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.

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.

NanI sialidase contributes to toxin expression and host cell binding of Clostridium perfringens type G strain CP56 in vitro.

Necrotic enteritis, caused by NetB producing Clostridium perfringens type G strains, is a globally important poultry disease. An initial step in the pathogenesis of necrotic enteritis is the colonization and degradation of the intestinal mucus layer, a process in which C. perfringens sialidases - such as NanI sialidase - may play an important role. Sialidases cleave terminal sialic acid from complex carbohydrates on glycoconjugates, such as mucins. This study shows that NE-associated C. perfringens strain CP56 is able to use sialic acid (Neu5Ac) as a carbon source for bacterial growth. It is shown that supplementation of Neu5Ac in the growth medium does not only induce the production of extracellular sialidases of strain CP56, but also increases the production of both alpha toxin and NetB toxin. Moreover, it was found that pre-treating avian hepatocellular carcinoma cells (LMH cells) with the recombinant NanI sialidase increases the adherence of C. perfringens type G strain CP56 to these cells. As such, the data suggest an important role for sialidases in the pathogenesis of the disease.

Protein Truncating Variants of colA in Clostridium perfringens Type G Strains.

Extracellular matrix (ECM) degrading enzymes produced by Clostridium perfringens may play an important role during the initial phases of avian necrotic enteritis by facilitating toxin entry in the intestinal mucosa and destruction of the tissue. C. perfringens is known to produce several ECM-degrading proteases, such as kappa toxin, an extracellular collagenase that is encoded by the colA gene. In this study, the colA gene sequence of a collection of 48 C. perfringens strains, including pathogenic (i.e. toxinotype G) and commensal (i.e. toxinotype A) chicken derived strains and strains originating from other host species, was analyzed. Although the colA gene showed a high level of conservation (>96% nucleotide sequence identity), several gene variants carrying different nonsense mutations in the colA gene were identified, leading to the definition of four truncated collagenase variant types (I-IV). Collagenase variant types I, III and IV have a (nearly) complete collagenase unit but lack parts of the C-terminal recruitment domains, whereas collagenase variant types II misses the N-terminal part of collagenase unit. Gene fragments encoding a truncated collagenase were mainly linked with necrotic enteritis associated C. perfringens type G strains with collagenase variant types I and II being the most prevalent types. Gelatin zymography revealed that both recombinant full-length and variant type I collagenase have active auto-cleavage products. Moreover, both recombinant fragments were capable of degrading type I as well as type IV collagen, although variant type I collagenase showed a higher relative activity against collagen type IV as compared to full-length collagenase. Consequently, these smaller truncated collagenases might be able to break down collagen type IV in the epithelial basement membrane of the intestinal villi and so contribute to the initiation of the pathological process leading to necrotic enteritis.

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.

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.

In-feed resin acids reduce matrix metalloproteinase activity in the ileal mucosa of healthy broilers without inducing major effects on the gut microbiota.

The chicken gut is constantly exposed to harmful molecules and microorganisms which endanger the integrity of the intestinal wall. Strengthening intestinal mucosal integrity is a key target for feed additives that aim to promote intestinal health in broilers. Recently, dietary inclusion of resin-based products has been shown to increase broiler performance. However, the mode of action is still largely unexplored. Coniferous resin acids are known for their anti-microbial, anti-inflammatory and wound-healing properties, all properties that might support broiler intestinal health. In the current study, the effect of pure resin acids on broiler intestinal health was explored. Ross 308 broilers were fed a diet supplemented with coniferous resin acids for 22 days, after which the effect on both the intestinal microbiota as well as on the intestinal tissue morphology and activity of host collagenases was assessed. Dietary inclusion of resin acids did not alter the morphology of the healthy intestine and only minor effects on the intestinal microbiota were observed. However, resin acids-supplementation reduced both duodenal inflammatory T cell infiltration and small intestinal matrix metalloproteinase (MMP) activity towards collagen type I and type IV. Reduced breakdown of collagen type I and IV might indicate a protective effect of resin acids on intestinal barrier integrity by preservation of the basal membrane and the extracellular matrix. Further studies are needed to explore the protective effects of resin acids on broiler intestinal health under sub-optimal conditions and to elaborate our knowledge on the mechanisms behind the observed effects.

Elevated faecal ovotransferrin concentrations are indicative for intestinal barrier failure in broiler chickens.

Intestinal health is critically important for the welfare and performance of poultry. Enteric diseases that cause gut barrier failure result in high economic losses. Up till now there is no reliable faecal marker to measure gut barrier failure under field conditions. Therefore, the aim of the present study was to identify a faecal protein marker for diminished intestinal barrier function due to enteric diseases in broilers. To assess this, experimental necrotic enteritis and coccidiosis in broilers were used as models for gut barrier failure. Ovotransferrin was identified as a marker for gut barrier failure using a proteomics approach on samples from chickens with necrotic enteritis. These results were confirmed via ELISA on samples derived from both necrotic enteritis and coccidiosis trials, where faecal ovotransferrin levels were significantly correlated with the severity of gut barrier failure caused by either coccidiosis or necrotic enteritis. This indicates that faecal ovotransferrin quantification may represent a valuable tool to measure gut barrier failure caused by enteric pathogens.

The 5-HT4 receptor agonist prucalopride does not facilitate cholinergic neurotransmission in circular and longitudinal smooth muscle preparations of equine mid-jejunum.

BACKGROUND: Postoperative ileus (POI) remains an important cause of death in horses. The recently developed selective 5-HT4 receptor agonists such as prucalopride target 5-HT4 receptors on myenteric cholinergic neurons to enhance acetylcholine release and GI motility. No clearcut in vitro evaluation whether highly selective 5-HT4 receptor agonists enhance submaximal cholinergic neurotransmission towards the muscle layer has been performed in horses. OBJECTIVES: To identify functional 5-HT4 receptors in equine jejunum. STUDY DESIGN: In vitro experimental study. METHODS: Circular and longitudinal smooth muscle strips (mid-jejunum) were mounted in organ baths between 2 platinum electrodes allowing electrical field stimulation (EFS). To delineate the conditions to obtain purely cholinergic responses, voltage-response curves were studied. To investigate the influence of prucalopride and 5-HT, submaximal cholinergic contractions at a single voltage were induced. RESULTS: In circular and longitudinal strips, EFS induced voltage-dependent neurogenic on-contractions when the bathing medium contained a NO-synthesis inhibitor and apamin to prevent inhibitory responses to NO and ATP. Contractions at a voltage inducing 50% of maximal amplitude were cholinergic, as they were blocked by atropine. These contractions were not influenced by prucalopride (up to 3µM), even in the presence of the phosphodiesterase inhibitor isobutyl-methyl-xanthine to inhibit breakdown of the second messenger of 5-HT4 receptors, cAMP. Also the full 5-HT4 receptor agonist 5-HT did not influence the EFS-induced submaximal cholinergic contractions. Moreover, prucalopride did not influence muscle tone continuously enhanced with KCl. CONCLUSIONS: There are no functional 5-HT4 receptors on myenteric cholinergic neurons nor muscular 5-HT4 receptors in equine jejunum.