Clostridium perfringens Produces an Adhesive Pilus Required for the Pathogenesis of Necrotic Enteritis in Poultry.

Clostridium perfringens type G strains cause necrotic enteritis (NE) in poultry, an economically important disease that is a major target of in-feed antibiotics. NE is a multifactorial disease, involving not only the critically important NetB toxin but also additional virulence and virulence-associated factors. We previously identified a C. perfringens chromosomal locus (VR-10B) associated with disease-causing strains that is predicted to encode a sortase-dependent pilus. In the current study, we sought to provide direct evidence for the production of a pilus by C. perfringens and establish its role in NE pathogenesis. Pilus structures in virulent C. perfringens strain CP1 were visualized by transmission electron microscopy (TEM) of immunogold-labeled cells. Filamentous structures were observed extending from the cell surface in wild-type CP1 but not from isogenic pilin-null mutant strains. In addition, immunoblotting of cell surface proteins demonstrated that CP1, but not the null mutant strains, produced a high molecular weight ladder-like pattern characteristic of a pilus polymer. Binding to collagen types I, II, and IV was significantly reduced (Tukey's test, P < 0.01) in all three pilin mutants compared to CP1 and could be specifically blocked by CnaA and FimA antisera, indicating that these pilins participate in adherence. Furthermore, fimA and fimB null mutants were both severely attenuated in their ability to cause disease in an in vivo chicken NE challenge model. Together, these results provide the first direct evidence for the production of a sortase-dependent pilus by C. perfringens and confirm its critical role in NE pathogenesis and collagen binding.IMPORTANCE In necrotic enteritis (NE), an intestinal disease of chickens, Clostridium perfringens cells adhere tightly to damaged intestinal tissue, but the factors involved are not known. We previously discovered a cluster of C. perfringens genes predicted to encode a pilus, a hair-like bacterial surface structure commonly involved in adherence. In the current study, we have directly imaged this pilus using transmission electron microscopy (TEM). We also show that inactivation of the pilus genes stops pilus production, significantly reducing the bacterium's ability to bind collagen and cause disease. Importantly, this is the first direct evidence for the production of a sortase-dependent pilus by C. perfringens, revealing a promising new target for developing therapeutics to combat this economically important disease.

Growth performance, breast yield, gastrointestinal ecology and plasma biochemical profile in broiler chickens fed multiple doses of a blend of red, brown and green seaweeds.

1. A total of 864 d old (male) Ross × Ross 708 broiler chicks were allocated to 48 floor pens (12 pens/treatment and 18 birds/pen) to investigate dose-response of a blend of seaweeds (SB) on growth performance, breast yield, jejunal histomorphology, microbial metabolites and community and plasma biochemical profile. 2. A maize-soybean meal diet was formulated with 0, 5, 10 or 20 g/kg of SB. Diets were formulated for a three-phase feeding programme (starter: d 0-10, grower: d 11-24, and finisher: d 25-42) and met or exceeded Aviagen nutrient specifications. Diets were allocated to pens (n = 12) balanced for body weight (BW). Birds had free access to feed and water, BW and feed intake (FI) were monitored by phase. One bird per pen was randomly selected on d 42, bled for plasma, and samples for intestinal tissue and caecal digesta were taken. Microbial DNA was extracted and submitted for microbial community profile using the Illumina Miseq® platform. 3. In the starter phase, SB linearly (P ≤ 0.01) improved BW, body weight gain (BWG), and FCR. However, the improvement was quadratic, such that there was no further improvement beyond 5 g/kg SB inclusion. Growth performance response to SB in the grower phase was similar to the starter phase, with the exception of FCR (P > 0.05). Overall, from d 0-42, a linear and quadratic (P < 0.01) response was observed for final BW (d 42), whereby birds fed 5, 10 and 20 g/kg SB were heavier than control by 166, 183 and 180 g, respectively. A quadratic (P = 0.03) effect was observed for breast yield in response to SB. There was a quadratic reduction (P < 0.05) in gamma-glutamyl transferase (GGT) and a linear increase in glutamate dehydrogenase (GDH) in response to SB. Supplemental SB linearly reduced (P ≤ 0.04) the relative abundance of phylum Bacteroidetes and Proteobacteria, and increased the abundance of phylum Firmicutes (linearly; P = 0.02) and Actinobacteria (quadratically; P = 0.03). 4. The data indicated that the optimal inclusion for SB was between 5 and 10 g/kg for improved growth performance and breast yield. However, increased abundance of Firmicutes and actinobacteria in the caecal digesta suggested that the higher doses enhanced prebiotic effects of seaweed components.

Immunization with subunits of a novel pilus produced by virulent Clostridium perfringens strains confers partial protection against necrotic enteritis in chickens.

Necrotic enteritis (NE) is an economically important disease of broiler chickens that is caused primarily by Clostridium perfringens strains that produce the NetB toxin. It is controlled in North America principally through the application of in-feed antimicrobials, but alternative control methods, such as vaccination, are urgently needed. We previously identified a cluster of C. perfringens genes prevalent in disease-causing strains, denominated VR-10B, that is predicted to encode a pilus. The current study evaluated the ability of three predicted pilin structural subunits (CnaA, FimA, FimB) to protect against NE in two immunization studies. In the first study, young broiler chickens were immunized twice intramuscularly (i.m.) with CnaA or FimA, which resulted in only a weak serum antibody response, and no reduction in the severity of intestinal lesions following experimental challenge with C. perfringens strain CP1. In the second study, chickens were injected subcutaneously (s.c.) with CnaA, FimB, or a combination of all three proteins, on days 7, 14 and 19, which resulted in a marked antibody response specific to each antigen. Chickens immunized with either CnaA or FimB had significantly reduced NE lesion severity, whereas immunization with all three proteins in combination did not provide protection. Western blot experiments using serum from immunized birds were also performed, providing the first experimental evidence to suggest that this locus may in fact encode a functional pilus structure.

Identification of accessory genome regions in poultry Clostridium perfringens isolates carrying the netB plasmid.

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.

Targeted microarray analysis of stationary phase Escherichia coli O157:H7 subjected to disparate nutrient conditions.

AIMS: To determine how stress response and virulence gene expression of stationary phase (SP) Escherichia coli O157:H7 are affected by nutrient levels. METHODS AND RESULTS: A targeted microarray (n=125 genes) was used to determine the impact of nutrient deprivation [15 min in 3-(N-Morpholino)propanesulfonic acid buffer] on SP E. coli O157:H7. In total, 24 genes were significantly affected (>1·5-fold; P <0·05) with 17 induced and seven attenuated. Additionally, 11 genes belonging to significantly affected stress response regulons were significantly induced (P<0·05), though <1·5-fold. Induced genes included global and specific stress response regulators, the mar operon, iron acquisition and virulence genes. In contrast, transcript for major porins and replicative genes were repressed. Comparison of the nutrient deprived transcriptome to that derived from nutrient replenished cells revealed a disparate transcriptome, with 44 genes expressed at significantly elevated levels in nutrient replenished cells, including all queried global and specific stress response regulators and key virulence genes. Genes expressed at elevated levels in nutrient deprived cells were related to σ(S) . The microarray data were validated by qRT-PCR. CONCLUSIONS: SP E. coli O157:H7 were affected by nutrient deprivation, with both starvation-related and unrelated networks induced, thereby demonstrating how the E. coli O157:H7 stress response transcriptome is fine-tuned to environmental conditions. Further, by comparison of starved cells to cells provided with fresh nutrients, it is clear starved E. coli O157:H7 undergo massive physiological reprogramming dominated initially by stress response induction to adapt to a nutrient rich environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated how σ(S) -induced SP E. coli O157:H7 remain highly sensitive and adaptable to environmental conditions. Further, by examining how starved cells respond to nutrient-rich conditions, we show preliminary adaptation to a nutrient rich environment is dominated by the induction of diverse stress response networks. Combined, this provides E. coli O157:H7 stress physiology-based knowledge that can be used to design more effective food safety interventions.

Transcriptional analysis of host responses to Marek's disease virus infection in genetically resistant and susceptible chickens.

Marek's disease virus (MDV) is a cell-associated oncogenic herpesvirus that targets B cells and T cells, inducing lymphoid tumours in chickens. Genetic resistance to Marek's disease (MD) is regulated in a polygenic fashion. In this study, we sought to compare the gene expression profiles following infection of birds that are genetically resistant or susceptible to MD (with the B21 and B19 haplotypes respectively at the MHC locus), including comparisons to uninfected controls. On days 4, 7, 14 and 21 post-infection, gene expression profiles in spleen tissue were obtained using a chicken immune-specific microarray. A number of genes showed significant (P or=2-fold in resistant compared with susceptible infected chickens. IgM and IgG were significantly induced on day 7 post-infection in susceptible chickens compared to resistant birds, whereas both of these genes were repressed in susceptible birds on day 14 post-infection. Overall, gene expression profiles in the chicken spleen observed after MDV infection were dependent on time and host genetic background. These gene expression profiles provide a platform for defining novel candidate genes for resistance or susceptibility to MD.

Molecular typing and distribution of Staphylococcus aureus isolates in Eastern Canadian dairy herds.

Macrorestriction analysis of SmaI-digested chromosomal DNA, using pulsed field gel electrophoresis (PFGE) was performed to type and estimate genetic relationships among 288 Staphylococcus aureus isolates recovered from 58 Eastern Canadian dairy herds. In addition, a subset of the collection was phage typed and evaluated for sensitivity to 10 antimicrobial compounds. Of 288 isolates recovered, 29 distinct PFGE types were identified. Based on estimates of genetic relationships, the PFGE types were assigned to six lineage groups, designated A through F. Of all of the isolates, ca. 93% were assigned to lineage groups A, D, or F. In 58.6% of herds, only a single PFGE type was recovered, while the remainder had two to four types. Of the 212 isolates evaluated for antimicrobial resistance, 24.5% were resistant to one or more antimicrobials. Resistance to penicillin (9.9%) was most common, followed by resistance to sulfadimethoxine (7.5%). Isolates resistant to multiple antibiotics were rare. A total of 63% of isolates responded to phages from groups 1 and 3, and 32.8% could not be typed with any of the phage strains used. The other 4.1% belonged to a variety of phage types. Most of the PFGE lineage group A and F isolates corresponded to phage groups 3 and 1, respectively, and most group D isolates were not typeable. PFGE typing had better discriminatory power than phage typing in defining the relatedness of the S. aureus isolates. Distribution of PFGE types and phage types was independent across regions and within herds.

Respiratory muscle training in Duchenne muscular dystrophy.

Eighteen boys with Duchenne muscular dystrophy were entered into trials to assess the effects of specific ventilatory strength and endurance training programmes. The findings showed an improvement in ventilatory muscle endurance but not in strength as a result of specific respiratory muscle training. The clinical significance of these findings is uncertain, however, and needs further evaluation.