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This study assessed the in vitro and in vivo activity of an ear solution containing a third-generation chelating agent (Tricide) as an antimicrobial potentiator for miconazole in chronic Malassezia otitis. Thirty-one ears from 20 dogs were enrolled in the study. Fungal culture, minimum inhibitory concentration (MIC), and minimum fungicidal concentration (MFC) testing of miconazole with and without Tricide were performed on all ears. In a randomized, controlled, and blinded treatment trial the ears were treated either with 0.9% saline solution containing 0.01% miconazole, 0.03% dexamethasone and 540 microg/mL Tricide or the same solution without Tricide. Cytologic and auroscopic examinations were conducted on day 0, 14 and 28 and evaluated for number of yeast organisms, degree of erythema, hyperplasia and amount of discharge. The in vitro data was compared with Wilcoxon signed-rank test. The cytologic and auroscopic scores were compared between the visits and treatment groups at day 0, 14 and 28 using a Wilcoxon-Mann-Whitney test and repeated measures analysis. MIC and MFC were significantly (P < 0.0001) reduced when miconazole was combined with the chelating agent versus miconazole alone. The cytologic scores were significantly lower on days 14 (P = 0.0156) and 28 (P = 0.0280) for the group treated with Tricide. The auroscopic scores decreased significantly by the end of the trial compared to day 0, but the difference between the two groups was not significant. This study suggests that Tricide enhances in vitro activity and in vivo efficacy against Malassezia sp. in dogs with yeast otitis.

Public pressure to reduce or eliminate antimicrobials as ingredients of feed for poultry and other agricultural animals is mounting, primarily due to the fear of multidrug-resistant bacteria in clinical infections in both animals and humans. Exploration of the occurrence of antibiotic resistance in the gut flora of wildlife avian flocks that presumptively do not receive antimicrobials will determine the rate of resistance in a naïve population. Fecal samples collected from a healthy population of the yellow-headed blackbirds (YHB) (Xanthocephalus xanthocephalus) in North Dakota were cultured to determine what genera and species of gram-negative facultative anaerobic bacteria these wild birds carry in their intestinal flora and to evaluate the antimicrobial susceptibility profiles. Isolates of Escherichia coli were further characterized for the presence of putative virulence factors and for pathogenic potential using the chicken embryo lethality assay (ELA). The ELA was performed in chicken embryos with challenges at both 12 days and 16 days of incubation to determine whether the 16-day-old embryos were better able to fight the infection and subsequent disease and also to determine whether the ELA could distinguish between primary and secondary avian Escherichia coli pathogens. After screening 33 isolates from the 21 fecal samples, only two E. coli isolates were identified. The predominant genus and species of bacterium identified was Pantoea agglomerans. Collectively, 12 of the 33 isolates (36%) exhibited no resistance to any antimicrobial tested. However, several multidrug-resistant isolates of varying genera were identified. Among the antimicrobial resistances observed, the most common was to ampicillin (60%), followed by cephalothin (33%). Neither E. coli isolate belonged to serogroups that are notorious for causing major outbreaks of colibacillosis in poultry, and only one E. coli isolate retained resistance to any antibiotics; nevertheless, the ELA results indicate that at least one of these E. coli may be a primary pathogen of chickens. This study demonstrates that antibiotic resistance occurs in the gut flora of natural populations of YHB despite the absence of antibiotic pressure. In addition, these results indicate that YHB will harbor E. coli isolates that are potentially pathogenic in poultry. However, these E. coli isolates are not a significant reservoir for multiple antibiotic resistances nor are they widespread in the population of YHB surveyed in North Dakota.

Objective: To determine whether a novel third-generation chelating agent (8 mM disodium EDTA dehydrate and 20 mM 2-amino-2-hydroxymethyl-1, 3-propanediol) would act as an antimicrobial potentiator to enhance in vitro activity of antifungal medications against fungal isolates obtained from horses with mycotic keratitis., Sample Population: Fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicillium isolate, 1 Cladosporium isolate, and 1 Curvularia isolate) obtained from horses with mycotic keratitis and 2 quality-control strains obtained from the American Type Culture Collection (ATCC; Candida albicans ATCC 90028 and Paecilomyces variotii ATCC 36257)., Procedure: Minimum inhibitory concentrations (MICs) against fungal isolates for 4 antifungal drugs (miconazole, ketoconazole, itraconazole, and natamycin) were compared with MICs against fungal isolates for the combinations of each of the 4 antifungal drugs and the chelating agent. The Clinical and Laboratory Standards Institute microdilution assay method was performed by use of reference-grade antifungal powders against the fungal isolates and quality-control strains of fungi., Results: Values for the MIC at which the antifungal drugs decreased the growth of an organism by 50% (MIC50) and 90% (MIC90) were decreased for the control strains and ophthalmic fungal isolates by 50% to 100% when the drugs were used in combination with the chelating agent at a concentration of up to 540 microg/mL., Conclusions and Clinical Relevance: The chelating agent increased in vitro activity of antifungal drugs against common fungal pathogens isolated from eyes of horses with mycotic keratitis.

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 formalin-fixed, 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.

In previous studies, the embryo lethality assay (ELA) discriminated between virulent and avirulent avian Escherichia coli isolates, and also proved to be highly correlated with mortality and morbidity results of the intravenous (IV) challenge model. In the current study, the same 20 avian E. coli isolates were used in subcutaneous (subQ) and intratracheal (IT) chicken challenge models in order to determine whether the results from the prior ELA challenges and/or the IV challenge model correlate with these models. The correlation observed between the two previous ELA trials and the combined mortality/morbidity percentages of the subQ challenge model were r = 0.792, P > 0.0001 for the first ELA trial and r = 0.738, P = 0.0002 for the second ELA trial. The IV challenge results were more highly correlated with the subQ challenge results (mortality/morbidity comparison, r = 0.894, P < 0.0001). The IV challenge mortality results were slightly correlated (r = 0.4810, P=0.0319) with the IT challenge results. Several of the isolates differed in their ability to produce mortality and/or morbidity with the different challenge models. The mortality/morbidity results of the IV and subQ challenges and the mortality results of the ELA were all positively correlated with the ability of an E. coli isolate to produce Colicin V (ColV) (r = 0.7131, P = 0.0004). The IT mortality results were slightly correlated with the production of ColV (r = 0.455, P = 0.049). The IT challenge results were only slightly correlated with resulting IV mortality and ColV production. Previous results indicate that the ELA correlates extremely well with the IV challenge model. The current study demonstrates that ELA also correlates well with the subQ challenge model. Overall, the conclusion of this study is that the ELA, IV, and subQ challenge models similarly demonstrate the ability to discriminate between virulent and avirulent avian E. coli isolates.

Prevention or resolution of microbial colonization of wounds is critical to rapid and uneventful healing. The use and misuse of antimicrobial agents continues to support the evolution of multidrug resistant organisms that can cause severe or life-threatening infections. Chelating agents have been shown to potentiate the effects of antimicrobial compounds. The third generation chelating agent. Tricide has been shown to be effective against many multidrug resistant pathogens, prevents pathogens from development resistance to the antimicrobials with which it is mixed and substantially reduces the amount of antimicrobials needed to kill bacteria and fungi.

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 amongthis 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.

In previous studies, the embryo lethality assay (ELA) was able to discriminate between virulent and avirulent avian Escherichia coli isolates and to predict percent mortality of the embryos resulting from an isolate based on three traits. The abilities to resist host complement, presence of Colicin V activity, and presence of the increased serum survival gene cluster (iss), were used together in a logistic regression analysis to predict the percentage of embryo deaths resulting from each of 20 avian E. coli isolates used in the ELA. In the present study, the same 20 isolates are used in an intravenous chicken challenge model in an effort to determine whether the ELA could be used to replace chicken challenge studies. Correlations between the mortality and a combination of mortality and morbidity (the survivors at trial termination with lesions suggestive of colibacillosis) and the previous ELA results and with selected isolate traits were performed. Additionally, resulting body weights in surviving chickens were compared between groups. The highest positive correlations were observed between the ELA and the combined mortality/morbidity of the intravenous challenge (r = 0.861, P < 0.0001 for the first ELA challenge, and r = 0.830, P < 0.0001 for the second ELA challenge). The IV challenge combined mortality/morbidity results had the highest correlation coefficients with the presence of iss (r = 0.864, P < 0.0001) and the expression of ColV (r = 0.878, P < 0.0001). The presence of tsh was slightly correlated with mortality (r = 0.465, P = .0389) but demonstrated a higher correlation with the combined mortality and morbidity of the IV challenge (r = 0.558, P = 0.0106). Even though the ELA results in a higher number of nonspecific deaths, the two challenge methods exhibit similar results and a high correlation with each other. Interestingly, some of the isolates showed differences in their ability to cause mortality between the ELA and the IV challenge model. Furthermore, some isolates reflected significant differences in body weights of surviving birds at IV trial termination.

Differentiating between virulent and avirulent avian Escherichia coli isolates continues to be a problem for poultry diagnostic laboratories and the study of colibacillosis in poultry. The ability of a laboratory to conduct one simple test that correlates with virulence would simplify studies in these areas; however, previous studies have not enabled researchers to establish such a test. In this study, the occurrence of certain phenotypic and genotypic traits purported to contribute to avian E. coli virulence in 20 avian E. coli isolates was correlated with the results of embryo challenge studies. This analysis was undertaken in an effort to determine which trait(s) best identified each avian E. coli isolate as virulent or avirulent. Traits selected were complement resistance, production of colicin V (ColV), motility, type F1 pili expression, presence of the temperature-sensitive hemagglutinin gene (tsh), and presence of the increased serum survival genetic locus (iss). ColV production, complement resistance, and presence of the iss genetic element were the three traits most highly correlated with high embryo lethality. A logistic regression model was used to predict the embryo lethality results on the basis of the most frequent isolate characteristics. Results indicate that ColV, complement resistance, and if are significant predictor variables for the percentage of embryo lethality resulting from challenge with a specific avian E. coli isolate. However, no single trait has the ability to predict virulent isolates 100% of the time. Such results suggest the possibility that the embryo lethality assay may prove to be the one test needed to determine if an avian E. coli isolate is virulent.

Minimum inhibitory concentrations for enrofloxacin were determined for 63 bacterial isolates from dogs with otitis externa or urinary tract infections. Development of resistant mutants was determined after exposing the isolates to enrofloxacin in vitro for up to five serial passages. Results indicated that Pseudomonas aeruginosa and Enterococcus spp isolates exposed to enrofloxacin developed resistance rapidly, whereas Klebsiella, Proteus, and Streptococcus spp were less likely to develop resistance. Despite the presence of enrofloxacin pressure, no resistant bacteria developed in the Escherichia coli and staphylococcal isolates. In many isolates, susceptibility patterns changed from susceptible to intermediate.

Studies have indicated variations in the degree of efficacy of certain commercial disinfectants used in poultry production facilities. We used an adequate method of in vitro testing to compare the efficacy of disinfectants while testing them in conditions similar to those of the poultry facilities. BioSentry 904, ethylenediaminetetracetic acid (EDTA)-Tris, and a combination of the two were tested by this method against five field isolates of Pseudomonas aeruginosa at 10(3), 10(6), and 10(9) colony-forming units (CFU)/ml. At the 10(9) CFU/ml concentration, most compounds failed to achieve a total kill with a contact time of 15 min. When tested at bacterial concentrations of 10(3) CFU/ml, the combination of EDTA-Tris mixed at a 1:1 ratio with BioSentry 904 killed the bacteria upon initial contact (< or = 0.05 min). This disinfectant mixture exhibited antagonistic, indifferent, or synergetic effects when exposed to different bacterial isolates at a concentration of 10(6) CFU/ml.

In this study, we assessed the pathogenic potential of Escherichia coli associated with a commercial competitive exclusion (CE) product by examining the phenotypic characteristics associated with E. coli virulent for humans and domestic animals. Most E. coli isolates were capable of proliferating in iron-deplete chicken sera. Interestingly, none of the E. coli isolates from the commercial CE product contained the bacterial adhesin Tsh characteristic of avian pathogenic E. coli associated with airsacculitis and colisepticemia. In terms of virulence potential for humans, most E. coli isolates (78%) were sensitive to killing by 12.5% human sera. Because of their sensitivity to human sera, the E. coli in the CE product are not likely to cause a serious systemic infection in humans and, therefore, do not present a risk of causing septicemia in humans. Because these isolates also lack the gene tsh, they are also less likely to cause systemic disease or airsacculitis in poultry than pathogenic strains commonly isolated from diseased birds.

A study was conducted to evaluate the antimicrobial activity of selected commercial mastitis medications with and without EDTA-Tris by comparing minimum inhibitory concentrations and minimum bactericidal concentrations in vitro. Solutions containing 5 mM EDTA/50 mM Tris potentiated the antimicrobial action of seven commercially available mastitis medications when reacted in vitro with Staphylococcus aureus, Streptococcus uberis, and Pseudomonas aeruginosa. Lesser potentiation was observed for Escherichia coli, whereas no potentiation was observed for Klebsiella pneumoniae. The incorporation of EDTA-Tris solutions into mastitis medications may reduce the amount of antibiotics necessary for therapy and may prevent formation of antibiotic-resistant forms.

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.

Previous work in our labs has shown that avian Escherichia coli virulence is correlated with resistance to complement. Also, our studies have revealed that the presence of the increased serum survival gene (iss), known to contribute to the complement resistance and virulence of mammalian E. coli, may predict the virulent nature of an avian E. coli isolate. This relationship warrants further research, but further clarification of the relationship among virulence, complement resistance, and iss sequences requires use of complement susceptibility assays. Such assays, unfortunately, are labor-intensive, expensive, and difficult to perform. In the present study, the results of two complement susceptibility assays for 20 E. coli isolates, 10 incriminated in avian colibacillosis and 10 from the intestinal tracts of apparently healthy birds, were compared in an attempt to determine if flow cytometric analysis was a reasonable alternative to a viable count assay. In addition, the virulence of these isolates for chick embryos was determined, and each isolate was examined for the presence of iss using amplification techniques. The flow cytometric method was found to be repeatable for most isolates, and its results showed moderate agreement with those obtained through viable counts. All intestinal isolates of healthy birds proved avirulent using the embryo lethality assay; however, not all isolates from sick birds were demonstrated to be virulent. Possible explanations of these results include that the methods originally used to isolate these organisms failed to detect the illness-inciting strains or that the virulence of these strains had declined following initial isolation. Additionally, we must consider the possibility that the embryo lethality assay of virulence used here might not be sensitive enough to detect differences between these two groups of isolates. Also, it should be noted that virulence assays, such as the one used here, fail to account for predisposing host or environmental conditions, enabling a less virulent isolate to cause disease under natural conditions. Interestingly, the complement resistance of a strain was significantly associated with its lethality in embryos, and iss-containing isolates were significantly more likely than those lacking iss to be classified as complement-resistant and virulent. Such results, at least for this group of avian E. coli, suggest that there is a compelling but imperfect relationship among complement resistance, virulence, and the presence of iss. These results also suggest that the flow cytometric assay may be a reasonable alternative to the viable count method of determining complement resistance.

Objective: To determine whether an inactivated culture of a microcin-producing avian Escherichia coli was capable of killing Salmonella isolates from reptiles in an in vitro test system., Sample Population: 57 Salmonella isolate from reptiles., Procedure: A wild-type avian E. coli electrotransformed with a plasmid coding for the production of microcin 24 was tested in an in vitro microassay system for its ability to kill 57 Salmonella spp isolated from reptiles. The reptile population included snakes, iguana, frilled lizards, turtles, other lizards, and unspecified reptiles., Results: 44 of the Salmonella isolates were inhibited strongly, compared with the in vitro assay controls; 12 had weak inhibition, and 1 was not inhibited by the microcin-producing E. coli. Thirteen of the 57 isolates had resistance to at least 1 antibiotic, primarily streptomycin. There were 9 O serogroups identified in the 57 isolates, with serogroup H being the most prevalent (18 to 57)., Conclusion and Clinical Relevance: Antibiotics are not recommended to eliminate Salmonella organisms from reptiles because of the development of antibiotic resistance. Further studies are necessary to determine whether the use of microcin-producing bacteria will be effective in controlling Salmonella infections in companion reptiles.

Solutions of ethylenediaminetetraacetic acid (EDTA)-Tris showed synergistic or additive effects on gram-negative bacteria when combined with hatchery disinfectants consisting of phenol and detergent (Magnaphen-100), quaternary ammonium compound (QAC) and glutaraldehyde (Synergize), QAC (BioSentry 904), and hydrogen peroxide. The gram-positive bacteria reacted less favorably, with reaction mixtures showing all three levels of potentiation (synergistie, additive, and antagonistic). Combinations of EDTA-Tris and a commercial glutaraldehyde solution (Glutracide), when mixed with the test organisms, showed mostly antagonistic effects. Solutions of EDTA-Tris decreased the concentration of hatchery disinfectants required for bacterial killing by 75% in those situations in which synergistic potentiation occurred. EDTA-Tris is nontoxic to 12-day-old embryos. Serial passage of the test organisms in solutions of EDTA-Tris did not result in the development of resistant forms.

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.

An avirulent, wild-type avian Escherichia coli (E. coli Av) was electrotransformed with a plasmid coding for the production of microcin 24 (pGOB18) and was designated E. coli AvGOB18. The transformant inhibited the growth of seven serotypes of Salmonella commonly associated with colonization and contamination of poultry products and seven strains of E. coli O157:H7 in the in vitro colicin/microcin assay. The transformant did not inhibit the replication of multiple isolates of Listeria monocytogenes or Campylobacter jejuni in similar assays. The transformant is nonconjugative, indicating that the plasmid would not be transmitted to other intestinal microflora in the environment. The transformant also survived in sterile tap and deionized water incubated at 25 C and 37 C in the laboratory for 30 days and was recovered from drinkers and birds in in vivo floor pen studies. In in vivo studies, E. coli AvGOB18 did not colonize the intestinal tract of broiler chicks when given as a single or multiple dose and did not reduce the Salmonella load in the broilers. But Salmonella typhimurium was reduced significantly in the intestinal tracts of broiler chickens when E. coli AvGOB18 was administered continually in the water supply.

Recombinant plasmid pHK11 was transformed into an avirulent, wild-type avian Escherichia coli (E. coli Av) in order to study the plasmid's effect on colonization of the chicken trachea. The transformant (E. coli Av + pHK11) produced colicin V (ColV), had type F1 fimbriae, and was motile. The E. coli Av recipient possessed type F1 fimbriae but was nonmotile; it did not produce ColV. Four-day-old chicks were inoculated in the trachea with 100 microliters of an overnight culture (approximately 10(8) colony-forming units) of E. coli Av, E. coli Av + pHK11, or sterile brain-heart infusion (BHI) broth. A group of uninoculated chicks was also included. Samples of the trachea were taken on days 4 and 10 postinoculation and compared histologically and bacteriologically. Birds inoculated with E. coli Av + pHK11 had enhanced tracheal colonization and showed increased histologic changes as compared with those inoculated with E. coli Av or BHI broth or uninoculated controls. These results indicate that production of ColV and motility enhance the colonization of the trachea and may be involved in the cause of pathologic lesions.

The purpose of this study was to determine the rate at which resistance developed in avian coliform bacteria when exposed to nalidixic acid, sarafloxacin, or enrofloxacin. In in vitro studies, the rates of mutation of avian isolates of Escherichia coli and Salmonella were determined following nalidixic acid, sarafloxacin, or enrofloxacin pressure. The rates of mutation were similar for nalidixic acid and sarafloxacin, whereas a lower rate of mutation was seen after enrofloxacin pressure. In in vivo studies, the quinolones were administered in the drinking water to broiler chickens at a concentration of 40 ppm for five consecutive days. Samples of feces were inoculated onto appropriate media and the frequency of resistance was determined. The frequency rates of resistance to nalidixic acid and sarafloxacin were similar. Enrofloxacin-medicated birds did not develop enrofloxacin-resistant coliform bacteria. The in vitro and in vivo data appear to correlate.

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Several plasmids that were isolated from complement-resistant Pasteurella multocida or Escherichia coli were evaluated for phenotypic markers. Plasmid p2267, isolated from a tetracycline-resistant, complement-resistant fowl cholera field isolate of P. multocida (PM2267), was used to transform a K-12 E. coli (C600); this resulted in increased complement resistance, which was eliminated by curing. Either of two plasmids (p1870 or p70-1, isolated from P. multocida and E. coli, respectively) conferred an increase in complement resistance and invasiveness to turkey epithelial cells when expressed in the Clemson University (CU) vaccine strain of P. multocida. Additionally, the CU strain containing p1870 was more virulent in turkey challenge, and the plasmid appeared amplified in vivo. No detectable differences in major outer-membrane proteins, capsule, or carbohydrate fermentation were found to be associated with the acquisition of these plasmids.

Two serotype 3,4:A strains of Pasteurella multocida that differ in virulence in turkeys were examined for their ability to invade epithelial cell monolayers grown in tissue culture. Both organisms were comparably adherent to cells of turkey kidney origin. However, the virulent strain (86-1913) penetrated primary turkey kidney epithelial cell monolayers at 10 times the level of the low-virulence vaccine strain. The virulent strain was also able to invade porcine epithelial cells (PK15) and feline epithelial cells (CRFK) in cell culture. Neither organism invaded rabbit epithelial cells (RK13). Invasion of turkey cells was prevented by inhibition of bacterial protein or RNA synthesis but not by pretreatment of the monolayers with periodate, trypsin, or neuraminidase. Invasion might be a mechanism of pathogenicity for this organism, contributing to colonization or virulence.

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.

An avirulent wild-type avian Escherichia coli strain (Av) was electrotransformed with plasmids coding for complement resistance (pKT107) and Colicin V (ColV) production (pHK11) in order to study the effects of complement resistance and ColV production on virulence. Transformants were also compared with the wild type for embryo lethality, uptake by macrophages, motility, growth rate, plasmid content, and hemolysis. Growth rates and complement resistance patterns of strain Av and transformant Av+pHK11 were similar, but Av+pHK11 caused a significantly greater number of deaths in embryos and acquired motility. Transformant Av+pKT107 had a lower rate of phagocytosis, a slower growth rate, and a greater sensitivity to complement, and it changed from being non-hemolytic to expressing alpha-hemolytic action. The 35-kb plasmid present in the wild type was not present in the transformants. Although some of the results demonstrate the difficulties encountered in using wild-type organisms as recipients in virulence studies, the results with Av+pHK11 indicate that ColV production plus the acquisition of motility contributes to the virulence of avian E. coli.

Colonization of the intestinal tracts of newly hatched chicks with Escherichia coli was attempted by swabbing test organisms onto the air-shell of 19-day-old embryos. Test organisms consisted of two virulent E. coli isolates, one avirulent isolate, and one laboratory-derived mutant of the avirulent isolate carrying a recombinant plasmid coding for Colicin V production. Chicks were cultured weekly for 3 weeks for total E. coli and for the test organisms using selective media. Control chicks were sampled on weeks 1 and 5, and the normal E. coli intestinal microflora were examined for the production of colicins. The two virulent E. coli isolates maintained colonization of the chicks for the 3-week test period, with titers decreasing from 10' to 10'- colony-forming units (CFU)/g of intestine. The avirulent isolate and laboratory mutant did not consistently colonize the intestinal tracts. The majority of intestinal samples taken from the control chicks at 1 and 5 weeks had colicin-producing E. coli that were inhibitory to the test organisms.

Avian isolates of Escherichia coli were classified as virulent based on their isolation from chickens with natural cases of colisepticemia, production of colicin V, and complement resistance. A second group of isolates was designated as avirulent based on their isolation from healthy chickens, their inability to produce colicin, and their classification as sensitive or intermediate to the action of complement. In vitro assays of phagocytosis and oxidant production were performed in an attempt to correlate these activities with the ability of each group of bacteria to escape the specific host defense mechanisms of phagocytosis and killing. Although oxidant production regressed with significant linearity on percent phagocytosis, neither group (virulent or avirulent) differed in ability to stimulate peritoneal macrophage phagocytic and oxidant activity when opsonized with normal chicken serum. These results differ from those in mammalian species.

A virulent, complement-resistant avian Escherichia coli isolate and its avirulent, complement-sensitive, transposon-insertion mutant were compared for the purpose of revealing structures associated with complement resistance. Both had a smooth lipopolysaccharide layer, contained traT, and lacked a capsule, but the mutant possessed a 16.2-kilodalton outer-membrane protein (OMP) not present in the wild-type. This protein may be the product of a coding region interrupted by transposon insertion. Such results suggest that an OMP greater than 16.2 kilodaltons in size may be responsible for the complement resistance and virulence of this wild-type E. coli.

A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production. These parameters were selected because of their reported association with complement resistance and virulence in E. coli. Lethality in chicken embryos has also been shown to be correlated with virulence of avian E. coli for chickens. The complement-resistant, virulent E. coli isolates did not possess a K-1 capsule. Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality. There was no correlation between the presence of traT and embryo lethality. These results suggest that complement resistance and virulence in avian E. coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.

Twenty avian Escherichia coli isolates from normal and diseased chickens were compared by use of three virulence tests. These tests included the uptake of Congo red dye, an embryo lethality test, and a quantitative microtiter complement resistance test. A direct correlation was seen between the results of the complement resistance test and the embryo lethality test. The results of the Congo red test did not correlate with the two other tests.

Complement resistance, antibiotic resistance profiles, and virulence profiles of 80 Escherichia coli isolates from the intestines of normal chickens (40 isolates) and chickens diagnosed as having colisepticemia (40 isolates) were compared. Differences were observed between the two groups for antibiotic resistance, siderophore production, presence of type 1 pili, complement resistance, motility, and size of plasmids. The systemic isolates were more likely to have siderophores and type 1 pili, and to be complement-resistant and motile than were the intestinal isolates. No differences between the two groups were observed for colicin production. Further comparison of the 10 most complement-resistant isolates from the systemic group and 10 most complement-sensitive isolates from the intestinal group revealed a correlation between an isolate's resistance to complement and its ability to kill embryos, express type 1 pili, and be motile. Virulence of avian E. coli strains appears to be correlated with complement resistance and the interaction of this resistance with the ability to produce type 1 pili and be motile.

Two avian strains of Pasteurella multocida, a vaccine strain and a virulent field isolate, were investigated to determine their propensity to release endotoxin from the cell surface. Both organisms released comparable amounts of endotoxin when plasma complement proteins were present, however the virulent strain did so without the loss of viability that occurred in the vaccine strain. Blocking complement activity decreased the ability of plasma to elicit endotoxin release from the bacteria. When the cells were treated with divalent metal chelators such as trans-1, 2-diaminocyclohexane-N,N,N1,N1-tetraacetic acid (CDTA), more endotoxin was released from the vaccine strain than from the virulent isolate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified lipopolysaccharide (LPS) from both strains revealed virtually identical patterns. Both had patterns considered typical of rough LPS. Challenge studies in 8 weeks old turkeys showed that the field strain induced endotoxemia of longer duration than the vaccine strain and produced greater mortality.

The role of complement resistance in the virulence of an avian Escherichia coli isolate was examined with transposon mutagenesis. A suicide plasmid containing a kanamycin-encoding mini-transposon was used to transform a virulent complement-resistant avian E. coli isolate. A less resistant mutant was identified that contained a transposon insertion in a plasmid and in the chromosome. This loss of complement resistance was associated with a drop in virulence in an embryo assay. No other phenotypic changes were detected in the mutant. These results suggest that complement resistance is associated with the virulence of this organism.

Three of four virulent avian Escherichia coli isolates transferred a single large molecular-weight R-plasmid to two recipient E. coli strains. Antibiotic resistances transferred included streptomycin (two isolates) and streptomycin-tetracycline-sulfa (one isolate). Production of colicin and siderophores, complement resistance, and embryo lethality present in the virulent isolates were not transferred to recipient organisms. From the results, it appears that the R-plasmids of these virulent avian E. coli are not associated with virulence.

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.

A quantitative microtiter method using chicken plasma is described for determining the degree of complement resistance or sensitivity of avian Escherichia coli isolates. Results obtained with the microtiter method using chicken plasma were compred with results obtained using commercially available standardized guinea pig serum as the source of complement. The test organisms consisted of five isolates of E. coli isolated from chickens. Three isolates were from flocks with colisepticemia; one was from a flock with omphalitis; and one isolate was a non-pathogenic control. Data were accumulated from the five avian E. coli isolates incubated at 35 C with either chicken plasma or guinea pig serum and with heat-inactivated chicken plasma or guinea pig serum. The microtiter results of the chicken plasma and guinea pig serum had a statistically positive correlation. The use of commercially available guinea pig serum in the test system will allow for standardization of this method.

A quantitative microtiter method for determining the degree of complement resistance or sensitivity of microorganisms is described. The microtiter method is compared with a quantitative automated system and the standard plate-count technique. Data were accumulated from 30 avian Escherichia coli isolates incubated at 35 C with either chicken plasma or heat-inactivated chicken plasma. Analysis of data generated by the automated system and plate-count techniques resulted in a classification of the microorganisms into three groups: those sensitive to the action of complement; those of intermediate sensitivity to the action of complement; and those resistant to the action of complement. Although the three methods studied did not agree absolutely, there were statistically significant correlations among them.

Phenotypic characteristics of 12 paired, Salmonella serotypes isolated from healthy and ill chickens were compared. Variables compared included antibiotic resistance profiles, production of colicins and siderophores, mannose-sensitive hemagglutination of erythrocytes, resistance to serum complement, carbon source utilization, presence and transmissibility of R plasmids, and invasiveness in primary chicken kidney cell culture. Differences were found between pairs for utilization of carbon sources, mannose-sensitive hemagglutination of erythrocytes, and invasiveness in cell culture.

Twenty-four isolates of Pasteurella multocida from clinical cases of fowl cholera and the Clemson University vaccine strain were surveyed for the presence of potential virulence markers. Membrane proteins, enzymatic activity of the membrane proteins, and carbohydrate fermentation patterns were also determined to demonstrate phenotypic relationships within the groups. Few differences were found in these phenotypic characteristics among the isolates. Almost all the organisms produced siderophore and were hemolytic on turkey red blood cells. No extracellular enzyme or bacteriocin activity was detected and little antibiotic resistance was found. However, many organisms contained plasmids and demonstrated some degree of resistance to complement. Both characteristics were correlative markers in Pasteurella multocida isolated from birds with fowl cholera.

Ten temperature-sensitive mutants of the Clemson University (CU) vaccine strain of Pasteurella multocida have been developed and were characterized by phenotypic attributes such as carbohydrate fermentation, antibiotic resistance, and membrane protein profiles. Some mutants were found to have lost the ability to utilize some substrates, notably xylose and gluconate, whereas others were able to ferment additional carbohydrates such as arabinose and rhamnose. CU was found to be resistant to sulfisoxazole, of intermediate resistance to bacitracin, and sensitive to rifampin; the sensitivity to these three antibiotics varied among the mutant strains, but 60% were resistant to rifampin. Membrane protein profiles demonstrated some changes in major bands, and there was variation in 50% of the mutants in proteins in the 31 kilodalton range. All strains were assayed for the presence of several virulence factors, and many were found to produce siderophore and to exhibit some degree of complement resistance.

Cecal samples from 100 broiler flocks were cultured for Escherichia coli and Salmonella. Samples were selected from flocks classified as either "good" or "poor" producers by a production formula. In an attempt to identify predictors of flock productivity, isolates were studied for differences in antibiotic resistances, hemagglutination of erythrocytes, production of colicins, production of siderophores, type of hemolysis, resistance to host complement, and presence of plasmids. S. typhimurium (copenhagen) was isolated from one poor producing flock and three good producers. Salmonella isolates showed no significant differences in the parameters studied. The E. coli isolates showed significant differences only for the presence of plasmids. These data indicate that differences in host intestinal E. coli from good and poor producing flocks do not predict flock productivity.

A study of the microbiological flora isolated from cultures of normal and lesional skin tissue samples collected from 19 bowhead whales (Balaena mysticetus) over a 4 yr period is presented. These cultures were obtained from 30 tissue samples (17 normal, 13 lesion) and 248 swab samples (157 normal, 91 lesion). Seven hundred-thirty bacterial and yeast isolations were made (285 normal, 445 lesion). Distribution revealed that 56% of the gram positive bacterial isolates, 75% of the gram negative bacterial isolates and 64% of the yeast isolates recovered were associated with lesional skin. It was found that 80% of one group of Corynebacterium sp. isolates, 90% of the Acinetobacter sp. isolates and 94% of the Moraxella sp. isolates were associated with lesional skin. Although the primary yeasts recovered were Candida spp., they were found on both normal and lesional skin. Enzymatic assays of isolates from normal and lesional skin demonstrated production of enzymes capable of causing necrosis. The majority of the microorganisms recovered were facultative anaerobes and many of them could be considered potential pathogens of mammalian hosts.

A microneutralization test (MNT) system utilizing cytopathic effect end points was effective in determing neutralization indexes for infectious bronchitis virus antibodies. The system is reproducible within 1 index unit at the 95% level of probability. Comparison of the MNT to tests in eggs resulted in a positive correlation (B =0.81), which was significant (P greater than 0.01). The quantitative dose-response relationship of the MNT is linear (P greater than 0.005), with the 95% prediction limits fitting between one 10-fold dilution.

Solutions of ethylenediaminetetraacetate (EDTA)-tris combined with antibiotics have been shown to be effective in treating selected cases of persistent bacterial infections. Basic techniques in microbial genetics, including mating frequencies, chemical elimination of R plasmids, isolation of plasmid DNA and agarose gel electrophoresis, were used to determine if EDTA-tris has a curing effect on an R plasmid as part of its clinical action. Results of this study indicated that EDTA-tris by itself eliminated an antibiotic resistance marker from a clinical isolate of Escherichia coli and when combined with another chemical curing agent altered the isolate's mating frequency.