Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation

Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining trays used to transport commercial chicks from the hatchery to the grow-out facility on the farm. These results suggest that C. perfringens contaminating the processed product could originate from facilities in the integrated poultry operation prior to grow out. In this study, samples were collected from the breeder farm, hatchery, previous grow-out flock, during grow out and after processing. In the first trial, C. perfringens was recovered from the breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, from processed carcasses, and from the breeder farm after processing in 4%, 30%, 4%, 0%, 2% and 16%, and 4% of the samples, respectively. In the second trial, the incidence of C. perfringens in samples collected from breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, and fromprocessed carcasses was 38%, 30%, 32%, 8%, 4%, and 8%, respectively. The genetic relatedness of the isolated strains as determined by ribotyping suggests that C. perfringens may be transmitted between facilities within the integrated broiler chicken operation.     

Incidence of Clostridium perfringens in broiler chickens and their environment during production and processing.

During a calendar year, a study was conducted involving 16 broiler flocks on four different farms, two farms belonging to each of two major U.S. poultry integrators. As determined by the detection of Clostridium perfringens in fecal or cecal samples, 15 (94%) of the flocks became positive for this bacterial enteropathogen, and only one remained negative throughout the 6-to-8-wk rearing period. Paper pads beneath chicks that were transported from the hatchery to the rearing house were contaminated with C. perfringens in 15 (94%) of the flocks. When sampled biweekly through grow out, 13 of the flocks were C. perfringens positive at 2 wk of age. These results suggest that colonization of the intestinal tract of broilers by C. perfringens is an early event. Of the environmental samples, all but feed in the hopper were contaminated before placement for at least one of the rearing periods. All sample types were contaminated at some point during the 6-to-8-wk grow-out period. Of the on-farm environmental samples, the highest incidences (percentage positive) of C. perfringens were detected in wall swabs (53%), fan swabs (46%), fly strips (43%), dirt outside the house entrance (43%), and swabs of workers' boots (29%). Birds were usually transported to the processing plant in coops that were already contaminated with C. perfringens. In the plant, C. perfringens was isolated more frequently from samples of scald water than from those of chill water. Clostridium perfringens was recovered from broiler carcasses after chilling in 13 (81%) of the 16 flocks. The proportion of C. perfringens-positive carcasses for the contaminated flocks ranged from 8% to 68% with a mean of 30%.     

Risk Factors Associated with Salmonella Status of Broiler Flocks Delivered to Grow‐Out Farms

In a prospective field observational study in the southeastern USA, we sampled gastrointestinal (GI) tracts from chicks of 65 broiler flocks delivered to conventional grow‐out farms for rearing. The flocks were hatched at seven broiler hatcheries. The mean within‐flock prevalence of Salmonella‐positive samples was 6.5% and ranged from 0% to 86.7%. Of the 65 flocks studied, 25 (38.5%) had at least one Salmonella‐positive sample. Accounting for confounding variability among the hatcheries and broiler companies, we tested whether the probability of detecting Salmonella in GI tracts of the chicks delivered was associated with certain characteristics of parent breeder flocks; hatchery production volume; hatchery ventilation system; hatchery egg‐room conditions; egg incubation, candling, hatching, eggshell and bird separation, and bird‐processing procedures; management of hatchery‐to‐farm transportation; day of week of hatch; weather conditions during transportation; or season of the hatch. Two risk factor models were adopted. The first model indicated that a greater number of parent flocks, manual separation of eggshell and bird, and a greater amount of fluff and feces on tray liners used during hatchery‐to‐farm transportation at delivery were associated with increased probability of detecting Salmonella in chick GI tracts, whereas a greater number of birds in the delivery vehicle was associated with decreased probability. The second model indicated that broiler flocks hatched on Tuesdays versus either Mondays or Thursdays (with no hatches on Wednesdays, Fridays or week‐ends), increased average hatchability of the eggs from the parent flocks, and greater amounts of fluff and feces on the transport tray liners at delivery were all associated with increased probability of detecting Salmonella in chick GI tracts. The results of this study suggest potential management decisions to lessen Salmonella contamination of broilers supplied by commercial hatcheries and areas for further research.     

Results of salmonella isolation from poultry products, poultry, poultry environment, and other characteristics

Five hundred sixty-nine Salmonella were isolated out of 4745 samples from poultry products, poultry, and poultry environment in 1999 and 2000 from the Pacific northwest. These Salmonella were identified to their exact source, and some were serogrouped, serotyped, phage typed, and tested for antibiotic sensitivity. Food product samples tested included rinse water of spent hens and broilers and chicken ground meat. Poultry environment samples were hatchery fluff from the hatcheries where eggs of grandparent broiler breeders or parent broiler breeder eggs were hatched and drag swabs from poultry houses. Diagnostic samples were of liver or yolk sac contents collected at necropsy from the young chicks received in the laboratory. Of these samples tested, 569 were Salmonella positive (11.99%). Ninety-two Salmonella were serogrouped with polyvalent somatic antisera A-I and the polymerase chain reaction. Somatic serogroups B and C comprised 95.25% of all the Salmonella. Out of a total of 569 positive samples, 97 isolates of Salmonella were serotyped. A total of 16 serotypes and an unnamed Salmonella belonging to serogroup C1 were identified. The Salmonella serotypes were heidelberg (25.77%); kentucky (21.64%); montevideo (11.34%); hadar and enteritidis (5.15% each); infantis, typhimurium, ohio, and thompson (4.12% each); mbandaka and cerro (3.09% each); senftenberg (2.06%); berta, istanbul, indiana, and saintpaul (1.03% each); and an unnamed monomorphic Salmonella (2.06%). Ninety-two Salmonella were tested for drug sensitivity with nine different antimicrobials. All of the 92 Salmonella were resistant to erythromycin, lincomycin, and penicillin except one sample (S. berta), which was moderately sensitive to penicillin. All of the tested Salmonella were susceptible to sarafloxacin and ceftiofur. The percentages of Salmonella susceptible to sulfamethoxazole-trimethoprim, gentamicin, triple sulfa, and tetracycline were 97.83%, 92.39%, 86.96%, and 82.61%, respectively.     

Determination of the incidence of Salmonella spp., Campylobacter jejuni, and Clostridium perfringens in wild birds near broiler chicken houses by sampling intestinal droppings

Several methods were evaluated for collecting fecal and intestinal samples from wild birds found near broiler chicken houses. A few intestinal samples and cloacal swabs were obtained from European starlings and house sparrows. Most of the samples collected consisted of wild bird droppings found on or near the houses. Samples were collected from each of four farms of a broiler integrator during a grow-out cycle: a cycle in the summer for farm A, fall for farm B, and spring, summer, fall, and winter for farms C and D. Of the 25 wild bird intestinal and fecal samples collected from a broiler house on farm A during a grow-out cycle in July-August 1997, 24% were positive for Salmonella spp., 4% for Campylobacter jejuni, and 28% for Clostridium perfringens. Of the nine fecal samples collected from broiler house B in a grow-out cycle in September-November 1997, 33% were positive for Salmonella spp., 11% for C. jejuni, and 22% for C. perfringens. For farms C and D, of the 23 samples collected in March-April 1998, 0 were positive for Salmonella spp., 11% for C. jejuni, and 52% for C. perfringens; of 27 samples collected in June-July 1998, 4% were positive for Salmonella spp., 0 for C. jejuni, and 13% for C. perfringens; of 24 samples collected in August-October 1998, 14% were positive for Salmonella spp., 5% for C. jejuni, and 4% for C. perfringens; of 14 samples collected December 1998-January 1999, 0 were positive for Salmonella, 50% for C. jejuni, and 14% for C. perfringens. The incidence of these bacterial enteropathogens in wild birds near the broiler chicken houses suggests that wild birds that gain entry to poultry grow-out houses have the potential to transmit these pathogens to poultry.     

Hatchery hygiene evaluation as measured by microbiological examination of samples of fluff

The microbial content of 1,180 samples of fluff from hatchers at 18 chick and 6 turkey hatcheries has been determined. The samples were taken (a) 12–15 hr before removal of birds from the hatchers, (b) just before or after theremoval of chicks orpoults, (c) after fumigation and (d) inreplicate from the same hatcher.

Fifty‐eight per cent of the samples of chick fluff contained one million or more bacteria but only 24 per cent contained more than 100,000 coli‐aerogenes bacteria. Moulds were not found in 68 per cent of the samples. Some samples of fluff were examined for coagulase positive staphylococci and fluorescent pseudomonads but these organisms were generally not detected. Bacillus cereus was recovered in very large numbers from a few samples. Bacterial and coli‐aerogenes counts from poult fluff were generally higher than those from chick fluff but the numbers of pseudomonads and coagulase positive staphylococci recovered were generally negligible.

In an intensive study of samples of fluff from one turkey hatchery, it was found that the level of contamination of fluff increased during hatching and the laying season and was accompanied by a decrease in poult quality. Sampling from any location in the hatcher appears to provide a representative indication of overall contamination at that time. Fumigation of fluff and debris generally caused a reduction in the level of contamination in the fluff.

A suggested assessment of hatchery hygiene based on the microbial examination of fluff is given and the advantages and disadvantages of this method are discussed.     

The occurrence of Clostridium perfringens in the intestine of chicks

Commercial broiler chicks brooded either on wire or on used or new litter demonstrated a 75% (62/75) incidence of recovery of "perfringens-like" colonies from the intestine during a 5-week period. Eleven Clostridium spp. were identified from among these "perfringens-like" organisms, which were cultured on SPS selective agar medium. Clostridium perfringens was positively identified only infrequently (five isolates) from among the "perfringens-like" colonies. In contrast, "perfringens-like" colonies were not recovered from the intestinal contents of specific-pathogen-free chicks reared in an isolation unit. However, C. perfringens was isolated from the yolk sac of one embryonated egg and from the intestine of a single 7-day-old chick, indicating the possibility of vertical transmission of this potential pathogen.     

Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes

This study was undertaken to determine the in vitro susceptibility of Clostridium perfringens, isolated from poultry to antimicrobials used in poultry production. The minimal inhibitory concentration (MIC) of eight antimicrobials, including the ionophoric coccidiostat narasin, was determined for 102 C. perfringens isolates, 58 from Sweden, 24 from Norway and 20 from Denmark. Susceptibility to each antimicrobial compound was determined by broth microdilution. The isolates were obtained from broilers (89), laying hens (9) and turkeys (4), affected by necrotic enteritis (NE) or by C. perfringens associated hepatitis (CPH), and from healthy broilers. All strains, regardless of origin, proved inherently susceptible to ampicillin, narasin, avilamycin, erythromycin and vancomycin. A low frequency of resistance to virginiamycin and bacitracin was also found. Resistance to tetracycline was found in strains isolated in all three countries; Sweden (76%), Denmark (10%) and Norway (29%). In 80% of the tetracycline-resistant isolates, the two resistance genes tetA(P) and tetB(P) were amplified by PCR whereas in 20% only the tetA(P) gene was detected. No tetM gene amplicon was obtained from any of the tetracycline-resistant isolates. The uniform susceptibility to narasin revealed in this study shows that the substance can still be used to control clostridiosis. In this study, C. perfringens also showed a low degree of resistance to most other antimicrobials tested. Despite the small amounts of tetracycline used in poultry, a considerable degree of resistance to tetracycline was found in C. perfringens isolates from Swedish broilers.     

Reproduction and treatment of necrotic enteritis in broilers.

A change in ration from one containing 50% fish meal to a standard chick starter containing 10(8) Clostridium perfringens/g of feed resulted in the production of necrotic enteritis in 2-week-old broiler chicks. Lincomycin added to the inoculated ration at a concentration of 20 g/907.2 kg significantly reduced mortality in the chicks. Inoculation of broth cultures of C perfringens directly into the duodenum, using a surgically inserted tetrafluoroethylene resin tube, indicated a relationship existed between the number of C perfringens inoculated and the gross lesions. The disease could be consistently produced by this technique.     

Direct polymerase chain reaction detection of Campylobacter spp. in poultry hatchery samples

A rapid, sensitive, and specific polymerase chain reaction (PCR) assay was developed for the direct detection of Campylobacter in environmental samples from hatcheries. PCR, with a set of primers specific for the Campylobacter flaA short variable region (SVR), detected the presence of Campylobacter in both fluff and eggshell samples; however, a determination of whether the organism was living or dead could not be made. Conventional cultural methods detected no Campylobacter from the same samples. An additional benefit of the direct PCR assay is it allows for the production of a product that can be sequenced to provide further epidemiologic information.     

产气荚膜梭菌多重PCR定型方法的建立及其应用

建立了多重PCR检测产气荚膜梭菌α、β、ε和ι毒素基因的方法。该方法具有良好的特异性和敏感性,只有产气荚膜梭菌呈现阳性,被检验的其他梭菌以及大肠杆菌、葡萄球菌均为阴性;将肉汤菌液样品10倍系列稀释后进行检测,检测灵敏度达到1.2×104CFU/mL。收集40份牛粪便样品,进行PCR检测,32份样品中成功扩增出589 bp的α毒素基因片段,阳性率为80%。结果显示,建立的多重PCR检测方法可取代血清中和试验,用于产气荚膜梭菌分型,同时表明A型产气荚膜梭菌在当地奶牛场中较为普遍。     

Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.     

A temporal study of Salmonella serovars from environmental samples from poultry breeder flocks in Ontario between 1998 and 2008

A temporal study was carried out to determine Salmonella prevalence, trends, major serovars, and their clusters from environmental samples, in poultry breeder flocks in Ontario between January 1998 and December 2008. Surveillance data were obtained from the Ontario Hatchery and Supply Flock Policy. Logistic regression with a random effect for flock was used to identify factors [poultry type, year (trend) and season] associated with the prevalence of Salmonella. A cluster detection test was used to identify clusters of common serovars. The period prevalence of Salmonella was 47.4% in broiler-breeder, 25.7% in layer-breeder, and 19.6% in turkey-breeder flocks. The overall trend in the prevalence of Salmonella was decreasing for all breeder types, due primarily to decreasing trends of Salmonella Heidelberg. The seasonal effects varied by year with the highest probability of Salmonella occurring in different seasons. The 4 most common serovars identified were Salmonella Heidelberg, Kentucky, Hadar, and Typhimurium in broiler-breeders; Salmonella Heidelberg, Brandenburg, Thompson, and Typhimurium in layer-breeders; and Salmonella Heidelberg, Saintpaul, Brandenburg, and Muenster in turkey-breeders. Salmonella Enteritidis was infrequently isolated in all poultry breeder types. Temporal clusters of different serovars were identified in all poultry breeder types. Clusters of Salmonella Heidelberg, Typhimurium, and Hadar from environmental samples from breeder flocks were detected during a similar period to clusters from hatchery fluff samples from the same population. Therefore, interventions at the breeder flock-level might help to reduce transmission of Salmonella from breeder flocks to hatcheries and possibly, to lower levels of the poultry production chain.     

Population-based study of fecal shedding of Clostridium perfringens in broodmares and foals

OBJECTIVE: To determine the percentage of broodmares and foals that shed Clostridium perfringens in their feces and classify the genotypes of those isolates. DESIGN: Prospective cross-sectional study. ANIMALS: 128 broodmares and their foals on 6 equine premises. PROCEDURES: Anaerobic and aerobic bacteriologic cultures were performed on feces collected 3 times from broodmares and foals. All isolates of C. perfringens were genotyped. RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: 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.     

THE USE OF FLUFF COUNTS IN A STUDY OF HYGIENE IN QUEENSLAND HATCHERIES

During 1972, 333 fluff samples were tested from 13 Queensland hatcheries and assessed according to the English standards for total count, coliforms and fungi. They were also assessed as being "standard" or "substandard", using a combination of the criteria of the English system. All hatcheries had at least 2 substandard samples and all the samples from 2 hatcheries were substandard.     

Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status

Due to the diminished use of growth-promoting antibiotics in the European Union, Clostridium perfringens induced necrotic enteritis and subclinical disease have become important threats to poultry health. A study was set up to genotypically and phenotypically characterise C. perfringens isolates from poultry flocks with different health status. Animals from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of animals suffering from necrotic enteritis were analysed. A total of 27 isolates was obtained from 23 broiler flocks without clinical problems and 36 isolates were obtained from 8 flocks with clinical problems. Using PFGE typing, high genetic diversity was detected between isolates from different flocks. Isolates derived from flocks where disease outbreaks occurred were clonal within each flock, but each flock harboured a different clone. All isolates were of toxin type A. Isolates from 5 out of 35 PFGE types carried the cpb2 gene, encoding the beta2 toxin, and isolates from 2 out of 35 PFGE types harboured the cpe gene, encoding the enterotoxin. In vitro alpha toxin production for all isolates was quantified by enzyme-linked immunosorbent assay. It was shown that in vitro alpha toxin production of C. perfringens isolates from diseased flocks was not higher than in vitro alpha toxin production from isolates derived from healthy flocks.     

Evaluation of Salmonella serotype distributions from commercial broiler hatcheries and grower houses

By conventional trayliner (hatcheries) and drag swab assembly (broiler houses) culture methods, the isolation distribution of Salmonella serotypes from five commercial broiler hatcheries (three sample times) and 13 broiler farms (eight sample times) was evaluated. A total of 11 different Salmonella serotypes were isolated from hatcheries, with Salmonella heidelberg (9/30) and Salmonella kentucky (6/30) accounting for 50% of the total isolations. Of 700 chick paperpad trayliners sampled, regardless of lot (breeder flock source) or hatchery, 12% were positive for Salmonella. When 10 individual trayliners were cultured from individual lots (same breeder flock source), Salmonella was detected in 24/57 lots (42%). Multiple serotypes were simultaneously isolated from the same lot on three occasions (6%). Of the 21 lots that were serially sampled, the Salmonella serotype detected was different within lots eight times (38%) on at least one occasion of two or more sampling times. Of the 196 individual broiler houses sampled, 44 were positive for Salmonella (42%). Twelve different serotypes were isolated from broiler houses during this study. The serotypes isolated most frequently were S. heidelberg (34/94) and S. kentucky (22/94). These two serotypes accounted for 59.6% (56/94) of the total broiler house isolations. Of the 38 houses that were serially sampled, two or more serotypes were detected in the same broiler house on 20 occasions (53%). Of the 38 serially sampled houses (four or more times), a consistent Salmonella serotype was detected in five houses (13%). In only 5 of the 38 (13%) serially sampled houses did we fail to detect Salmonella on four or more samplings. No significant difference in Salmonella isolation frequency was observed between poultry houses using new or used litter. These data support previous findings indicating that paratyphoid Salmonella serotypes are prevalent in some broiler hatcheries and houses. Further, the observation of multiple serotypes simultaneously and serially isolated from the same breeder hatchery lots suggests that breeder flocks may be infected with more than one serotype, possibly providing a source for multiple serotype infections in progeny grower flocks.     

Cecal carriage of Clostridium perfringens in broiler chickens given Mucosal Starter Culture.

Day-of-hatch broiler chicks housed in isolation units were each given, by oral gavage, 0.1 ml of Mucosal Starter Culture (MSC) or saline control. Each of the treated and control chicks was subsequently given a composite culture of three strains of bacitracin-resistant Clostridium perfringens (Cp) previously isolated from chickens with symptoms of necrotic enteritis. Some chicks were maintained on a corn-based diet provided ad libitum. Others were given the feed supplemented with 50% rye (a predisposing factor for necrotic enteritis). At 7, 14, and 21 days after receiving Cp, chicks were euthanatized, and cecal contents were diluted and plated on selective agar containing bacitracin. For chicks on corn feed, Cp numbers were similar in control birds and birds given MSC in three of four trials. In two of the trials that demonstrated no effect of MSC on Cp numbers, enterotoxin presence was determined. The number of birds with detectable Cp enterotoxin in their small intestine and the mean toxin levels were lower in the MSC-treated birds. In a fourth trial with birds on corn-based feed, mean Cp numbers and the number of Cp-positive birds were lower in the MSC-treated birds. For the two trials involving chickens on rye-supplemented feed, Cp numbers and the percentage of Cp-positive birds were significantly reduced in MSC-treated birds compared with control birds. Enterotoxin in birds receiving the 50% rye diet was at low levels or not detected in control and MSC-treated birds. Results suggest that MSC may reduce intestinal proliferation of Cp, a causative agent of necrotic enteritis in poultry and of foodborne disease in humans.     

Necrotic hepatitis due to Clostridium perfringens infection in newly hatched broiler chicks

Multiple necrotic hepatitis lesions of 5 newly hatched broiler chicks in three flocks derived from two hatcheries were examined pathologically. The livers were brittle, and multiple yellowish or green foci were scattered on the surface and cut surface. The main histological finding was well demarcated multi-focal necrosis in the liver. Many Gram-positive large bacilli that reacted positively with polyclonal anti-Clostridium perfringens serum were observed in necrotic areas.     

Clostridium perfringens in Animal Disease: A Review of Current Knowledge

The diseases caused by various types of Clostridium perfringens are critically reviewed in the light of current knowledge. Particular emphasis is placed on information concerning these diseases in Canadian livestock.There are two etiologically clearly-defined acute C. perfringens diseases recognized in Canada: hemorrhagic enteritis of the new born calf, caused by C. perfringens type C, and enterotoxemia of sheep, caused by type D. Clostridium perfringens type A may play a role as a secondary pathological agent in various disease conditions, such as necrotic enteritis of chickens. It may also cause wound infections and may provide a source for human food poisoning outbreaks.There appears to be a considerable lack of knowledge regarding the distribution of C. perfringens types, their pathogenesis, diagnosis and the incidence of diseases caused by this organism.