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Beth Houser

Toxigenic Clostridium difficile in the Calf Gut: Association with Enteric Disease in Calves and Significance to Public Health. Beth Houser. Background. Background. Clostridium difficile Gram positive, spore forming, anaerobic bacterium. Produces three toxins

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Beth Houser

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  1. Toxigenic Clostridium difficile in the Calf Gut: Association with Enteric Disease in Calves and Significance to Public Health Beth Houser

  2. Background

  3. Background • Clostridium difficile Gram positive, spore forming, anaerobic bacterium • Produces three toxins • TcdA (308 kDal) and TcdB (270 kDal) • Monoglucosylation of Rho proteins • Disruption of tight junctions allows penetration of colon epithelium • Inflammatory cascade leads to tissue damage • CDT (150 kDal) • Binary toxin, ADP-ribosyltransferase • Targets actin and disrupts cytoskelleton

  4. Background • Clostridium difficile associated disease (CDAD) in humans • Antibiotic associated diarrhea • Colitis • Toxic mega colon • CDAD mainly associated with antimicrobial use in healthcare settings • Severity of disease associated with toxin levels • Increasing mortality rate Rupnik et al., 2009, Nature Rev Microbiol Redelings et al., 2007, EID

  5. Background • Real-time PCR assays are superior for detection of C. difficile in fecal samples (van den Berg et al., 2007) • No real-time PCR assays have been reported that are capable of detecting all three toxins of C. difficile • C. difficile is associated with enteric disease in calves (Hammitt et al., 2007; Rodriguez-Palacios et al., 2006) • ~60% of preweaned calf mortality is attributed to diarrhea (USDA-APHIS-NAHMS, 2002) • Holes exist in data • Examination for other enteric pathogens • Histology and gross pathology

  6. Background • Animals are a reservoir for toxigenic C. difficile(Jhung et al., 2008; Rodriguez-Palacios et al., 2006; Rupnik, 2007). • C. difficile isolated from animals genotypically similar to those isolated from animals (CDC, 2005; Jhung et al., 2008; Rodriguez-Palacios et al., 2006; Rupnik, 2007) • Community acquired cases in humans are on the rise (CDC, 2005) • Food animal products may be a source of infection • Toxigenic C. difficile isolated from ground meat • 18% of beef and veal, Canada(Rodriguez-Palacios et al., 2007) • 42% of cooked and uncooked meat, Tucson, Arizona (Songer et al., 2009)

  7. Objectives • 1. Develop, standardize, and validate a high throughput assay for detection toxigenic C. difficile using multiplex real-time PCR directly from fecal and food samples • 2. Investigate C. difficile and/or its toxins in the calf gut and enteric disease in young calves • Determine the association of the presence of C. difficile and /or its toxins in the calf gut and enteric disease in young calves • Characterize toxigenic C. difficile isolated from calves using genotypic and phenotypic typing methods • 3. Determine the public health significance of toxigenic C. difficile shed in feces of veal calves

  8. Overview ofCompleted Research

  9. Chapter 1: Real-time multiplex PCR assay for rapid detection of Clostridium difficile toxin encoding strains Objective: develop, standardize, and validate a high throughput assay for detection toxigenic C. difficile using multiplex real-time PCR directly from fecal and food samples Study design: Standardization Validation Standard Strain Cultures CDa, tcdA-/B-/cdt- CDb, tcdA+/B-/cdt- CDc, tcdA+/B+/cdt+ Laboratory: C. difficile spiked samples of Ground veal Calf feces Field: Unknown Samples Calf feces (n=71) Ground meat (n=53) Serial dilutions of DNA DNA isolation directly from samples Sample Enrichment DNA isolation Culture Generate Standard Curve Real-time QPCR (triplicate) tcdA and tcdB (Taqman) cdtA and cdtB (Taqman) Compare PCR results to Culture results Determine Specificity and Sensitivity In feces and meat

  10. Chapter 1: Real-time multiplex PCR assay for rapid detection of Clostridium difficile toxin encoding strains Results: Assay Standardization

  11. Chapter 1: Real-time multiplex PCR assay for rapid detection of Clostridium difficile toxin encoding strains Results: Laboratory Validation Field Validation * Note: Although not included in this publication, toxigenic C. difficile was later detected by the multiplex real-time PCR assay in ground veal and beef samples collected for Chapter 3 .

  12. Chapter 2: Prevalence of toxigenic Clostridium difficile and its association with enteric disease in young calves • Objectives: Investigate C. difficile and/or toxins TcdA and TcdB in the calf gut and enteric disease in young calves • Determine the association of the presence of C. difficile and /or its toxins in the calf gut and enteric disease in young calves • Characterize toxigenic C. difficile isolated from calves using genotypic and phenotypic typing methods • Examine the effect of sub-inhibitory concentrations of oxytetracycline on TcdA/B expression of C. difficile • Study design: Calves submitted to PSU-ADL for necropsy (>5 weeks of age, n=71) • Data from routine Necropsy supplied by PSU-ADL • Evidence of enteric disease • Presence of enteric pathogens Fecal Samples: Culture for C. difficile TcdA/B EIA Isolate Characterization: tcdCdeletion Antimicrobial resistance Toxin production in-vitro Exclude cases where intestinal lesions were noted if: Pathogenic Salmonella present at site of lesion Virulent E. coli present at site of lesion C. perfringens and hemorrhage present at site of lesion • Logistic Regression Analysis: • Presence of C. difficile vs. Evidence of Disease • Presence of TcdA/B vs. Evidence of Disease

  13. Chapter 2: Prevalence of toxigenic Clostridium difficile and its association with enteric disease in young calves • Results: • Determine the association of the presence of C. difficile and /or its toxins in the calf gut and enteric disease in young calves

  14. Chapter 2: Prevalence of toxigenic Clostridium difficile and its association with enteric disease in young calves • Results: • Characterize toxigenic C. difficile isolated from calves using genotypic and phenotypic typing methods • 81 C. difficile isolates from the 21 culture positive calves were collected over the course of the study • 67 C. difficile isolates were examined, 14 isolates were not able to be revived from frozen stock.

  15. Chapter 2: Prevalence of toxigenic Clostridium difficile and its association with enteric disease in young calves • Results: • 2. Characterize toxigenic C. difficile isolated from calves using genotypic and phenotypic typing methods • 81 C. difficile isolates from the 21 culture positive calves were collected over the course of the study • 67 C. difficile isolates were examined, 14 isolates were not able to be revived from frozen stock.

  16. Chapter 3: Prevalence of toxigenic Clostridium difficile in the veal industry from the farm to the grocery shelf • Objectives: Determine the public health significance of toxigenic C. difficile shed in feces of veal calves • Determine the prevalence of toxigenic C. difficile among veal calves over time • Determine the effect of the presence of toxigenic C. difficile on calf weight gain • Determine if the prevalence of toxigenic C. difficile is influenced by farm, season, or age • Examine the possible dissemination of toxigenic C. difficile from veal calves to humans through ground veal meant for human consumption • Study design: Veal Growers: 4 herds N= 600; n=200 Calves receive same diet and vaccines Ground veal product of slaughter house sampled from local grocery store Fecal samples collected every 4-6 weeks from arrival at veal grower to 1-2 weeks before slaughter Calves from 2 herds (n=100) followed to slaughter house, carcass swabs collected pre and post wash (2.5% citric acid) Enrichment and PCR Enrichment and Culture PCR Enrichment and Culture PCR Prevalence Statistics Compare weight gain: t-test Farm, season, age influence: Test of independence Prevalence statistics Prevalence Statistics

  17. Chapter 3: Prevalence of toxigenic Clostridium difficile in the veal industry from the farm to the grocery shelf • Results: • Determine the prevalence of toxigenic C. difficile among veal calves over time

  18. Chapter 3: Prevalence of toxigenic Clostridium difficile in the veal industry from the farm to the grocery shelf • Results: • 2. Determine the effect of the presence of toxigenic C. difficile on calf weight gain No significant difference in weight gain observed (t-test)

  19. Chapter 3: Prevalence of toxigenic Clostridium difficile in the veal industry from the farm to the grocery shelf • Results: • Determine if the prevalence of toxigenic C. difficile is influenced by farm, season, or age • Farm: • χ2: 2.22 p: 0.33 • Season: • χ2 : 33.30 p: 0.28e-6 • Age:

  20. Chapter 3: Prevalence of toxigenic Clostridium difficile in the veal industry from the farm to the grocery shelf • Results: • Examine the possible dissemination of toxigenic C. difficile from veal calves to humans through ground veal meant for human consumption • Carcass swabs: • * 3 swabs were positive for non-toxigenic C. difficile • Ground meat:

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