Effects of Clostridium perfringens iota toxin in the small intestine of mice.

Iota toxin is a binary toxin solely produced by Clostridium perfringens type E strains, and is structurally related to CDT from C. difficile and CST from C. spiroforme. As type E causes hemorrhagic enteritis in cattle, it is usually assumed that associated diseases are mediated by iota toxin, although evidence in this regard has not been provided. In the present report, iota toxin intestinal effects were evaluated in vivo using a mouse model. Histological damage was observed in ileal loops treated with purified iota toxin after 4 h of incubation. Luminal iota toxin induced fluid accumulation in the small intestine in a dose dependent manner, as determined by the enteropooling and the intestinal loop assays. None of these changes were observed in the large intestine. These results suggest that C. perfringens iota toxin alters intestinal permeability, predominantly by inducing necrosis and degenerative changes in the mucosal epithelium of the small intestine, as well as changes in intestinal motility. The obtained results suggest a central role for iota toxin in the pathogenesis of C. perfringens type E hemorrhagic enteritis, and contribute to remark the importance of clostridial binary toxins in digestive diseases.

Effects of a blend of chestnut and quebracho tannins on gut health and performance of broiler chickens.

Antimicrobial restrictions prompted the search for cost and biologically effective alternatives to replace antimicrobial growth promoters (AGPs) in food-producing animals. In addition, the efficacy of this alternatives needs to be contrasted in field/commercial trials under different challenge conditions. However only a few studies describing the impact of tannins or others AGP-alternatives in commercial poultry production conditions are actually available. The aim of the present work is to study how the inclusion of a blend of chestnut and quebracho tannins can affect broiler productive performance and health under commercial conditions. Three experiments with different approaches were conducted: (1) a trial comparing the effects of both additives (tannins vs AGP) on different commercial farms at the same time; (2) the follow-up of one farm during an entire productive year; and (3) an experimental trial using a C. perfringens challenge model in broiler chickens. Although productive results from field trials were similar among treatments, evaluations of gut health indicators showed improvements in the tannins treated flocks. Frequency and severity of intestinal gross lesions were reduced in jejunum (42% vs 23%; p<0.05-1.37 vs. 0.73; p<0.01, respectively) and ileum (25% vs. 10%; p<0.0.5-1.05 vs. 0.58; p<0.01) in tannins treated birds. Results from 16S studies, show that cecal microbiota diversity was not differentially affected by AGPs or tannins, but changes in the relative abundance of certain taxa were described, including Lactobacillus and Bifidobacterium groups. Results from experimental C. perfringens necrotic enteritis showed that tannins treated birds had reduced incidence of gross lesions in jejunum (43.75 vs. 74.19%; p<0.01) and ileum (18.75% vs. 45.16%; p<0.05) compared with control. These results suggest that AGPs can be replaced by tannins feed additives, and contribute in the implementation of antimicrobial-free programs in broilers without affecting health or performance.

Overview of the role of Shiga toxins in porcine edema disease pathogenesis.

Shiga toxin-producing Escherichia coli (STEC) have been implicated as the cause of enterotoxemias, such as hemolytic uremic syndrome in humans and edema disease (ED) of pigs. Stx1 and Stx2 are the most common types found in association with illness, but only Stx2e is associated with disease in the animal host. Porcine edema disease is a serious affection which can lead to dead causing great losses of weaned piglets. Stx2e is the most frequent Stx variant found in porcine feces and is considered the key virulence factor involved in the pathogenesis of porcine edema disease. Stx2e binds with higher affinity to Gb4 receptor than to Gb3 which could be due to amino acid changes in B subunit. Moreover, this subtype also binds to Forssman glycosphingolipids conferring upon Stx2e a unique promiscuous recognition feature. Manifestations of edema disease are caused by systemic effects of Stx2e with no significant morphologic changes in enterocytes. Endothelial cell necrosis in the brain is an early event in the pathogenesis of ED caused by Stx2e-producing STEC strains. Further studies are needed to generate techniques and tools which allow to understand the circulation and ecology of STEC strains in pigs even in resistant animals for diagnostic and epidemiological purposes.

Simultaneous Carriage of mcr-1 and Other Antimicrobial Resistance Determinants in Escherichia coli From Poultry.

The use of antimicrobial growth promoters (AGPs) in sub-therapeutic doses for long periods promotes the selection of resistant microorganisms and the subsequent risk of spreading this resistance to the human population and the environment. Global concern about antimicrobial resistance development and transference of resistance genes from animal to human has been rising. The goal of our research was to evaluate the susceptibility pattern to different classes of antimicrobials of colistin-resistant Escherichia coli from poultry production systems that use AGPs, and characterize the resistance determinants associated to transferable platforms. E. coli strains (n = 41) were obtained from fecal samples collected from typical Argentine commercial broiler farms and susceptibility for 23 antimicrobials, relevant for human or veterinary medicine, was determined. Isolates were tested by PCR for the presence of mcr-1, extended spectrum ß-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) coding genes. Conjugation and susceptibility patterns of the transconjugant studies were performed. ERIC-PCR and REP-PCR analysis showed a high diversity of the isolates. Resistance to several antimicrobials was determined and all colistin-resistant isolates harbored the mcr-1 gene. CTX-M-2 cefotaximase was the main mechanism responsible for third generation cephalosporins resistance, and PMQR determinants were also identified. In addition, co-transference of the qnrB determinant on the mcr-1-positive transconjugants was corroborated, which suggests that these resistance genes are likely to be located in the same plasmid. In this work a wide range of antimicrobial resistance mechanisms were identified in E. coli strains isolated from the environment of healthy chickens highlighting the risk of antimicrobial abuse/misuse in animals under intensive production systems and its consequences for public health.

Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes.

Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions.

Clostridium perfringens type E virulence traits involved in gut colonization.

Clostridium perfringens type E disease in ruminants has been characterized by hemorrhagic enteritis or sudden death. Although type E isolates are defined by the production of alpha and iota toxin, little is known about the pathogenesis of C. perfringens type E infections. Thus far, the role of iota toxin as a virulence factor is unknown. In this report, iota toxin showed positive effects on adherence and colonization of C. perfringens type E while having negative effect on the adherence of type A cells. In-vitro and in-vivo models suggest that toxinotype E would be particularly adapted to exploit the changes induced by iota toxin in the surface of epithelial cells. In addition, type E strains produce metabolites that affected the growth of potential intra-specific competitors. These results suggest that the alteration of the enterocyte morphology induced by iota toxin concomitantly with the specific increase of type E cell adhesion and the strong intra-specific growth inhibition of other strains could be competitive traits inherent to type E isolates that improve its fitness within the bovine gut environment.

Carriage of Clostridium perfringens by benthic crabs in a sewage-polluted estuary.

The Estuary of Bahía Blanca (EBB), Argentina, is an important wetland under intense sewage pollution. We investigated the occurrence of Clostridium perfringens (CP) in populations of two benthic crabs (Neohelice granulata and Cyrtograpsus angulatus) and in sediment from the EBB. CP was found in 49.1% of the crabs and all of the isolates were identified as type A. The alpha (cpa) and enterotoxin (cpe) encoding genes were identified. Genetic analyses identified 13 novel sequence types, and found no clustering among isolates, suggesting that CP is not part of the crabs' commensal flora. CP carriage was 51 times more likely in crabs from the area nearest sewage outfalls compared with crabs from a reference site. Our in vitro experiments suggest that the carriage of CP in crabs is transient. The use of these benthic crabs as monitoring organisms of sewage pollution in coastal habitats is proposed.

Perspectives in the use of tannins as alternative to antimicrobial growth promoter factors in poultry.

Antibiotics have been included in the formulation of feed for livestock production for more than 40 years as a strategy to improve feed conversion rates and to reduce costs. The use of antimicrobials as growth-promoting factors (AGP) in sub-therapeutic doses for long periods is particularly favorable for the selection of antimicrobial resistant microorganisms. In the last years, global concern about development of antimicrobial resistance and transference of resistance genes from animal to human strains has been rising. Removal of AGP from animal diets involves tremendous pressure on the livestock and poultry farmers, one of the main consequences being a substantial increase in the incidence of infectious diseases with the associated increase in the use of antibiotics for therapy, and concomitantly, economic cost. Therefore, alternatives to AGP are urgently needed. The challenge is to implement new alternatives without affecting the production performances of livestock and avoiding the increase of antimicrobial resistant microorganisms. Plant extracts and purified derived substances are showing promising results for animal nutrition, either from their efficacy as well as from an economical point of view. Tannins are plant derived compounds that are being successfully used as additives in poultry feed to control diseases and to improve animal performance. Successful use of any of these extracts as feed additives must ensure a product of consistent quality in enough quantity to fulfill the actual requirements of the poultry industry. Chestnut (hydrolysable) and Quebracho (condensed) tannins are probably the most readily available commercial products that are covering those needs. The present report intends to analyze the available data supporting their use.