Isolation and identification of AIDA-I receptors in porcine intestinal mucus.

Porcine AIDA-I positive Escherichia coli causes diarrhea in neonatal piglets and AIDA-I adhesin is an important virulence factor involved in intestinal colonization with biofilm formation. This biofilm consists of AIDA-I(+)E. coli bacteria stratified within mucus layers covering the intestinal mucosa. Based on the intimate interaction between AIDA-I(+)E. coli and mucus within the intestinal biofilm, we hypothesized that porcine intestinal mucus contains receptor(s) for AIDA-I adhesin. Since porcine AIDA-I receptors have not been identified, we employed affinity chromatography and in vitro adhesion assays to investigate AIDA-I binding proteins in porcine intestinal mucus that might serve as receptors for attachment of AIDA-I positive E. coli. We demonstrated that porcine mucus contains 65 and 120kDa proteins (p65 and p120) that bind with high affinity to purified AIDA-I adhesin and that AIDA-I positive E. coli binds to these proteins with higher affinity than do AIDA-I negative mutant. The identity of p65 was not determined based on LC-MS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.

Tularemia in deer mice (peromyscus maniculatus) during a population irruption in Saskatchewan, Canada.

Type B tularemia caused by Francisella tularensis subsp. holarctica was diagnosed in deer mice (Peromyscus maniculatus) found dead at four sites in west-central Saskatchewan during April and May 2005. The occurrence of tularemia coincided with a decline in the number of deer mice in part of a large area (>22000 km(2) ) in which deer mice had been extremely abundant during the autumn of 2004 and spring of 2005, and in which mice caused damage to crops in the autumn of 2004. This is apparently the first report of tularemia as a cause of death of wild deer mice. The bacterium isolated from deer mice was atypical in that cysteine was not required in the media used for isolation. Three isolates tested were genotypes not previously identified in Canada. There were no reports of human disease in the area.

Characterization and immuno-detection of AIDA-I adhesin isolated from porcine Escherichia coli.

A relatively high percentage of porcine Escherichia coli isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). This gene and its corresponding protein were first identified and characterized in E. coli strain 2787 isolated from human infantile diarrhea. Little is known about the properties of the AIDA-I protein and its immuno-detection on surface of AIDA-I positive porcine E. coli isolates. In this study, we demonstrated that the AIDA-I adhesin isolated from porcine AIDA-I positive E. coli is an acidic protein consisting of five isoforms. It has a similar molecular weight (100 kDa) and relatively high amino acid homology (78-87%) with the AIDA-I adhesin expressed by human AIDA-I positive E. coli strain 2787. Based on limited comparison, it appears that there is a very high homology among AIDA-I proteins expressed by porcine AIDA-I positive E. coli isolates. Sensitivity of detection of surface AIDA-I adhesin of PCR-positive AIDA-I E. coli by immuno-dot-blot and coagglutination tests was 76 and 71%, respectively, whereas specificity was 89 and 84%, respectively. These tests are unlikely to be used for diagnostic detection of AIDA-I positive E. coli due to the relatively low sensitivity; however, they may be potentially useful for identification of false positive reactions generated by other diagnostic tests.

A longitudinal study on enteropathogenic infections of livestock in Trinidad.

A longitudinal study was conducted on selected livestock farms to determine the prevalence of enteropathogens in diarrhoeic and non-diarrhoeic animals. The enteropathogens assayed from faecal samples and rectal swabs were bacteria (Escherichia coli, Campylobacter spp. Salmonella spp. and Yersinia enterocolitica), parasites (coccidia, gastrointestinal nematodes and Cryptosporidium spp.) and viruses (group A rotavirus and parvovirus). The prevalence of the enteropathogens in various animal species was related to age and month of the year. Generally, younger animals presented a higher prevalence of infection by enteropathogens than older animals while most infections occurred between the months of January and April.

A longitudinal study on enteropathogenic infections of livestock in Trinidad

A longitudinal study was conducted on selected livestock farms to determine the prevalence of enteropathogens in diarrhoeic and non-diarrhoeic animals. The enteropathogens assayed from faecal samples and rectal swabs were bacteria (Escherichia coli, Campylobacter spp. Salmonella spp. and Yersinia enterocolitica), parasites (coccidia, gastrointestinal nematodes and Cryptosporidium spp.) and viruses (group A rotavirus and parvovirus). The prevalence of the enteropathogens in various animal species was related to age and month of the year. Generally, younger animals presented a higher prevalence of infection by enteropathogens than older animals while most infections occurred between the months of January and April.

Um estudo longitudinal foi realizado em fazendas de criação selecionadas, para determinar a prevalência de enteropatógenos em animais com ou sem diarréia. Os enteropatógenos analisados de amostras fecais e swabs retais foram: bactérias (Escherichia coli, Campylobacter spp, Salmonella spp e Yersinia enterocolitica); parasitas (coccídeos, nematóides gastrintestinais e Cryptosporidium spp ) e vírus (Rotavírus grupo A e parvovírus). A prevalência dos enteropatógenos em várias espécies de animais foi relacionada à idade e mês do ano. Geralmente, a prevalência de infecção por enteropatógenos foi maior entre os animais mais jovens que entre os animais mais velhos, enquanto a maioria das infecções ocorreu entre os meses de janeiro e abril.

A longitudinal study on enteropathogenic infections of livestock in Trinidad

A longitudinal study was conducted on selected livestock farms to determine the prevalence of enteropathogens in diarrhoeic and non-diarrhoeic animals. The enteropathogens assayed from faecal samples and rectal swabs were bacteria (Escherichia coli, Campylobacter spp. Salmonella spp. and Yersinia enterocolitica), parasites (coccidia, gastrointestinal nematodes and Cryptosporidium spp.) and viruses (group A rotavirus and parvovirus). The prevalence of the enteropathogens in various animal species was related to age and month of the year. Generally, younger animals presented a higher prevalence of infection by enteropathogens than older animals while most infections occurred between the months of January and April.

Retrospective study on Escherichia coli infection in broilers subjected to postmortem examination and antibiotic resistance of isolates in Trinidad.

An 8-yr retrospective study was conducted to evaluate the rate of Escherichia coli infection and antibiotic resistance of isolates from diseased broilers submitted for diagnosis in Trinidad from 1990 to 1997. Of a total of 906 cases of diseased birds subjected to postmortem examination, 603 (66.6%) had E. coli infection. The number of cases increased over the years from 16 in 1990 to a peak of 294 in 1996. For every year, at least 50% of all broiler cases had E. coli infection. The rate of infection was significantly higher during the rainy season (74.1 +/- 6.9%) than during the dry season (57.8 +/- 7.0%). Approximately 50% of all E. coli isolates were resistant to 9 out of a total of 11 antimicrobial drugs selected for the study. The isolates showed an increasing trend of resistance to amoxicillin, apramycin, gentamicin, nitrofurantoin, norfloxacin, and sulfamethoxazole-trimethoprim. However, only the trends of resistance to apramycin and norfloxacin were statistically significant. Overall, of the antimicrobial drugs selected, norfloxacin relatively appeared as the best choice for treatment. From this study, we conclude that the high rate of E. coli infection in broilers submitted for diagnosis along with the high resistance of isolates to antimicrobial drugs constitute a threat to the poultry industry on the island.

F165(1) fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils.

The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae. In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.

Escherichia coli cellulitis in broiler chickens: clonal relationships among strains and analysis of virulence-associated factors of isolates from diseased birds.

Thirty-nine Escherichia coli isolates from broiler chickens with cellulitis were serotyped and analyzed for clonal relationships by multilocus enzyme electrophoresis. The isolates were further characterized with respect to hemagglutination (HA); serum resistance; antibiotic susceptibility; production of aerobactin, colicin V, and hemolysin; expression of K1 or K5 capsule; sensitivity to cloacin DF13 after treatment with diphenylamine; expression of iron-regulated outer membrane proteins; and virulence in 1-day-old chickens. In addition, the isolates were examined for the presence of DNA sequences related to F1A (fim) and P (pap) fimbriae, aerobactin synthesis (iuc) and transport (iut), hemolysin operon hly, and TraT lipoprotein-induced serum resistance (traT). Only 38.4% of the isolates were typeable with standard O antisera, and of these, serogroups O25 and O78 were the most frequently observed. Multilocus enzyme electrophoresis, based on 20 enzymes, resolved 17 electrophoretic types, forming seven clusters. Isolates from four of these clusters fell into E. coli clone complexes that have been previously reported to be commonly associated with avian colibacillosis. All isolates expressed two to five iron-regulated outer membrane proteins, were resistant to serum and cloacin DF13, and possessed DNA sequences homologous to fim and iuc/iut. Most isolates (72%) were positive for traT, and a majority produced colicin V and aerobactin (92 and 82%, respectively). Assays for the presence of fim and pap DNA sequences, for HA, and for virulence gave variable results but suggest that cellulitis isolates may express F1A and/or other mannose-resistant HA fimbriae different from P and may be virulent in 1-day-old chickens. Our results support the hypothesis that cellulitis in broilers in many cases is caused by E. coli clones identical to other pathogenic avian E. coli strains. Certain clones may be specific to cellulitis, because 25% of the isolates tested belong to clusters not related to known clone complexes.

Septicemia-inducing Escherichia coli O115:K"V165"F165(1) resists killing by porcine polymorphonuclear leukocytes in vitro: role of F165(1) fimbriae and K"V165" O-antigen capsule.

Escherichia coli O115:K"V165":F165(1) wild-type strain 5131 survives in the bloodstream of experimentally inoculated gnotobiotic pigs and induces septicemia, whereas its afimbriate (F165(1)-negative) TnphoA mutant M48 and its acapsular (K"V165"-negative) spontaneous mutant 5131a are both nonpathogenic. We evaluated the role of the mannose-resistant F165(1) fimbrial system and of the O-antigen K"V165" capsule in resistance to phagocytosis by porcine polymorphonuclear leucocytes (PMNLs) in vitro. F165(1)-positive strains (5131 and 5131a) attached to and were ingested by PMNLs at a significantly higher level than afimbrial mutant M48 (P < 0.001) after 1 h of incubation. During incubation of these strains with PMNLs for up to 6 h, parental strain 5131 resisted killing whereas afimbriate mutant M48 and acapsular mutant 5131a were gradually killed and were found at significantly lower numbers than the parental strain 5131 at 2 (P < 0.05) and 6 (P < 0.001) h. When bacteria were opsonized with normal pig serum, the afimbriate and acapsular mutants survived less well than when the bacteria were nonopsonized. Upon examination by electron microscopy of PMNLs after 2 h of incubation with bacteria, structurally normal bacteria were observed more often within phagosomes of PMNLs incubated with the parental strain than within phagosomes of PMNLs incubated with the afimbriate or the acapsular mutant. The extracellular oxidative response (as tested by release of hydrogen peroxide) of PMNLs stimulated by phorbol myristate acetate was completely inhibited by F165(1)-positive strains but only partially inhibited by the afimbriate mutant. These results suggest that the F165(1) fimbrial system may mediate adherence of E. coli O115 to PMNLs. Survival of the parental strain in the presence of PMNLs, which may be intracellular, is at least partially due to the presence of the F165(1) fimbrial system and of the O-antigen capsule K"V165". Furthermore, the presence of the F165(1) fimbrial system may contribute to the bacterial inhibition of the oxidative response of porcine PMNLs.

Pathogenicity of an Escherichia coli O115:K"V165" mutant negative for F165(1) fimbriae in septicemia of gnotobiotic pigs.

To evaluate the role of the F165(1) fimbrial system in the pathogenesis of septicemia, 2-day-old germfree pigs were inoculated intragastrically with Escherichia coli O115:K"V165":F165 wild-type strain 5131, its F165(1)-negative TnphoA mutant M48, or E. coli O115:K(-):F165(-) wild-type strain 862B. Pigs were sacrificed at different times (3, 6, 12, 24, 48, and 96 h) postinfection (p.i.). Pigs inoculated with strain 5131 developed clinical signs (anorexia, lameness, reluctance to move, or lack of motor coordination) and were moribund within 48 h p.i., and, at necropsy, infecting bacteria were isolated in various extraintestinal organs. Strain 5131 was isolated as early as 6 h p.i. from the blood of inoculated pigs. Pigs inoculated with mutant M48 developed only mild clinical signs at 96 h p.i. Mutant M48 colonized extraintestinal organs of pigs but to a lesser extent than the parent strain did. In contrast to the parent strain, this mutant was not isolated in the blood of inoculated pigs. Pigs inoculated with strain 862B remained normal during the experiment. All of the strains colonized the mucus layer of the intestine, but no histological changes of intestinal mucosa were observed by either light or electron microscopy. The parent strain, but not the mutant M48, expressed F165(1) in vivo. In a competitive study in which the parent strain and its afimbrial mutant were inoculated simultaneously, clinical signs of septicemia developed 24 h after inoculation, and only the parent strain 5131 was isolated from the blood of inoculated pigs. Our results suggest that the F165(1) fimbrial system of E. coli O115:K"V165" strains may play an important role in the ability of the bacteria to survive in the blood and spread systemically through the porcine host.

Characterization of a polysaccharide capsular antigen of septicemic Escherichia coli O115:K "V165" :F165 and evaluation of its role in pathogenicity.

Escherichia coli strains of serogroup O115:K(-):F165 have been associated with septicemia in calves and piglets. These strains express a capsular antigen referred to as K"V165" which inhibits agglutination of the O antigen by anti-O115 serum. We used hybrid transposon TnphoA mutants M48, 18b, and 2, and a spontaneous O-agglutinable mutant, 5131a, to evaluate the role of K"V165" in the pathogenicity of E. coli O115. Mutant M48 was as resistant to 90% rabbit serum and as virulent in day-old chickens as the parent strain 5131, mutants 18b and 5131a were less resistant to serum and less virulent in chickens, and mutant 2 was serum sensitive and avirulent. Analysis of outer membrane protein and lipopolysaccharide profiles failed to show any difference between the transposon mutants and the parent strain. In contrast, the spontaneous O-agglutinable mutant showed additional bands in the 16-kDa region of the polysaccharide ladder-like pattern. Mutants 2 and 5131a produced significantly less K"V165" capsular antigen than the parent strain, as demonstrated by a competitive enzyme-linked immunosorbent assay with adsorbed anti-K"V165" serum. In addition, electron microscopic analysis revealed that mutants 2 and 5131a had lost the capsular layer observed in the parent strain after fixation with glutaraldehyde-lysine. This capsule contained carbohydrate compounds and resembled an O-antigen capsule since it prevented O-antigen agglutination before the bacteria were heated at 100 degrees C and induced bacterial serum resistance. The capsule-defective mutants colonized the intestinal epithelium of experimentally infected gnotobiotic pigs but failed to induce clinical signs of septicemia. We concluded that E. coli strains of serogroup O115 expressed a polysaccharide capsular antigen which induced serum resistance and consequently contributed to the pathogenicity of the bacteria.

Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165.

Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea. Putative factors important in the pathogenicity of E. coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin. Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E. coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant. In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence. Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165. Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.

Endotoxin increases the nephrotoxic potential of gentamicin and vancomycin plus gentamicin.

To assess the possible role of endotoxin as an amplification factor for experimental nephrotoxicity due to gentamicin plus vancomycin, rats were given continuous intravenous (iv) endotoxin or saline followed by twice-daily intraperitoneal (ip) saline, vancomycin (20 mg/kg ip), gentamicin (15 mg/kg subcutaneously), or both gentamicin and vancomycin. After 5 or 8 days of treatment, functional and histologic parameters of renal function were evaluated: cortical drug levels, tritiated thymidine incorporation into cellular DNA, creatinine clearance, and appearance by light and electron microscopy. In animals not given endotoxin, only rats that received gentamicin plus vancomycin developed measurable abnormalities. Endotoxin did not cause nephrotoxicity in vancomycin-treated rats. However, in endotoxin-infused rats treated with gentamicin or gentamicin plus vancomycin for 8 days, the increase in blood urea nitrogen, decrease in creatinine clearance, and rise in renal cortical DNA synthesis were more severe than those in non-endotoxin-infused rats (P less than .01). In these studies, endotoxin amplified the nephrotoxic potential of gentamicin alone and gentamicin plus vancomycin.

Intrarenal distribution of vancomycin in endotoxemic rats.

We previously observed that the intrarenal distribution of aminoglycosides was modified by Escherichia coli endotoxin in the absence of any major renal physiological disturbance or histological changes. In the present study, we evaluated the role of E. coli endotoxin on the intrarenal distribution of vancomycin. At the beginning of the experiment, female Sprague-Dawley rats were infused intravenously with saline (control) or endotoxin (0.25 mg/kg) during 15 min. Thereafter, saline was constantly infused for the following 4 h. Two hours after the beginning of the infusion, animals were injected intravenously with a single dose of vancomycin (20 mg/kg). The drug levels in serum; renal cortex, medulla, and papilla; and urine were evaluated from 0.08 to 24 h after injection. Analysis of the area under the curve of the drug concentration in the different kidney components versus time showed a higher accumulation of vancomycin in the renal cortex and medulla in the endotoxin-infused rats than in the normal rats (P less than 0.01). Endotoxin was associated with an increase in the half-life in serum (P less than 0.01) and a lower elimination rate constant. The total clearance of vancomycin from plasma was significantly decreased in endotoxin-treated rats (P less than 0.01). These results demonstrate that endotoxin modifies the renal handling of vancomycin.