The incidence of necrotic enteritis in turkeys is associated with farm, season and faecal Eimeria oocyst counts.

BACKGROUND: Specific studies on the epidemiology of necrotic enteritis in turkeys are absent in the literature. Necrotic enteritis is common in turkeys and a leading cause of use of therapeutic antibiotics. This study describes the incidence of necrotic enteritis in turkey farms, and the association between incidence and bird age, season, faecal oocyst counts, grow-out size and feed mill. RESULTS: Necrotic enteritis was diagnosed post mortem in 20.2 % of 545 grow-outs of commercial female and male B.U.T. 10 turkeys started during the years 2010-2016. 80 % of all cases occurred at four to seven weeks of age. Median (minimum-maximum) age at disease detection was 37 (18-115) days. Turkey age at detection was influenced by season, and varied from 33 days among grow-outs hatched in February to 42 days among those hatched in July-August. The incidence also varied with season, showing peak occurrence among grow-outs hatched during February-March and the lowest incidence in turkeys hatched in July-August. 59 % of all cases were detected in 25 % of the farms. The incidence per farm varied from below 4 to 59 %. A multilevel mixed-effects logistic regression model indicated clear impacts of farm and season on incidence, and border-line impacts of grow-out size and feed mill. Grow-outs diagnosed with necrotic enteritis had higher counts of faecal Eimeria oocysts than grow-outs without a diagnosis. This difference was particularly clear during the high-risk period at five to seven weeks of age. Necrotic enteritis was the cause of treatment with therapeutic antibiotics in 88.2 % of all cases of treatment. CONCLUSIONS: Our data indicate that necrotic enteritis incidence in turkeys can be substantially influenced by risk factors at farm level. The incidence showed two seasonal peaks; a moderate peak in turkeys hatched in October/November and a marked peak in turkeys hatched during February/March. Mitigation measures at the farm may therefore be of particular importance during these months in farms located in the Northern temperate zone. Measures which effectively reduce counts of faecal Eimeria oocyst are likely to be among the more promising actions to take both at the farm and at population level.

Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions.

BACKGROUND: Necrotic enteritis is a significant problem to the poultry industry globally and, in Norway up to 30% of Norwegian turkey grow-outs can be affected. However, despite an awareness that differences exist between necrotic enteritis in chickens and turkeys, little information exists concerning the pathogenesis, immunity, microbiota or experimental reproduction of necrotic enteritis in turkeys. In particular, it is important to determine the appearance of the gross lesions, the age dependency of the disease and the role of netB toxin of Clostridium perfringens. To this end, we report our findings in developing an in vivo experimental model of necrotic enteritis in turkeys. RESULTS: A four tier (0-3) scoring system with clearly defined degrees of severity of macroscopic intestinal lesions was developed, based on 2312 photographic images of opened intestines from 810 B.U.T. 10 or B.U.T. Premium turkeys examined in nine experiments. Loss of macroscopically recognizable villi in the anterior small intestine was established as the defining lesion qualifying for a score 3 (severe intestinal lesions). The developed scoring system was used to identify important factors in promoting high frequencies of turkeys with severe lesions: a combined Eimeria meleagrimitis and Clostridium perfringens challenge, challenge at five rather than 3 weeks of age, the use of an Eimeria meleagrimitis dose level of at least 5000 oocysts per bird and finally, examination of the intestines of 5-week-old turkeys at 125 to 145 h after Eimeria meleagrimitis inoculation. Numbers of oocysts excreted were not influenced by Clostridium perfringens inoculation or turkey age. Among three different lesion score outcomes tested, frequency of severe lesions proved superior in discriminating between impact of four combinations of Clostridium perfringens inoculation and turkey age at challenge. CONCLUSIONS: This study provides details for the successful establishment of an in vivo model of necrotic enteritis in turkeys.

Epidemiologic aspects of necrotic enteritis in broiler chickens - disease occurrence and production performance.

Since future conventional broiler production can no longer rely upon in-feed antimicrobials (anticoccidials and antibiotic growth promoters), understanding the most important non-antimicrobial factors influencing occurrence of necrotic enteritis (NE) in poultry will become urgent. Solid population-based data on NE occurrence are scarce. Additionally, data on cholangiohepatitis (CPH) at slaughter is a useful indirect measurement of NE occurrence. Existing data suggest that coccidiosis and nutritional factors are among the most important determinants of NE occurrence. Dietary cereal contents and dietary level of animal proteins can both influence NE occurrence, but cereal composition may be more important because cereals constitute a larger portion of the diet. Losses associated with NE vary depending on the severity of the disease, but data indicate that the farmers' profit may be reduced on average with as much as one third during an epidemic of clinical disease.

Substitution of dietary fish oil with plant oils is associated with shortened mid intestinal folds in Atlantic salmon (Salmo salar).

BACKGROUND: Fish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds. RESULTS: Morphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes. CONCLUSIONS: Shortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietary fish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon.

The noncommensal bacterium Methylococcus capsulatus (Bath) ameliorates dextran sulfate (Sodium Salt)-Induced Ulcerative Colitis by influencing mechanisms essential for maintenance of the colonic barrier function.

Dietary inclusion of a bacterial meal has recently been shown to efficiently abolish soybean meal-induced enteritis in Atlantic salmon. The objective of this study was to investigate whether inclusion of this bacterial meal in the diet could abrogate disease development in a murine model of epithelial injury and colitis and thus possibly have therapeutic potential in human inflammatory bowel disease. C57BL/6N mice were fed ad libitum a control diet or an experimental diet containing 254 g/kg of body weight BioProtein, a bacterial meal consisting of Methylococcus capsulatus (Bath), together with the heterogenic bacteria Ralstonia sp., Brevibacillus agri, and Aneurinibacillus sp. At day 8, colitis was induced by 3.5% dextran sulfate sodium (DSS) ad libitum in the drinking water for 6 days. Symptoms of DSS treatment were less profound after prophylactic treatment with the diet containing the BioProtein. Colitis-associated parameters such as reduced body weight, colon shortening, and epithelial damage also showed significant improvement. Levels of acute-phase reactants, proteins whose plasma concentrations increase in response to inflammation, and neutrophil infiltration were reduced. On the other, increased epithelial cell proliferation and enhanced mucin 2 (Muc2) transcription indicated improved integrity of the colonic epithelial layer. BioProtein mainly consists of Methylococcus capsulatus (Bath) (88%). The results that we obtained when using a bacterial meal consisting of M. capsulatus (Bath) were similar to those obtained when using BioProtein in the DSS model. Our results show that a bacterial meal of the noncommensal bacterium M. capsulatus (Bath) has the potential to attenuate DSS-induced colitis in mice by enhancing colonic barrier function, as judged by increased epithelial proliferation and increased Muc2 transcription.

Gizzard erosion and ulceration syndrome in chickens and turkeys: a review of causal or predisposing factors.

Gizzard erosion and ulceration syndrome (GEU) was described for the first time in the 1930s. The main focus of early studies was on nutritional deficiencies and peroxidation of highly polyunsaturated fatty acids as causative factors. During the 1970s and 1980s the focus was moved towards toxic substances in the feed. Scott's review in 1985 concluded that overproduction of gastric acid induced by gizzerosine was a major cause of GEU. During the last decades, serotype 1 of fowl adenovirus A and Clostridium perfringens have been implicated as important pathogenic agents in the development of GEU in chickens. Although GEU is globally distributed and its subclinical form appears to be common in commercial poultry flocks, the condition is rarely mentioned in standard textbooks on poultry health. This regrettable fact is probably due in part to the lack of one definitive cause of the syndrome.

Extensive genome analysis identifies novel plasmid families in Clostridium perfringens.

Globally, the anaerobic bacterium Clostridium perfringens causes severe disease in a wide array of hosts; however, C. perfringens strains are also carried asymptomatically. Accessory genes are responsible for much of the observed phenotypic variation and virulence within this species, with toxins frequently encoded on conjugative plasmids and many isolates carrying up to 10 plasmids. Despite this unusual biology, current genomic analyses have largely excluded isolates from healthy hosts or environmental sources. Accessory genomes, including plasmids, also have often been excluded from broader scale phylogenetic investigations. Here we interrogate a comprehensive collection of 464 C. perfringens genomes and identify the first putative non-conjugative enterotoxin (CPE)-encoding plasmids and a putative novel conjugative locus (Bcp) with sequence similarity to a locus reported from Clostridium botulinum. We sequenced and archived 102 new C. perfringens genomes, including those from rarely sequenced toxinotype B, C, D and E isolates. Long-read sequencing of 11 C. perfringens strains representing all toxinotypes (A-G) identified 55 plasmids from nine distinct plasmid groups. Interrogation of the 464 genomes in this collection identified 1045 plasmid-like contigs from the nine plasmid families, with a wide distribution across the C. perfringens isolates. Plasmids and plasmid diversity play an essential role in C. perfringens pathogenicity and broader biology. We have expanded the C. perfringens genome collection to include temporal, spatial and phenotypically diverse isolates including those carried asymptomatically in the gastrointestinal microbiome. This analysis has resulted in the identification of novel C. perfringens plasmids whilst providing a comprehensive understanding of species diversity.

Anticoccidial Vaccination Is Associated with Improved Intestinal Health in Organic Chickens.

Eimeria spp. and Clostridium perfringens (CP) are pathogens associated with coccidiosis and necrotic enteritis (NE) in broiler chickens. In this study we evaluated the effect of anticoccidial vaccination on intestinal health in clinically healthy organic Ross 308 chickens. On each of two farms, one unvaccinated flock (A1 and B1) was compared to one vaccinated flock (A2 and B2) until ten weeks of age (WOA). Faecal oocysts were counted weekly, and species were identified by PCR (ITS-1 gene). Lesion scoring, CP quantification and PCR targeting the CP NetB toxin gene were performed at three, four, and six WOA and chickens were weighed. Necropsies were performed on randomly selected chickens to identify coccidiosis/NE. Oocyst shedding peaked at three WOA in all flocks. Later oocyst shedding (E. tenella/E. maxima) in unvaccinated flocks at 5-7 WOA coincided with coccidiosis/NE. Although results differed somewhat between farms, vaccination was associated with lower intestinal lesion scores, reduced caecal CP counts, lower proportions of netB-positive CP, lower body weight at three-four WOA, and similar or slightly increased body weight at six WOA. In conclusion, the intestinal health of organic broilers can benefit from anticoccidial vaccination when oocyst exposure levels are high.

Effect of Feed Additives as Alternatives to In-feed Antimicrobials on Production Performance and Intestinal Clostridium perfringens Counts in Broiler Chickens.

Numerous non-antibiotic feed additives (alternatives to antibiotics, ATAs) have been marketed, but few have been evaluated under uniform testing conditions modelling commercial flocks. We compared 24 ATA treatments and the ionophorous coccidiostat narasin against a diet without any feed additives. Feed conversion ratio and body weight gain were registered from day 0 to 28 in Ross 308 chickens housed on litter floor. The chickens were challenged with Eimeria spp., and cecal Clostridium perfringens (CP) counts were investigated. Active components from all ATA classes had a positive impact on intestinal health or production performance. Whereas narasin had a strong CP-reducing effect in combination with performance-promoting impact, only two ATA treatments achieved significantly beneficial effects on CP counts as well as feed conversion during the time span following Eimeria challenge. Active components present in these two treatments include a Bacillus subtilis probiotic strain, short- and medium-chain fatty acids and Saccharomyces cerevisiae components. Different ATA classes had beneficial impact during distinct rearing phases and on specific performance targets, suggesting that optimizing combinations and use of active components can make ATAs even more useful tools in broiler rearing without the use of in-feed antimicrobials. Further studies of promising ATAs and ATA combinations are required.

Significant reduction of vancomycin resistant E. faecium in the Norwegian broiler population coincided with measures taken by the broiler industry to reduce antimicrobial resistant bacteria.

Vancomycin resistant enterococci (VRE) belong to the most common causes of nosocomial infections worldwide. It has been reported that use of the glycopeptide growth promoter avoparcin selected for a significant livestock-reservoir of VRE in many European countries, including Norway. However, although avoparcin was banned as a feed-additive in 1995, VRE have for unknown reasons consistently been reported in samples from Norwegian broilers. When avoparcin was banned, broiler-feed was supplemented with the polyether ionophore narasin in order to control the diseases coccidiosis and the frequent sequela necrotic enteritis. A potential link between transferrable vancomycin resistance and reduced susceptibility to narasin was recently reported. The use of narasin as a feed additive was abolished by the Norwegian broiler industry in 2016 and since then, broilers have been reared without in-feed antibacterial supplements. In this study, we demonstrate that all VRE isolates from Norwegian broilers collected in 2006-2014 displayed reduced susceptibility to narasin. Surveillance data collected two years after the narasin abolishment show a significant reduction in VRE, below the detection limit of the surveillance method, and a concurrent marked reduction in Enterococcus faecium with reduced susceptibility to narasin. The significant decline of E. faecium with reduced susceptibility to these antimicrobial compounds also coincided with an increased focus on cleaning and disinfection between broiler flocks. Furthermore, data from a controlled in vivo experiment using Ross 308 broilers indicate that the proportion of E. faecium with reduced susceptibility to narasin was heavily reduced in broilers fed a narasin-free diet compared to a diet supplemented with narasin. Our results are consistent with that the abolishment of this feed additive, possibly in combination with the increased focus on cleaning and disinfection, has had a substantial impact on the occurrence of VRE in the Norwegian broiler population.

A survey of the economic impact of subclinical Eimeria infections in broiler chickens in Norway.

The objective of this work was to examine the impact of subclinical coccidial infection on commercial performance, expressed as a modified European Production Index, in broilers. Performance data, and litter and faecal samples, were collected from two independent observational surveys of Norwegian broilers receiving in-feed narasin during 2000 to 2004. Numbers of oocysts per gram (OPG) of litter collected during rearing (Study 1) or faecal samples collected at slaughter (both studies), and relative frequencies of Eimeria species categories (both studies) were calculated. Polymerase chain reaction-based identification of Eimeria species was performed in Study 2. A definition of flocks at risk of impaired performance associated with coccidia ("risk flock"), using the predominant species and OPG level as criteria, was tested. Coccidia had a significant effect on performance in the first, but not the second study. In Study 1 the following coccidia variables were found to be associated with impaired performance in multivariate models: OPG at slaughter (ordinal), mean OPG during rearing (ordinal) and "risk flock" (binomial). The European Production Index was approximately 9% lower in flocks with infection levels >50 000 OPG at slaughter in Study 1. The composition of coccidial populations shifted between Study 1 and Study 2, from a dominance of medium and large oocysts to a dominance of small oocysts. There was a substantial increase in prevalence of coccidial infection from Study 1 to Study 2, but mean infection levels were similar in the two surveys. The "risk flock" definition was useful as an indicator of coccidia-associated performance loss in Study 1, where subclinical coccidiosis was an important factor. The results suggest that the economic importance of subclinical coccidiosis may vary substantially with time, and they emphasize the need for population studies on the importance and dynamics of specific coccidial infections under different field conditions.

Coccidial infections in commercial broilers: epidemiological aspects and comparison of Eimeria species identification by morphometric and polymerase chain reaction techniques.

The objective of this study was to add to existing knowledge of the epidemiology and the aetiology of coccidial infections in commercial broiler flocks. Polymerase chain reaction (PCR) and morphometric identification of the Eimeria species were compared as means of differentiation in the field samples of faeces and litter. For morphometry, the Eimeria species were categorized into three groups based on lengths of the oocysts. Two random samples of commercial broilers were studied, one during 2000/01 and the other during 2003/04. The prophylactic regime (in-feed narasin), husbandry and methods applied were broadly the same for both subpopulations. Coccidial infection prevalence increased from approximately 45% to approximately 75% during this period, but infection levels (oocysts per gram of faeces) did not significantly change. There were substantial geographical differences in both prevalence and infection levels. A change in Eimeria species profile occurred during the study period. Five Eimeria species were identified at slaughter, by PCR targeting the ITS-1 region of the genome; Eimeria acervulina (100%), Eimeria tenella (77%), Eimeria maxima (25%), Eimeria praecox (10%) and Eimeria necatrix (2%). PCR and morphometric tentative identification were in complete agreement in only 49% of the cases.

Dietary Deoxynivalenol (DON) May Impair the Epithelial Barrier and Modulate the Cytokine Signaling in the Intestine of Atlantic Salmon (Salmo salar).

Impaired growth, immunity, and intestinal barrier in mammals, poultry, and carp have been attributed to the mycotoxin deoxynivalenol (DON). The increased use of plant ingredients in aquaculture feed implies a risk for contamination with mycotoxins. The effects of dietary DON were explored in 12-month-old Atlantic salmon (Salmo salar) (start weight of 58 g) that were offered a standard feed with non-detectable levels of mycotoxins (control group) or 5.5 mg DON/kg feed (DON group). Each group comprised two tanks with 25 fish per tank. Five fish from each tank were sampled eight weeks after the start of the feeding trial, when mean weights for the control and DON groups were 123.2 g and 80.2 g, respectively. The relative expression of markers for three tight junction proteins (claudin 25b, occludin, and tricellulin) were lower, whereas the relative expression of a marker for proliferating cell nuclear antigen was higher in both the mid-intestine and the distal intestine in fish fed DON compared with fish from the control group. The relative expression of markers for two suppressors of cytokine signaling (SOCS1 and SOCS2) were higher in the distal intestine in fish fed DON. There was no indication of inflammation attributed to the feed in any intestinal segments. Our findings suggest that dietary DON impaired the intestinal integrity, while an inflammatory response appeared to be mitigated by suppressors of cytokine signaling. A dysfunctional intestinal barrier may have contributed to the impaired production performance observed in the DON group.

What has happened in norway after the ban of avoparcin? Consumption of antimicrobials by poultry.

When avoparcin was prohibited for use as feed additive in poultry in Norway on 31 May 1995, an increased incidence of Clostridium perfringens-associated necrotic enteritis (NE) and an increase in the use of antibacterial (AB) drug therapy in meat-type poultry was expected. The consumption of AB drugs for use against NE in poultry in the period 1990-2001 was investigated by use of sales statistics at the drug-wholesaler level. Defined daily dose (DDD) per kg live weight poultry was the unit of measurement for drug use (to correct for differences in the dosages). Sales figures of the AB drugs were converted to number of DDDpoultry sold for the numbers of broilers at risk (broilers were 97% of the slaughter poultry). Estimated annual percentages of the broilers treated against NE increased abruptly after the avoparcin ban--but in 1996, this figure declined to the same level as before the ban and has remained at that low level since then. In November 1995, narasin was approved temporarily as an ionophore feed additive (IFA) in broilers. The usage patterns of IFAs in broilers were measured as the weight of feed to which an IFA was added per broiler chicken produced. In 1996-2001, the IFAs used in broilers were predominantly narasin. We note that the temporary increase in NE after the avoparcin ban coincide with the period before narasin became available. The increase in the consumption of AB drugs for the treatment of NE in poultry following the avoparcin ban has been negligible.

Type C and C/D toxigenic Clostridium botulinum is not normally present in the intestine of healthy broilers.

Toxigenic Clostridium botulinum spores are widely distributed in wetland environments and are frequently recovered from healthy wild birds, where ingestion of toxic maggots from carcasses is considered a major initiating factor for botulism outbreaks. Toxic carcasses can perpetuate an existing outbreak also in poultry, but their importance as an initiating factor for poultry botulism is less clear. Although toxigenic C. botulinum can be recovered from healthy broilers during outbreaks, there are almost no data on the prevalence in healthy broiler flocks (and, by extension, carcasses) and their environment. To test the hypothesis that toxigenic C. botulinum is frequently present in healthy broilers, we examined 100 healthy broiler flocks and environmental samples from 30 broiler houses 3 to 4 years after an epidemic of botulism in the broiler population. None of the 100 flocks yielded type C or C/D botulinum toxin genes using real time PCR whereas a flock house that had remained empty since the most recent outbreak yielded type C (or C/D) toxin genes. The absence of toxigenic C. botulinum in all examined flocks indicates that botulism is a sporadic and exogenously acquired event in this broiler population and unlikely to mirror the epidemiology in wild birds.

Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens.

Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a ß-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a ß-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

Genetic relatedness and netB prevalence among environmental Clostridium perfringens strains associated with a broiler flock affected by mild necrotic enteritis.

In a previous study we investigated pulsed-field gel electrophoresis (PFGE) genotype diversity and prevalence of the netB toxin gene in Clostridium perfringens (CP) isolates recovered from a broiler flock (flock 1) affected by necrotic enteritis (NE). In this follow-up work, we examined samples collected before placement of flock 1, to see if NE during rearing could be traced back to the cleaned and empty building or the day-old chicks. Litter from the next flock in the same building (flock 2) was also examined. We detected 25 different PFGE genotypes, five of which were found only in litter from flock 2. Six genotypes which had been found in flock 1 during rearing were detected in samples collected before placement. NetB positive isolates belonging to two of these genotypes had been recovered from NE lesions during rearing, suggesting that virulent strains were transmitted from the cleaned and disinfected broiler house. NetB frequency among isolates from the empty building was 45%, indicating that netB positive strains were prevalent in a building that previously had housed a healthy flock offered in-feed narasin (flock 0). NetB frequency among isolates from litter used by flock 2 was 22%, indicating that netB positive strains were present in the environment of a 14-days-old healthy flock offered in-feed narasin. Two prevalent genotypes were consistently either netB negative or netB positive. However, the presence of genotypes represented by both negative and positive isolates may suggest that the gene can spread horizontally among different CP strains.

Genetic diversity and prevalence of netB in Clostridium perfringens isolated from a broiler flock affected by mild necrotic enteritis.

This study was undertaken to examine the genetic diversity of Clostridium perfringens isolated from a single broiler flock reared without in-feed antimicrobials (antibacterial growth promoters and anticoccidials) and affected by mild necrotic enteritis (NE). We used pulsed-field gel electrophoresis (PFGE) to investigate the genetic diversity of C. perfringens isolates from broilers of varying disease status, and from litter. The prevalence of the toxin gene netB was also investigated. Altogether 32 PFGE genotypes were found among 88 isolates. Several genotypes were detected in C. perfringens-associated organ lesions from chickens that were sampled at random and alive without clinical symptoms, suggesting that these genotypes proliferated concurrently in such lesions. More than 90% of all isolates from NE-specific organ lesions carried netB which codes for a recently described pore-forming toxin. NetB positive isolates were less predominant in non-lesion samples from broilers affected by NE, and found infrequently or not at all in healthy birds and isolates from litter. These findings show that the presence of netB in C. perfringens strains is associated with NE and suggest that mild NE differs from severe NE with regard to C. perfringens genotype diversity.

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

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