Clostridium perfringens antigens recognized by broiler chickens immune to necrotic enteritis

Little is known about immunity to necrotic enteritis (NE) in chickens. A recent study of broiler chickens showed that protection against NE was associated with infection-immunization with virulent but not with avirulent Clostridium perfringens.In the current study, six secreted antigenic proteins unique to virulent C. perfringens that reacted to serum antibodies from immune birds were identified by mass spectrophotometry; three of these proteins are part of the VirR-VirS regulon.     

Enzootic outbreak of necrotic gastritis associated with Clostridium perfringens in broiler chickens

Clinical morphological investigations were carried out in a flock of 22,000 Ross 308 broiler chickens at the age of 38 days that experienced a sudden increase in mortality rates. Morbidity and mortality rates were followed and gross anatomical examination of 150 bodies (7%) of all 1541 dead chickens was performed. In all necropsied birds, without exception, the typical macroscopic lesions were observed only in the gizzard. Focal or diffuse pseudomembranous deposits were found subcuticularly and on the gizzard mucous coat. Microscopically, hyalinization, desquamated epithelial cells and single foci of microorganisms were present among the formed pseudomembranes. Among the fibrin networks of coagulated exudate, a single bacilli were detected. Clostridium perfringens was isolated from all gastric samples investigated. Polymerase chain reactions were positive for α-toxin and negative for β-toxin and β₂-toxin.     

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.     

Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens

The Clostridium perfringens alpha-toxin has previously been implicated as the major virulence factor in necrotic enteritis in chickens, although definitive proof has not been reported. In this study an alpha-toxin mutant was constructed in a virulent chicken isolate and shown to retain full virulence in a chicken disease model. These results demonstrated that alpha-toxin is not an essential virulence factor in the pathogenesis of necrotic enteritis in chickens.     

Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials

Clostridium perfringens strains isolated in 2002 from the intestines of broiler chickens from 31 different farms located in Belgium were tested for susceptibility to 12 antibiotics used for therapy, growth promotion or prevention of coccidiosis. All strains were uniformly sensitive to the ionophore antibiotics monensin, lasalocid, salinomycin, maduramycin and narasin. All were sensitive to avilamycin, tylosin and amoxicillin, while flavomycin (bambermycin) showed low or no activity. Chlortetracycline and oxytetracycline were active at very low concentrations, but low-level acquired resistance was detected in 66% of the strains investigated. Fifty percent of these strains carried the tetP(B) resistance gene, while the tet(Q) gene was detected in only one strain. One strain with high-level resistance against tetracyclines carried the tet(M) gene. Sixty-three percent of the strains showed low-level resistance to lincomycin. The lnu(A) and lnu(B) genes were each only found in one strain. Compared with a similar investigation carried out in 1980, an increase was seen in resistance percentages with lincomycin (63% against 49%) and a slight decrease with tetracycline (66% against 74%).     

Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.     

Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens

Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3' end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain chi8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10(9) CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE.     

Highly conserved alpha-toxin sequences of avian isolates of Clostridium perfringens

Clostridium perfringens causes necrotic enteritis in chickens, and alpha-toxin has been suggested to be a key virulence determinant. Analysis of the alpha-toxin of 25 chicken-derived C. perfringens strains demonstrated high homology to mammal-derived strains rather than to the only avian-derived C. perfringens alpha-toxin sequence reported previously.     

Comparison of virulence plasmids among Clostridium perfringens type E isolates

Clostridium perfringens type E isolates produce iota-toxin, which is encoded by iap and ibp genes. Using Southern blot analyses, the current study identified iap/ibp plasmids of approximately 97 or approximately 135 kb among eight type E isolates. For most of these isolates, their iap/ibp plasmid also encoded urease and lambda-toxin. However, the beta2-toxin gene, if present, was on a different plasmid from the iap/ibp plasmid. For all isolates, the iap/ibp plasmid carried a tcp locus, strongly suggesting that these plasmids are conjugative. Overlapping PCR analyses demonstrated some similarity between the iap/ibp plasmids and enterotoxin-encoding plasmids of type A isolates. Additional PCR analyses demonstrated that the iap/ibp locus is located near dcm sequences, an apparent plasmid hot spot for toxin gene insertion, and that two IS1151-related sequences are present in the iap/ibp locus. To begin testing whether those IS1151-like sequences can mobilize iap/ibp genes, a PCR assay was performed that amplifies a product only from circular DNA forms that could represent transposition intermediates. This PCR assay detected circular forms containing iap/ibp genes and silent enterotoxin gene sequences, with or without an IS1151-like sequence. Collectively, these results suggest that a mobile genetic element carrying iap/ibp has inserted onto a tcp-carrying enterotoxin plasmid in a type A isolate, creating a progenitor iap/ibp plasmid. That plasmid then spread via conjugation to other isolates, converting them to type E. Further iap/ibp plasmid diversity occurred when either the iap/ibp genes later remobilized and inserted onto other conjugative plasmids or some iap/ibp plasmids acquired additional DNA sequences.     

Identification and antimicrobial resistance patterns of clinical isolates of Clostridium clostridioforme, Clostridium innocuum, and Clostridium ramosum compared with those of clinical isolates of Clostridium perfringens.

Clostridium ramosum, C. innocuum, and C. clostridioforme are frequently isolated from clinical specimens including blood. Because of Gram stain variability, a lack of spores, and atypical colonial morphology, identification of these species is often difficult. Three anaerobe identification kits were evaluated for their abilities to identify these species. For comparison, 11 strains of C. perfringens were evaluated in parallel. By using profile numbers and codebooks, the correct genus and species were identified, as follows: with the RapID ANA II kit, 100% (20 of 20) of C. ramosum isolates, 24% (5 of 21) of C. innocuum isolates, and 50% (10 of 20) of C. clostridioforme isolates; with the AnIDent kit, 60% (12 of 20) of C. ramosum isolates, 28% (6 of 21) of C. innocuum isolates, and 90% (18 of 20) of C. clostridioforme isolates; with the ATB32A kit, 70% (14 of 20) of C. ramosum isolates, 0% (0 of 21) of C. innocuum isolates, and 40% (8 of 20) of C. clostridioforme isolates. Profile numbers that overlapped several species were obtained as follows: with the RapID ANA II kit, 0% of C. ramosum isolates, 76% of C. innocuum isolates, and 40% of C. clostridioforme isolates; with the AnIDent kit 40% of C. ramosum isolates, 62% of C. innocuum isolates, and 5% of C. clostridioforme isolates; with the ATB32A kit, 15% of C. ramosum isolates, 52% of C. innocuum isolates, and 25% of C. clostridioforme isolates. One strain of C. innocuum was misidentified by the AnIDent kit, and the remainder yielded profile numbers that were not listed in the codebooks. The MICs of 11 antimicrobial agents including penicillin G, metronidazole, clindamycin, cefoxitin, cefotetan, imipenem, meropenem, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, and vancomycin were determined by the agar dilution method. All C. perfringens strains were susceptible to all antimicrobial agents tested. Various levels of resistance to cefoxitin, cefotetan, and penicillin G were noted with C. ramosum, C. clostridioforme, and C. innocuum. In addition, resistance to clindamycin was noted with C. ramosum (5%) and C. innocuum (10%). Most strains of C. innocuum were only moderately susceptible to vancomycin (MIC at which 90% of strains are inhibited, 4 micrograms/ml).     

Cholangiohepatitis in chickens induced by bile duct ligations and inoculation of Clostridium perfringens

The association of bile duct ligation with the development of cholangiohepatitis due to Clostridium perfringens was examined. Chickens with only the cystoenteric duct ligated had no significant liver lesions even if C. perfringens was inoculated into the cystoenteric duct. Chickens with both the cystoenteric and hepatoenteric ducts ligated had enlargement of the liver with an apparent acinar pattern. Histopathologically, proliferation of bile ductules was seen. Bile ductules proliferated extensively, and fibrosis formed bridges between triads. The gall bladder and extrahepatic bile ducts were thickened and distended with yellow inspissated material in chickens that had both ducts ligated and had C. perfringens inoculated into the cystoenteric duct. Multiple granulomas and intrahepatic cholangitis were frequently observed. Infiltration of lymphocytes and plasma cells with germinal centres, and heterophilic extramedullary haematopoiesis were seen in the portal areas. Thus, cholangiohepatitis similar to field cases can only be induced by both ligation of bile ducts and inoculation of C. perfringens.     

Exogenous lysozyme influences Clostridium perfringens colonization and intestinal barrier function in broiler chickens

Necrotic enteritis is a worldwide poultry disease caused by the overgrowth of Clostridium perfringens in the small intestine. An experiment with a 2×2 factorial design (supplementation with or without 40 mg lysozyme/kg diet for chickens challenged with or without C. perfringens) was conducted to investigate the inhibitory efficacy of exogenous lysozyme against intestinal colonization by C. perfringens in chickens subject to oral inoculation of C. perfringens type A on days 17 to 20. The C. perfringens challenge resulted in significant increase of C. perfringens, Escherichia coli and Lactobacillus populations in the ileum, bacteria translocation to the spleen, the intestinal lesion scores , There was significantly lower intestinal lysozyme activity in the duodenum and jejunum and weight gain during days 14 to 28 of the experiment. The addition of exogenous lysozyme significantly reduced the concentration of C. perfringens in the ileum and the intestinal lesion scores, inhibited the overgrowth of E. coli and Lactobacillus in the ileum and intestinal bacteria translocation to the spleen, and improved intestinal lysozyme activity in the duodenum and the feed conversion ratio of chickens. These findings suggest that exogenous lysozyme could decrease C. perfringens colonization and improve intestinal barrier function and growth performance of chickens.     

Fatal necrotic enteritis associated with Clostridium perfringens in wild crows (Corvus macrorhynchos)

Sporadic outbreaks of fatal enteritis occurred among free-living wild crows (‘large billed’ or ‘wok’ crow; Corvus macrorhynchos) in an open-air park in Japan in 2002. Eight crows were found dead during February, followed by two more in September, and five of the eight were examined histopathologically. At necropsy, all cases showed a markedly dilated small intestine, especially the jejunum and ileum, with large amounts of gas, and dark red to greenish–brown soft content. The necrotic intestinal wall was markedly thickened with multifocal haemorrhages. All cases had multifocal white foci in the liver, and four cases showed marked splenomegaly. Histologically, there was severe necrotic enteritis characterized by extensive mucosal necrosis and multifocal haemorrhages, as well as inflammatory cell infiltrations. A prominent pseudo-membrane formation was noted in the affected intestine. Severe adhesive peritonitis was also observed in three cases. Gram-positive bacilli were present in large numbers in the lumen, and in and around necrotic lesions in the affected intestine. The bacilli were positive for Clostridium perfringens enterotoxin type A by immunohistochemistry, and were also positive for C. perfringens type A using the immunofluorescence method. C. perfringens was isolated by anaerobic culture from the intestinal contents. The present enteritis was thought to be induced by proliferated C. perfringens in the intestine, and to be the cause of death.     

Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates

Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.     

Molecular subtyping of poultry-associated type A Clostridium perfringens isolates by repetitive-element PCR

Clostridium perfringens strains (type A) isolated from an integrated poultry operation were subtyped using repetitive-element PCR with Dt primers. Isolates were obtained from fecal, egg shell, fluff, and carcass rinse samples as part of a previously reported temporally linked epidemiological survey. A total of 48 isolates of C. perfringens were obtained from different stages of the broiler chicken production chain from two separate breeder farms that supplied a single hatchery that in turn provided chicks to a single grow-out farm whose flocks were processed at a single plant. All 48 isolates were typeable (100% typeability) by repetitive-element PCR with Dt primers. This subtyping method was highly reproducible and discriminatory. By repetitive-element PCR with Dt primers, isolates were classified into four major branches with 12 subgroups or clades. The Simpson's index of discrimination was calculated to be 0.96 for groupings of >95% correlation. Toxin gene profiles of the isolates indicated that all of the isolates were C. perfringens alpha-toxin gene positive and 46 of 48 isolates were beta2-toxin gene positive. All strains were negative for beta- and epsilon-toxin genes. Repetitive sequence-based PCR was found to be a technically practical and reproducible means of subtyping C. perfringens libraries from specific epidemiological or production environment settings.     

Association between gizzard lesions and increased caecal Clostridium perfringens counts in broiler chickens.

The aim of this study was to investigate the relationship between mucosal gizzard lesions and caecal Clostridium perfringens counts. Gross pathological changes in the gizzard and small intestine, and caecal C. perfringens counts from 1217 meat-type chickens were recorded during the course of six experiments and were statistically analysed. C. perfringens counts increased significantly (P < 0.001) with the severity of mucosal gizzard lesions. Mucosal gizzard lesions were more prevalent than necrotic enteritis. Correcting for the pen and necrotic enteritis within experiment, mucosal gizzard lesions explained 31.8% of the variation in C. perfringens counts. Mucosal gizzard lesions and age together explained 59.1% of the variation in C. perfringens counts. The mean ages of birds with moderate and severe mucosal gizzard lesions were 1.7 and 0.8 days lower than the mean age of birds with necrotic enteritis, respectively. The association between mucosal gizzard lesions and high C. perfringens counts might be of importance when attempting to improve production efficiency, health and the welfare of the chickens.     

Enterotoxin plasmid from Clostridium perfringens is conjugative

Clostridium perfringens enterotoxin is the major virulence factor involved in the pathogenesis of C. perfringens type A food poisoning and several non-food-borne human gastrointestinal illnesses. The enterotoxin gene, cpe, is located on the chromosome of food-poisoning isolates but is found on a large plasmid in non-food-borne gastrointestinal disease isolates and in veterinary isolates. To evaluate whether the cpe plasmid encodes its own conjugative transfer, a C. perfringens strain carrying pMRS4969, a plasmid in which a 0.4-kb segment internal to the cpe gene had been replaced by the chloramphenicol resistance gene catP, was used as a donor in matings with several cpe-negative C. perfringens isolates. Chloramphenicol resistance was transferred at frequencies ranging from 2.0 x 10(-2) to 4.6 x 10(-4) transconjugants per donor cell. The transconjugants were characterized by PCR, pulsed-field gel electrophoresis, and Southern hybridization analyses. The results demonstrated that the entire pMRS4969 plasmid had been transferred to the recipient strain. Plasmid transfer required cell-to-cell contact and was DNase resistant, indicating that transfer occurred by a conjugation mechanism. In addition, several fragments of the prototype C. perfringens tetracycline resistance plasmid, pCW3, hybridized with pMRS4969, suggesting that pCW3 shares some similarity to pMRS4969. The clinical significance of these findings is that if conjugative transfer of the cpe plasmid occurred in vivo, it would have the potential to convert cpe-negative C. perfringens strains in normal intestinal flora into strains capable of causing gastrointestinal disease.     

Multilocus sequence typing subtypes of poultry Clostridium perfringens isolates demonstrate disease niche partitioning

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.     

Clostridium perfringens and other anaerobes isolated from bile.

Clostridium perfringens was isolated from bile in 13 cases of 150 patients examined. The serotypes of C perfringens strains isolated from bile and faeces were investigated using antisera to Hobbs' type 1-17. Two or more serological types were often found in a single specimen, but in the same patient the serotypes of C perfringens strains isolated from the bile were identical with those from the faeces. Beta-glucuronidase production in these C perfringens serotypes was tested with the API-Strep system. Strains agglutinated with Hobbs' antisera produced beta-glucuronidase, but non-agglutinated strains did not.