Perfringens Populations Research Articles

Emergence of genetic diversity and multi-drug resistant Clostridium perfringens from wild birds

BackgroundClostridium perfringens (C. perfringens) is an important zoonotic microorganism that can cause animal and human infections, however information about the prevalence status in wild birds of this pathogenic bacterium is currently limited.ResultIn this study, 57 strains of C. perfringens were isolated from 328 fecal samples of wild birds. All the isolates were identified as type A and 70.18% of the isolates carried the cpb2 gene. Antimicrobial susceptibility testing showed that and 22.80% of the isolates were classified as multidrug-resistant strains. The MLST analysis of the 57 isolates from wild birds was categorized into 55 different sequence types (STs) and clustered into eight clonal complexes (CCs) with an average of 20.1 alleles and the Simpson Diversity index (Ds) of 0.9812, and revealed a high level of genetic diversity within the C. perfringens populations. Interestingly, the isolates from swan goose were clustered in the same CC while isolates from other bird species were more scattered suggesting that a potential difference in genetic diversity among the C. perfringens populations associated with different bird species.ConclusionC. perfringens exhibits a wide range of host adaptations, varying degrees of antimicrobial resistance, and a high degree of genetic diversity in wild birds. Understanding the prevalence, toxin type, antimicrobial resistance, and genetic diversity of C. perfringens in wildlife populations is essential for developing effective strategies for disease control and management.

Multiple factorial analysis of growth performance, gut population, lipid profiles, immune responses, intestinal histomorphology, and relative organ weights of Cobb 500 broilers fed a diet supplemented with phage cocktail and probiotics

Previous studies on probiotics in chickens have shown varying efficacy, and the use of phage as a supplement for improving chicken performance and health has gained interest. However, the combination of these supplements has not been thoroughly investigated. This study aimed to assess the effects of an Escherichia coli phage cocktail, both alone and in combination with probiotics, on specific gut populations, lipid profiles, heterophil-to-lymphocyte ratio, serum immunoglobulins, villus height to crypt depth ratio, and relative organ weights in chickens at day 21 and 35. The experiment followed a completely randomised design with factorial arrangements, consisting of three levels of phage cocktail dosage (0 g/kg, 1 g/kg, and 2 g/kg) and two levels of probiotics dosage (0 g/kg and 1 g/kg), with a total of 288 one-day-old male Cobb 500 broilers. Our study demonstrated that supplementing chicken diets with 1 g/kg phage cocktail and 1 g/kg probiotics resulted in significant (p < 0.05) improvements in growth performance and reduced Clostridium perfringens populations. It also reduced triglyceride levels and did not cause physiological stress, as indicated by the heterophil-to-lymphocyte ratio. Overall, this study provides important insights into the potential benefits of combining phages and probiotics in broiler chicken diets, which have not been extensively studied.

Validating the use of a newly developed cinnamaldehyde product in commercial broiler production

Essential oils (EOs) have been considered as an alternative to antibiotics for animal production. In the current study, 4 trials were conducted on a commercial broiler farm to investigate the effects of dietary supplementation of an encapsulated cinnamon EO product (NE-OFF) on the bird growth performance, gut health, and gene expression in the ileum, spleen, and liver relating to the host response to heat and other stresses, including potential NE challenge. In each trial, approximately 30,000 Cobb or Ross broilers were randomly allocated to 4 treatments: a raised without antibiotics (RWA) commercial diet as positive control, an adjusted RWA commercial diet as negative control, and the negative control diet supplemented with 2 different dosages of NE-OFF, which was added during feed pelleting. Although the final average body weight did not differ significantly among treatment groups, birds fed NE-OFF had an increased ratio of villus height and crypt depth in the jejunum, and reduced fecal oocyst counts. Trial 2 was conducted in the summer and had a necrotic enteritis (NE) outbreak. The supplementation of NE-OFF reduced the NE incidence and bird mortality. The samples from Trial 2 were hence selected for the analyses of Clostridium perfringens and NetB toxin gene abundance in the ileum, and host responses. The C. perfringens population appeared to be positively correlated with the NetB gene abundance. The gene expression analysis suggested that NE-OFF supplementation improved nutrient absorption and transportation as well as antioxidant activities to help the birds against stress. These on-farm trial results support the hypothesis that the use of NE-OFF as a feed additive can improve bird gut health and performance in commercial broiler production, especially for preventing NE outbreaks when birds are under stress.

A calf with hind limb paralysis and dysstasia and a genome sequence analysis of an isolated Clostridium perfringens toxinotype E strain.

Clostridium perfringens toxinotype E infections are rare in calves, and the development of intestinal lesions were commonly observed. In 2012, a 6-day-old calf in Japan exhibited swelling with emphysema on the right gluteal region, sudden paralysis of the hind limb and dysstasia. A pathological examination revealed myositis of the gluteal muscle and neuritis of the ischiatic nerve. C. perfringens type E strain CP118 was isolated from the affected muscle. However, the intestinal symptoms and lesions that commonly develop in type E infections in calves were not detected in the present case. Genome analyses revealed that CP118 possessed 16 virulence-related genes, including enterotoxin, and was closely related to other type E and F strains. Particularly, CP118 was more closely related to type E strains from humans, including a food poisoning case, than calf isolates, suggesting its potential to cause food poisoning in humans and, thus, its importance as a potential risk to public health. Since CP118 did not possess the reported toxin genes associated with neuropathy, pyogenic inflammation caused by CP118 and/or other bacteria may have damaged the ischiatic nerve, resulting in neuropathy. Alternatively, unidentified CP118 toxins may have caused the neuropathy. This is the first study to report C. perfringens type E infection with peripheral neuropathy. The distribution of all the reported virulence-related genes in the C. perfringens population as well as the details of this rare case will provide further insights into C. perfringens type E infections.

Effects of dietary inclusion of Xanthoceras sorbifolia Bunge leaves on growth performance, gastrointestinal development, digestive function and gut microbial flora of rabbits

This study aimed to investigate the effects of dietary inclusion of Xanthoceras sorbifolia Bunge (X sorbifolia) leaves (XSL) or solid-state fermented X sorbifolia leaves (FXSL) on the growth performance, gastrointestinal tract development, serum metabolites, intestinal mucosa morphology, cecal fermentation and microflora enumeration of rabbits. A total of 192 healthy New Zealand White male rabbits weighing (940 ± 11 g) were randomly allocated to 3 dietary treatments, with 8 replicates of 8 rabbits each and designated as control, XSL, and FXSL groups. The rabbits in the XSL and FXSL groups were fed diets containing 150 g/kg XSL and FXSL in the basal diets to replace the same level of alfalfa hay, respectively for 42 days. Compared with the control or XSL group, the FXSL rabbits showed increased average daily weight gain (P < 0.05) and final body weight, as well as a decreased feed to gain ratio (P < 0.05). They had increased small intestine weight, small intestine relative weight (P < 0.05), cecum weight, cecum relative weight, and decreased urea nitrogen (UN) content (P < 0.05). Rabbits in the XSL and FXSL groups had lower serum alanine aminotransferase (ALT) (P < 0.05) and aspartate aminotransferase (AST) activities, as well as higher serum alkaline phosphatase (AKP) activity (P < 0.05). Compared to the other two groups, the total volatile fatty acids, the ratio of acetic acid, as well as Lactobacilli and Bifidobacterium populations in the cecal content was increased (P < 0.05) in the FXSL group, while the cecal pH, NH3-N, the ratio of butyric acid, Escherichia coli as well as Clostridium perfringens populations were decreased (P < 0.05). Further, the rabbits from the FXSL group showed higher (P < 0.05) villus height (VH) and VH/crypt depth (CD) both in the jejunum and duodenum. In conclusion, adding 150 g/kg XSL to the diet can replace the same amount of alfalfa powder, simultaneously, adding 150 g/kg FXSL can improve the growth and gastrointestinal tract development, as well as the cecal environment and microbiota balance.

Effects of feeding diets containing increasing content of purified lignocellulose supplied by sugarcane bagasse to early-weaned pigs on growth performance and intestinal health

A total of 96 weaned piglets, with mean age of 21 days and initial body weight (BW) of 6.67 ± 0.63 kg, were used in a 42-day experiment to evaluate the effects of supplementation of dietary purified lignocellulose (LNC) on growth performance and intestinal health. Piglets were blocked by initial BW and allotted to one of 32 pens, each pen containing three pigs. Dietary treatments consisted of a corn-soybean meal-based control diet with no added LNC and three dietary inclusion rates (10, 20, and 30 g/kg) of LNC. Average daily feed intake (ADFI), average daily gain (ADG), and gain-to-feed ratio (G:F) were determined over 42 days postweaning. On day 14 postweaning, one pig from each pen was euthanized to determine gastrointestinal tract organ weight, small intestinal morphology, and digesta pH, viscosity, fermentation end-products, and bacterial enumeration. Total tract retention time was measured by adding 10 g/kg ferric oxide in the morning feed on day 42. From day 1–14 postweaning, G:F tended to linearly increase (P = 0.088) as the dietary LNC contents increased. Although increasing the dietary LNC supplementation quadratically increased (P = 0.008) ADFI and tended to quadratically decrease (P = 0.072) G:F from day 30–42 postweaning, overall growth performance was unaffected by the treatments. The weight of the small intestine adjusted for kg of BW on day 14 postweaning increased quadratically (P = 0.041) as the dietary LNC contents increased. Increasing the dietary LNC supplementation linearly increased (P = 0.027) crypt depth and tended to linearly reduce (P = 0.075) villus:crypt ratio in the jejunum. Dietary treatments did not affect total tract retention time as well as pH and viscosity of both cecum and colonic digesta. Increasing the dietary LNC supplementation quadratically increased (P = 0.001) cecal butyrate formation, whereas cecal acetate and propionate concentrations did not differ among treatments. Although ileal counts of Lactobacillus spp. and Escherichia coli were similar among treatments, the pathogenic Clostridium perfringens population exhibited a quadratic decrease (P = 0.041) as the dietary LNC contents increased. These results indicate that feeding pig diets containing up to 30 g/kg purified LNC enhanced gut health through the inhibition of harmful bacteria proliferation and the expansion of butyrate producers, without compromising overall growth performance.

The effects of Pediococcus acidilactici and Saccharomyces cerevisiae on broiler chickens challenged with Clostridium perfringens induced subclinical necrotic enteritis

The objective of this study was to evaluate the effects of Pediococcus acidilactici (P. acidilactici) and Saccharomyces cerevisiae (S. cerevisiae) on the growth performance, intestinal lesions and ileal Clostridium perfringens (C. perfringens) count of broiler chickens challenged with C. perfringens induced sub-clinical necrotic enteritis (NE). A total of 150 broiler chicks, allocated into five treatment groups (6 replicates of 5 chicks/cage), were reared in cages for 29 days: T0 (control group, not infected, not supplemented); T1 (infected with C. perfringens, not supplemented); T2 (infected with C. perfringens and coccidia, not supplemented); T3 (infected with C. perfringens and coccidia, supplemented with P. acidilactici); T4 (infected with C. perfringens and coccidia, supplemented with S. cerevisiae). The parameters analyzed were: body weight gain, feed intake, feed conversion ratio, mortality, intestinal lesion score and ileal C. perfringens enumeration by quantitative real-time PCR. The challenge resulted in impairment of growth performance, increased lesion score (≤ 2) and overgrowth of the C. perfringens population. However, the dietary inclusion of P. acidilactici or S. cerevisiae caused a significant improvement in feed conversion, net reduction of gut lesions, as well as a decrease in the intestinal C. perfringens population. In conclusion, these results suggest that dietary supplementation with probiotics (P. acidilactici or S. cerevisiae) could be beneficial to alleviate the negative effects of subclinical NE in broiler chickens.

Supplementing Garlic Nanohydrogel Optimized Growth, Gastrointestinal Integrity and Economics and Ameliorated Necrotic Enteritis in Broiler Chickens Using a Clostridium perfringens Challenge Model.

Simple SummaryNecrotic enteritis (NE) is one of the most serious diseases in terms of economic losses. Aspects related to application of nanotechnology to control outbreaks of NE due to Clostridium perfringens (C. perfringens) are not completely understood. Hence, the purpose of this study was to evaluate the beneficial effects of garlic nano-hydrogel (G-NHG) on the performance, intestinal integrity, economic returns and alleviation of the intestinal C. perfringens levels using an NE challenge model in broiler chickens. Dietary inclusion of 400 mg/kg of G-NHG improved body weight gain (BWG) and feed conversion ratio (FCR). The digestive enzymes and tight junction and gut barrier-related genes expression were negatively impacted post C. perfringens challenge, resulting in a decrease in BWG with an increase in FCR. Meanwhile, G-NHG supplementation decreased C. perfringens levels, mortality rates and intestinal lesion score, and thereby improved intestinal permeability measurements, which consequently resulted in improved growth performance parameters. In conclusion, G-NHG markedly ameliorated the negative effects of C. perfringens challenge, which positively reflected on the growth performance of challenged birds, suggesting its beneficial effects in controlling C. perfringens infection in broiler chickens.Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds’ intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

Impacts of Short-Term Antibiotic Withdrawal and Long-Term Judicious Antibiotic Use on Resistance Gene Abundance and Cecal Microbiota Composition on Commercial Broiler Chicken Farms in Québec.

The ever-increasing problem of antibiotic resistance makes routine use of antibiotics in animal production no longer considered as a reasonable and viable practice. The Chicken Farmers of Canada have developed and are implementing an Antimicrobial Use Reduction Strategy, which has the ultimate goal of eliminating the preventive use of medically important antibiotics in broiler chicken and turkey production. However, very little is known about the real overall impact of an antibiotic use reduction strategy in complex ecosystems, such as the bird intestine or the commercial broiler chicken farm. The main objectives of the present study were to compare the abundance of antibiotic resistance-encoding genes, characterize the intestinal microbiota composition, and evaluate the presence of Clostridium perfringens, in six commercial poultry farms adopting short-term antibiotic withdrawal and long-term judicious use strategy. Implementing an antibiotic-free program over a 15-months period did not reduce the abundance of many antibiotic resistance-encoding genes, whereas the judicious use of antibiotics over 6 years was found effective. The short-term antibiotic withdrawal and the long-term judicious use strategy altered the intestinal microbiota composition, with the Ruminococcaceae and Lachnospiraceae families being negatively impacted. These findings are in agreement with the lower production performance and with the increased C. perfringens populations observed for farms phasing out the use of antibiotics. Adopting a conventional rearing program on commercial broiler chicken farms selected for specific antibiotic resistance-encoding genes in many barns. This study highlights the potential impacts of different rearing programs in poultry production and will help guide future policies in order to reduce the use of antibiotics while maintaining production performance.

Control of Clostridium perfringens spore germination and outgrowth by potassium lactate and sodium diacetate in ham containing reduced sodium chloride

The effect of potassium lactate and sodium diacetate mixture (Opti.Form PD4®) on Clostridium perfringens spore germination and outgrowth during abusive cooling of reduced sodium ham was evaluated. A ham formulation containing NaNO2 (0 or 100 ppm), NaCl (1 or 2%) and Opti.Form PD4® (0, 1.5 or 2.5%) was prepared. Meat (10 g) was vacuum-packaged, stored under refrigeration (5 °C) for 3 or 24 h and inoculated with a three-strain C. perfringens spore cocktail. Samples were vacuum-packaged, heat treated (75 °C, 20 min) and cooled from 54.4 °C to 7.2 °C within 9, 15 or 21 h. C. perfringens population increases of 5.18 and 4.26 log CFU/g were observed in non-cured ham (control; stored for 3 h) formulated with 1 or 2% of NaCl, respectively and cooled exponentially within 21 h. Incorporation of Opti.Form PD4® (1.5%) inhibited C. perfringens spore germination and outgrowth regardless of the cooling rate (9, 15 or 21 h). Addition of NaNO2 (100 ppm) enhanced antimicrobial activity of Opti.Form PD4®. In general, abusive cooling (≥15 h) of ham after 24 h of storage resulted in greater populations of C. perfringens. Addition of NaNO2, Opti.Form PD4® and processing immediately resulted in greater inhibition of C. perfringens spore germination and outgrowth.

Effects of Nitrite and Erythorbate on Clostridium perfringens Growth during Extended Cooling of Cured Ham

To control the growth of Clostridium perfringens in cured meat products, the meat and poultry industries commonly follow stabilization parameters outlined in Appendix B, "Compliance Guidelines for Cooling Heat-Treated Meat and Poultry Products (Stabilization)" ( U.S. Department of Agriculture, Food Safety and Inspection Service [USDA-FSIS], 1999 ) to achieve cooling (54.4 to 4.4°C) within 15 h after cooking. In this study, extended cooling times and their impact on C. perfringens growth were examined. Phase 1 experiments consisted of cured ham with 200 mg/kg ingoing sodium nitrite and 547 mg/kg sodium erythorbate following five bilinear cooling profiles: a control (following Appendix B guidelines: stage A cooling [54.4 to 26.7°C] for 5 h, stage B cooling [26.7 to 4.4°C] for 10 h), extended stage A cooling for 7.5 or 10 h, and extended stage B cooling for 12.5 or 15 h. A positive growth control with 0 mg/kg nitrite added (uncured) was also included. No growth was observed in any treatment samples except the uncured control (4.31-log increase within 5 h; stage A). Phase 2 and 3 experiments were designed to investigate the effects of various nitrite and erythorbate concentrations and followed a 10-h stage A and 15-h stage B bilinear cooling profile. Phase 2 examined the effects of nitrite concentrations of 0, 50, 75, 100, 150, and 200 mg/kg at a constant concentration of erythorbate (547 mg/kg). Results revealed changes in C. perfringens populations for each treatment of 6.75, 3.59, 2.43, -0.38, -0.48, and -0.50 log CFU/g, respectively. Phase 3 examined the effects of various nitrite and erythorbate concentrations at 100 mg/kg nitrite with 0 mg/kg erythorbate, 100 with 250, 100 with 375, 100 with 547, 150 with 250, and 200 with 250, respectively. The changes in C. perfringens populations for each treatment were 4.99, 2.87, 2.50, 1.47, 0.89, and -0.60 log CFU/g, respectively. Variability in C. perfringens growth for the 100 mg/kg nitrite with 547 mg/kg erythorbate treatment was observed between phases 2 and 3 and may have been due to variations in treatment pH and NaCl concentrations. This study revealed the importance of nitrite and erythorbate for preventing growth of C. perfringens during a much longer (25 h) cooling period than currently specified in the USDA-FSIS Appendix B.

Recurring Necrotic Enteritis Outbreaks in Commercial Broiler Chicken Flocks Strongly Influence Toxin Gene Carriage and Species Richness in the Resident Clostridium perfringens Population.

Extensive use of antibiotic growth promoters (AGPs) in food animals has been questioned due to the globally increasing problem of antibiotic resistance. For the poultry industry, digestive health management following AGP withdrawal in Europe has been a challenge, especially the control of necrotic enteritis. Much research work has focused on gut health in commercial broiler chicken husbandry. Understanding the behavior of Clostridium perfringens in its ecological niche, the poultry barn, is key to a sustainable and cost-effective production in the absence of AGPs. Using polymerase chain reaction and pulsed-field gel electrophoresis, we evaluated how the C. perfringens population evolved in drug-free commercial broiler chicken farms, either healthy or affected with recurring clinical necrotic enteritis outbreaks, over a 14-month period. We show that a high genotypic richness was associated with an increased risk of clinical necrotic enteritis. Also, necrotic enteritis-affected farms had a significant reduction of C. perfringens genotypic richness over time, an increase in the proportion of C. perfringens strains harboring the cpb2 gene, the netB gene, or both. Thus, necrotic enteritis occurrence is correlated with the presence of an initial highly diverse C. perfringens population, increasing the opportunity for the selective sweep of particularly virulent genotypes. Disease outbreaks also appear to largely influence the evolution of this bacterial species in poultry farms over time.

The impact of fruit and soybean by-products and amaranth on the growth of probiotic and starter microorganisms

The ability of different fruit by-products, okara, and amaranth flour, to support the growth of probiotic and non-probiotic strains was evaluated. The tests were conducted with three commercial starter cultures (Streptococcus thermophilus), ten probiotic strains (seven Lactobacillus spp. and three Bifidobacterium spp. strains), and two harmful bacteria representative of the intestinal microbiota (Escherichia coli and Clostridium perfringens). In vitro fermentability assays were performed using a modified MRS broth supplemented with different fruits (acerola, orange, passion fruit, and mango), and soy (okara) by-products or amaranth flour. Orange and passion-fruit by-products were the substrates that most promoted the growth of bacterial populations, including pathogenic strains. On the other hand, the acerola by-product was the substrate that showed the highest selectivity for beneficial bacteria, since the E. coli and Cl. perfringens populations were lower in the presence of this fruit by-product. Although the passion fruit by-product, okara, and amaranth stimulated the probiotic strains, the growth of the pathogenic strains studied was higher compared to other substrates. Different growth profiles were verified for each substrate when the different strains were compared. Although pure culture models do not reflect bacterial interaction in the host, this study reinforces the fact that the ability to metabolize different substrates is strain-dependent, and acerola, mango, and orange by-products are the substrates with the greatest potential to be used as prebiotic ingredients.

Effects of dietary yeast β-glucans supplementation on growth performance, gut morphology, intestinal Clostridium perfringens population and immune response of broiler chickens challenged with necrotic enteritis

Necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens that results in substantial economic losses to the global poultry industry. This study was conducted to investigate the effects of dietary yeast β-glucans (YG) on growth performances, gut morphology, intestinal C. perfringens population, endogenous antimicrobial peptide expression, and humoral immune response of broiler chickens infected with NE. A total of 240 one-day-old male broiler chicks were randomly assigned to a 2×2 factorial arrangements of treatments with two dietary glucan levels (0 or 200mg/kg of diet) and two disease challenge status (control or NE challenged). NE model was induced via oral inoculation of mixed strains of Eimeria species at 12 d of age, followed by an oral inoculation with C. perfringens at 16, 17 and 18 d of age. Infected birds fed the glucan-supplemented diet had significantly increased body weight (13 to 21 d, 0 to 42 d), significantly improved feed efficiency (13 to 21 d, 21 to 42 d), increased antibody levels against C. perfringens and improved villi height and villi height to crypt depth ratio. In addition, infected birds that were supplemented with YG had markedly reduced intestinal C. perfringens numbers, and NE gut lesion scores compared with infected birds fed unsupplemented diet. Moreover, YG supplementation increased gene expression of Cath-2, AvBD-4, and AvBD-10 expression at the early infection stage and Cath-1, Cath-2, and AvBD-1 at the later infection stage in the gut compared with unsupplemented birds. Decreased AvBD-10 and LEAP-2 mRNA levels were observed at the later infection stage in the glucan-supplemented birds when compared with that in the non-supplemented control group. In conclusion, yeast β-glucans supplementation improved intestinal health of broiler birds challenged with C. perfringens-induced necrotic enteritis.

Implications of Decreased Nitrite Concentrations on Clostridium perfringens Outgrowth during Cooling of Ready-to-Eat Meats

Increased popularity of natural and organic processed meats can be attributed to the growing consumer demand for preservative-free foods, including processed meats. To meet this consumer demand, meat processors have begun using celery juice concentrate in place of sodium nitrite to create products labeled as no-nitrate or no-nitrite-added meat products while maintaining the characteristics unique to conventionally cured processed meats. Because of flavor limitations, natural cures with celery concentrate typically provide lower ingoing nitrite concentrations for ready-to-eat processed meats than do conventional cures, which could allow for increased growth of pathogens, such as Clostridium perfringens, during cooked product cooling such as that required by the U.S. Department of Agriculture. The objective of this study was to investigate the implications associated with reduced nitrite concentrations for preventing C. perfringens outgrowth during a typical cooling cycle used for cooked products. Nitrite treatments of 0, 50, and 100 ppm were tested in a broth system inoculated with a three-strain C. perfringens cocktail and heated with a simulated product thermal process followed by a typical cooling-stabilization process. The nitrite concentration of 50 ppm was more effective for preventing C. perfringens outgrowth than was 0 ppm but was not as effective as 100 ppm. The interaction between nitrite and temperature significantly affected (P < 0.05) C. perfringens outgrowth in both total population and number of vegetative cells. Both temperature and nitrite concentration significantly affected (P < 0.05) C. perfringens spore survival, but the interaction between nitrite and temperature did not have a significant effect (P > 0.05) on spore outgrowth. Results indicate that decreased nitrite concentrations (50 ppm) have increased potential for total C. perfringens population outgrowth during cooling and may require additional protective measures, such as faster chilling rates.

Low Prevalence of netB and tpeL in Historical Clostridium perfringens Isolates from Broiler Farms in Alabama.

The discovery of novel Clostridium perfringens toxins NetB and TpeL has initiated questions regarding their role in the pathogenesis of disease. However, data showing the prevalence of these genes in C. perfringens populations are limited to certain geographical areas. If netB and tpeL are important virulence factors for disease worldwide, one would expect to find these genes in isolates from other regions as well. To address this hypothesis, C. perfringens isolates collected from Alabama broiler farms over 15 yr ago were toxin genotyped using PCR. Each isolate was screened for netB and tpeL; the major lethal toxin genes cpa, cpb, etx, and ia; and the enterotoxin gene cpe. Results of the assay showed all isolates presumed to be C. perfringens were genotypically type A, cpe negative except for one broiler litter isolate, which was genotypically type C. Only two isolates were positive for netB. Similarly, only two isolates were positive for tpeL, one of which was also netB positive. The low incidence observed for netB and tpeL indicates that these genes are not significant virulence factors for the sampled population.

Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling

The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5 °C for 3 or 24 h after preparation) were transferred to a vacuum bag and inoculated with a three-strain C. perfringens spore cocktail to obtain an inoculum of ca. 2.5 log spores/g. The bags were vacuum-sealed, and the meat was heat treated (75 °C, 20 min) and cooled within 15 h from 54.4 to 7.2 °C. Residual nitrite was determined before and after heat treatment using ion chromatography with colorimetric detection. Cooling of ham (control) stored for 3 and 24 h, resulted in C. perfringens population increases of 1.46 and 4.20 log CFU/g, respectively. For samples that contained low NaNO2 concentrations and were stored for 3 h, C. perfringens populations of 5.22 and 2.83 log CFU/g were observed with or without sodium erythorbate, respectively. Residual nitrite was stable (p > 0.05) for both storage times. Meat processing ingredients (sodium nitrite and sodium erythorbate) and their concentrations, and storage time subsequent to preparation of meat (oxygen content) affect C. perfringens spore germination and outgrowth during abusive cooling of ham.

Growth Potential of Clostridium perfringens from Spores in Acidified Beef, Pork, and Poultry Products during Chilling

The ability of Clostridium perfringens to germinate and grow in acidified ground beef as well as in 10 commercially prepared acidified beef, pork, and poultry products was assessed. The pH of ground beef was adjusted with organic vinegar to achieve various pH values between 5.0 and 5.6; the pH of the commercial products ranged from 4.74 to 6.35. Products were inoculated with a three-strain cocktail of C. perfringens spores to achieve ca. 2-log (low) or 4-log (high) inoculum levels, vacuum packaged, and cooled exponentially from 54.4 to 7.2°C for 6, 9, 12, 15, 18, or 21 h to simulate abusive cooling; the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) recommends a cooling time of 6.5 h. Total germinated C. perfringens populations were determined after plating on tryptose-sulfite-cycloserine agar and incubating the plates anaerobically at 37°C for 48 h. In addition, C. perfringens growth from spores was assessed at an isothermal temperature of 44°C. Growth from spores was inhibited in ground beef with a pH of 5.5 or below, even during extended cooling from 54.4 to 7.2°C in 21 h. In ground beef with a pH of 5.6, the growth was >1 log after 18 h of cooling from 54.4 to 7.2°C. However, 15 h of cooling controlled the growth to <1 log, regardless of the inoculum level. In addition, no growth was observed in any product with a pH ranging from 4.74 to 5.17, both during exponential abusive cooling periods of up to 21 h and during storage for 21 h at 44°C. While <1-log growth of C. perfringens from spores was observed in the pH 5.63 product cooled exponentially from 54.4 to 7.2°C in 15 h or less, the pH 6.35 product supported growth, even after 6 h of cooling from 54.4 to 7.2°C. These challenge tests demonstrate that adjustment of ground beef to pH of 5.5 or less and of barbeque products to pH of 5.63 or less inhibits C. perfringens spore germination and outgrowth during extended cooling periods from 54.4 to 7.2°C up to 15 h. Therefore, safe cooling periods for products with homogeneous, lower pHs can be substantially longer.

Effect of phosphate and meat (pork) types on the germination and outgrowth of Clostridium perfringens spores during abusive chilling.

The effect of phosphate blends and pork meat type (pale, soft, and exudative [PSE]; normal; and dark, firm, and dry [DFD]) on the germination and outgrowth of Clostridium perfringens during abusive exponential chilling times was evaluated. Two phosphates were used: tetrasodium pyrophosphate (TSPP) and sodium acid pyrophosphate (SAPP; from two different sources, SAPP(1) and SAPP(2)). The pork loins representing each meat type were ground (1/8-in. [0.3-cm] plate), and one of the three phosphate blends (SAPP(1)+SAPP(2), TSPP+SAPP(1), or TSPP+SAPP(2)) was added (0.3% total, equal proportions of 0.15% each type) with salt (1.0%). The pork was then mixed with a three-strain C. perfringens spore cocktail to obtain a final concentration of ca. 2.0 to 2.5 log spores per g. The inoculated product was heat shocked for 20 min at 75 degrees C and chilled exponentially from 54.4 to 4 degrees C in a period of 6.5, 9, 12, 15, 18, or 21 h. In control samples (PSE, normal, and DFD), the increase in C. perfringens population was <1 log CFU/g within the 6.5-h chilling period, and longer chilling times resulted in greater increases. C. perfringens population increases of 5.95, 4.73, and 5.95 log CFU/g of meat were observed in normal, PSE, and DFD pork, respectively, during the 21-h abusive chilling period. The combination of SAPP(1)+SAPP(2) was more effective than the other treatments for inhibiting C. perfringens. The types of phosphate and their blends and the meat type affected the germination and outgrowth of C. perfringens spores in cooked pork during abusive chilling periods.

The effect of feeding a commercial essential oil product on Clostridium perfringens numbers in the intestine of broiler chickens measured by real-time PCR targeting the α-toxin-encoding gene ( plc)

Proliferation of Clostridium perfringens type A in the broiler intestinal tract is related to poor growth and litter quality, and can under certain conditions lead to the development of necrotic enteritis (NE), a severe gastrointestinal disease in broilers. The aim of the present study was to investigate the influence of a commercial essential oil blend, CRINA ® Poultry, on the intestinal C. perfringens population in broilers vaccinated against coccidiosis. The observed C. perfringens levels were relatively high in general and peaked at 27 days of bird age with numbers ranging from 7 to 8 log cells per g of ileal and caecal content; these numbers correspond to those typically found in birds suffering from NE. No C. perfringens-related pathological changes were observed, however, indicating low levels of virulent strains among the C. perfringens or lack of other predisposing factors. Dietary concentrations of 100 and 200 mg/kg feed of the essential oil blend did not reduce the intestinal numbers of C. perfringens compared to a non-supplemented control group ( P>0.05). Further, the essential oil blend failed to improve ( P>0.05) both the growth and feed conversion ratio of the broilers. For rapid quantification of C. perfringens type A in broilers, a real-time PCR assay, targeting the α-toxin-encoding plc gene, was developed for use in ileal and caecal samples and was shown to be a fast and reliable alternative to conventional plate counting.