The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were >50-fold more resistant to free chlorinc when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.

Avian colibacillosis is a costly disease for the poultry industry. The mechanisms of virulence employed by the etiologic agent of this disease remain ill defined. However, accumulated evidence suggests that complement resistance and the presence of the increased serum survival gene (iss) in an avian Escherichia coli isolate may be indicative of its ability to cause disease. This association of iss with the E. coli implicated in avian disease may mean that iss and/or, perhaps, the genes associated with it are important contributors to avian E. coli virulence. For this reason, we have begun a search for iss's location in the bacterial genome. Thus far, iss in an avian E coli isolate has been localized to a conjugative R plasmid and estimated to be about 100 kilobase (kb) in size, encoding resistance to tetracycline and ampicillin. Hybridization studies have revealed that this plasmid contains sequences with homology to tsh, a gene associated with virulence of avian E coli; intI 1, a gene encoding the integrase of Class 1 integrons; and certain genes of the aerobactin- and CoIV-encoding operons. Sequences homologous to merA, a gene of the mercury resistance operon, were not identified on this R plasmid. This plasmid, when transferred into an avirulent, recipient strain by conjugation, enhanced the transconjugant's resistance to complement but not its virulence, in spite of the plasmid's possession of several putative virulence genes and traits. Such results may reflect the multifactorial nature of virulence, the degree of the recipient's impairment for virulence, or an inability of the embryo assay used here to detect this plasmid's contribution to virulence. Additionally, this plasmid contains genes encoding antimicrobial resistances, which may provide a selective advantage to virulent E. coli in the production environment. Further study will be needed to determine whether this plasmid is widespread among virulent E. coli and to ascertain the implications that this link between virulence and antimicrobial resistance genes may have for poultry management.

Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E. coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence. The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test. This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E. coli isolates. Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates. Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates. Analysis of data indicated that the length of the test should be 4 days. In the virulent group, day 2 postinoculation had the most significant death patterns. Sample size calculations indicated that 11 embryos are sufficient for the assay. On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29%. An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.

Abstract.A reproducible, experimental model of columnaris disease was developed to study the pathogenesis of cutaneous disease associated with Flavobacterium columnare infection in koi (Cyprinus carpio). In experimental infections, lesions were usually restricted to skin and fins; gill necrosis was not a consistent finding. Cytologic and histopathologic examinations provided a presumptive diagnosis of columnaris disease. Specific detection of F. columnare was done using the polymerase chain reaction and DNA in situ hybridization (ISH). Polymerase chain reaction allowed the detection of F. columnare in fresh biological material and in formalinfixed, paraffin-embedded tissues. The DNA ISH technique allowed the identification and localization of F. columnare in formalin-fixed, paraffin-embedded tissues. Using these molecular techniques, F. columnare was readily detected in skin specimens from infected fish; however, the bacterium was infrequently detected in specimens of liver, kidney, and spleen. These observations suggest that columnaris disease generally presents as a cutaneous disease that is unassociated with systemic infection in koi. Hematologic studies indicated that most infected koi developed microcytic, normochromic, nonregenerative anemia and leukopenia characterized by lymphopenia, mild neutrophilia, and monocytosis. Biochemical changes in diseased fish included significant hyperglycemia, hyponatremia, and hypochloridemia.

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Combinations of EDTA-Tris and two aminoglycoside antibiotics (amikacin and neomycin) were tested for synergistic activities against the microorganisms associated with otitis externa in dogs and for the solutions' stability over time. Synergistic activity was observed when EDTA-Tris plus amikacin and EDTA-Tris plus neomycin were tested against Staphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli, but not against Candida albicans. Stability studies over a 3-month period indicated that the test solutions were stable at room temperature and that their antimicrobial activity was maintained.

Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos, and the isolate of healthy birds that contained all the genes killed the most embryos among this group. These genes were not found among the non-E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.

Results with four pathogenic avian Escherichia coli isolates and one avirulent isolate in a complement resistance test, a chicken lethality test, and a chicken embryo lethality test were compared. Results of the complement resistance test with these isolates were highly correlated to results of the chicken lethality test of virulence. The chicken embryo test yielded results that were of a medium positive correlation with the chicken lethality results. The results of the complement resistance and chicken embryo lethality tests were highly correlated.