Strains Of Clostridium Perfringens Research Articles

A blend of microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25% citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlusP]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n = 23–26) and evaluated in a NE challenge model (n = 3). Birds were administered 2X cocci vaccine on d 14 and challenged with a cocktail of Clostridium perfringens strains (107) on d 17 to 19. On d 20 to 21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n = 5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t test and lesion scores analyzed using F-test two-sample for variances (P < 0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P = 0.004). Lesions scores were lower (P = 0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P = 0.19) to be heavier (848.6 g) than controls (796.2 g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.

Pathogens Detected in 205 German Farms with Porcine Neonatal Diarrhea in 2017.

Neonatal diarrhea (ND) is still a frequently observed problem in modern industrial pig production. ND is predominantly caused by bacterial and viral pathogens. The objective of this study was to give an overview of different pathogens involved in ND in Germany. In 2017, a total number of 555 litters from 205 German pig farms with clinical ND were sampled with pooled fecal samples. All samples were analyzed regarding bacterial pathogens by culture and viral pathogens by polymerase chain reaction (PCR). Isolated strains of Clostridium (C.) perfringens, Escherichia (E.) coli, and C. difficile were further characterized by molecular techniques (e.g., PCR). There were 200 litters (36%), out of 555 sampled litters of 205 farms, which were positive for at least one, while most of them were positive for two or more pathogens. Toxin-producing C. perfringens type A could be detected in 122 farms (59.2%), C. difficile in 116 (56.1%), pathogenic E. coli in 79 (38.6%), and Rotavirus type A in 72 (35%). Among E. coli isolates, enterotoxigenic (8.8%) (F4 fimbriae positive (60.0%)) and necrotoxigenic E. coli (5.3%) were the most frequently detected pathotypes. In conclusion, in most of the farms with porcine ND it turned out to be a disease mainly caused by multiple pathogens, predominantly C. perfringens type A, pathogenic E. coli, and Rotavirus type A. Nevertheless, C. difficile and necrotoxigenic E. coli might be emerging pathogens in ND.

Tracking Clostridium perfringens strains from breeding duck farm to commercial meat duck farm by multilocus sequence typing

Clostridium perfringens (C. perfringens) is an important zoonotic food-borne pathogenic microorganism. Currently, there are many reports on the prevalence of C. perfringens in poultry farms, while few studies on the prevalence and infection source of C. perfringens in egg hatcheries. The present study was undertaken to investigate and track C. perfringens from one breeding duck farm, one duck egg hatchery and one commercial meat duck farm along the production chain. A total of 334 isolates were obtained from 788 samples, including 316 type A strains (94.61 %), 11 type F strains (3.29 %) and seven type G strains (2.10 %). Antimicrobial susceptibility testing revealed that 61.87 % of the tested isolates were multidrug-resistant. Multilocus sequence typing showed that 66 representative isolates encompassed 60 different sequence types (STs), clustered in ten clonal complexes (CCs) and 20 singletons. CC2 was the most popular CC, accounting for 58.82 % (10/17) of the strains from breeding duck farm. Some strains of duck embryos were distributed in the same ST or CC as strains from breeding duck farm and duck egg hatchery, indicating close genetic relationship between them. The study showed that isolates from breeding duck farm had formed a dominant CC through long-term evolution, some duck embryos had been infected with C. perfringens during the incubation period and the infected strains should be from the duck egg hatchery or breeding duck farm with the possibility of vertical transmission. The close relationship between strains from breeding ducks and duck embryos, the high antibiotic resistance of isolates and the presence of cpe-positive or netB-positive isolates indicated that alert should be paid to such risks associated with these.

Effects of Treatment with Lactobacilli on Necrotic Enteritis in Broiler Chickens.

Growth promoter antibiotics have been commonly used for the control of necrotic enteritis (NE) in broilers for decades. However, due to a ban on the use of these antibiotics, alternatives such as probiotics have been tested widely for NE control. The present study tested the efficacy of four different species of lactobacilli (two isolates of Lactobacillus johnsonii and one of Ligilactobacillus (L.) salivarius, Limosilactobacillus (L.) reuteri, and L. crispatus) against NE. Day-old male broiler chickens were divided into six groups and orally inoculated with 1 × 107 or 1 × 108 colony-forming units (CFU) of lactobacilli on 1, 7, 14, and 20days of age. While negative and positive control groups did not receive lactobacilli, the latter was challenged with Clostridium perfringens (CP). Chickens, at 21days old, were challenged for 3days with 3 × 108CFU of a virulent strain of CP. Tissues were collected for immune system gene expression, immunophenotyping, intestinal histomorphometry, and microbiota analysis. Lactobacilli inoculation conferred partial protection in chickens against NE, marked by lowered lesion scores and improved villus:crypt ratio. Immunomodulatory effects were demonstrated by the significant alteration of interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-12p35, IL-17, and transforming growth factor beta (TGF-β) gene transcription in the duodenum and jejunum as well as subtle changes in the frequency of CD8 + T cells and B cells in the cecal tonsil of the treated chickens. Microbiota analysis showed increased levels of some bacterial phyla including Actinobacteria, Lactobacillaceae, and Firmicutes. In conclusion, these findings suggest that the use of certain lactobacilli can reduce NE severity and modulate immune responses and intestinal microbiota composition in chickens.

A stress-induced model of acute necrotic enteritis in broiler chickens using dietary corticosterone administration

Mounting evidence indicates that stress can predispose chickens to disease. The objective of the current study was to develop a method that utilized physiological stress to predispose Ross 308 broiler chickens to acute necrotic enteritis (NE). Stress was mediated through the administration of the stress hormone, corticosterone. At 11 d posthatch (p.h.), corticosterone (20 mg kg−1) administration commenced. At 12 and 13 d p.h., birds were orally inoculated with a virulent strain of Clostridium perfringens, and at 14 d p.h., birds were euthanized. Birds administered corticosterone exhibited decreased weight gain, and birds co-challenged with C. perfringens and corticosterone were affected to a higher degree. Necrotic lesions were present in birds inoculated with C. perfringens (33%), but a substantially higher prevalence of birds treated with C. perfringens and corticosterone in combination exhibited lesions (100%). Clostridium perfringens densities were correlated with necrotic lesion and histopathologic scores. Both C. perfringens and corticosterone challenge altered mRNA immune responses in the small intestine. In this regard, birds infected with the pathogen showed higher relative mRNA concentrations of toll-like receptor 2A (TLR2A), transforming growth factor beta 2 (TGFβ2), and inducible nitric oxide synthase (INOS). Birds co-challenged with C. perfringens and corticosterone showed hindered TLR2A mRNA expression. A reduction in TLR2A responses mediated by corticosterone administration suggests that the glucocorticoid suppresses immune stimulation in jejunal mucosa, which may be the underlying cause for the increased prevalence and intensity of disease observed in corticosterone treated birds. Overall, the corticosterone stress model resulted in levels of NE comparable to other models of NE that currently exist without the use of a co-infection agent. This model may facilitate the exploration of mechanisms of stress-induced NE, and the development of effective alternatives to antibiotics.

Toxigenic Profile of Clostridium perfringens Strains Isolated from Natural Ingredient Laboratory Animal Diets.

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium that ubiquitously inhabits a wide varietyof natural environments including the gastrointestinal tract of humans and animals. C. perfringens is an opportunistic enteropathogen capable of producing at least 20 different toxins in various combinations. Strains of C. perfringens are currentlycategorized into 7 toxinotypes (A, B, C, D, E, F, and G) based on the presence or absence of 6 typing-toxins (α, β, epsilon, iota, enterotoxin, and netB). Each toxinotype is associated with specific histotoxic and enteric diseases. Spontaneous enteritis due to C. perfringens has been reported in laboratory animals; however, the source of the bacteria was unknown. The Quality Assurance Laboratory (QAL) at the National Institute of Environmental Health Sciences (NIEHS) routinely screens incoming animal feeds for aerobic, enteric pathogens, such as Salmonella spp. and E. coli. Recently, QAL incorporated anaerobic screening of incoming animal feeds. To date, the lab has isolated numerous Clostridium species, including C. perfringens, from 23 lots ofnatural ingredient laboratory animal diets. Published reports of C. perfringens isolation from laboratory animal feeds couldnot be found in the literature. Therefore, we performed a toxin profile screen of our isolated strains of C. perfringens usingPCR to determine which toxinotypes were present in the laboratory animal diets. Our results showed that most C. perfringens strains we isolated from the laboratory animal feed were toxinotype A with most strains also possessing the theta toxin. Two of the C. perfringens strains also possessed the β toxin. Our results demonstrated the presence of C. perfringens in nonsterile, natural ingredient feeds for laboratory animals which could serve as a source of this opportunistic pathogen.

Antimicrobial Effects of Plant Extracts against Clostridium perfringens with Respect to Food-Relevant Influencing Factors

The application of plant extracts (PEs) could be a promising option to satisfy consumers' demand for natural additives to inhibit growth of variable pathogenic bacteria. Thus, the aim of this study was to develop a standardized microdilution method to examine the antimicrobial effects of 10 hydrophilic PEs against two strains of Clostridium perfringens facing various food-relevant influencing factors. Because of the high opacity of PEs, resazurin was used as an indicator for bacterial growth instead of pellet formation. The highest value of the MIC of the replications of each PE was defined as effective plant extract concentration (EPC), whereas the next concentration beneath the lowest MIC was defined as the ineffective plant extract concentration (IEPC). The EPCs of seven PEs, allspice, cardamom, cinnamon, clove, coriander, ginger, and mace, were between 0.625 and 10 g/kg, whereas extracts of caraway, nutmeg, and thyme showed no antimicrobial activity up to the maximum concentration tested (10 g/kg) against C. perfringens in vitro. Two intrinsic factors, sodium chloride (NaCl) and sodium nitrite (NaNO2), displayed either synergistic or additive effects or no interaction with most PEs. By combination with PEs at their IEPC (0.08 to 1.25 g/kg), MIC of NaCl and NaNO2 decreased from between 25 and 50 g/kg to between 6 and 25 g/kg and from more than 200 mg/kg to between 0.2 and 100 mg/kg, respectively. In contrast, lipid (sunflower oil) at a low concentration inhibited the antimicrobial effects of all tested PEs. For extrinsic factors, only allspice, ginger, and coriander could maintain their antimicrobial effects after being heated to 78°C for 30 min. The synergistic effect between PEs and pH values (5.0 and 5.5) was also found for all PEs. The established screening method with resazurin and defining EPC and IEPC values allows the verification of antimicrobial effects of PEs under various food-relevant influencing factors in a fast and reproducible way.

Survival of Clostridium perfringens, Staphylococcus aureus and Salmonella enterica in alternatively cured bacon during cooking and process deviations

Pork bellies were injected with four different alternative curing brines. The bellies were inoculated on the surface and at a depth of 1 cm with multiple strains of Clostridium perfringens, Staphylococcus aureus and Salmonella enterica. The bellies were processed using either a standard process cycle or an interrupted process cycle to simulate a process deviation. Additionally, laboratory simulation of the same cycles was conducted where surface inoculated pork belly samples (22 ± 1 g) were processed in a circulating water bath. Microbiological populations were determined at the beginning, mid-point and end of the cycles, and the change in population was calculated for each bacterium at each time point, by comparing the population to the initial inoculated population. Irrespective of the brine or process cycle, the populations of all of the inoculated bacteria on both the surface and interior samples had decreased by the end of the process. There was no difference in the reductions in bacterial populations for all of the inoculated bacteria by brine type or by sample location (P > 0.30). There were differences in the microbial population reductions for C. perfringens attributable to the processing cycle (P < 0.001), with less population reductions associated with the standard cycle when compared to the interrupted cycle. However, no differences (P > 0.10) were observed in the population reductions between the two processing cycles for either S. aureus or S. enterica.

Tracing Clostridium perfringens strains from beef processing of slaughter house by pulsed-field gel electrophoresis, and the distribution and toxinotype of isolates in Shaanxi province, China

The purpose of this study was to investigate the distribution and specify the transmission and cross-contamination of Clostridium perfringens (C. perfringens) in the beef slaughtering and butchering process. The prevalence of 21.2% (150/708) yielded 208 isolates of C. perfringens, including 80.8% type A and 19.2% type D, 0.4% (3/708) samples carried both type A and D strains, and 72.5% type D isolates carried both cpe and atyp.cpb2 genes. C. perfringens were identified through the whole slaughtering process but no type F (cpe and cpa isolates) was found. 69 isolates were further analyzed and classified into 28 PFGE genotypes and clade I contained 94.2% isolates and 24 PFGE genotypes, which showed the genetic diversity and epidemic correlation. Our study traced C. perfringens contamination along the handling processes and showed a gradually ascending contamination rate during the whole process, revealing widespread cross-contamination from the feces and hides of slaughtered cattle to the carcass in the slaughtering workshop, so as from tools and personnel to meat of the cutting workshops. Strains from different slaughterhouses (regions) have high homology, and type A is the predominant toxinotype. It is necessary to monitor and control several key points of cross-contamination during slaughtering process to reduce a risk of C. perfringens infection.

Novel ex vivo screening assay to preselect farm specific pre- and probiotics in pigs

A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.

A Rapid and Simple Assay Correlates In Vitro NetB Activity with Clostridium perfringens Pathogenicity in Chickens.

Necrotic enteritis is an important enteric disease in poultry, caused by NetB-producing Clostridium (C.) perfringens strains. As no straight-forward method to assess the NetB activity of C. perfringens was available, we aimed to develop an easy, high-throughput method to measure the NetB activity produced by C. perfringens. First, the appearance of C. perfringens on different avian blood agar plates was assessed. Based on the size of the haemolysis surrounding the C. perfringens colonies, NetB-positive strains could phenotypically be discriminated from NetB-negative strains on both chicken and duck blood agar. Additionally, strains producing the consensus NetB protein induced more pronounced haemolysis on chicken blood agar as compared to the weak outer haemolysis induced by A168T NetB-variant-producing C. perfringens strains. Next, a 96-well plate-based haemolysis assay to screen NetB activity in the C. perfringens culture supernatants was developed. Using this assay, a positive correlation between the in vitro NetB activity and virulence of the C. perfringens strains was shown. The developed activity assay allows us to screen novel C. perfringens isolates for their in vitro NetB activity, which could give valuable information on their disease-inducing potential, or identify molecules and (bacterial) metabolites that affect NetB expression and activity.

Pathogenic Characterization of Clostridium perfringens Strains Isolated From Patients With Massive Intravascular Hemolysis.

Sepsis caused by Clostridium perfringens infection is rare but often fatal. The most serious complication leading to poor prognosis is massive intravascular hemolysis (MIH). However, the molecular mechanism underlying this fulminant form of hemolysis is unclear. In the present study, we employed 11 clinical strains isolated from patients with C. perfringens septicemia and subdivided these isolates into groups H and NH: septicemia with (n = 5) or without (n = 6) MIH, respectively. To elucidate the major pathogenic factors of MIH, biological features were compared between these groups. The isolates of two groups did not differ in growth rate, virulence-related gene expression, or phospholipase C (CPA) production. Erythrocyte hemolysis was predominantly observed in culture supernatants of the strains in group H, and the human erythrocyte hemolysis rate was significantly correlated with perfringolysin O (PFO) production. Correlations were also found among PFO production, human peripheral blood mononuclear cell (PBMC) cytotoxicity, and production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by human PBMCs. Analysis of proinflammatory cytokines showed that PFO induced tumor necrosis factor-α (TNF-α), IL-5, IL-6, and IL-8 production more strongly than did CPA. PFO exerted potent cytotoxic and proinflammatory cytokine induction effects on human blood cells. PFO may be a major virulence factor of sepsis with MIH, and potent proinflammatory cytokine production induced by PFO may influence the rapid progression of this fatal disease caused by C. perfringens.

Genotyping Toxins of Clostridium perfringens Strains of Rabbit and Other Animal Origins

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Buffered formic acid and a monoglyceride blend coordinately alleviate subclinical necrotic enteritis impact in broiler chickens

The objective of this study was to evaluate the effect of 2 different doses of a partially buffered formic acid product (Amasil NA; 61% formic acid, 20.5% sodium formate), and a monoglyceride blend of short- and medium-chain fatty acids (BalanGut LS P) on necrotic enteritis (NE) infected broilers in terms of performance, intestinal microbial population and short-chain fatty acids concentrations in the gastrointestinal tract. A total of 528-day-old as hatched Ross 308 broilers were allocated to 48 pens with 11 birds in each pen. Six dietary treatments applied in the study were: T1) nonsupplemented diet (Control); T2) antibiotic supplemented diets; T3) and T4) high (Starter: 0.5%; Grower and Finisher: 0.5%) and low (Starter: 0.3%; Grower and Finisher: 0.2%) dose of Amasil NA; and groups T5) and T6) high (Starter: 0.3%; Grower and Finisher: 0.2%) and low dose (Starter: 0.3%; Grower: 0.15%; Finisher: 0.075%) of (BalanGut LS P). All birds in this study were fed starter (d 0-10), grower (d 11–24) and finisher (d 25–35) diets and challenged with NE. To induce subclinical NE, oral administrations of Eimeria oocysts (d 9) followed by inoculation of Clostridium perfringens strains (d 14 and 15) were applied.Results showed that birds fed the high dose of Amasil NA, had a higher feed conversion ratio (FCR,P < 0.05) compared to the nonsupplemented group during the starter period. Antibiotic supplementation reduced FCR during the grower (P < 0.001), finisher (P < 0.05) and overall (P < 0.001) periods of the experiment. Both levels of BalanGut LS P and low levels of Amasil NA enhanced overall FCR (P < 0.05) compared to the birds in the nonsupplemented group. Compared to the nonsupplemented group, high levels of Amasil NA and low levels of BalanGut LS P improved FCR in the finisher stage (P < 0.05). On d 16, cecum digesta of birds fed with antibiotic supplemented diets showed a significantly lower number of C. perfringens (P < 0.001) compared to the nonsupplemented and high level of BalanGut LS P group. Bacillus (P < 0.01) and Ruminococcus numbers were significantly lower in the birds fed with high level of Amasil NA (P < 0.05) compared to the antibiotic supplemented diets. High doses of Amasil NA, showed the highest propionate concentration in the cecum (P < 0.001). The study suggests that supplementation of BalanGut LS P and Amasil NA at different feeding phases may achieve optimal performance improvement in broilers under NE challenge.

Taxonomic characteristics of micro-organisms isolated from the Baikal seal

The relevance of the ecological problem of the mass death of the Baikal seal (Pusa sibirica Gm.) forces researchers to establish causes of this phenomenon. The article presents the results of microbiological studies of swabs isolated from the nasal, oral cavities and rectum. The taxonomic characteristics of microorganisms and their virulence were studied. 14 Baikal seals found in the nerpinaria of Listvyanka were tudied. It was established that in the seal there are the following microorganisms: Clostridium perfringens, Escherichia coli, Enterococcus faecalis, Staphylococcus warneri, Pseudomona aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, Proteus hauseri, Proteus vulgaris bellisella, Proteus migarisella, rabitemorganii, Psychrobactersanguinis, Enterobacter cloacae. When studying the virulence of microorganisms, it was found that 58% of the strains do not cause the death of laboratory animals, they are mainly the representatives of opportunistic microflora. 42% of the strain were pathogenic, of which two strains of Plesi omonas shigelloides and 42 strains of Clostridium perfringens were identified.

Over-processed meat and bone meal and phytase effects on broilers challenged with subclinical necrotic enteritis: Part 3. Bone mineralization and litter quality.

This study was conducted to determine the effect of necrotic enteritis (NE), phytase level and meat and bone meal (MBM) processing on bone mineralization of broilers and litter quality. Ross 308 male broiler chicks (n = 768) were allotted to 48 pens with 16 birds each. There were 8 dietary treatments in a 2 × 2 × 2 factorial arrangement. Factors were NE challenge (no or yes), phytase level (500 or 5,000 FTU/kg), and MBM (as-received or over-processed). Half of the birds were challenged with field strains of Eimeria spp. at d 9 and 108 CFU per mL of Clostridium perfringens strain EHE-NE18 on d 14 and 15. The middle toe, tibia and femur of 2 birds per pen were excised at d 16 and 29 for determination of ash, breaking strength (BS) and bone mineralization. At d 42, all were assessed for hock burns and litter was scored and assessed for dry matter (DM). At d 16, challenged birds had lower toe ash (P < 0.01), femur ash (P < 0.001), tibia ash (P < 0.001) and tibial BS (P < 0.001) than unchallenged birds. At d 16, challenged birds fed high phytase and over-processed MBM had higher toe Mn than those fed low phytase and as-received MBM. At d 29 unchallenged birds fed high phytase and as-received MBM had a higher toe Mn than those fed over-processed MBM. At d 16, a phytase × MBM interaction was detected for femur Zn concentration (P < 0.05), where a higher level of Zn was observed in the high phytase group fed over-processed MBM. At d 16, tibial Ca (P < 0.05) and P (P < 0.05) were lower in the challenged whereas the femur K (P < 0.001), Mn (P < 0.01) and Na (P < 0.001) were higher in the challenged at d 16. At d 42, challenged birds had higher litter DM (P = 0.058) and fewer hock burns than those unchallenged (P < 0.05). In conclusion, NE impaired bone traits but high phytase and over-processed MBM increased bone mineral contents. Cases of hock burns may be lower under NE incidences due to lower livability of birds reducing litter wetness.

Differential expression of intestinal genes in necrotic enteritis challenged broiler chickens with 2 different Clostridium perfringens strains

The primary cause of necrotic enteritis (NE) disease in chickens is the NetB-positive Clostridium perfringens bacterium. Many factors are known to affect the severity of NE in the challenge models of broiler chickens, and one of these factors is the virulence of C. perfringens strain. This study was conducted to evaluate the effect of 2 pathogenic C. perfringens strains in a NE challenge model on gut health and mRNA expression of genes encoding apoptosis, tight junction, immunity, and nutrient transporters in broilers. Day-old Ross-308 male broilers (n = 468) were allocated in a 2 × 3 factorial arrangement of treatments with in-feed antibiotics (no or yes) and challenge (Non, C. perfringens strain NE18, and C. perfringens strain NE36) as the factors. The birds in the challenged groups were inoculated with Eimeria species on day 9 and with a fresh suspension of C. perfringens NE18 or NE36 on day 14 and 15. Sample collection was performed on 2 birds of each pen on day 16. Necrotic enteritis challenge, impaired feed conversion ratio during day 0 to 16 compared with the control group where the effect of the NE36 challenge was more severe than that with NE18 (P < 0.001). The mRNA expression of mucin-2, immunoglobulin-G, occludin (P < 0.001), and tight junction protein-1 (P < 0.05) genes were downregulated in both challenged groups compared with the nonchallenged counterparts. Antibiotic supplementation, on the other hand, increased weight gain, and feed intake in all challenged birds (P < 0.01), but upregulated mucin-5ac and alanine, serine, cysteine, and threonine transporter-1 (P < 0.05) only in the NE18 challenged birds. The challenge with NE36 significantly upregulated caspase-8 and claudin-1 (P < 0.001), but downregulated glucose transporter-2 (P < 0.001) compared with the NE18 challenge. These results suggest that NE challenge is detrimental to the performance of broilers through compromised intestinal health, and different C. perfringens strains can affect the severity of the disease through modulating the expression of intestinal genes encoding proteins responsible for apoptosis, gut integrity, immunity, mucus production, and nutrient transporters.

Gas gangrene-associated gliding motility is regulated by the Clostridium perfringens CpAL/VirSR system

Clostridium perfringens strains cause a wide variety of human and animal disease, including gas gangrene or myonecrosis. Production of toxins required for myonecrosis, PFO and CPA, is regulated by the C. perfringens Agr-like (CpAL) system via the VirSR two-component system. Myonecrosis begins at the site of infection from where bacteria migrate deep into the host tissue likely using a previously described gliding motility phenotype. We therefore assessed whether gliding motility was under the control of the CpAL/VirSR regulon. The migration rate of myonecrosis-causing C. perfringens strain 13 (S13) was investigated during a 96 h period, including an adaptation phase with bacterial migration (∼1.4 mm/day) followed by a gliding phase allowing bacteria faster migration (∼8.6 mm/day). Gliding required both an intact CpAL system, and signaling through VirSR. Mutants lacking ΔagrB, or ΔvirR, were impaired for onward gliding while a complemented strain S13ΔagrB/pTS1303 had the gliding phenotype restored. Gene expression studies revealed upregulated transcription of pili genes (pilA1, pilA2 and pilT) whose encoded proteins were previously found to be required for gliding motility and CpAL/VirSR-regulated pfoA and cpa toxin genes. Compared to S13, transcription of cpa and pfoA significantly decreased in S13ΔagrB, or S13ΔvirR, strains but not that of pili genes. Further experiments demonstrated that mutants S13ΔpfoA and S13Δcpa migrated at the same rate as S13 wt. We demonstrated that CpAL/VirSR regulates C. perfringens gliding motility and that gliding bacteria have an increased transcription of toxin genes involved in myonecrosis.

Characterization of a novel bacteriophage φCJ22 and its prophylactic and inhibitory effects on necrotic enteritis and Clostridium perfringens in broilers

High necrotic enteritis (NE) incidence and mortality rates in poultry can be caused by Clostridium perfringens (CP) coinfected with Eimeria spp., a causative agent of coccidiosis. Banning of prophylactic use of antibiotics in feed has been accompanied by increased NE outbreaks, resulting in economically devastating losses to the broiler industry. To determine alternatives for controlling NE, we isolated CP-specific bacteriophages (BP), characterized their properties, evaluated their inhibitory effects on pathogenic CP, selected a highly effective phage (φCJ22), and used φCJ22 as a dietary supplement in experimental NE-afflicted broiler chickens. Male broilers (n = 780) were randomly assigned to 60 pens (n = 13 broilers/pen) and into 5 groups [CP-uninfected negative control (NC), basal diet (BD) without CP and BP; CP-infected positive control (PC), BD + CP; and 3 BP groups receiving low- (LP; BD + CP+105 BP), medium- (MP; BD + CP+106 BP), and high-phage (HP; BD + CP+107 BP plaque-forming units/kg) concentrations]. The results showed that MP and HP groups presented an antimicrobial activity toward clinical CP isolate strains, and the groups decreased NE lesions and mortality rates without changes in chicken performance at the end of the experimental period. After CP-challenge body weight gain and feed efficiency were significantly lower in phage-fed groups than that in the PC group (P < 0.05), and NE-associated mortality was the lowest in the HP group (P < 0.001). Moreover, histopathology revealed lesser gastrointestinal mucosal damage in the NC and BP-treated (LP, MP, and HP) groups than that in the PC group, and MP and HP significantly lowered viable CP number in the cecum content by up to 1.24log10 relative to only CP-infected PC group (P < 0.05). These findings suggest that addition of φCJ22 to chicken feed might effectively ameliorate NE, which is accompanied by reduced CP strains in the gut and compensate the performance of NE-afflicted broilers.

Molecular Characterization of Clostridium perfringens Strains Isolated in Italy.

Clostridium (C.) perfringens is the causative agent of several diseases and enteric infections in animals and humans. The pathogenicity of the bacterium is largely mediated by the production of a wide range of toxins. Individual C. perfringens strains produce only subsets of this toxin repertoire, which permits the classification in seven toxinotypes (A–G). In addition, a variety of minor toxins further characterizes the single strains. The aim of this work was to evaluate, using Polymerase Chain Reaction (PCR) assays, the diversity of 632 C. perfringens strains isolated in Italy over 15 years. The genotyped strains were analyzed to determine the presence of major and minor toxins (cpe, consensus, and atypical cpb2), their geographical origins, and the source of isolation (animal species or food). Our study shows that toxinotype A had the greatest representation (93%) and correlated mainly with consensus cpb2 in a variety of animal species, as well as with atypical cpb2 in the five food samples. Type D, associated with cpe and atypical cpb2 minor toxins, was identified in 3% of the cases, and type F was identified in 2.5%. Seven type C isolates (1.1%) were detected in cattle, whereas the only type B atypical cpb2 isolated in Italy was detected in a goat, and one type E cpe+atypical cpb2 was detected in a sheep. Type G was not detected.