Abstract not available

The objective of this study was to determine the effect of time, temperature and organic matter on disinfectant efficacy. Staphylococcus aureus (SA) and Salmonella Typhimurium (ST) were used as organisms to represent Gram positive and Gram negative bacteria, respectively, commonly found in poultry housing. Three independent experiments evaluated the effect of temperature, time, and organic matter on the efficacy of working concentrations of disinfectants against representative organisms found in commercial poultry housing. Quaternary ammonium, chlorhexidine, phenolic and binary ammonium based solutions represented disinfectants commonly used within the poultry industry. Results from these experiments indicated that long term storage of disinfectants will reduce their efficacy against SA. However, a reduction (p ≤ 0.05) in efficacy was observed with the phenolic compound against ST at elevated temperatures. Following the inclusion of organic matter (OM), reduced (p ≤ 0.05) efficacy of all disinfectants was observed in a dose dependent manner against both organisms, with the exception of the phenolic compound against SA. Fresh disinfectant performed better (p ≤ 0.05) in the presence of OM than 30 wk old disinfectant. These results emphasize the need to use fresh disinfectants and that OM should be removed prior to disinfection. We also evaluated the effect of bismuth citrate, lactose and citric acids on the development of necrotic enteritis in broilers. Clostridium perfringens’ associated necrotic enteritis in poultry causes significant loss and increased morbidity in the industry. Due to the reduced usage of antibiotic growth promoters, the incidence of necrotic enteritis has increased. These experiments evaluated different levels of bismuth citrate and bismuth citrate with lactose or citric acid added, on lesion development, bacterial intestinal colonization of C. perfringens and pH levels in the gut of broilers orally challenged with C.perfringens. Results from this investigation indicate that bismuth citrate at 100 ppm and 200 ppm caused a reduction (p ≤ 0.05) in C. perfringens colonization and intestinal lesion development. The addition of dietary lactose to bismuth citrate enhanced the effect of bismuth citrate on intestinal lesion development. These data suggest that bismuth citrate alone or in combination with dietary lactose will reduce intestinal lesion development in broilers with necrotic enteritis.

La enterotoxemia, causada por el Clostridium perfringens, es la enfermedad infecciosa más importante que afecta a las alpacas, debido a que ocasiona elevadas tasas de mortalidad neonatal de hasta 70%. A pesar de esto, existe poca información sobre los factores de virulencia (toxinas) del C. perfringens que participan en la etiopatogénesis de la enfermedad. El presente estudio tuvo como objetivo determinar el genotipo de los C. perfringens aislados de casos de enterotoxemia en base a la presencia de los genes (cpa, cpb, etx e iap) codificantes de las toxinas principales (α, β, ε y ι) así como el subtipo en base a la presencia de los genes cpe y cpb2 codificantes de la enterotoxina (CPE) y la toxina β2, respectivamente. En el estudio se analizaron 47 aislamientos de C. perfringens obtenidos de intestino de crías muertas de alpaca con signos clínicos y lesiones anatomopatológicas e histopatológicas correspondientes a enterotoxemia. El ADN de estos aislados fue extraído y analizado por la técnica de PCR Múltiple conteniendo iniciadores específicos para los genes codificantes de las toxinas mencionadas, encontrándose en 33/47 (70.2%) aislamientos sólo al gen cpa (genotipo A subtipo cpe-cpb2-), en 13/47 (27.7%) a los genes cpa y cpb2 (genotipo A subtipo cpe-cpb2+) y en 1/47 (2.1%) a los genes cpa, cpb y cpe ( genotipo A subtipo cpe+cpb2-). Estos resultados evidencian principalmente a la toxina α, así como a la β2 y β participar en la etiopatogénesis de la enterotoxemia en las alpacas. Palabras Clave: Clostridium perfringens, genotipificación, enterotoxemia, alpacas. / Enterotoxemia, caused by the Clostridium perfringens, is the most important infectious disease which affects alpacas, causing up to 70% neonatal mortality. In spite of this, little information exists on the virulence factors (toxins) of C. perfringens which play an important role in the etiopathogenesis of the disease. The objective of the present study was to determine the genotype of C. perfringens isolated from cases of enterotoxemia based on the presence of genes (cpa, cpb, etx and iap) which encode the main toxins (α, β, ε and ι), as well as the subtypes based on the presence of genes cpe and cpb2 which encode the enterotoxin (CPE) and β2-toxin respectively. A total of 47 isolations of C. perfringens were obtained from the small intestine of neonatal alpaca mortalities which had both clinical signs and gross and histological injuries typical of enterotoxemia. The DNA was extracted from these isolates and analyzed by PCR Multiplex using specific primers for the toxin genes. The cpa gene genotype A subtype cpe-cpb2-) was the only gene found in 70.2% (33/47) of the isolations. In 27.7% (13/47) of the cases the cpa and cpb2 (genotype A subtype cpe-cpb2+) genes were found and in 2.1% (1/47) the cpa, cpb and cpe (genotype C subtype cpe+cpb2-) genes were present. These results demonstrate the primary role of α-toxin, as well as the presence of β2 and β-toxins in the etiopathogenesis of enterotoxemia in alpacas. Key Words: Clostridium perfringens, genotypification, enterotoxemia, alpacas.

published_or_final_version / Microbiology / Master / Master of Science

Clostridium perfringens is an important pathogen in veterinary and medical fields. Understanding epidemiology of C. perfringens diseases and evolution of virulence within C. perfringens necessitates an efficient, time and cost effective strain typing method. Multiple-locus variable-number tandem repeat analysis (MLVA) has been applied to typing of other pathogens and we describe here the development of a MLVA scheme for C. perfringens. We characterized five VNTR loci, and screened 112 C. perfringens isolates to evaluate typability, reproducibility, and discriminatory power of the scheme. All isolates were assigned a MLVA genotype and the technique has excellent reproducibility, with a numerical index of discrimination of 0.995. Thus, MLVA is an efficient tool for C. perfringens strain typing, and being PCR based makes it rapid, easy, and cost effective. In addition, it can be employed in epidemiological, ecological, and evolutionary investigations of the organism.Virulence of this species is not fully understood and it does seem that distribution of the toxin/enzyme genes is erratic within the population. We used the MLVA scheme to investigate evolution of virulence and population structure of this species. Analysis of the phylogenetic signal indicates that acquisition of the major toxin genes and other plasmid-borne toxin genes is a recent evolutionary event, and their maintenance is essentially a function of the selective advantage they confer to strains carrying them in certain micro-niches under different conditions. In addition, it indicates the ability of virulent strains to cause disease in different hosts. More interestingly, there is evidence that certain normal flora strains are virulent when they gain access to a different host species. Analysis of the population structure indicates that recombination events are the major tool that shapes the population and this panmixia is interrupted with frequent clonal expansion that mostly corresponds to disease processes. Signature of positive selection was detected in the alpha toxin gene, suggesting the possibility of adaptive alleles on the other chromosomally-encoded determinants. Finally, C. perfringens proved to have a dynamic population, and availability of more genome sequences, use of comparative proteomics and of animal models would provide more insight into the pathogenicity of this organism.

This study sought to understand the pathogenesis of hemorrhagic abomasitis in calves by characterizing a type A Clostridium perfringens isolate. The complete genome sequence of an isolate from an outbreak of hemorrhagic abomasitis was compared to the three complete C. perfringens genomes currently available in GenBank. Unique findings included the presence of an integrated plasmid sequence and a frameshift mutation in the virS gene, which encodes the main sensor kinase that controls virulence gene regulation. An ~ 55 kb plasmid similar to pCW3 was found, in addition to two smaller plasmids with no significant similarity to available C. perfringens plasmid sequences. A number of plasmid-related fragments were also identified. Neither genomic nor proteomic approaches identified novel toxins, but an alternate and unexpected picture of virulence has emerged suggesting that anomalous virulence gene regulation might contribute to pathogenicity in this isolate. / Natural Sciences and Engineering Research Council, the Ontario Graduate Scholarship, the Animal Health Strategic Initiative of the Ontario Ministry of Agriculture, Food and Rural Affairs

The main objective of this work was to develop an antigen-capture enzyme-linked immunosorbent assay for detection of beta2-toxin in the intestine of neonatal piglets. The format of the assay comprised of capture antibodies (polyclonal), antigen (beta2-toxin), detecting antibody (labeled monoclonal) and a substrate. The ELISA was optimized using recombinant protein. After intestinal content samples were applied, the test protocol needed to be adjusted because of the presence of high background signal in some samples consistent with intestinal proteases. This was overcome by processing the samples at 4oC and using citrate buffer pH 6.1 containing 5% bovine serum albumin. The second objective was to identify cpb2 in Clostridium perfringens type A isolates from selected animal species and to examine genotype-phenotype corelation. The study concluded that consensus cpb2, if present, was almost always expressed. In contrast, only about three-quarters of atypical cpb2, mostly was present in isolates of non-porcine origin, were expressed. / Ontario Ministry of Agriculture, Food and Rural Affairs; The Natural Sciences and Engineering Research Council of Canada

To study the distribution of Clostridium perfringens and toxin genes, 48 swine farms were visited and 354 fecal samples were collected. The isolates recovered from lactating sows, gestating sows, grower-finishers, and manure pits were less likely to possess consensus gene cpb2 compared to those from suckling pigs (P<0.05). The relative importance of different pathogens associated with neonatal piglet diarrhea was identified. A total of 237 neonatal diarrhea cases were submitted to the Animal Health Laboratory, University of Guelph between 2001 and 2010. The combined frequencies for cases involving enterotoxigenic Escherichia coli, Clostridium perfringens type A, rotavirus, and Clostridium difficile accounted for 56% of the total cases. A survey was administered to 22 practitioners and 17 pathologists for the diagnosis of C. perfringens type A. The majority (95%) of practitioners were moderately to very confident of their diagnosis, but almost half (41%) of the pathologists were not confident of their diagnosis. / Ontario Ministry of Agriculture Food and Rural Affairs

Necrotic enteritis (NE) is an economically important enteric disease of broiler chicken primarily caused by a-toxin (Cpa) secreted by C. perfringens type A. Mice immunized with recombinant C-terminal domain of Cpa (CpaCD) had transient and fewer localized lesions upon challenge with C. perfringens type A. These results demonstrate the usefulness of CpaCD as an immunogen for vaccine development against NE for chickens. Chicken CD40 (chCD40) is mainly expressed on the surface of chicken antigen-presenting cells (APCs), and the interaction of chCD40 and chCD40L (natural ligand for chCD40) provides crucial activation signals for chicken B-cells. A hypothesis was proposed that in ovo vaccination with an adenovirus-vectored CpaCD vaccine capable of targeting immunogen to APCs through the CD40 pathway will improve protection against NE in chickens. One agonistic monoclonal anti-chCD40 antibody (designated 2C5) was produced and characterized. 2C5 not only detected expression of chCD40 on chicken APCs, but also induced NO synthesis in chicken HD11 macrophages and enhanced proliferation of serum-starved chicken DT40 B-cells. This demonstrated substantial functional equivalence of 2C5 with chCD40L. The potential of 2C5 as an immunological adjuvant was further assessed by targeting a hapten to chicken APCs in hopes of enhancing an effective IgG response. Seven-week old chickens were immunized subcutaneously once with a complex consisting of 2C5 and peptide, and relative quantification of the peptide-specific IgG response showed that this complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. An agonistic anti-chCD40 single-chain variable fragment (designated DAG1) was combined with an adenoviral delivery system to create a vaccine, Ad-(DAG1-Cp aCD-FLAG), for in ovo administration. The efficacy of in ovo vaccination of broilers with Ad-(DAG1-Cp aCD-FLAG) in controlling NE was evaluated by C. perfringens type A challenge at 18 days post-hatch. Neither statistically significant IgA / IgG response nor protection against C. perfringens type A challenge was found in the vaccinated birds. These preliminary data suggest that a super-optimal dose of Ad-(DAG1-Cp aCD-FLAG) may be the main issue, because Cpa-specific B-cells may undergo apoptosis through the CD40 pathway.

Universiẗat, Diss., 2000--Kiel.