Perfringens Isolates Research Articles

Susceptibility of Clostridium perfringens to antimicrobials produced by lactic acid bacteria: Reuterin and nisin

The effectiveness as antimicrobials of lactic acid bacteria produced compounds reuterin and nisin was assessed against vegetative cells and spores of Clostridium perfringens isolates (from ovine milk obtained in farms with diarrheic lambs) and C. perfringens CECT 486 (type A toxin producer). We also tested the inhibitory effect of lysozyme and sodium nitrite on Clostridium. Minimal inhibitory concentrations (MIC) of antimicrobials were determined in modified RCM (mRCM) and milk by using a broth microdilution method, after 7 d at 37 °C under anaerobic conditions. The sensitivity of C. perfringens to the tested antimicrobials was strain and culture medium-dependent. In general, vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 2.03–16.25 mM) inhibited the growth of vegetative cells and the outgrowth of spores of all tested C. perfringens, both in mRCM and milk, with higher resistance in milk. Nisin (MIC values 0.78–12.5 μg/ml) was also effective against vegetative cells and spores of tested C. perfringens in both culture media. However, lysozyme (up to 400 μg/ml) did not control the growth of any of the tested Clostridium. Sodium nitrite only inhibited the outgrowth of spores of two C. perfringens isolates at the maximum concentration assayed (300 μg/ml) exclusively in mRCM medium. These results suggest that reuterin and nisin have the potential to control the growth of C. perfringens, and might help to ensure safety at different stages of the food chain. Future studies in food/feed products would be necessary to further corroborate this hypothesis.

BACTERIOLOGICAL STUDIES OF CLOSTRIDIUM PERFRINGENS IN FISH

The current research was carried out on one hundred and thirteen random fish samples (56 processed and 57 unprocessed) collected from different localities in El-Sharkia Governorate, Egypt to obtain a complete picture of C. perfringens. Bacteriological and biochemical examination was done on the isolates. Nagler's test was applied on the recovered. C. perfringens isolates. The total incidence of positive samples from 56 processed fish samples were 32 isolates (57.1%) and from 57 unprocessed fish samples were 34 isolates (59.6%). The. Typing of C. perfringens isolates revealed that the incidence of toxigenic and non-toxigenic isolates were 84.8 and 15.2% respectively. Typing of toxigenic strains of C. perfringens revealed that C. perfringens type A was the most predominant one comparing with type D. Immune diffusion test showed that 17 and 11 toxins of types A and D gave identified reaction with incidences of 47.2 and 55% respectively.

The prevalence and molecular typing of Clostridium perfringens in ground beef and sheep meats

This study was aimed at the investigation of the prevalence of Clostridium perfringens in ground beef and sheep meats and to detect the cpa, cpb, etx, iA, cpe and cpb2 toxin genes by multiplex PCR. Obtained from retail markets and butchers, 100 ground meat samples, 50 of which were beef and 50 of which were sheep meat samples, were examined. Out of the 50 ground beef samples 48 (96 %) and out of the 50 ground sheep meat samples 44 (88 %) were contaminated with C. perfringens. According to microscopic examination and the results of biochemical tests, 262 positive isolates obtained from these samples were identified as C. perfringens. Multiplex PCR results demonstrated that, out of the 262 isolates, 203 (77.4 %) possessed only the cpa gene (type A), while 20 (7.6 %) carried the cpa and cpb2 toxin genes (type A-cpb2), and 6 (2.2 %) the cpa and cpe toxin genes (type A-cpe). Furthermore, it was determined that, out of the 262 C. perfringens isolates, 4 (1.5 %), 10 (3.8 %) and 19 (7.2 %) were of the types B, C and D, respectively.

Distribution of <i>Clostridium perfringens</i> Isolates from Piglets in South Korea

ABSTRACTClostridium perfringens causes various digestive system disease symptoms in pigs. In the present study, 11 C. perfringens isolates were obtained from diarrheic piglets and 18 from healthy piglets. All of the C. perfringens isolates were shown to be type A using a multiplex PCR assay. The β2 toxin gene was detected in 27/29 C. perfringens isolates, i.e., 81% (9/11) of diarrheic piglets and 100% (18/18) of healthy piglets, and all of the genes had the same sequence. In conclusion, the β2 toxin gene of C. perfringens was distributed widely in Korean piglets regardless of the incidence of diarrhea, and there was no clear relationship with enteric disease. A pulsed-field gel electrophoresis analysis of DNA digested using SmaI demonstrated the non-clonal spread of C. perfringens isolates from piglets.

Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl–l-alanine amidase as a potential antimicrobial to control the bacterium

Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal, and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl-L-alanine amidase or MurNAc-LAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and L-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction, and the gene product (PlyCpAmi) was expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact, and injured compared to the untreated cells. Among a variety of C. perfringens strains, there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results further demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens.

Characterization of Clostridium perfringens isolates from healthy turkeys and from turkeys with necrotic enteritis

Clostridium perfringens is an important bacterial pathogen, especially in poultry, where it can lead to both subclinical and clinical disease. The aim of this study was to present data on pathological findings at outbreaks of necrotic enteritis (NE) in turkey production in Finland during the period from 1998 to 2012. Furthermore, C. perfringens isolates from healthy and diseased turkeys were characterized and their genetic diversity was investigated using pulsed-field gel electrophoresis (PFGE). Isolates (n = 212) from birds with necrotic gut lesions and from healthy flocks of 30 commercial turkey farms were characterized for the presence of cpa, cpb, iA, etx, cpb2, and cpe and netB genes. A total of 93 C. perfringens isolates, including 55 from birds with necrotic gut lesions and 38 from healthy birds from 13 different farms, were analyzed with PFGE. All contract turkey farmers (n = 48) of a turkey company that produces 99% of domestic turkey meat in Finland were interviewed about background information, management at the farm, and stress factors related to NE outbreaks. Pulsed-field gel electrophoresis analysis with SmaI restriction enzyme resulted in 30 PFGE patterns among the 92 C. perfringens isolates of high diversity. Out of all isolates, 212 (100%) were α-toxin-positive and one isolate (0.5%) was both α- and β2 toxin-positive. Fourteen isolates (6.6%) were necrotic enteritis toxin B (NetB) positive; all were recovered from turkeys with NE. In none of the isolates obtained from healthy turkeys was the netB toxin identified. In conclusion, a high diversity of C. perfringens isolates from turkeys with different health status was shown. All isolates produced α toxin, whereas only low percentages of isolates carried the netB toxin gene. The role of the netB toxin in NE in turkeys needs to be further investigated.

Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: Responses to antibiotic challenge

Antibiotics are important to equine medicine, but antibiotic-associated diarrhea (AAD) can lead to poor performance and even mortality. AAD is attributed to disruption of the hindgut microbiota, which permits proliferation of pathogenic microbes. The goal of this study was to evaluate the effects of common antibiotics on cellulolytic bacteria, lactobacilli, and AAD-associated pathogens in the feces of healthy horses. Fifteen horses were assigned to three treatment groups (blocked by age and sex): control (no antibiotics), trimethoprim-sulfadiazine (PO), or ceftiofur (IM). Fecal samples (n=8 per horse) were taken during dietary adaptation (3 weeks), antibiotic challenge (1 week), and withdrawal (1 week). Bacteria were enumerated by serial dilution and viable count. Cellulolytic bacteria decreased by >99% during administration of either antibiotic (P<0.0001) and were still less than controls at the end of the withdrawal period (P<0.0001). Fecal samples from horses challenged with ceftiofur had 75% fewer lactobacilli than those from control horses at the end of the antibiotic challenge period (P<0.05). Antibiotic challenged horses also shed more salmonella than control horses (P<0.05). Antibiotics had no effect on the number of Clostridium perfringens isolates. There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P<0.0001) to approximately 10(4)cfu/g when horses were challenged with antibiotics, and were still detectable 1 week after withdrawal. These results indicate that antibiotics can disrupt the normal gastrointestinal microbiota and allow proliferation of Salmonella spp. and C. difficile.

Detection of A/B toxin and isolation of Clostridium difficile and Clostridium perfringens from foals

Toxin detection and screening could contribute to knowledge of the transmission patterns, risk factors and epidemiology of Clostridium difficile and Clostridium perfringens. To isolate C. difficile and C. perfringens and to detect A/B toxins in faecal samples from diarrhoeic and nondiarrhoeic foals. Cross-sectional observational study. A total of 153 samples from foals were collected: 139 samples from farms and 14 samples from diarrhoeic foals admitted to a veterinary hospital. The A/B toxins were detected by cytotoxicity assay. All suspected colonies of C. perfringens were subjected to polymerase chain reaction for detection of the major toxin genes (α, β, ε and ι) and for detection of β2-, NetB- and enterotoxin-encoding genes. Furthermore, C. difficile and C. perfringens isolates were evaluated for in vitro antimicrobial susceptibility. Seven of 153 (4.6%) samples, all from diarrhoeic foals, were positive for C. difficile A/B toxin. Of these, 5 of 14 (35.7%) were from hospitalised foals, and only 2 of 63 (3.2%) diarrhoeic foal samples were from farms (P = 0.002). Clostridium perfringens was isolated from 31 (20.3%) foals, of which 21 of 76 (27.6%) were diarrhoeic and 10 of 76 (13.2%) were nondiarrhoeic, demonstrating a difference between these 2 groups (P = 0.045). Only 4 strains were positive for the β2-encoding gene (cpb2). All C. difficile and C. perfringens isolates were susceptible to metronidazole and vancomycin. The present report highlights the need for laboratory diagnostics to differentiate C. difficile-associated infection in foals from other causes of diarrhoea to facilitate adequate antimicrobial therapy. More studies are needed to clarify the role of C. perfringens as a primary agent of diarrhoea in foals.

Molecular Studies on Clostridium Perfringens Resistant to Tetracycline Group

Clostridia are spore-forming bacteria causing health hazards for human beings and animals ranging from wound infection to necrotic enteritis. In the present work, 60 dropping samples were collected from diarrhoeic broilers of 7 different farms in Sharkia and Cairo Governorates for isolation and identification of C.perfringens by conventional methods. The results showed that incidence of C. perfringens was 41% (25/60), 23 out of them were proved to be C. perfringens type A, while only two isolates were type C. Antibiotic susceptibility test revealed that 88% of C. perfringens isolates were sensitive to amoxicillin, 84% were sensitive to cefotaxime, cefuroxime, cefotraxion, piperacillin and bacitracin, 80% were sensitive to erythromycin, 76% were sensitive to fuscidic acid, 64% were sensitive to tetracycline, doxycycline and clindamycin, while 24% of the isolates were sensitive to lincomycin. Furthermore, MICs for tetracycline resistant isolates were ranged from 8-32 µg/ml. PCR was applied to detect α-toxin gene of C. perfringens and tetracycline resistant genes (tetB, tetM and tetK). All isolates gave positive amplification for alpha toxin gene, while tetB gene was detected in two out of 5 isolates , but tetK and tetM genes weren’t detected in any isolates.

Molecular Typing of Clostridium perfringens Isolates from Soil, Healthy, and Diseased Sheep in Egypt by Multiplex PCR

In this study multiplex PCR was used for typing of Clostridium perfringens isolates from soil, clinically healthy and diseased sheep. Clostridium perfringens was isolated from 41 out of 100 soil samples, 12 out of 100 clinically healthy sheep and 118 out of 200 sheep with enterotoxaemia signs. Genotyping of 41 isolates from soil indicated that 29 (70.73%) were type A, 3 (7.31%) were type B and 9 (21.95%) were type D. Of 12 isolates from clinically healthy sheep 6 (50%) were type A and 6(50%) were type D. Of 118 isolates from diseased sheep 42 (35.59%) were type A, 22 (18.64%) were type B and 54 (45.76%) were type D. This result indicates that Clostridium perfringens type A, B and D are the main types causing enterotoxaemia in sheep in Egypt and Clostridium perfringens type A must be included in any vaccine programme to ensure optimum protection.

Lesion Development in a New Intestinal Loop Model Indicates the Involvement of a Shared Clostridium perfringens Virulence Factor in Haemorrhagic Enteritis in Calves

Clostridium perfringens-associated enterotoxaemia is a fatal disease in fast growing suckler and veal calves. An intestinal loop model was developed to study the pathogenesis of the disease. Loops were injected with stationary and logarithmic C. perfringens cultures with or without, a milk protein-based commercial milk replacer for calves. Isolates tested were from cases of bovine enterotoxaemia and from calves without signs of enterotoxaemia, in addition to netB-positive and -negative isolates from poultry, a type C isolate from piglets and the human isolate JIR325. All isolates induced necrohaemorrhagic lesions in combination with milk replacer, while all control loops (i.e. medium plus milk replacer) remained histologically normal. In addition, time-course experiments were conducted using an isolate from an outbreak of bovine enterotoxaemia. Histological examination showed that the earliest lesion was congestion of the capillaries, starting within 30min of inoculation. Haemorrhage and mucosal necrosis began at the tips of the villi 3-4h after bacterial inoculation. These lesions are similar to those observed in natural cases of bovine enterotoxaemia. Therefore, in this model, necrohaemorrhagic lesions can be induced by C. perfringens isolates from diverse origins, suggesting that the lesions may be caused by one or more virulence factors that are shared by these isolates.

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.

Antibiotic resistance of Clostridium perfringens isolates from broiler chickens in Egypt.

The use of antibiotic feed additives in broiler chickens results in a high prevalence of resistance among their enteric bacteria, with a consequent emergence of antibiotic resistance in zoonotic enteropathogens. Despite growing concerns about the emergence of antibiotic-resistant strains, which show varying prevalences in different geographic regions, little work has been done to investigate this issue in the Middle East. This study provides insight into one of the world's most common and financially crippling poultry diseases, necrotic enteritis caused by Clostridium perfringens. The study was designed to determine the prevalence of antibiotic resistance in C. perfringens isolates from clinical cases of necrotic enteritis in broiler chickens in Egypt. A total of 125 isolates were obtained from broiler flocks in 35 chicken coops on 17 farms and were tested using the disc diffusion method. All 125 isolates were resistant to gentamicin, streptomycin, oxolinic acid, lincomycin, erythromycin and spiramycin. The prevalence of resistance to other antibiotics was also high: rifampicin (34%), chloramphenicol (46%), spectinomycin (50%), tylosin-fosfomycin (52%), ciprofloxacin (58%), norfloxacin (67%), oxytetracycline (71%), flumequine (78%), enrofloxacin (82%), neomycin (93%), colistin (94%), pefloxacin (94%), doxycycline (98%) and trimethoprim-sulfamethoxazole (98%). It is recommended that C. perfringens infections in Egypt should be treated with antibiotics for which resistant isolates are rare at present; namely, amoxicillin, ampicillin, cephradine, fosfomycin and florfenicol.

Molecular Evaluation of the Enterotoxigenicity of &amp;lt;i&amp;gt;Clostridium difficile&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;Clostridium perfringens&amp;lt;/i&amp;gt; Swine Isolates by PCR Assays

Clostridium difficile and C. perfringens are enteric pathogens affecting a variety of mammals. This study evaluated the molecular enterotoxigenicity of Clostridium swine isolates by PCRs. One hundred and ten swine faeces were analyzed by culture assay. The faecal samples were from sixty-seven healthy animals and 43 with gastrointestinal tract disease. C. difficile strains were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes. All C. perfringens isolates were tested for the characterization of the toxinotype. Overall, sixty-five swine resulted positive: 38 for C. difficile and 17 for C. perfringens. One sample tested C. perfringens and C. difficile-positive, at the same time: on the whole, 39 C. difficile strains were isolated. Thirty-eight C. difficile isolates (all from healthy animals) resulted tcdA/tcdB and cdtA/cdtB-negative by PCRs and toxins A/B-negative by immunological tests. All C. perfringens strains were type A; eight were also cpb2-positive. In the sample (diarrhoeic), with double infection, C. difficile tested tcdA/tcdB and cdtA/cdtB-positive by PCRs and toxins A/B-positive by immunoassays; C. perfringens resulted cpb2-positive. The molecular genotypeing/toxinotyping should be applied to establish a final diagnosis and to assess properly the full implications and the epidemiological impact of these findings in particular in samples of healthy animals and aid in the development of effective intervention methods for controlling clostridial disease outbreaks.

Occurrence of Beta2 toxigenic Clostridium perfringens isolates with different toxin types in Iran

Clostridium perfringens is an important cause of enteric diseases in both human and animals. The bacteria produce several toxins which play key roles in the pathogenesis of diseases and are classified into five toxin types, on the basis of the differential production of Alpha, Beta, Epsilon and Iota toxins. In this study a single PCR assay was developed and used for detection of cpb2 gene to identify the Beta2 harboring isolates among different types of C. perfringens isolated from animal enteric diseases in Iran. It was found that cpb2 presents among C. perfringens isolates types A, B, C and D with 54.5% (6/11), 62% (13/21), 42.8% (6/14), 69.25% (9/13), respectively. Totally 34 of 59 (56.7%) isolates screened by PCR were cpb2positive. This is the first report of cpb2 positive isolates of C. perfringens causing enteric diseases of animals in Iran. Further studies to demonstrate the exact role of Beta2 toxin in pathogenesis of the bacterium is suggested.

Occurrence and Expression of Toxin Genes in Clostridium Perfringens Isolates from Pigs

Abstract Clostridium perfringens isolates were obtained from pigs of five porcine farms in Poland. The presence of C. perfringens was detected in 92% of faeces samples and its number ranged from 1.0 x 101 cfu/g to 1.2 x 107 cfu/g. All the isolates belonged to type A and 48.7% of them contained cpb2 gene. The qualitative assessment of toxin genes expression by type A subtype β2 isolates showed expression of cpa gene in 100% of strains and cpb2 gene in 71% of the analysed strains. The isolate from one-day-old piglets demonstrated also the expression of cpa and cpb2 genes.

Prevalence of Clostridium perfringens type A isolates in commercial broiler chickens and parent broiler breeder hens in Egypt

The aim of this study was to determine the presence of genes coding for alpha (cpalpha), beta (cpbeta), epsilon (epsilontx), iota (iotaA), enterotoxin (cpepsilon) and beta2 (cpbeta2) toxins in Clostridium perfringens isolates from broiler chickens and parent broiler breeder hens, using multiplex polymerase chain reaction (PCR) assay. The prevalence of C. perfringens in the intestinal segments and the effects of age were also investigated. The highest isolation rate was from the duodenum, at 41.7% in broiler chickens and 58.4% in parent broiler breeder hens; the lowest isolation rates came from the ileum, at 15.6% and 27.1%, respectively. Chickens harboured C. perfringens in the intestine and this increased with age. Clostridium perfringens was detected in 35.4% (17/48) of asymptomatic broiler chickens and 22.1% (17/77) of asymptomatic parent broiler breeder hens. The bacterium was detected in 100% of the broiler chickens and parent broiler breeder hens with clinical signs (31/31 and 60/60, respectively). The multiplex PCR assay indicated that in 99 (79.2%) of the 125 samples that tested positive for C. perfringens the strains isolated were type A and were shown to carry the cpalpha gene (99/99, or 100%). The gene encoding cpbeta2-toxin was present in 62.6% (62/99) of the isolates. A significant association was found between C. perfringens possessing the beta2-toxin gene and necrotic enteritis in broiler chickens and parent broiler breeder hens, suggesting that this gene might play a key role in the pathogenesis of the disease in Egypt. The authors suggest that the presence of the cpbeta2-toxin gene in C. perfringens isolates found in broiler chickens and parent broiler breeder hens during this study poses a risk of transmission to humans through the food chain.

Characterization of clostridial species and sulfite-reducing anaerobes isolated from foie gras with respect to microbial quality and safety

Anaerobic sulfite-reducing bacteria are generally considered as indicators of clostridial contamination in meat products. We reconsidered the relevance of this indicator for crude and pasteurized foie gras. A three-year study was conducted to recover anaerobic sulfite-reducing bacteria from foie gras manufactures. Under anaerobic culture at 37 °C, 98 isolates were obtained from crude or pasteurized foie gras, surfaces and pepper used in the recipe and further identified. Heat-treated products lead to the exclusive isolation of Clostridium strains, but other samples demonstrated a high diversity of non-spore forming bacterial species. The diversity in the Clostridium group was also high, with 14 different species represented from the 49 clostridial isolates, including Clostridium perfringens and psychrotolerant species. All the 12 C. perfringens isolates belonged to type A, but none carried the enterotoxin gene. In addition, none of them was able to grow in foie gras at 8 °C over a 130-days period. Other species were also tested for their ability to grow in these conditions: only three isolates, identified as Clostridium sordellii, Clostridium tertium and Clostridium algidicarnis/putrefaciens, grew at 8 °C, exhibiting 5 to 6 log of population increase in 70 days. Consequently, regarding the risk of product spoilage, we recommend anaerobes enumeration at 37 °C for pasteurized foie gras products, or after a thermal treatment aiming at spore selection, for crude foie gras.

Comparative Genomic Hybridization Analysis Shows Different Epidemiology of Chromosomal and Plasmid-Borne cpe-Carrying Clostridium perfringens Type A

Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH) analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands

Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.