It is likely that NetB will be an important antigen to include in an effective, commercially viable, necrotic enteritis vaccine.

Outbreaks of Acute Necrotic Enteritis and Haemolytic Crisis and Renal Disease Associated with Clostridium perfringens in Captive Toucans (Ramphastos spp).

Four toco toucans (Ramphastos toco), one channel-billed toucan (Ramphastos vitellinus) and one white-throated toucan (Ramphastos tucanus) died in two disease outbreaks in the same aviary in 2011 and 2016. Post-mortem examination revealed diffuse necrotic enteritis (NE) as the cause of death of five of these six birds. Clostridium perfringens was identified by culture and real-time multiplex PCR for C. perfringens α-, ß-, ε- and ι-toxin genes in ligated intestine of one toucan from each outbreak. At another aviary, two keel-billed toucans (Ramphastos sulfuratus) died peracutely from severe haemolytic crisis with haemoglobinaemic nephrosis and cholestasis and acute tubulointerstitial nephritis. Mild NE was present in these birds and C. perfringens was demonstrated in liver by bacterial culture and real-time multiplex PCR for C. perfringens α-, ß-, ε- and ι-toxin genes. To the authors' knowledge, this is the first description of outbreaks of NE associated with C. perfringens in captive toucans. Although haemolytic crisis has been reported in humans with C. perfringens type A septicaemia and hepatic abscesses, this presentation appears not to have been described in C. perfringens infections in toucans or other avian species. The factors causing C. perfringens proliferation and disease in the toucans were not identified. PCR for C. perfringens NetB toxin and enterotoxin genes performed retrospectively on one of the C. perfringens isolates from the second outbreak and on paraffin-embedded tissues from one dead toucan from the first outbreak was negative. With the current C. perfringens toxin typing scheme, C. perfringens type A was identified in the first two outbreaks.

Role of Clostridium perfringens Necrotic Enteritis B-like Toxin in Disease Pathogenesis.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G that impacts the global poultry industry by compromising the performance, health, and welfare of chickens. Coccidiosis is a major contributing factor to NE. Although NE pathogenesis was believed to be facilitated by α-toxin, a chromosome-encoded phospholipase C enzyme, recent studies have indicated that NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, is the primary virulence factor. Since the discovery of NetB toxin, the occurrence of NetB+ C. perfringens strains has been increasingly reported in NE-afflicted poultry flocks globally. It is generally accepted that NetB toxin is the primary virulent factor in NE pathogenesis although scientific evidence is emerging that suggests other toxins contribute to NE. Because of the complex nature of the host-pathogen interaction in NE pathogenesis, the interaction of NetB with other potential virulent factors of C. perfringens needs better characterization. This short review will summarize the primary virulence factors involved in NE pathogenesis with an emphasis on NetB toxin, and a new detection method for large-scale field screening of NetB toxin in biological samples from NE-afflicted commercial broiler flocks.

Metagenomic Analysis of 16S Clostridium perfringens Amplicons Corroborates C. perfringens Counts on Select Agar and C. perfringens PCR Analyses of Bacteria in Broiler Farm Litter.

The purpose of this study was twofold-first, to determine whether analysis of bacterial 16S ribosomal RNA (rRNA) in poultry litter corroborated standard Clostridium perfringens counts and PCR assay, and second, to find whether a correlation between 16S rRNA analysis and netB or Tpel toxin PCR intensity with chick mortality existed. At three time points of growout (0, 2, and 4 wk) litter samples were collected from 23 broiler houses representing eight farms during a coccidiosis vaccine control program. DNA extracted from these samples was used for microbiota determination by sequencing the hypervariable V3-V4 region of bacterial 16s rRNA. Obtained sequences were analyzed by QIIME 2 and the Greengenes database for taxonomic composition and relative abundance of C. perfringens in the litter bacterial population. Clostridium perfringens counts on select agar and semiquantitative PCR for C. perfringens were compared with 16S analysis for equivalence testing. Relative abundance of C. perfringens estimated by 16S analysis and semiquantitative PCR for netB and Tpel toxin DNA were analyzed by Pearson linear correlation and statistical equivalence analyses with cumulative chick mortality at 4 and 9 wk growout. When data from all time points were combined, abundance estimates by C. perfringens 16S were statistically equivalent (α = 0.10) to both C. perfringens PCR and C. perfringens counts. Yet, no correlations were observed between any estimate of C. perfringens abundance and cumulative percent chick mortality at 4 or 9 wk growout. However, correlation analyses revealed a significant linear relationship between netB signal at 0 wk (r = 0.55) and 4 wk (r = 0.46) and cumulative mortality at 9 wk growout (P < 0.05). Similarly, abundance of Tpel at 0 and 2 wk showed a linear relationship with cumulative percent mortality at both 4 and 9 wk growout (0.44 ≤ r ≤ 0.54, P < 0.05). No correlations were observed between any other genera or species determined by 16S and cumulative percent chick mortality.

Research Note: First report on the detection of necrotic enteritis (NE) B-like toxin in biological samples from NE-afflicted chickens using capture enzyme-linked immunosorbent assay.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type G. One of the pore-forming toxins, NE B-like (NetB) toxin, secreted by pathogenic C. perfringens type G, has been proposed to be the main virulent factor in NE pathogenesis. The present study aimed to detect the presence of NetB toxin in biological samples of NE-afflicted chickens using NetB-specific monoclonal-based enzyme-linked immunosorbent assay (ELISA). Biological samples, including serum, digesta, and fecal droppings, were obtained from three previous NE studies (designated as Trials 1 to 3). In Trials 1 and 2, broiler chicks were infected with Eimeria maxima strain 41A on day 1 and followed by the netB-positive C. perfringens on day 18. Serum samples were obtained at 20 d post-hatch (i.e., 2 d post C. perfringens infection). In addition, various samples, including serum, gut digesta, and fecal droppings, that had been collected 0, 6, 24, and 30 h post C. perfringens infection were obtained. In Trial 3, broiler chicks were indirectly infected with litter-contaminated E. maxima on d 14 and followed by netB-positive C. perfringens via drinking water on days 18, 19, and 20. Serum samples and fecal droppings were obtained 21 d post-hatch (i.e., 1 d post last C. perfringens infection). The results showed that NetB toxin was not detected in serum samples in Trials 1 and 3. No NetB toxin was detected in all samples obtained before C. perfringens infection in Trial 2. Low but detectable amounts of NetB toxin were found in the serum samples obtained 6 h post C. perfringens infection in Trial 2. While NetB toxin in digesta and fecal droppings was detected 6 h post C. perfringens infection, its level plateaued 24 and 30 h post C. perfringens infection. In Trial 3, NetB toxin was detected in fecal droppings from the NE group, and its concentration ranged from 2.9 to 3.1 ng/g of wet feces. In Trial 2, NE-specific lesions were not seen 0 and 6 h post C. perfringens infection but exhibited lesions were moderate to severe 24 h post infection, leading to a moderate association (r = +0.527) between NE lesions and NetB toxin in the gut digesta. This is the first study to use NetB-specific monoclonal-based capture ELISA to determine and report the presence of native NetB toxin in biological samples from NE-induced chickens.

Correlation Between Clostridium perfringens Alpha- and NetB-Toxin and Chick Mortality in Commercial Broiler Farms During Different Anticoccidial Control Programs.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).

Detection of Necrotic Enteritis B-like Toxin Secreted by Clostridium perfringens Using Capture Enzyme-Linked Immunosorbent Assay.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G, which affects global poultry industry by compromising the performance, health, and welfare of chickens. The causative main virulent factor responsible for NE pathogenesis has been shifted from a phospholipase C portion of an α-toxin, to an NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, in NE development. Therefore, the ability to detect NetB toxin will enable early diagnosis of field NE. Because the NetB protein can only be detected by western blot analysis with polyclonal anti-NetB antiserum, we developed a NetB-specific monoclonal antibody (mAb)-based capture enzyme-linked immunosorbent assay (ELISA). Twenty mAbs reacting with Escherichia coli-expressed NetB protein were selected, isotyped, and conjugated with horseradish peroxidase for antibody pair tests. Multiple mAb pairs were found to detect E. coli NetB protein and native NetB protein secreted by netB-positive C. perfringens isolates. The developed capture (sandwich) ELISA could be useful to identify in vitro production of native NetB protein secreted from netB-positive field C. perfringens isolates and to conduct a large field test of commercial chickens undergoing NE infection. Here, we first report that native NetB toxin can be detected in C. perfringens NetB-specific mAb-based capture ELISA.

Comparison of multiple methods for induction of necrotic enteritis in broilers: II. Impact on the growth curve.

Necrotic enteritis (NE) is a disease that has gained relevance in the poultry industry with both immediate and sustained effects on BW of broilers. The objective of the 3 experiments was to evaluate the impact of NE, induced by methods that reflect common broiler production systems, on the growth curve throughout the growth period. In addition, the impact of Eimeria maxima (EM) on NE, as well as the long-term impact of Clostridium perfringens (CP) on BW, were analyzed. In experiment 1, a dual infection model of EM and CP was compared to a non-challenged control, while experiment 2 evaluated 2 different strains of EM dual infection, as well as 6 CP-only groups. Similarly, experiment 3 tested dual infection and both high and low dose CP-only groups. Both NetB and non-NetB strains of CP were used to evaluate whether NetB toxin may potentially play a role in NE induction. In all 3 experiments, BW was measured immediately before infection on day 16, then weekly through the end of the test period. In all 3 experiments, a decrease (p < 0.05) in BW was observed immediately following the acute NE disease period of day 21 to day 23, with a negative impact also observed of BW gain during NE disease period (p < 0.05). A long-term effect on BW was most clearly detected in the EM + CP dual infection models, as well as when high levels of CP-only were administered. In these cases, BW was impacted long-term, with a requisite week or more to return to a BW similar to the non-challenged control. The separation in BW, though not significant, was nearly parallel with the non-challenged control throughout the growth period, indicating a shift in the growth curve. In addition to showing the long-term impact of various forms of NE on broiler growth, these shifts in the growth curve can be used to measure the effects of treatments on prevention and recovery of broilers impacted by NE.