Enterobacteriaceae and Enterococcaceae are the dominant bacterial families translocating to femur heads in broiler chicks.

RESEARCH HIGHLIGHTS: Large number of bacteria isolated from femoral heads of clinically healthy broilers.The prevailing taxa in femoral heads were Escherichia/Shigella and Enterococcus spp.Continuous presence of bacteria in blood and liver of clinically healthy broilers.Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae prevail in blood and liver.

Prevalence and serotype of poultry salmonellosis in Africa: a systematic review and meta-analysis.

RESEARCH HIGHLIGHTS: The pooled sample prevalence of poultry salmonellosis in Africa is high (14.4%).The highest PPE was recorded in meat and meat products.Salmonella serotypes of zoonotic importance were found in all sample types.Salmonella Enteritidis and Typhimurium are common serotypes spreading in Africa.

Oral vaccination of young broilers with a live Salmonella Typhimurium vaccine reduces caecal and internal organ colonization following a Salmonella Infantis challenge in a seeder-bird model.

Poultry products are an important source of foodborne Salmonella infections in humans. Amongst these, the prevalence of S. Infantis is rising. In this study, the protection efficacy of an authorized live-attenuated S. Typhimurium vaccine against S. Infantis, was examined using a seeder-bird model in broilers. Vaccinated birds displayed a significantly lower colonization of S. Infantis bacteria in the caeca compared to the non-vaccinated counterparts (P = 0.017), with no significant differences observed in the spleen among the groups, three days post-infection. Thirty-two days post-infection, the disparity in average S. Infantis concentration between all-vaccinated and non-vaccinated birds was significant in both caeca (P = 0.0003) and spleen (P = 0.0002). Interestingly, a third group, consisting of seeder birds that were not vaccinated but housed with vaccinated penmates, exhibited significantly lower S. Infantis levels in both caeca (P = 0.0014) and spleen (P < 0.0001) compared to the non-vaccinated group. These findings underscore the potential of a live-attenuated S. Typhimurium vaccine administered to 2-day-old chicks in conferring protection against S. Infantis in broilers up to slaughter age.

Bacterial chondronecrosis with osteomyelitis related Enterococcus cecorum isolates are genetically distinct from the commensal population and are more virulent in an embryo mortality model.

Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of broiler lameness. Bacteria that are found in BCO lesions are intestinal bacteria that are proposed to have translocated through the intestinal epithelium and have spread systemically. One of the specific bacterial species frequently isolated in BCO cases is Enterococcus cecorum. In the current study, caecal isolates were obtained from birds derived from healthy flocks (12 isolates from 6 flocks), while isolates derived from caeca, colon, pericardium, caudal thoracic vertebrae, coxo-femoral joint, knee joint and intertarsal joint (hock) were obtained from broilers derived from BCO outbreaks (111 isolates from 10 flocks). Pulsed field gel electrophoresis was performed to determine similarity. Clonal E. cecorum populations were isolated from different bones/joints and pericardium from animals within the same flock, with intestinal strains carrying the same pulsotype, pointing to the intestinal origin of the systemically present bacteria. Isolates from the intestinal tract of birds from healthy flocks clustered away from the BCO strains. Isolates from the gut, bones/joints and pericardium of affected animals contained a set of genes that were absent in isolates from the gut of healthy animals, such as genes encoding for enterococcal polysaccharide antigens (epa genes), cell wall structural components and nutrient transporters. Isolates derived from the affected birds induced a significant higher mortality in the embryo mortality model as compared to the isolates from the gut of healthy birds, pointing to an increased virulence.

Broiler performance correlates with gut morphology and intestinal inflammation under field conditions.

Maintaining optimal gut health is a key driver for a well-performing broiler flock. Histology of intestinal sections and quantification of villus structure can be used to evaluate gut health. While these measurements have been used in experimental models to evaluate gut health, less is known about the associations of these parameters with performance in commercial broiler farms. The objective of the present study was to evaluate possible associations of intestinal villus structure and the inflammatory condition of the gut with Ross 308 broiler performance in 50 commercial farms. On day 28 of the production round, 20 randomly selected broilers per farm were weighed, euthanized, and a duodenal section was collected to determine villus length, crypt depth and the CD3+ T-lymphocytes area percentage (CD3+ %). We found a relatively low coefficient of variance (CV) for the villus length (between farms; 9.67%, within farms; 15.97%), while the CD3+ (%) had a high CV (between farms; 29.78%, within farms; 25.55%). At flock level, the CD3+ (%) was significantly correlated with the villus length (r = -0.334), crypt depth (r = 0.523) and the villus-to-crypt ratio (r = -0.480). The crypt depth was significantly correlated with the European production index (EPI) (r = -0.450) and feed conversion ratio (FCR) (r = 0.389). At broiler level, a significant association was found between the individual body weight (day 28), CD3+ (%) and villus-to-crypt ratio. These data thus show that gut villus structure is significantly associated with bird performance under commercial conditions. RESEARCH HIGHLIGHTSGut histology parameters vary between and within farms.Broiler performance is associated with gut morphology.

Digital PCR: a tool in clostridial mutant selection and detection.

The ClosTron mutagenesis system has enabled researchers to efficiently edit the clostridial genome. Since site-specific insertion of the mobile ClosTron insert may cause errors, validation is key. In this paper we describe the use of digital PCR (dPCR) as an alternative tool in selecting clostridial mutant strains. Clostridium perfringens chitinase mutant strains were constructed in which the mobile ClosTron intron was inserted into one of the chitinase genes. On-target insertion of the mobile intron was validated through conventional PCR. In order to confirm the absence of off-target insertions, dPCR was used to determine the amount of the ClosTron intron as well as the amount of a reference gene, located in close proximity to the interrupted gene. Subsequently, mutant strains containing an equivalent amount of both genes were selected as these do not contain additional off-target mobile ClosTron inserts. The outcome of this selection procedure was confirmed through a validated PCR-based approach. In addition to its application in mutant selection, dPCR can be used in other aspects of clostridial research, such as the distinction and easy quantification of different types of strains (wildtype vs. mutant) in complex matrices, such as faecal samples, a process in which other techniques are hampered by bacterial overgrowth (plating) or inhibition by matrix contaminants (qPCR). This research demonstrates that dPCR is indeed a high-throughput method in the selection of clostridial insertion mutants as well as a robust and accurate tool in distinguishing between wildtype and mutant C. perfringens strains, even in a complex matrix such as faeces. KEY POINTS: • Digital PCR as an alternative in ClosTron mutant selection • Digital PCR is an accurate tool in bacterial quantification in a complex matrix • Digital PCR is an alternative tool with great potential to microbiological research.

Hemorrhagic bowel syndrome in dairy cattle: Gross, histological, and microbiological characterization.

Hemorrhagic bowel syndrome (HBS) is a sporadic and fatal disease of predominantly lactating dairy cattle, characterized by segmental hemorrhage and luminal clot formation in the small intestine. Although, Clostridium perfringens and Aspergillus fumigatus have been associated with HBS, the pathogenesis and cause are currently unknown. In this study, 18 naturally occurring cases of HBS (7 necropsied immediately following euthanasia, 11 with 12-48 hour postmortem intervals) were investigated to characterize the pathology and the intestinal microbiome. Hemorrhagic bowel syndrome was characterized by a single small-intestinal, intramucosal hematoma with dissection of the lamina muscularis mucosae. In most cases necropsied immediately after euthanasia (4/7), the intestinal mucosa proximal to the hematoma contained 9 to 14, dispersed, solitary or clustered, erosions or lacerations measuring 4 to 45 mm. In 77% (37/48) of these mucosal lesions, microscopic splitting of the lamina muscularis mucosae comparable to the hematoma was present. These findings suggest the intramucosal hematoma to originate from small mucosal erosions through dissecting hemorrhage within the lamina muscularis mucosae. No invasive fungal growth was observed in any tissue. Bacteriological cultivation and nanopore sequencing showed a polymicrobial population at the hematoma and unaffected intestine, with mostly mild presence of C perfringens at selective culture. Gross and microscopic lesions, as well as the culture and sequencing results, were not in support of involvement of C perfringens or A fumigatus in the pathogenesis of HBS.

The in vitro effect of lactose on Clostridium perfringens alpha toxin production and the implications of lactose consumption for in vivo anti-alpha toxin antibody production.

Necro-hemorrhagic enteritis in calves, caused by Clostridium perfringens type A, is a fatal disease, mostly affecting calves in intensive rearing systems. The lack of development of active immunity against α toxin, an essential virulence factor in the pathogenesis, has been proposed as a main trigger. In this experimental study, the effect of a set of milk replacer components on α toxin production, and the effect of lactose on in vivo antibody production, were investigated. For the latter, Holstein-Friesian bull calves (n = 18) were fed an all liquid diet that contained either a milk replacer with high-lactose content (45% DM) or the same milk replacer that was lactase treated, resulting in a lactose-free equivalent. Antibody levels against α toxin were monitored from 2 to 12 wk of age. In the in vitro part of the study, a concentration-dependent inhibitory effect of lactose on in vitro C. perfringens α toxin activity was observed, whereas protein did not influence α toxin activity. The in vivo experiment then showed from the age of 10 wk onwards, that anti-α toxin antibody levels of high-lactose animals declined, whereas antibody levels of the animals consuming lactose-free milk replacer remained the same throughout the trial. This points to a natural decline in maternal immunity of lactose-consuming animals, that is not compensated by the development of an active immunity, resulting in inferior protection. This study suggests that dietary lactose reduces C. perfringens α toxin production in vivo, which may lead to a decreased antigen presentation and thus lower serum antibody levels against the toxin. Consequently, any event causing massive α toxin production puts lactose-consuming calves at higher risk of developing necro-hemorrhagic enteritis.

The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction.

BACKGROUND: Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. RESULTS: Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. CONCLUSION: Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

Omics technologies in poultry health and productivity - part 2: future applications in the poultry industry.

The increasing global demand for poultry products, together with the growing consumer concerns related to bird health and welfare, pose a significant challenge to the poultry industry. Therefore, the poultry industry is increasingly implementing novel technologies to optimize and enhance bird welfare and productivity. This second part of a bipartite review on omics technologies in poultry health and productivity highlights the implementation of specific diagnostic biomarkers based on omics-research in the poultry industry, as well as the potential integration of multi-omics in future poultry production. A general discussion of the use of multiple omics technologies in poultry research is provided in part 1. To date, approaches focusing on one or more omics type are widely used in poultry research, but the implementation of these omics techniques in poultry production is not expected in the near future. However, great potential lays in the development of diagnostic tests based on disease- or gut health-specific biomarkers, which are identified through omics research. As the cost of omics technologies is rapidly decreasing, implementation of multi-omics measurements in routine poultry monitoring systems might be feasible in the more distant future. Therefore, the opportunities, challenges and requirements to enable the integration of multi-omics-based monitoring of bird health and productivity in future poultry production are discussed.

Omics technologies in poultry health and productivity - part 1: current use in poultry research.

In biology, molecular terms with the suffix "-omics" refer to disciplines aiming at the collective characterization of pools of molecules derived from different layers (DNA, RNA, proteins, metabolites) of living organisms using high-throughput technologies. Such omics analyses have been widely implemented in poultry research in recent years. This first part of a bipartite review on omics technologies in poultry health and productivity examines the use of multiple omics and multi-omics techniques in poultry research. More specific present and future applications of omics technologies, not only for the identification of specific diagnostic biomarkers, but also for potential future integration in the daily monitoring of poultry production, are discussed in part 2. Approaches based on omics technologies are particularly used in poultry research in the hunt for genetic markers of economically important phenotypical traits in the host, and in the identification of key bacterial species or functions in the intestinal microbiome. Integrative multi-omics analyses, however, are still scarce. Host physiology is investigated via genomics together with transcriptomics, proteomics and metabolomics techniques, to understand more accurately complex production traits such as disease resistance and fertility. The gut microbiota, as a key player in chicken productivity and health, is also a main subject of such studies, investigating the association between its composition (16S rRNA gene sequencing) or function (metagenomics, metatranscriptomics, metaproteomics, metabolomics) and host phenotypes. Applications of these technologies in the study of other host-associated microbiota and other host characteristics are still in their infancy.

Acute Endotoxemia-Induced Respiratory and Intestinal Dysbiosis.

Systemic inflammatory response syndrome (SIRS) is a severe condition characterized by systemic inflammation, which may lead to multiple organ failure, shock and death. SIRS is common in burn patients, pancreatitis and sepsis. SIRS is often accompanied by intestinal dysbiosis. However, the mechanism, role and details of microbiome alterations during the early phase of acute SIRS are not completely understood. The current study aimed to characterize the dynamic alterations of both the intestinal and respiratory microbiome at two timepoints during the early phase of acute SIRS (4 and 8 h after LPS) and link these to the host response in a mouse model of a LPS-induced lethal SIRS. Acute SIRS had no effect on the microbiome in the large intestine but induced a rapid dysbiosis in the small intestine, which resembled the microbiome alterations commonly observed in SIRS patients. Later in the disease progression, a dysbiosis of the respiratory microbiome was observed, which was associated with the MMP9 expression in the lungs. Although similar bacteria were increased in both the lung and the small intestine, no evidence for a gut-lung translocation was observed. Gut dysbiosis is commonly observed in diseases involving inflammation in the gut. However, whether the inflammatory response associated with SIRS and sepsis can directly cause gut dysbiosis was still unclear. In the current study we provide evidence that a LPS-induced SIRS can directly cause dysbiosis of the small intestinal and respiratory microbiome.

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

Dietary muramidase degrades bacterial peptidoglycan to NOD-activating muramyl dipeptides and reduces duodenal inflammation in broiler chickens.

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

Effect of vitamin E level and dietary zinc source on performance and intestinal health parameters in male broilers exposed to a temperature challenge in the finisher period.

The objective of this study was to evaluate the interaction of zinc source (ZnSO4 vs. zinc amino acid complex) and vitamin E level (50 IU vs. 100 IU) on performance and intestinal health of broilers exposed to a temperature challenge in the finisher period. A total of 1224 day old male Ross 308 broilers were randomly distributed among 4 dietary treatments (9 replicates per treatment). Dietary treatments were organized in a 2 × 2 factorial arrangement: two sources of zinc, 60 mg/kg of Zn as ZnSO4 .7H2 O or 60 mg/kg of Zn as zinc amino acid complexes (ZnAA) combined with two levels of vitamin E (50 or 100 IU/kg). Zinc and vitamin E were added to a wheat/rye-based diet that was designed to create a mild nutritional challenge. From day 28 until day 36 (finisher period), all birds were subjected to chronic cyclic high temperatures (32°C ± 2°C and RH 55-65% for 6 h daily). The combination of ZnAA and 50 IU/kg of vitamin E improved weight gain in the starter (day 0-10), finisher (day 28-36) and overall period (day 0-36) and feed conversion ratio in the starter (day 0-10) and finisher phase (day 28-36). Providing Zn as ZnAA significantly improved villus length and villus/crypt ratio in the starter, grower and finisher period and decreased infiltration of T-lymphocytes and ovotransferrin leakage in the finisher period. In conclusion, providing broilers with a diet supplemented with ZnAA and a vitamin E level of 50 IU/kg, resulted in better growth performance as compared to all other dietary treatments. Interestingly, under the conditions of this study, positive effects of ZnAA on performance did not occur when vitamin E was supplemented at 100 IU/kg in feed. Moreover, providing zinc as zinc amino acid complex improved intestinal health.

Effect of in feed administration of different butyrate formulations on Salmonella Enteritidis colonization and cecal microbiota in broilers.

Butyrate has been used extensively as a feed additive to improve gut health and to decrease Salmonella colonization in poultry. Salmonella mainly colonizes the ceca so butyrate concentrations should be increased in this gut segment. Discrepancies on the effects of butyrate on Salmonella colonization, described in the scientific literature, could thus be due to butyrate release location effects. In this study, newly developed butyrate formulations were evaluated for their effect on cecal butyrate concentrations and on colonization by Salmonella Enteritidis. In a first trial, broilers were randomly allocated to 7 dietary treatment groups with formulations based on different approaches to modify the butyrate release profile: release from wax matrices based on diffusion/erosion; micropellets supposedly release butyrate around pH 7 in the colon; tributyrin is based on the hydrolysis of esters in the small intestine. Fat-protected butyrate was included as a reference, because of its known effect on reduction of Salmonella colonization. Four days after infection, the number of cfu Salmonella per g cecal content and spleen were determined. Butyrate formulations in a wax matrix significantly reduced the Salmonella colonization in cecal content. In a second trial, wax and fat-protected butyrate treatments were replicated and results from the first trial were confirmed. Compared to the control group a higher proportion of butyrate concentration was observed in ceca for those groups with reduced Salmonella colonization. This was associated with a beneficial shift in the cecal microbiota. In conclusion, formulations that increase cecal butyrate concentrations are superior in protecting against Salmonella Enteritidis colonization.

C. perfringens challenge reduces matrix metalloproteinase activity in the jejunal mucosa of Eimeria-infected broiler chickens.

Matrix metalloproteinases (MMPs) play an important role in intestinal extracellular matrix homeostasis. An overexpression of MMPs results in tissue destruction and local inflammation and has been associated with multiple inflammatory diseases. These host proteases might also be important in tissue damage caused by infectious agents, such as in intestinal damage in Clostridium perfringens-induced avian necrotic enteritis (NE). The aim of the present study was to elucidate the effect of a C. perfringens infection on the MMP activity in the small intestine of birds with a pre-disposing coccidial infection to obtain a more thorough understanding of the pathogenesis of NE. For this purpose, the gelatinolytic activity present in jejunal tissue of Eimeria infected birds which were challenged with either a pathogenic C. perfringens type G strain or a commensal C. perfringens type A strain was analyzed using substrate zymography. The results show that infection of broilers with Eimeria and different C. perfringens strains, independent of their pathogenicity, decreases the expression of a 40-45 kDa host collagenase in the jejunum, as compared to the expression in Eimeria-infected control birds. It was also shown that the expression of 2 MMPs with molecular weights of approximately 50-60 and 60-70 kDa was significantly lower in necrotic tissue as compared to the activity in macroscopically healthy tissue adjacent to the lesion. These results indicate that host collagenases are not elicited by the C. perfringens infection for permeabilizing the host mucosa to allow penetration of the NetB toxin in Eimeria infected broilers.

A study on risk factors for macroscopic gut abnormalities in intensively reared broiler chickens.

Intensification of broiler production has coincided with an increase in enteric disorders. Enteric syndromes of unknown aetiology are often associated with an increased feed conversion ratio and are given the general term "dysbiosis". Despite the importance of dysbiosis, information on factors contributing to this condition are scarce. Therefore, the aim of this study was to describe dysbiosis in broilers (Ross 308) during one production round and to identify risk factors. Fifteen farms in Flanders (Belgium) were followed up, with visits at days 10, 17, 20, 24 and 28 of production. At every visit, 10 random birds were inspected for footpad lesions, hock burns and breast blisters. Also, coccidiosis and enteric abnormalities were scored after necropsy. A gut appearance score (GAS) was given based on 10 macroscopically visible parameters, where a higher GAS equalled more enteric abnormalities. Footpad lesions were seen in 14 farms and increased in prevalence with the age of the birds. Hock burns were seen less frequently, and no breast blisters were detected. Eimeria acervulina lesions were most frequently observed, followed by E. maxima and E. tenella lesions. The average GAS increased from day 10 until day 20. The strong correlations between the GAS at days 10, 17 and 20 indicate that prevalence of gut abnormalities at day 10 can be predictive for scores later on. A higher amount of intestinal defects was seen in older female birds, in the presence of a conceivable E. tenella infection and at farms with a higher productivity and sanitary status.RESEARCH HIGHLIGHTSGut lesions found in young broilers can predict further development of defects.Caecal lesions showed the strongest correlation with GAS in a multivariate model.

Incidence and associated risk factors of necrotic enteritis in Belgian layer pullet flocks.

Necrotic enteritis (NE) caused by Clostridium perfringens is commonly reported in broilers. Recently, increased NE prevalence in layer breeds was reported in the Indian subcontinent. NE is also frequently observed by veterinary practitioners in Europe, mainly during the pullet rearing phase. In this study, data from layer pullet flocks in Belgium over a 5-year period (2013-2017) were used to assess the incidence of NE and identify potential risk factors for NE in layer pullets. NE was observed in 26% of the layer pullet flocks receiving veterinary intervention. This accounts for an overall estimated NE incidence of 12.3% in Belgian layer pullet flocks. Occurrence of NE was significantly associated with coccidiosis, with flocks being diagnosed with coccidiosis being two-fold more likely to develop NE. Additionally, birds kept in aviary houses were less prone to NE than flocks reared in floor systems or enriched cages. At necropsy, necrotic lesions in the small intestine were comparable to NE in broilers. A single strain of C. perfringens was isolated from the necrotic lesions of three different birds from the same flock; however, no NetB could be detected.

A comparative study on the use of selective media for the enumeration of Clostridium perfringens in poultry faeces.

Isolation and fast detection of Clostridium perfringens is essential in veterinary medical diagnostics and veterinary research, as it allows to recommend suitable treatment options after antimicrobial resistance determination, and is essential to study pathogenesis. In this study four selective media were tested for the enumeration of, and selectivity towards C. perfringens in faecal samples from poultry. The routinely used Columbia agar with 5% sheep (CBA), Shahidi-Ferguson-perfringens agar (SFP), tryptose sulphite cycloserine agar (TSC), and a novel chromogenic medium, CHROMagar™ C. perfringens (CHCP), were tested. Overall, no difference in C. perfringens recovery could be observed between the selective media. The limit of quantification was 103 CFU/mL for all agars. CHCP showed the highest specificity, especially when low C. perfringens loads were present in the faeces, with TSC being the second most specific selective medium. Both CBA and SFP allowed considerable growth of other faecal microbiota and were not specific for C. perfringens. On CHCP, differentiation of C. perfringens from other faecal bacteria was straightforward due to the appearance of C. perfringens as orange colonies, with other bacteria being absent or appearing as blue/green colonies. On TSC, C. perfringens appeared as black colonies, but longer incubation periods were sometimes needed for the black colour to develop. Therefore, CHCP can be recommended when timely and easy identification and enumeration of C. perfringens from complex samples, such as faeces, is needed.