This research evaluates the effectiveness of a concentrated, buffered vinegar product (CBV) and a simple buffered vinegar product (BV) for controlling Clostridium perfringens outgrowth during extended cooling times of ready-to-eat roast turkey and roast beef respectively. Whole turkey breasts and beef inside rounds were injected with a typical brine, then ground and mixed with CBV (0.0, 2.01, 2.70 and 3.30% wt/wt) or BV (0.0, 1.75, 2.25, and 3.75% wt/wt) and a three-strain C. perfringens spore cocktail to a detectable level of ca. 2-3 log CFU/g. The meat was divided into 10g portions and vacuum packaged and stored frozen until tested. The meat was cooked in a programmable water bath to 71.6°C (160.8°F) in 5 hours. The meat was then cooled exponentially with the times between 48.9°C and 12.8°C (120°F and 55°F) lasting 6, 9, 12, 15, and 18 hours for the five different cooling treatments. The cooling continued until the temperature reached 4.4°C (40°F). C. perfringens counts were taken at 54.4°C (130°F) and 4.4°C (40°F). At a 2.01% concentration, CBV effectively limited C. perfringens growth to 1-log or less up to a 9-hour cooling treatment, while 2.70 and 3.30% concentrations were effective up to the 18 hour cooling treatment. BV had an inhibitory effect on C. perfringens outgrowth in roast beef, but did not limit growth to 1-log or less at any concentration tested for any of the cooling treatments.

Clostridium perfringens (CP) was evaluated as an additional indicator in assessing impacts and sources of microbial pollution in the Idaho-Utah Cache Valley . Point , nonpoint, river water, and animal fecal samples were analyzed for CP, total coliforms, fecal collforms, and fecal streptococci. Monthly river samples consistently contained <20 CP/100 mL , but concentrations of the other indicators varied significantly by location and date. Two sample stations consistently had CP concentrations greater than 20 / 100 mL . One of these stations was influenced by an upstream wastewater discharge . Chlorinated effluent from this trickling filter plant contained greater than 103 CP / 100 mL, but met a 400 FC/100 mL discharge standard. A consistent decrease in CP concentrations in samples taken downstream from this wastewater source were fo und, despite significant impact from adjacent nonpoint pollution. Lagoon and oxidation ditch wastewater effluents sampled contained <20 CP/100 mL. Nonpoint sources sampled (e.g . , cattle feedlot runoff) contained <20 CP / 100 mL and 102-104/100 mL coliforms and fecal streptococcus. Cattle, horse, and sheep feces analyzed contained 104-107/g coliforms and fecal streptococcus, but less than 102 CP/g. Nonpoint pollution from such animals may contribute significant coliforms and streptococci but not CP. Wastewater treatment effluents may or may not contain elevated levels of CP depending on factors such as wastewater residence time and particular treatment process employed. The occurrence of relatively high, i.e., >102 CP/100 mL, in areas impacted by nonpoint sources may suggest a municipal wastewater input. Coliform and streptococci indicators may not be able to distinguish municipal or domestic microbial loading in the presence of nonpoint source interferences in many circumstances.

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