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      Pathogenesis and clinical significance of AIDA-I-positive E. coli in diarrhea of pigs

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      Date
      2006-06-12
      Author
      Ravi, Madhu Babu
      Type
      Thesis
      Degree Level
      Masters
      Metadata
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      Abstract
      Escherichia coli remains a significant cause of diarrhea worldwide and in recent years a relatively high number of E. coli carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarrEscherichia coli remains a significant cause of diarrhea worldwide and in recent years a relatively high number of E. coli carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarrhea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in E. coli isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic E. coli isolates and that from a human E. coli isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I E. coli virotype are unknown in humans or in animals. First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) E. coli; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic E. coli strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains.Second, 110 F4 negative E. coli isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ E. coli isolates. The clinical significance of the AIDA-I+ E. coli was studied using clinical data available for 35 of the 110 E. coli isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ E. coli and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic E. coli strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ E. coli. In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs. .hea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in E. coli isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic E. coli isolates and that from a human E. coli isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I E. coli virotype are unknown in humans or in animals. First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) E. coli; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic E. coli strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains. Second, 110 F4 negative E. coli isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ E. coli isolates. The clinical significance of the AIDA-I+ E. coli was studied using clinical data available for 35 of the 110 E. coli isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ E. coli and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic E. coli strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ E. coli. In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs.
      Degree
      Master of Science (M.Sc.)
      Department
      Veterinary Pathology
      Program
      Veterinary Pathology
      Supervisor
      Middleton, Dorothy M.; Simko, Elemir
      Committee
      Ngeleka, Musangu; Mainar-Jaime, Raul
      Copyright Date
      June 2006
      URI
      http://hdl.handle.net/10388/etd-06292006-124512
      Subject
      AIDA-I E. coli Electron microscopy
      AIDA-I Biofilm
      E. coli diarrhea pathogenesis
      Immunogold electron-microscopy
      E. coli
      E. coli diarrhea pigs
      AIDA-I positive E. coli
      AIDA-I E. coli
      AIDA-I E. coli mutant
      E. coli virulence factors
      multiplex PCR
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