The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

Comparative transcriptome analysis by RNAseq of necrotic enteritis Clostridium perfringens during in vivo colonization and in vitro conditions.

BACKGROUND: Necrotic enteritis (NE) caused by netB-positive type A Clostridium perfringens is an important bacterial disease of poultry. Through its complex regulatory system, C. perfringens orchestrates the expression of a collection of toxins and extracellular enzymes that are crucial for the development of the disease; environmental conditions play an important role in their regulation. In this study, and for the first time, global transcriptomic analysis was performed on ligated intestinal loops in chickens colonized with a netB-positive C. perfringens strain, as well as the same strain propagated in vitro under various nutritional and environmental conditions. RESULTS: Analysis of the respective pathogen transcriptomes revealed up to 673 genes that were significantly expressed in vivo. Gene expression profiles in vivo were most similar to those of C. perfringens grown in nutritionally-deprived conditions. CONCLUSIONS: Taken together, our results suggest a bacterial transcriptome responses to the early stages of adaptation, and colonization of, the chicken intestine. Our work also reveals how netB-positive C. perfringens reacts to different environmental conditions including those in the chicken intestine.

Experimental reproduction of necrotic enteritis in chickens: a review.

This review discusses key factors important in successful experimental reproduction of necrotic enteritis (NE) in chickens, and how these factors can be adjusted to affect the severity of the lesions induced. The critical bacterial factor is the need to use virulent, netB-positive, strains of Clostridium perfringens; disease severity can be enhanced by using netB-positive C. perfringens strains that are also tpeL-positive, by the use of young rather than old broth cultures, and by the number of days of inoculation and the number of bacteria used. Use of cereals rich in non-starch polysaccharides can enhance disease, as does use of animal proteins. Administration of coccidia, including coccidial vaccines, combined with netB-positive C. perfringens, increases the severity of experimentally-induced NE. Dietary manipulation may be less important in coccidia-based models since the latter are so effective. Disease scoring systems and welfare considerations are discussed.

The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review.

This review summarizes advances in understanding the pathogenesis of necrotic enteritis of chickens caused by netB-positive Clostridium perfringens. The discovery of NetB as the essential toxin trigger for the disease was followed by recognition that it forms part of a large plasmid-encoded 42 kb pathogenicity locus (NELoc-1). While the locus is critical for toxin production, it likely has additional functions related to colonization and degradation of the mucus barrier, which are essential both to multiplication and to bringing NetB close to the intestinal epithelium. Two "chitinases" (glycoside hydrolases (GHs)) present on NELoc-1 are predicted to be involved in mucin degradation, as is the large carbohydrate-binding metalloprotease, shown to be involved in mucinase activity in other clostridia. A second pathogenicity locus found in netB-positive C. perfringens, NELoc-2, also encodes a GH likely involved in mucin degradation. Upon reaching a sufficient cell density on the intestinal mucosa, the Agr-like quorum-sensing system is triggered, which in turn up-regulates the VirR/VirS regulon. This regulon includes NetB. Where NetB initiates damage is unresolved, but it may be deep in the intestinal mucosa, rather than superficially. As the disease progresses, C. perfringens line what remains of the intestinal epithelium in large numbers. This likely involves a number of different bacterial adhesins, including additional NELoc-1-encoded bacterial surface proteins, some of which may adhere to epithelial cell ligands exposed by bacterial sialidases. Further studies of the pathogenesis of necrotic enteritis should lead to development of novel ways to control the infection.

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

How do swine practitioners and veterinary pathologists arrive at a diagnosis of Clostridium perfringens type A enteritis in neonatal piglets?

A questionnaire was administered to 22 veterinary practitioners and 17 veterinary pathologists to investigate the methods used for diagnosis of Clostridium perfringens type A enteritis in neonatal pigs. Practitioners generally diagnosed C. perfringens type A associated enteritis by age of onset of diarrhea (between 1 to 7 days of age). Most practitioners (95%) were moderately to very confident in their diagnosis. Pathologists generally diagnosed C. perfringens type A associated enteritis by combinations of isolation of the organism, genotyping or detecting the toxins of the organism, and ruling out other pathogens through histopathology. Almost half (41%) of the pathologists were not confident of their diagnosis. This study reports that the current diagnostic method for C. perfringens type A enteritis is not specific, and although many pathologists expressed reservations about making a diagnosis of C. perfringens type A enteritis, most practitioners were confident in their diagnosis, even though reported clinical signs of clostridial diarrhea are similar to those of a number of other enteric diseases.

Comment les praticiens porcins et les pathologistes vétérinaires parviennent-ils à un diagnostic d'entérite àClostridium perfringenstype A chez les porcelets nouveau-nés? Un questionnaire a été administré à 22 praticiens vétérinaires et à 17 pathologistes vétérinaires afin de faire enquête sur les méthodes utilisées pour le diagnostic de l'entérite à Clostridium perfringens type A chez les porcs nouveau-nés. Les praticiens diagnostiquaient généralement une entérite associée à C. perfringens type A selon l'âge à l'apparition de la diarrhée (âge d'entre 1 et 7 jours). La plupart des praticiens (95 %) ont déclaré un niveau de confiance de modérément confiant à très confiant à l'égard de leur diagnostic. Les pathologistes diagnostiquaient généralement l'entérite associée à C. perfringens type A par une combinaison d'isolement de l'organisme, du génotypage ou de la détection des toxines de l'organisme et éliminaient les autres agents pathogènes à l'aide d'une histopathologie. Près de la moitié (41 %) des pathologistes n'étaient pas confiants de leur diagnostic. Cette étude signale que la méthode diagnostique actuelle pour l'entérite à C. perfringens type A n'est pas spécifique et, même si beaucoup de pathologistes ont exprimé des réserves à propos d'un diagnostic d'entérite à C. perfringens type A, la plupart des praticiens éprouvaient de la confiance face à leur diagnostic, même si les signes cliniques signalés de la diarrhée clostridiale sont semblables à plusieurs autres maladies entériques.(Traduit par Isabelle Vallières).

The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review.

Necrotic enteritis (NE) is one of the most important enteric diseases in poultry and is a high cost to the industry worldwide. It is caused by avian-specific, Necrotic Enteritis Beta toxin (NetB)-producing, strains of Clostridium perfringens that also possess in common other virulence-associated genes. In Europe the disease incidence has increased since the ban on in-feed "growth promoting" antibiotics. Because of this, many recent studies of NE have focused on finding different ways to control the disease, and on understanding its pathogenesis. Frustratingly, reproduction of the disease has proven impossible for some researchers. This review describes and discusses factors known to be important in reproducing the disease experimentally, as well as other considerations in reproducing the disease. The critical bacterial factor is the use of virulent, netB-positive, strains; virulence can be enhanced by using tpeL- positive strains and by the use of young rather than old broth cultures to increase toxin expression. Intestinal damaging factors, notably the use of concurrent or preceding coccidial infection, or administration of coccidial vaccines, combined with netB-positive C. perfringens administration, can also be used to induce NE. Nutritional factors, particularly feeding high percentage of cereals containing non-starch polysaccharides (NSP) (wheat, rye, and barley) enhance disease by increasing digesta viscosity, mucus production and bacterial growth. Animal proteins, especially fish meal, enhance C. perfringens proliferation and toxin production. Other factors are discussed that may affect outcome but for which evidence of their importance is lacking. The review compares the different challenge approaches; depending on the aim of particular studies, the different critical factors can be adjusted to affect the severity of the lesions induced. A standardized scoring system is proposed for international adoption based on gross rather than histopathological lesions; if universally adopted this will allow better comparison between studies done by different researchers. Also a scoring system is provided to assist decisions on humane euthanasia of sick birds.

The epidemiology of Clostridium perfringens type A on Ontario swine farms, with special reference to cpb2-positive isolates.

BACKGROUND: There is poor understanding of most aspects of Clostridium perfringens type A as a possible cause of neonatal diarrhea in piglets, and the prevalence and types of C. perfringens present on Ontario swine farms is unknown. To study the prevalence of fecal C. perfringens and selected toxin genes, 48 Ontario swine farms were visited between August 2010 and May 2011, and 354 fecal samples were collected from suckling pigs, lactating sows, weanling pigs, grower-finisher pigs, and gestating sows, as well as from manure pits. The fecal samples were cultured quantitatively, and toxin genes were detected by real-time multiplex polymerase chain reaction (PCR). RESULTS: In mixed multivariable linear analysis, log(10) C. perfringens in fecal samples from suckling pigs were higher than that of weanling pigs, grower-finisher pigs, and manure pit samples (P <0.05). In mixed multivariable logistic analysis, the C. perfringens isolates recovered from lactating sows (OR = 0.069, P <0.001), gestating sows (OR = 0.020, P <0.001), grower-finishers (OR = 0.017, P <0.001), and manure pits (OR = 0.11, P <0.001) were less likely to be positive for the consensus beta2 toxin gene cpb2 compared to the isolates from suckling pigs. The prevalence of cpb2 in the isolates recovered from weanlings did not differ significantly from suckling pigs. C. perfringens isolates that were positive for cpb2 were more likely to carry the atypical cpb2 gene (atyp-cpb2) (OR = 19, P <0.001) compared to isolates that were negative for cpb2. Multivariable analysis did not identify farm factors affecting the presence of consensus cpb2 and atyp-cpb2 genes. CONCLUSIONS: This study provides baseline data on the prevalence of C. perfringens and associated toxin genes in healthy pigs at different stages of production on Ontario swine farms. The study suggests that if C. perfringens type A are involved in neonatal enteritis, there may be strains with specific characteristics that cannot be identified by the existing genotyping system.

Clostridium perfringens type A fatal acute hemorrhagic gastroenteritis in a dog.

The morning after participating in a dog show, a 2-year-old Pomeranian dog was found dead in a pool of bloody feces. Necropsy revealed hemorrhagic gastroenteritis of the entire gastrointestinal tract, with many Gram-positive bacilli on the surface and in the lumen and crypts of the intestine. Enterotoxin-positive type A Clostridium perfringens were isolated in large numbers. This dramatic case of fatal C. perfringens gastroenteritis highlights the need to better understand the role of this bacterium in enteric disease of dogs.

Out-patient antimicrobial drug use in dogs and cats for new disease events from community companion animal practices in Ontario.

This study investigated oral and parenteral antimicrobial use in dogs and cats, and evaluated antimicrobial use in feline upper respiratory tract disease (FURTD), feline lower urinary tract disease (FLUTD), and canine infectious tracheobronchitis. Study journals (n = 1807) were submitted by 84 veterinarians. Sixty-five percent of the antimicrobials prescribed in dogs and 67% in cats were ß-lactams. Most frequently prescribed in dogs were cephalexin (33%) and amoxicillin-clavulanic acid (16%), and in cats, amoxicillin-clavulanic acid (40%) and cefovecin (17%); 7% of the prescriptions in dogs and 12% in cats were for fluoroquinolones. Sixty-seven percent of the disease events associated with canine infectious tracheobronchitis, and 70% and 74% associated with FURTD and FLUTD, respectively, were treated with antimicrobials. These results suggest that cefovecin and fluoroquinolones may be over-used and antimicrobial use for the treatment of FURTD, FLUTD, and canine infectious tracheobronchitis could probably be reduced to lessen resistance selection pressure without compromising patient health.

Clostridium perfringens in retail chicken.

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% "atypical" genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.

Escherichia coli and selected veterinary and zoonotic pathogens isolated from environmental sites in companion animal veterinary hospitals in southern Ontario.

Hospital-based infection control in veterinary medicine is emerging and the role of the environment in hospital-acquired infections (HAI) in veterinary hospitals is largely unknown. This study was initiated to determine the recovery of Escherichia coli and selected veterinary and zoonotic pathogens from the environments of 101 community veterinary hospitals. The proportion of hospitals with positive environmental swabs were: E. coli--92%, Clostridium difficile--58%, methicillin-resistant Staphylococcus aureus (MRSA)--9%, CMY-2 producing E. coli--9%, methicillin-resistant Staphylococcus pseudintermedius--7%, and Salmonella--2%. Vancomycin-resistant Enterococcus spp., canine parvovirus, and feline calicivirus were not isolated. Prevalence of antimicrobial resistance in E. coli isolates was low. Important potential veterinary and human pathogens were recovered including Canadian epidemic strains MRSA-2 and MRSA-5, and C. difficile ribotype 027. There is an environmental reservoir of pathogens in veterinary hospitals; therefore, additional studies are required to characterize risk factors associated with HAI in companion animals, including the role of the environment.

NetF-producing Clostridium perfringens and its associated diseases in dogs and foals.

The role of type A Clostridium perfringens in canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis is poorly characterized. However, a highly significant association between the presence of novel toxigenic C. perfringens and these specific enteric diseases has been described. These novel toxigenic strains produce 3 novel putative toxins, which have been designated NetE, NetF, and NetG. Although not conclusively demonstrated, current evidence suggests that NetF is likely the major virulence factor in strains responsible for canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis. NetF is a beta-pore-forming toxin that belongs to the same toxin superfamily as CPB and NetB toxins produced by C. perfringens. The netF gene is encoded on a conjugative plasmid that, in the case of netF, also carries another putative toxin gene, netE. In addition, these strains consistently also carry a cpe tcp-conjugative plasmid, and a proportion also carry a separate netG tcp-conjugative plasmid. The netF and netG genes form part of a locus with all the features of the pathogenicity loci of tcp-conjugative plasmids. The netF-positive isolates are clonal in origin and fall into 2 clades. Disease in dogs or foals can be associated with either clade. Thus, these are strains with unique virulence-associated characteristics associated with serious and sometimes fatal cases of important enteric diseases in 2 animal species.

Rhodococcus equi virulence-associated protein A is required for diversion of phagosome biogenesis but not for cytotoxicity.

Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysosomes and did not stain with the acidotropic fluor LysoTracker similar to those containing virulent wild-type R. equi. However, vapA mutant phagosomes had a lower average pH. Late in infection, phagosomes containing the vapA mutant were as frequently positive for LysoTracker as phagosomes containing plasmid-cured, avirulent bacteria, whereas those with virulent wild-type R. equi were still negative for the fluor. Macrophage necrosis after prolonged infection with virulent bacteria was accompanied by a loss of organelle staining with LysoTracker, suggesting that lysosome proton gradients had collapsed. The vapA mutant still killed the macrophages and yet did not affect the pH of host cell lysosomes. Hence, VapA is not required for host cell necrosis but is required for neutralization of phagosomes and lysosomes or their disruption. This is the first report of an R. equi mutant with altered phagosome biogenesis.

Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis using purified recombinant immunogenic proteins.

This study identified and assessed secreted proteins of Clostridium perfringens additional to those previously described for their ability to protect broiler chickens against necrotic enteritis (NE). Secreted proteins of virulent and avirulent C. perfringens were electrophoretically separated and reacted with serum of chickens immune to NE. Three immunoreactive protein bands unique to the virulent C. perfringens were identified by mass spectrometry as the toxin C. perfringens large cytotoxin (TpeL), endo-beta-N-acetylglucosaminidase (Naglu), and phosphoglyceromutase (Pgm). The genes encoding Naglu and Pgm proteins were cloned, and their gene products were purified as histidine-tagged recombinant proteins from Escherichia coli and used in immunizing chickens. Immunized and nonimmunized control broiler chickens were then challenged with two different strains (CP1, CP4) of C. perfringens and assessed for the development of NE. Of the two immunogens, Pgm immunization showed significant protection of broiler chickens against experimental NE, although protection reduced as challenge severity increased. However, birds immunized with Naglu were protected from challenge only with strain CP4. Birds immunized with these proteins had antigen-specific antibodies when tested in an enzyme-linked immunosorbent assay. In conclusion, this study demonstrated the partial efficacy of additional secreted proteins in immunity of broiler chickens to NE. The study also showed that there may be differences in the protective ability of immunogens depending on the infecting C. perfringens strain.

Assessment of laboratory and biosafety practices associated with bacterial culture in veterinary clinics.

OBJECTIVE: To investigate bacterial culture practices in veterinary clinics, with an emphasis on laboratory biosafety and on quality of laboratory practices. DESIGN: Survey-based prospective study. SAMPLE POPULATION: 166 veterinarians. PROCEDURES: Veterinarians were recruited through the Veterinary Information Network (an Internet-based network restricted to veterinary personnel). All Network-registered veterinarians were eligible to participate. A standardized questionnaire regarding bacterial culture practices in veterinary clinics was completed electronically by study participants. RESULTS: 720 veterinarians completed the survey; 166 (23%) indicated that bacterial culture was performed in his or her clinic. Clinic practices ranged from preliminary aerobic bacterial culture only with submission of isolates to a diagnostic laboratory for further testing (93/160 [58%]) to bacterial culture, identification, and antimicrobial susceptibility testing (19/160 [12%]). Most commonly, urine samples were cultured (151/162 [93%] clinics). Several problematic practices were identified regarding quality and quality control, including inadequate facilities, equipment, supervision, interpretation of data, and culture methods. Biosafety infractions were also common, including inadequate laboratory location, lack of biosafety protocols, and dangerous disposal practices. Ninety-four percent of respondents stated that continuing education regarding culture practices and laboratory safety would be useful. CONCLUSIONS AND CLINICAL RELEVANCE: Data confirmed that bacterial culture was commonly performed in clinics, but that major deficiencies in laboratory methods were widespread. These could result in negative effects on testing quality and increased risk of laboratory-acquired infections among clinic personnel. Veterinary practices in which bacterial cultures are performed must ensure that adequate equipment, facilities, personnel, and training are provided to enable accurate and safe sample testing.