No description available.

A study on cuticle-degrading enzymes (CDE) of three hyphomycete entomopathogens has produced information on enzyme types, levels, characteristics, mode of action, regulation, sequence of production, cellular localisation and production during host penetration. This is the first critical work on CDE of any entomopathogen. Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as sole carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular and bound enzymes corresponding to the major components of insect cuticle e.g. 3 endo-proteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase and N-acetylglucosaminidase. Considerable variations occurred in levels of production between spp. and even within a sp., but endo-proteases were exceptional in being produced in large amounts by all the isolates. CDE were produced rapidly and sequentially in culture. The first activities to appear (< 24 h) were those of the proteolytic complex, chitinases were always produced substantially later. Properties of CDE were investigated in terms of pH and tem- erature optima, substrate specificity, molecular weight, iso-electric point, mechanism of substrate degradation and the effect of specific inhibitors. Studies with culture filtrates and purified CDE revealed that substrates in intact cuticles are amenable to degradation but the prior action of protease is necessary for significant degradation of the chitin. Staining of chitin by a fluorescent lectin (FITC-WGA) and calcofluor only in cuticles from which protein has been removed (by protease or KOH) also suggests initial masking of chitin. This and determination of amino acid composition of peptides solubilised by endo-protease revealed the potential of CDE in studying the physicochemical structure of insect cuticles. The apparently localised action of CDE during host penetration may result from molecular sieving, binding to fungal walls or binding to cuticle. The first possibility is lessened by the small size of the endo-enzymes (<34 K daltons) which could allow diffusion via the various canals which traverse cuticle. However, cuticle effectively binds (ionically) CDE, and also activities of several CDE remain partly bound in various ways to hyphae and conidia (by ionic binding to walls, by disulphide bonds, and on or within membrane structures). The involvement of proteolytic enzymes in infection was suggested by their presence in conidia, penetration structures, and infected cuticle (detected histochemically and following extraction from cuticles). Also the constitutive production of endo- and exo-proteases lends weight to their possible significance in parasitism as synthesis will be subject only to catabolite repression. Chitinase is induced by N-acetylglucosamine and was not detected in infected cuticle. Possible mechanisms and significance of enzymic degradation of cuticle during infection are discussed, particularly in comparison to host penetration by phytopathogenic fungi.

A microfluidic device, based on electrophoretic transport and electrostatic trapping of charged particles, has been developed for continuous capture and concentration of microorganisms from water. A generic design, utilizing mobility and zeta potential measurements of various microorganisms exposed to different environmental conditions and physiological states, was employed. Water and buffer samples at pH values ranging from 5.2–7.0 were seeded with bacteria (E. coli, Salmonella, and Pseudomonas) and viruses (MS-2 and Echovirus). Negative control and capture experiments were performed simultaneously using two identical devices. Both culture based methods and real-time PCR analysis were utilized to characterize the capture efficiency as a function of time, flowrate, and applied electric field. Based on differences between the capture and negative control data, capture efficiencies of 90% to 99% are reported for E. coli, Salmonella, Pseudomonas, and MS-2, while the capture efficiency for Echovirus was around 75%. Overall, the device exhibits 16.67 fold sample volume reduction within an hour at 6 mL/hr. This results in a concentration factor of 15 at 90% capture efficiency. Direct quantification of capture on the anode of the prototype microfluidic device was also performed by particle tracking using fluorescent microscopy. Based on image processing, the capture data at different locations on the electrode surface is quantified as a function of the wall shear stress at these locations, which is calculated using CFD simulations. Finally, the Faradaic processes in the microchannel due to electrochemical reactions are studied to predict the amount of electrophoresis in the system. Scaling of the device to sample 5 L/hr can be achieved by stacking 835 identical microchannels. Power and wetted volume for the prototype and scaled devices are presented. The device can thus function either as a filtration unit or as a sample concentrator to enable the application of real-time detection sensor technologies. The ability to continuously sample water without chemical additives facilitates the use of this device in drinking water distribution systems. This work constitutes the first step in our development of a continuous, microbial capture and concentration system from large volumes of potable water.

The purpose of this work was to examine the effectiveness of several replacements for antibiotics in broiler chickens using bacterial challenge models. For this work, pathogen challenge models were developed using three model pathogens; two human pathogens (<i>Salmonella enteritidis</i> and <i>Campylobacter jejuni</i>), and one poultry pathogen (<i>Clostridium perfringens</i>). The first set of experiments involved the selection and use of 2 model probiotics; <i>Bifidobacterium animalis</i> and <i>Lactobacillus fermentum</i>. Oral administration of either probiotic did not significantly reduce (P < 0.05) the level of intestinal colonization by either <i>S. enteritidis</i> or <i>C. jejuni</i> in experimentally infected broiler chickens. The next set of experiments examined the effectiveness of orally administered, pathogen-specific antibodies obtained from hyperimmunizing laying hens in controlling bacterial infections with <i>S. enteritidis</i>, <i>C. jejuni</i> or <i>Clostridium perfringens</i> in broiler chickens. Regardless of the concentration, or mode of administration, anti-<i>S. enteritidis</i> hen-egg antibodies or anti-<i>C. jejuni</i> hen-egg antibodies were unable to significantly reduce (P < 0.05) the intestinal colonization by either pathogen in experimentally infected broiler chickens. Likewise, administration of anti-<i>C. perfringens</i> hen-egg antibodies did not reduce intestinal colonization by <i>C. perfringens</i>, and actually exacerbated the clinical outcome of this important poultry pathogen by significantly increasing (P < 0.05) intestinal lesions scores compared to negative control birds. Lastly, the effect of dietary protein source on intestinal <i>C. perfringens</i> populations was investigated. In broiler chickens experimentally infected with <i>C. perfringens</i> and fed diets which varied in the source of dietary protein, it was shown that birds fed fish meal, meat/bone meal, feather meal and potato protein concentrate had significantly higher intestinal <i>C. perfringens</i> counts than the birds fed corn gluten meal, soy or pea protein concentrates or the control diet (P < 0.05). Further, it was shown that the glycine content of the diets and ileal contents was significantly, positively correlated with <i>C. perfringens</i> numbers in ileum and cecum. It is concluded that although the intervention strategies employed in these studies show promise, diet composition clearly had the largest effect on intestinal bacterial populations. Further studies are required to examine both the impact that diet and these intervention strategies have on the factors which control intestinal colonization by pathogens on a case by case basis.

The purpose of this work was to examine the effectiveness of several replacements for antibiotics in broiler chickens using bacterial challenge models. For this work, pathogen challenge models were developed using three model pathogens; two human pathogens (<i>Salmonella enteritidis</i> and <i>Campylobacter jejuni</i>), and one poultry pathogen (<i>Clostridium perfringens</i>). The first set of experiments involved the selection and use of 2 model probiotics; <i>Bifidobacterium animalis</i> and <i>Lactobacillus fermentum</i>. Oral administration of either probiotic did not significantly reduce (P < 0.05) the level of intestinal colonization by either <i>S. enteritidis</i> or <i>C. jejuni</i> in experimentally infected broiler chickens. The next set of experiments examined the effectiveness of orally administered, pathogen-specific antibodies obtained from hyperimmunizing laying hens in controlling bacterial infections with <i>S. enteritidis</i>, <i>C. jejuni</i> or <i>Clostridium perfringens</i> in broiler chickens. Regardless of the concentration, or mode of administration, anti-<i>S. enteritidis</i> hen-egg antibodies or anti-<i>C. jejuni</i> hen-egg antibodies were unable to significantly reduce (P < 0.05) the intestinal colonization by either pathogen in experimentally infected broiler chickens. Likewise, administration of anti-<i>C. perfringens</i> hen-egg antibodies did not reduce intestinal colonization by <i>C. perfringens</i>, and actually exacerbated the clinical outcome of this important poultry pathogen by significantly increasing (P < 0.05) intestinal lesions scores compared to negative control birds. Lastly, the effect of dietary protein source on intestinal <i>C. perfringens</i> populations was investigated. In broiler chickens experimentally infected with <i>C. perfringens</i> and fed diets which varied in the source of dietary protein, it was shown that birds fed fish meal, meat/bone meal, feather meal and potato protein concentrate had significantly higher intestinal <i>C. perfringens</i> counts than the birds fed corn gluten meal, soy or pea protein concentrates or the control diet (P < 0.05). Further, it was shown that the glycine content of the diets and ileal contents was significantly, positively correlated with <i>C. perfringens</i> numbers in ileum and cecum. It is concluded that although the intervention strategies employed in these studies show promise, diet composition clearly had the largest effect on intestinal bacterial populations. Further studies are required to examine both the impact that diet and these intervention strategies have on the factors which control intestinal colonization by pathogens on a case by case basis.

During the harvesting process, meat products can become contaminated with enteric pathogens, such as Escherichia coli O157:H7 and Salmonella Typhimurium. Surrogates for these pathogens would be beneficial for validating carcass decontamination treatments. Surrogate organisms are organisms that behave similarly to specific pathogens but are non-pathogenic and can be used to determine efficacy of decontamination regimes for pathogens. The surrogates proposed are non-pathogenic, ampicillin-resistant E. coli biotype I strains that were previously isolated from beef cattle hides. Each E. coli strain was transformed to express a fluorescent protein (red: EcRFP; green: EcGFP; yellow: EcYFP) that is detectable under an ultraviolet light source. Surface areas on hot boned beef carcasses (clod, brisket, outside round) were inoculated with a fecal slurry containing EcRFP, EcGFP, EcYFP and rifampicin-resistant E. coli O157:H7 and S. Typhimurium. Surface regions were then treated in a model spray cabinet using an initial water wash (28ºC) followed by treatments using 2% L-lactic acid (55ºC), hot water (95ºC at source) or a combination of the two. Treatments were compared for their effectiveness at reducing populations of inoculated (4.7 to 6.7 log CFU/cm2) E. coli, S. Typhimurium, EcRFP, EcGFP and EcYFP. Log reductions for inoculated organisms were calculated individually and then total and average surrogate cocktail values were calculated. All decontamination treatments reduced the inoculated numbers of pathogens and surrogates to near or below the detection limit of 0.5 log CFU/cm2. The combined treatment resulted in the greatest log reductions. The three individual surrogate organisms varied in log reductions according to the different decontamination treatments applied; however, log reductions for the total surrogate cocktail did not differ significantly from that of E. coli O157:H7. With the exception of EcYFP, the individual surrogates and average surrogate cocktail were significantly more resistant to microbial interventions including lactic acid than S. Typhimurium. Because abattoirs utilize different carcass decontamination treatments, it is difficult for one single fluorescent protein-producing isolate to accurately represent the behavior of E. coli O157:H7 or S. Typhimurium. Instead, surrogates should be used as a total cocktail to accurately represent the effectiveness of different treatments for reduction of enteric pathogens.

A microfluidic device, based on electrophoretic transport and electrostatic trapping of charged particles, has been developed for continuous capture and concentration of microorganisms from water. A generic design, utilizing mobility and zeta potential measurements of various microorganisms exposed to different environmental conditions and physiological states, was employed. Water and buffer samples at pH values ranging from 5.2–7.0 were seeded with bacteria (E. coli, Salmonella, and Pseudomonas) and viruses (MS-2 and Echovirus). Negative control and capture experiments were performed simultaneously using two identical devices. Both culture based methods and real-time PCR analysis were utilized to characterize the capture efficiency as a function of time, flowrate, and applied electric field. Based on differences between the capture and negative control data, capture efficiencies of 90% to 99% are reported for E. coli, Salmonella, Pseudomonas, and MS-2, while the capture efficiency for Echovirus was around 75%. Overall, the device exhibits 16.67 fold sample volume reduction within an hour at 6 mL/hr. This results in a concentration factor of 15 at 90% capture efficiency. Direct quantification of capture on the anode of the prototype microfluidic device was also performed by particle tracking using fluorescent microscopy. Based on image processing, the capture data at different locations on the electrode surface is quantified as a function of the wall shear stress at these locations, which is calculated using CFD simulations. Finally, the Faradaic processes in the microchannel due to electrochemical reactions are studied to predict the amount of electrophoresis in the system. Scaling of the device to sample 5 L/hr can be achieved by stacking 835 identical microchannels. Power and wetted volume for the prototype and scaled devices are presented. The device can thus function either as a filtration unit or as a sample concentrator to enable the application of real-time detection sensor technologies. The ability to continuously sample water without chemical additives facilitates the use of this device in drinking water distribution systems. This work constitutes the first step in our development of a continuous, microbial capture and concentration system from large volumes of potable water.

No description available.

Cyathocotyle bushiensis (Digenea) and Sphaeridiotrema globulus (Digenea) are gastrointestinal pathogens of waterfowl, and are known to co-occur in salvaged ducks. The intensity and time-dependent pathogenesis induced by single infections of the two digeneans, and concurrent infections were studied in two species of dabbling ducks. Gross tissue pathology by single C. bushiensis infection was found to be a function of both the intensity and the duration of infection. Infection with single species infections of C. bushiensis and S. globulus was associated with decreased weight gain, tendencies for increased body temperatures and increased hematological parameters in Pekin ducklings. The hematological parameters in blue winged teal infected with C. bushiensis were found to decrease. Infection with S. globulus did not induce any significant systemic changes in the blue winged teal. A preliminary study of the effects of concurrent infection on the duck hosts suggests that in general, concurrent infection enhances the detrimental aspects of single infection, decreasing weight gain, increasing body temperatures, and decreasing hematological parameters in both species.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1991. / Includes bibliographical references (leaves 159-175). / Microfiche. / xiii, 175 leaves, bound ill. (some col.) 29 cm