Enteric Diseases of Swine and Cattle: Prevention, Control and Food Safety

NON-TECHNICAL SUMMARY: Certain agricultural practices contribute to the problem of oysters accumulating Salmonella and therefore, exposing humans to Salmonella upon ingestion. Campylobacter jejuni continues to cause disease in many humans. Shellfish and water surveillance may identify novel important routes of human exposure to Salmonella. PilA, if confirmed important in adherence to cells and to colonize chicks, will validate this protein as a novel vaccine candidate for the prevention of Campylobacter.
<P>APPROACH: Microbial pathogens present in agricultural runoff impose a significant hazard on human health when acquired directly via the fecal-oral route or indirectly as a waterborne contaminant. Salmonella enterica is a common cause of systemic and diarrheal disease in livestock and causes approximately 2 million cases of diarrhea per year in humans in the US with up to 1000 deaths and an economic loss estimated to be $12 billion. Over the past few years, the CDC has noted a rapid increase in the number of laboratory confirmed Salmonella Newport infections (126%). This raises concern for two reasons: 1) the spectrum of illness due to this serovar tends to be more severe, and 2) an increasing number of Newport isolates have recently been shown to be multi-drug resistant. Multi-drug resistance prevalence increased from 1% in 1998 to 26% in 2001, with some isolates being resistant to ceftriaxone, a drug commonly used to treat invasive Salmonella infections. The emergence of a multi-drug resistant S. Newport has been attributed to various factors including intensive farming practices, the movement of cattle between farms and the overuse of antimicrobial agents in dairy operations (Gupta et al., 2003). Shellfish and environmental water sources will be surveyed for potential foodborne enteric pathogens and sequence analysis will be used to infer their host species origin. Campylobacter spp. (primarily C. jejuni) cause an estimated 2.4 million cases of gastroenteritis per year in the U. S. (Mead and Dubreuil, 2002) yet their pathogenic mechanisms remain largely unknown. A novel pilus expressed by C. jejuni upon colonization of abiotic and biotic surfaces during biofilm formation has been discovered in the lab. This pilin (Pil) protein has been sequenced and the gene identified in the genomic sequence of NCTC11168 and a mutant with disrupted pilA has been prepared. Gene complementation in C. jejuni is difficult but possible (e.g., Day et al. (2000). AZ will complement the "fla "pilA mutant in trans to restore piliation using a published method (Mixter et al., 2003). To obtain purified protein for further characterization of the pilus and to produce antisera specific for the pilin protein, the pilA gene will be over-expressed in an E. coli expression vector (Dabrowski and Kur, 1999). AZ will examine the ability of the pilus mutant to adhere to epithelial cells and to colonize chicks. NCTC 11168, a human clinical strain, nevertheless has been shown (Gaynor (2004) to be able to colonize chicks. Finally, we will identify genes involved in biofilm formation using expression arrays. Genes up-regulated during biofilm formation will be mutated and pilus expression will be examined by electron microscopy and through in vitro biofilm formation to allow us to fully characterize the pilus formation by identifying genes involved in regulation and pilus expression.<P>PROGRESS: 2007/01 TO 2007/12
OUTPUTS: From previous studies, Yaquina Bay has had oysters contaminated with Salmonella, mostly Salmonella Newport. Salmonella Newport is an emerging pathogen, and is of particular interest to CDC due to its multi-drug resistant nature. Oysters and water from the Yaquina Bay and Tillamook Bay have both been sent every 2nd month to the laboratory at the University of Arizona from Oregon State University. These oysters are homogenized and enriched to isolate for Salmonella Newport. Additionally, oysters from retail outlets such as restaurants in Arizona are being tested every second month for Salmonella. Campylobacter spp. (primarily C. jejuni) cause an estimated 2.4 million cases of gastroenteritis per year in the U. S. yet their pathogenic mechanisms remain largely unknown. A novel pilus expressed by C. jejuni upon colonization of abiotic and biotic surfaces during biofilm formation has been discovered in the lab. This pilin protein has been sequenced and the gene identified in the genomic sequence of NCTC11168 and a mutant with disrupted pilA has been prepared. The ability of the pilus mutant to adhere and invade epithelial cells was analyzed with HEp2 cells. <br>
PARTICIPANTS: Lynn Joens: PI, to supervise all aspects of project Bibiana Law, Assistant Research Professor, to design protocols, train students, and perform research Crystal Brillhart and Alex Armstrong: Graduate students, to conduct experiments<br>TARGET AUDIENCES: Project provided laboratory training for students.

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IMPACT: 2007/01 TO 2007/12
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As we did obtain some Salmonella positives from Tillamook Bay during the study, we are currently processing the samples on a monthly basis. We are analyzing rainfall events and environmental factors to increase our understanding of the contamination. Some oyster samples from the restaurants have also been positive. The Salmonella isolates will be analyzed via Pulsed Field Gel Electrophoresis, and tested for antibiotic resistance. The pilA mutant showed decreased adherence and no invasion in the in vitro assay, indicating that the gene is involved in adherence and invasion.
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PROGRESS: 2006/01/01 TO 2006/12/31
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Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries, and a source of public health and economic burden. C. jejuni is consistently present in the intestinal tract of commercial broiler chickens, which are probably the main source of human infections. In fact biofilm formation on watering nipples may play a role in the colonization of broiler chickens. We have sampled watering nipples from two more broiler houses and currently have cultured over 250 isolates of bacteria. We are currently performing 16S PCR on each culture and the products will be sent to the sequencing facility. Experiments will be performed to characterize the biofilm bacterial community. We are also currently investigating the conditions required for C. jejuni to form biofilms in well water. Various conditions such as various timepoints, and adding nutrients or organisms such as Pseudomonas will be assessed for their affect on the growth of C. jejuni biofilms. Ultimately, the goal is to understand how biofilms are formed by C. jejuni and if this is an environmental reservoir involved in the infection of poultry.
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IMPACT: 2006/01/01 TO 2006/12/31
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Understanding how biofilms are formed by C. jejuni will contribute to how C. jejuni can be prevented from the formation of C. jejuni biofilms in broiler houses and ultimately result in reducing the colonization of chickens with C. jejuni.

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PROGRESS: 2005/01/01 TO 2005/12/31
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Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries, and a source of public health and economic burden. C. jejuni is consistently present in the intestinal tract of commercial broiler chickens, which are probably the main source of human infections. In fact biofilm formation on watering nipples may play a role in the colonization of broiler chickens. By using a staining procedure to visualize biofilms, we have determined that 1) C. jejuni can form biofilms on abiotic surfaces such as polyvinyl chloride plastic (PVC), acrylonitrile-butadiene-styrene plastic (ABS), copper, polystyrene, and polypropylene when grown in Mueller-Hinton broth, 2) protein synthesis maybe required for the early stages of biofilm formation as chloramphenicol inhibits biofilm formation, 3) as nutritional and environmental conditions of the medium changes, it can have varying affects on the ability of C. jejuni to form biofilms, and 4) C. jejuni mutants defective in the putative virulence genes and a quorum sensing gene had varying affects on biofilm formation. Therefore, with the combination of genetic, molecular and microscopy techniques, we have been able to initiate investigations into the mechanisms that control C. jejuni biofilm formation and development. Based on the results, we can see that C. jejuni biofilm formation can be a set of complex actions. To form complex communities, C. jejuni must integrate environmental signals by determining density and nutrient availability. We have sampled a biofilm off a water pipe from a broiler house and over 100 isolates of bacteria were cultured and 16S PCR performed on each culture. The PCR products will be sent to the sequencing facility and experiments to characterize the biofilm bacterial community will be performed. The actions and gene expressions that drive biofilm development will continue to be the point of investigation.
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IMPACT: 2005/01/01 TO 2005/12/31
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Campylobacter jejuni: A microbial community on abiotic surfaces: Understanding how biofilms are formed by C. jejuni will contribute to how C. jejuni can be prevented from the formation of C. jejuni biofilms in broiler houses and ultimately result in reducing the colonization of chickens with C. jejuni.
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PROGRESS: 2004/01/01 TO 2004/12/31
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Presence, potential virulence and antibiotic susceptibility of Campylobacter jejuni isolated from food and companion animals: Infection with Campylobacter jejuni is associated with exposure to numerous animal related sources. In this study, we report on the recovery, potential for virulence and antibiotic resistance levels of C. jejuni isolated from food and companion animals. Three hundred seventy-eight fecal samples from food and companion animals and beef carcass swabs were tested for the presence of Campylobacter jejuni. Campylobacter jejuni was isolated from 13.8% (11/80) of dogs, 5% (1/20) of goats, 30.5% (53/174) of cattle and 94.7% (18/19) of beef carcasses. Forty-two of these isolates were tested for susceptibility to four antibiotics, each representing a class of antibiotic approved for use in both humans and animals. No isolates were found to be resistant to erythromycin or gentamicin, 2.4% of isolates were resistant to ciprofloxacin and 28.6% of isolates were resistant to tetracycline. The virulence of these 42 isolates was assessed using in vitro macrophage survival and epithelial cell association assays. Of these, 34 were able to survive within macrophages through 72 hrs, 39 were able to attach to epithelial cells and 31 were capable of invading epithelial cells. Data from these studies suggests that many of the isolates recovered from the non-poultry animal sources included this study have the capacity to cause disease if transmitted to humans. Campylobacter jejuni: A microbial community on abiotic surfaces: Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries, and a source of public health and economic burden. C. jejuni is consistently present in the intestinal tract of commercial broiler chickens, which are probably the main source of human infections. In fact biofilm formation on watering nipples may play a role in the colonization of broiler chickens. By using a staining procedure to visualize biofilms, we have determined that 1) C. jejuni can form biofilms on abiotic surfaces such as polyvinyl chloride plastic (PVC), acrylonitrile-butadiene-styrene plastic(ABS), copper, polystyrene, and polypropylene when grown in Mueller-Hinton broth, 2) protein synthesis maybe required for the early stages of biofilm formation as chloramphenicol inhibits biofilm formation, 3) as nutritional and environmental conditions of the medium changes, it can have varying affects on the ability of C. jejuni to form biofilms, and 4) C. jejuni mutants defective in the putative virulence genes and a quorum sensing gene had varying affects on biofilm formation. Therefore, with the combination of genetic, molecular and microscopy techniques, we have been able to initiate investigations into the mechanisms that control C. jejuni biofilm formation and development. Based on the results, we can see that C. jejuni biofilm formation can be a set of complex actions. To form complex communities, C. jejuni must integrate environmental signals by determining density and nutrient availability. Therefore, these actions and gene expressions that drive biofilm development will continue to be the point of investigation.
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IMPACT: 2004/01/01 TO 2004/12/31
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Presence, potential virulence and antibiotic susceptibility of Campylobacter jejuni isolated from food and companion animals: Campylobacter jejuni has been isolated from many cattle and livestock animals. The presence of such a reservoir in animals contributes to the zoonotic spread of diseases such as C. jejuni to man. Determining the prevalence of C. jejuni in a broad range of species, the virulence of isolates, and antibiotic resistance profiles will add to our understanding of transmission and treatment options. Campylobacter jejuni: A microbial community on abiotic surfaces: Understanding how biofilms are formed by C. jejuni will contribute to how C. jejuni can be prevented from the formation of C. jejuni biofilms in broiler houses and ultimately result in reducing the colonization of chickens with C. jejuni.
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PROGRESS: 2003/01/01 TO 2003/12/31
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Cattle and livestock have been shown to shed Campylobacter jejuni indicating that they are potential reservoirs of infection for man. In this study bovine, and other livestock feces or intestinal swabs from carcasses were cultured for C. jejuni using broth enrichment followed by selective plating. Isolates were initially selected based on colony morphology, microscopy, and hippurate hydrolysis and subsequently confirmed as C. jejuni by PCR. To date 213 have been evaluated for C. jejuni, with 21 confirmed positives. The ability of the isolates to survive in macrophages was evaluated in J774A.1 cell line using the gentamicin protection assay. Results indicate the ability of the isolates to survive for 72 hours at levels comparable to C. jejuni M129, a human clinical strain. All isolates were tested against selected antibiotic to test for resistance levels.
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IMPACT: 2003/01/01 TO 2003/12/31
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Campylobacter jejuni has been isolated from many cattle and livestock animals. The presence of such a reservoir in animals contributes to the zoonotic spread of diseases such as C. jejuni to man. Determining the prevalence of C. jejuni in a broad range of species, the virulence of isolates, and antibiotic resistance profiles will add to our understanding of transmission and treatment options.
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PROGRESS: 2001/01/01 TO 2001/12/31
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To date, 15 adult milk cows and 30 unbred heifer calves aged less than 3 mos. have been sampled. Two fecal swabs were taken directly from each subject and transported to the laboratory in Cary-Blair media on ice. At the laboratory, one swab from each subject was processed for Salmonella and the other processed for Campylobacter. Four cows have tested positive for C. jejuni based on culture, hippurate hydrolysis, and PCR using C. jejuni specific primers. No subjects have tested positive for Salmonella. All C. jejuni isolates obtained in this study will be tested for pathogenicity via cellular invasion and survival assays.

IMPACT: 2001/01/01 TO 2001/12/31
Sampling of cattle represent an initial effort to determine the coloniztion of multiple animal sources including chickens. Such efforts will be useful in determining the sources of contamination in livestock and may have an impact in both animal health and food safety.

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PROGRESS: 2000/01/01 TO 2000/12/31
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1.Campylobacter Intracellular Survival Kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies suggest that bacterial factors which combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, sequenced, and isogenic katA mutant strains were constructed. Kinetic studies of bacterial viability indicate that catalase provides resistance to hydrogen peroxide in vitro but does not have a role in intraepithelial cell survival as growth curves for the katA mutant and wild type strains generated from long term culture within HEp2 cells are roughly identical. Catalase does however contribute to intramacrophage survival as katA mutants were recovered from cultured peritoneal macrophages at significantly reduced numbers (p less 0.05) relative to the wild type strain after 72 hour incubation with these cells. Additionally, inhibitors of nitric oxide production and the respiratory burst in J774A1 cells increased the ability of the katA mutant to survive within these cells. 2.Bacterial Contamination of Shellfish Recently, there has been an increase in the incidence of bacterial enteric disease from the consumption of raw shellfish. We examined clams and oysters collected from U.S. Coastal waters for the prevalence of fecal coliforms, Campylobacter spp. and Salmonella spp. Shellfish were harvested and shipped to Arizona on ice for bacterial examination. The shellfish meat was collected aseptically, homogenized, and cultured on selective agar following enrichment. DNA was extracted from a portion of each homogenate for PCR analysis. Shellfish samples (n equals 162) from six different bays were examined to date. Coliforms were detected in samples from each site with a prevalence ranging from 3 to 75 percent. Salmonella spp. were also detected in samples from each of the six sites, with a prevalence as high as 74percent. In contrast, Campylobacter spp. were only detected in samples from two different bays. Surprisingly, 21 of 89 samples collected from three of the six sites contained Salmonella spp., only. This result demonstrates that current coliform testing standards alone do not identify other potentially widespread foodborne pathogens.
<P>IMPACT: 2000/01/01 TO 2000/12/31
1. <br>
C. jejuni is able to survive in macrophages and get to deeper tissue.If it can be determined how C. jejuni can survive in macrophages then it would be possible to find new ways to treat infection before it can invade deeper tissue. 2. Current fecal detection methods include coliforms only. We show that there is no direct correlation between Salmonella and Campylobacter with coliforms. We need to start looking for these other organisms as well as coliforms.
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PROGRESS: 1999/01/01 TO 1999/12/31
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We have continued to look at the kinetics of C. jejuni intracellular survival within macrophages. In addition to previous survival studies performed on the isogenic katA mutant of C. jejuni in mouse peritoneal macrophages, studies in J774 and porcine peritoneal macrophages demonstrated a significant reduction in the number of viable katA mutant cells over a 72 hour period when compared to the wild type control. Invasion studies in epithelial cells with the sodB mutant from Dr. Pickett at the University of Kentucky confirmed her previous results. However, we also found the sodB mutant attached at a significantly lower rate than the wild type control. This finding, in addition to our work shows that this mutant exhibits almost identical survival kinetics to the sodB wild type and M129 strain C. jejuni in macrophages and has led us to believe that disruption of the sodB gene may hinder the organism's ability to adhere to epithelial cells and subsequently invade the cell. Since the bacteria are actively phagositized in macrophages we did not see reduced numbers of the sodB mutant when infecting J774 macrophages. Inhibitors were used in the above studies involving the katA mutant to determine what is actually mediating the destruction of this mutant in macrophages. The two inhibitors used were NG-L-monomethyl arginine, which competes for nitric oxide synthase production, and apocynin which inhibits NADPH oxidase and the respiratory burst. We were able to recover the katA mutant in numbers analogous to the wild type when one or both of the inhibitors were used in J774 macrophage studies. Further studies are being done utilizing confocal microscopy to determine the intracellular trafficking pattern of C. jejuni within macrophages. We will be looking at phagolysosome development of C. jejuni infected J774 cells in addition to changes in phagolysosomal pH.
<P>IMPACT: 1999/01/01 TO 1999/12/31
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The pathogenic mechanisms by which Campylobacter jejuni cause disease are currently unclear. Phenotypic traits such as invasion and survival within epithelial and phagocytic cells appear to play an important part in C. jejuni virulence. Therefore, studies dealing with C. jejuni intracellular survival may bring us closer to understanding the mechanisms by which the organism causes disease.
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PROGRESS: 1997/10/01 TO 2002/09/30
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Cattle and livestock have been shown to shed Campylobacter jejuni indicating that they are potential reservoirs of infection for man. In this study bovine, and other livestock feces or intestinal swabs from carcasses were cultured for C. jejuni using broth enrichment followed by selective plating. Isolates were initially selected based on colony morphology, microscopy, and hippurate hydrolysis and subsequently confirmed as C. jejuni by polymerase chain reaction. To date, 212 swabs have been evaluated, yielding 21 confirmed C. jejuni isolates. The ability of isolates to survive in macrophages was evaluated in the J774A.1 cell line using the gentamicin protection assay. Results indicate ability of isolates to survive for 72 hours at levels comparable to C. jejuni M129, a human clinical strain.
<P> IMPACT: 1997/10/01 TO 2002/09/30
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Campylobacter jejuni has been isolated from many cattle and livestock animals. The presence of such a reservoir in animals contributes to the zoonotic spread of diseases due to C. jejuni to man. Determining the prevalence of C. jejuni in a broad range of species, the virulence of isolates, and antibiotic resistance profiles will add to our understanding of transmission and treatment options.

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PROGRESS: 1997/01/01 TO 1997/12/31
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Campylobacterosis: Inhibition of transcription and translation of de novo expressed proteins in C. jejuni blocks its ability to invade host cells in vitro. In order to detect recombinant expressed C. jejuni OMPs, rabbit polyclonal anti-serum to sarkosyl extracted proteins derived from host-cell exposed C. jejuni were produced. Screening of a C. jejuni cosmid library with the anti-serum identified a single clone harboring 34 kb insert DNA. Lysates of this clone did not bind anti-serum specific for plate grown C. jejuni indicating that this epitope was expressed only while cultured with Hep-2 cells. Subclonal analysis identified the serum binding epitope encoded within a 6.6 kb ClaI fragment. Non-reactive subcloned fragments all mapped within a 3 kb portion of the fragment. Sequence and alignment analysis of this DNA identified three open reading frames comprising an operon of high homology to an iron uptake system (ferrichrome uptake operon fhuABCD) in other bacteria. We have also examined the role of catalase in C. jejuni intracellular survival. Initial studies using zymographic native gel analysis identifies a single pattern of catalase activity in C. jejuni lysates indicating the presence of only one catalase gene in this organism. To clone the C. jejuni strain M129 catalase gene a plasmid genomic library was introduced into the double catalase negative E. coli strain UM255. Clones containing the C. jejuni M129 catalase gene were identified using a bubble assay. Sequence and alignment analysis of the insert DNA revealed an ORF encoding catalase with high homology to numerous heme containing catalases present in other proteobacteria. A katA minus strain (JD900) of C. jejuni M129 was constructed via insertion mutagenesis. This strain has no catalase activity verified by spectrophotometric and zymographic analysis and is hyper-sensitive to even low concentrations of hydrogen peroxide (1mM) compared to the wild-type strain. Porcine proliferative intracellularis: Monoclonal antibodies were produced against L. intracellularis to better characterize the antigens involved in invasion of epithelial cells. Ten Mabs were recently produced using gradient purified L. intracellularis grown in cell culture. These are currently being cloned and characterized for their ability to neutralize growth or invasion of the bacterium. These include clones: 1C2=115 kDa, 2A2=60, 43, 41 kDa, 2B6=60, 43, 41 kDa, 2C1=60 kDa, 3A1=41 kDa, and 3D4= 71 kDa. Non-conserved primers to the L. intracellularis 16s rRNA gene were constructed and examined for their ability to amplify L. intracellularis DNA using standard PCR methods. Essentially, an intense 205 bp product was produced when DNA extracted from PPE tissue or HIE cells infected L. intracellularis was used as template. The 16s rRNA reaction correlated with the results from using C, D primers to insert 78 from L. intracellularis.
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PROGRESS: 1996/01 TO 1996/12
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Lipooligosaccharide (LOS) from isolates B204 (wildtype) and tly- (hemolysin minus mutant) s. Hyodysenteriae was sent for monosaccharide composition analysis. The results of the analysis demonstrated major reductions in the weight percent of carbohydrates in the LOS and also a reduction in the amount of muramic acid in the peptidoglycan layer in the hemolysin minus mutant. A cosmid library of S. Hyodysenteriae isolate B204 was made using a modified pRY107 shuttle vector (containing the SuperCos 1 plasmid) and XL-1 blue MR as the parent E. Coli strain. We have obtained approximately 1000 clones with inserts averaging to cover the genome about 1.5 times. Prelimary screening of the library with monoclonal antibodies specific for LOS carbohydrates have been negative. We are currently screening this expression library with B204 convalescent sera absorbed with S. Inncens or absorbed with a serotype 1 strain of S. Hyodyssenteriae. We are also awaiting the LOS structural data of S. Hyodysenteriae to initiate complementation studies with strains of Salmonella with mutanted biosynthetic genes for the expression of LPS.

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PROGRESS: 1996/01 TO 1996/12
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Lipooligosaccharide (LOS) from isolates B204 (wildtype) and tly- (hemolysin minus mutant) s. Hyodysenteriae was sent for monosaccharide composition analysis. The results of the analysis demonstrated major reductions in the weight percent of carbohydrates in the LOS and also a reduction in the amount of muramic acid in the peptidoglycan layer in the hemolysin minus mutant. A cosmid library of S. Hyodysenteriae isolate B204 was made using a modified pRY107 shuttle vector (containing the SuperCos 1 plasmid) and XL-1 blue MR as the parent E. Coli strain. We have obtained approximately 1000 clones with inserts averaging to cover the genome about 1.5 times. Prelimary screening of the library with monoclonal antibodies specific for LOS carbohydrates have been negative. We are currently screening this expression library with B204 convalescent sera absorbed with S. Inncens or absorbed with a serotype 1 strain of S. Hyodyssenteriae. We are also awaiting the LOS structural data of S. Hyodysenteriae to initiate complementation studies with strains of Salmonella with mutanted biosynthetic genes for the expression of LPS. Campylobacteriosis We continue work on identifying invasion-related genes/proteins of Campylobacter jejuni. Outer membrane proteins (OMP) were extracted from Hep-2 cell-exposed and agar plate-grown C. Jejuni isolate M129. SDS-PAGE analysis demonstrated a protein at 80 Kda in the profile of Hep-2 exposed bacteria that was not present in plate grown C. jejuni.
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PROGRESS: 1995/01 TO 1995/12
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Further information on S. hyodysenteriae-specific LOS in providing immunity to swine dysentery has been gained. Monoclonal antibodies specific for LOS can neutralize the growth of the spirochete in vitro & may provide some protection in orally immunized mice challenge exposed to S. hyodysenteriae. We have examined field-case sera from pigs with porcine proliferative enteritis (PPE) for antibody to the ISI agent using the Western blot technique. Although experimentally infected pigs often show no serological response to the ISI antigen, pigs with a chronic-type infection do develop an adequate antibody response. We have also demonstrated the presence of the ISI agent in mesenteric lymph nodes, as well as intestinal tissue from experimentally infected pigs. Infected lymph nodes were detected by both PCR and culture. Using zymography (substrate gels) we have demonstrated the presence of a single catalase protein produced by C. jejuni. The gene encoding catalase was cloned by complementing the double catalase mutant E. coli UM255 with a C. jejuni genomic library in the shuttle vector pRY107. Six catalase positive clones containing identical insert DNA were isolated. The catalase positive recombinant was found to harbor a 4.4 kilobase insert which encodes a catalase protein that co-migrates with the C. jejuni catalase on substrate gels. Subcloning analysis has revealed HincII and EcoRI sites within the catalase gene to be used for cassette mutagenesis to determine if catalase is a virulence factor.

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PROGRESS: 1994/01 TO 1994/12
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Growth of the PPE agent in continuous cell lines is extremely hard to monitor. Determination of actual numbers by culture or light microscopy is not possible due to the intracellular nature and size of the organism. The purpose of this work was to develop a competitive PCR assay that could be used to quantitate the organism in cells and in feces. This was accomplished through the development of a competitive PCR involving the internal primers (C/D) and an internal standard (mimic). The mimic (159 bp, was constructed by ligating linkers containing the C/D primer sequences to a 119 bp Xba-I/BssHII fragment from Bluescript (Stratagene). The product was quantitated, 10-fold diluted and the dilutions added to subsequent ileitis DNA templates to quantitate the organism in cell cultures. The product was determined to be 5ng/ul after ligation and amplification. Template DNA from the ileitis agent was co-amplified with the mimic DNA and quantitated by visual comparison. The age of the pig used in the PPE model and the effect of dexamethasone on lesion production in the pig was examined for future protection studies. Each pig received cells and spent media from a 225 cm2 flask infected for 12 days at 37C under an atmosphere of 5% CO2. Reproduction of PPE (75%) was accomplished in the older animal (7.5 weeks of age) with or without the administeration of dexamethasone. Gross lesions consisted of edema, hemorrhage, and fibrin cast formation in both the small and large intestine. Microscopic lesions demons.

<P>PROGRESS: 1993/01 TO 1993/12
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Porcine Proliferative Enteritis:. 1. Cell culture of the ileitis agent has been expanded to a Celligen system which is essentially a cell fermentor system. This system employs beads which provide the epithelial cells an expanded surface area for growth. Oxygen consumption is tracked over a 15 day period and the organism is harvested after consumption levels off. This unit has allowed for the production of a large mass of antigen for vaccine trials which are currently underway. 2. Random primers (10 bp) are currently being used with PCR to screen for the presence of the agent in inoculated cells. Products were obtained with two recently examined primers using low stringency conditions (37(degree)C). Specific amplified products were detected in extracted DNA from cells containing the ileitis agent, but not in noninfected Henle cells. These primers will be used to evaluate various cell lines for the establishment and growth of the organism. In addition, products specific for the agent will be sequenced, primers developed to the specific sequence and used to detect the agent in fecal preparations from infected pigs in the field.
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PROGRESS: 1992/01 TO 1992/12
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A "knockout mutant" of the Surpulina hyodysenteriae hemolysin (Tly) was producedfollowing recombination by using a cassette to disrupt the hemolysin gene (tly). Both the mutant strain and parent strain, T42 S. hyodysenteriae, were inoculated into 3 week-old pigs. Of the five three-week old pigs inoculated with the mutant strain, none exhibited symptoms of dysentery during the 30-day study period. Three of the T42 control pigs developed clinical signs of swine dysentery and died during the study period. This study suggests that one of the putative virulence factors of the spirochete may be the Tly hemolysin protein. In an attempt to reproduce porcine proliferative enteritis (PPE) with a definitive agent, enterocytes from PPE affected pigs were enzymatically removed, subjected to antibiotics for 3 hours at 37(degree)C, and lysed with 0.5% deoxycholate. The lysate was inoculated onto Henle cells and the cells examined daily for cytopathic effect. A strict intracellular organism of 1.4-2.5 (mu)m in length and 0.24-0.29 (mu)m in diameter was identified in infected Henle cells and intragastrically inoculated into 3-week old piglets. The intracellular agent produced clinical signs and proliferative lesions in both the small and large intestine of orally inoculated piglets. Control pigs, which received normal uninfected Henle cells, had no gross or microscopic lesions typical of PPE at necropsy.

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PROGRESS: 1991/01 TO 1991/12
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The hemolysin from Serpulina hyodysenteriae was further characterized by examining the inhibitory effects on the toxin by different factors including pH, temperature, cations and various blocking agents. The hemolysin was found to be active at temperatures ranging from 20-42(degree)C and from 3.0-9.0 pH. There was no inhibitory effect by calcium, magnesium, BSA, sucrose or 50mM EDTA, but while both zinc and copper were inhibitory. The activity of the hemolysin was partially blocked by the addition of components with a diameter of 1.0-1.1 nm (PEG 1000 or dextran 1500). Neutralization of the hemolysin by newborn calf serum but not fetal calf serum was also observed. The inhibitory effect was shown to be related to the high concentration of high density lipids in the newborn serum. Purification of the hemolysin protein was accomplished using a combined method of HPLC Ion-exchange chromatography and free-flow isoelectric focusing. The purified material was composed of three isomers having isoelectric points ranging between 5.0-5.5 pl.

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PROGRESS: 1990/01 TO 1990/12
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The expression of Iak molecules on murine peritoneal macrophages were significantly induced by lipopolysaccharide-like substance (LPSLS) from Treponema hyodysenteriae, but not with LPSLS from Treponema innocens. These results have potentially important implications for the pathogenicity of T. hyodysenteriae, since the lesions of swine dysentery are essentially inflammatory in nature. Hemolysin, extracted from t. hyodysenteriae and partially purified by QAE Ion-exchange chromatography, appears to have enzymatic action. The substrate appears to be a minor lipid present in the cell wall of the erythrocytes. In addition, the lipid contains no phosphate or primary amine groups and does not consist of cholesterol or sphinogomyelin.

<P>PROGRESS: 1989/01 TO 1989/12
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Envelope extracts (EE) of T. hyodysenteriae have been examined for immunogenicity in CF-1 mice and pigs. EE which were highly reactive to swine immune sera in the ELISA protected animals against challenge. Treatment with either periodate or proteinase K was shown to alter the immunogenicity of EE. Serum from vaccinated animals identified low molecular weight antigens in the Western blot analysis. In addition, the protection noted in vaccinated pigs appeared to be serotype specific. Lipooligosaccharides (LOS) from T. hyodysenteriae and T. innocens have been analyzed by high performance thin layer chromatography (HPTLC) and gas chromatography (GC). Pipopolysaccharide (LPS) from S. typhimurium was used for comparative purposes. The results indicated that differences in LOS among Treponemal isolates may account for differences in pathogenicity, and that Treponemal LOS is a substantially different compound from LPS. Outer-membrane proteins (OMP) and axial filaments of T. hyodysenteriae were extracted, separated by SDS-PAGE and transblotted to immobilon for Western blot analysis. All seven proteins in OMP preparations and six proteins in axial filament preparations reacted to swine immune sera. These antigens were shown to be carbohydrate in nature as determined by periodate oxidation.
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PROGRESS: 1988/01 TO 1988/12
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Two T. hyodysenteriae estrachromosomal DNA libraries have been constructed in E.coli DH5 using the cloning vector pUC 18. From these libraries we have identified two recombinant DNA probes. Hybridization experiments were conducted with cells and DNA from a battery of T. hyodysenteriae and T. innocens isolates, and demonstrated that both probes hybridized to DNA from 11 strains of T. hyodysenteriae, including each of the seven serotypes, but not to any of the 4 T. innocens strains. Western blot analysis of lipooligosaccharide (LOS) from T. hyodysenteriae implies that different epitopes of the LOS are being reorganized when natural infection is contrasted to whole cell immunization. In addition, the LOS from T. hyodysenteriae, when subjected to mild acid hydrolysis, does not resemble the hydrosylate from Salmonella typhimurium (Lipid A). This finding is of key importance as the major biological activity of LOS lies in the Lipid A molecule.
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PROGRESS: 1987/01 TO 1987/12
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Further work on the development of a DNA probe for the detection of swine dysentery has shown that the probe is as sensitive as standard methods currently used in the diagnosis of swine dysentery. A comparison of lipopolysaccharide extracted from various isolates of T. hyodysenteriae & T. innocens has indicated that an important difference between pathogenic and non-pathogenic isolates may lie in their carbohydrate content. Further work on porcine proliferative enteritis (PPE) has indicated that in addition to Campylobacter spp., at least two viruses are present in the intestine of pigs with PPE.

<P>
PROGRESS: 1986/01 TO 1986/12
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A method has been developed to test the efficacy of various antibiotics against swine dysentery (SD). Lincomycin and Dimetridazole, incorporated into feed at 1000 ppm, both prevented lesions of SD in orally challenged CF-1 mice. This method will be used to study new antibiotic compounds for anti-treponemal activity. Restriction digests of chromosomal DNA have indicated that there is gene homology within serotypes of T.hyodysenteriae. Using the plasma isolated from T. hyodysenteriae is a cDNA probe, 10 spirchetes/ml were detectable in porcine feces. This method may prove more rapid and sensitive than selective culture.
<P>
PROGRESS: 1985/01 TO 1985/12
<br>Further work on the in vitro attachment of T. hyodysenteriae to human embryonic intestinal (HEI) cells has shown that a sialic-acid receptor may be involved and that this receptor is located on the spirochete and not on the host cell. A plasmid has been isolated from T. hyodysenteriae and T. innocens. All plasmids migrated at approximately 8300 Bp. Restriction enzyme testing has shown that the plasmid from T. hyodysenteriae differs from that of T. innocens.
<P>PROGRESS: 1984/01 TO 1984/12
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An exotoxin has been detected in culture supernatant fluids from pathogenic isolate B204 T. hyodysenteriae. The exotoxin has been shown to be protein in nature, heat-labile, lethal to mice when inoculated intraperitoneally (IP), and toxic to Vero cells. Mice inoculated IP with the toxin revealed microscopic lesions in the cecum including dilation of the crypts and vacuolation of the goblet cells. Partial purificatin on Sephacryl 200 has yielded one fraction that is lethal for mice. Porcine proliferative ileitis was produced experimentally in conventional pigs five weeks of age. Lesions were present in the ileum of 3 of 5 pigs twenty-two days after oral inoculation with infective porcine ileal scrapings. Campylobacter spp. were found in the epithelial cells lining the crypts of the small intestine of all five inoculated pigs.
<P>PROGRESS: 1983/01 TO 1983/12
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LPS from 3 untypable strains of pathogenic Treponema hyodysenteriae was extracted using the Westphal hot phenol-water technique. These were examined serologically in a gel diffusion system against corresponding rabbit antisera. Results indicate that these 3 strains, B8044, B6933, and ACK 300/8, should be considered new serotypes of pathogenic T. hyodysenteriae. Results of attachment studies indicate that pathogentic T. hyodysenteriae probably adheres to intestinal epithelial cells in order to produce disease. Enzymatic treatment of the host cells with periodate and protease reduced attachment of T. hyodysenteriae to the cells; N-acetyl neuraminic acid competitively blocks attachment, while N-acetyl glucoronic acid does not. In vitro assays studying the effects of pig serum on the growth of T. hyodysenteriae and T. innocens indicate that inactivation of nonpathogenic and avirulent strains requires complement alone, while pathogenic T. hyodysenteriae required specific antibody in addition to complement for killing. Passive protection against swine dysentery was demonstrated in colonic loops instilled in normal pigs. Loops receiving homologous or heterologuous convalescent pig serum with active complement and cells were normal at necropsy, whereas loops which received heat-inactivated immune serum and isolates developed lesions typical of swine dysentery.

<P>PROGRESS: 1981/01 TO 1981/12
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Initial phases of a prevalence survey for swine dysentery were planned with other members of the Swine Dysentery Committee of Livestock Conservation Institute. The survey is to be carried out in 1982-83. <P>

PROGRESS: 1980/01 TO 1980/12 <br>
Work during the year past has been limited to participation in the preparation of a literature review in conjunction with the Diagnosis of Swine Dysentery Committee of the American Association of Veterinary Laboratory Diagnosticians. The review, entitled, "Diagnosis of Swine Dysentery," is presently under study by committee members.

<P>PROGRESS: 1979/01 TO 1979/12
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Contact pigs were exposed directly to feces from Treponema hyodysenteriae-infected mice. Swine dysentery occurred in contact pigs 11 to 13 days after exposure. Contact of pigs with non-swine reservoirs may, therefore, result in transmission of swine dysentery.