Cloning and sequence of the groESL heat-shock operon of Pasteurella multocida.

By using degenerate oligodeoxyribonucleotide primers for conserved regions of groEL, a 0.6-kb fragment of Pasteurella multocida genomic DNA was amplified using PCR. The amplified fragment was then used as a probe to isolate a genomic fragment containing the entire GroESL operon. The isolated genomic fragment was found to contain two open reading frames, the sequences of which were highly homologous to the prokaryotic groES and groEL families of genes.

Tissues and exudates contain sufficient thymidine for growth of anaerobic bacteria in the presence of inhibitory levels of trimethoprim-sulfamethoxazole.

Obligate anaerobes are susceptible in vitro to trimethoprim-sulfonamides. Clinical efficacy of this drug combination for treatment of infectious processes in which anaerobes are involved is uncertain. We hypothesize that this uncertainty is a result of thymidine in tissues and exudates which abrogates the inhibitory effect of trimethoprim-sulfonamides. We shown herein that species of anaerobic bacteria commonly encountered in infectious processes grew on thymidine-containing medium despite the presence of levels of trimethoprim-sulfamethoxazole (S x T) previously shown to be inhibitory. Biologic fluids and tissues, where anaerobic bacteria are commonly encountered in diseased states, were shown to contain thymidine levels that were sufficient to reverse the inhibitory effect of S x T upon these species of bacteria. These observations suggest that the use of trimethoprim-sulfonamides is not a rational choice for treatment of infectious processes in which an obligate anaerobe is a component.

Possession of identical nonconjugative plasmids by different isolates of Pasteurella multocida does not imply clonality.

Experiments were performed to test the hypothesis that possession of the same nonconjugative R plasmid by different isolates of Pasteurella multocida implied that they were of the same clone. Seven isolates of P. multocida were studied, two possessed an identical nonconjugative R plasmid (pVM109), four possessed another (pVM110), and one isolate possessed a nonconjugative R plasmid related to pVM110. Phenotypic and genotypic characteristics of the isolates were determined and compared. Isolates possessing the same nonconjugative R plasmid were shown to be different, and isolates possessing a different nonconjugative R plasmid were shown to be the same. We conclude that possession of an identical nonconjugative R plasmid by two isolates of P. multocida does not imply clonality.

Serum resistance is correlated with encapsulation of avian strains of Pasteurella multocida.

Encapsulated avian strains of Pasteurella multocida possessing an A-type capsule were shown to be resistant to the bactericidal action of turkey serum, whereas unencapsulated variants as well as other unencapsulated strains were not. Removal of the capsule from serum-resistant strain P1059-1 resulted in this strain becoming susceptible to the bactericidal effects of turkey serum. Since complement was consumed when encapsulated or unencapsulated strain P1059-1 was incubated in turkey serum, we conclude that the capsule acts to shield the outer membrane rather than prohibiting the generation of an effective membrane attack complex.

Characteristics of Escherichia coli isolated from septic foals.

Fifteen Escherichia coli isolates from the blood and tissue of foals with septicemia were compared with 15 from the feces of clinically normal horses. Comparisons were made with respect to survival in normal equine serum, production of aerobactin, and production of hemolysin. Isolates from the blood and tissues of septic foals were more likely to be resistant to equine serum than were isolates from feces of clinically normal horses. There were minimal differences between the isolates with respect to aerobactin and hemolysin production, almost all being nonhemolytic and aerobactin negative. Serum resistance is probably a virulence determinant for invasive E. coli in horses.

Comparative molecular characterization of Corynebacterium pseudotuberculosis of different origin.

Ribotyping and susceptibility to 17 antimicrobial agents were used to compare 37 isolates of Corynebacterium pseudotuberculosis (28 from horses, 1 from cattle, 3 from sheep and 5 from goats) derived from various types of lesions, and different geographic locations. According to the presence of nitrate reductase, all but one isolate from horses reduced nitrate (nitrate-positive), whereas all isolates from sheep and goats were unable to reduce nitrate (nitrate-negative). The ribotype of the nitrate-negative isolate from a horse with ulcerative lymphangitis was identical to all the other isolates from horses, and different than the ribotype of nitrate-negative isolates from sheep and goats. Ribotyping with one of the restriction endonucleases, Apa 1, revealed differences between, but not within, the two biotypes. However, ribotyping with Pst 1 endonuclease revealed one variant within the equine biotype and one variant within the ovine biotype. The minimum inhibitory concentration (MIC; microgram/ml) of antimicrobial agents against isolates from nitrate-negative and nitrate-positive groups was very similar, with the exception of isolates from sheep and goats which had a higher MIC for amikacin than isolates from horses and cattle.

Reduced microbicidal activity of peripheral mononuclear phagocytic cells infected with Pasteurella multocida.

Pasteurella multocida inhibits the uptake and killing of Candida albicans and P. multocida by avian mononuclear phagocytic cells. The toxic outer membrane protein of P. multocida, which has been previously described, also inhibited the uptake and killing of C. albicans. Antibody specific for the toxic outer membrane protein reversed this effect resulting not only in an increase in uptake of C. albicans and P. multocida, but also in intracellular killing of P. multocida. This antibody, however, only partially restored killing of C. albicans. These data support the hypothesis that P. multocida is capable of intracellular survival in avian mononuclear phagocytic cells and that the toxic outer membrane protein is totally or partly responsible for this occurrence.

Pasteurella multocida enters polarized epithelial cells by interacting with host F-actin.

We investigated the interaction of an avian strain of Pasteurella multocida with the cytoskeleton of MDCK cells, which formed a polarized epithelium when grown on type I collagen coated filters. Bacteria were incubated with MDCK cells for 30 min. 2, 4 and 6 hours and their location and association with the cell cytoskeleton determined by double-label immunofluorescence confocal microscopy. Cells were stained with a polyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) actin. Confocal microscopy revealed that bacteria entered the cells by 30 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Electron microscopy demonstrated that by 2 hours, each bacterium was associated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection disrupted the actin cytoskeleton and inhibited entry of P. multocida.

Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks.

Avian cellulitis in broiler chickens is primarily caused by Escherichia coli. Previous research found that the E. coli isolates of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. To test the hypothesis that the E. coli associated with cellulitis are endemic in the litter of the broiler house, we designed a study to determine whether E. coli DNA fingerprints associated with cellulitis persist over successive flocks that are grown in the same house. In addition, we assessed the impact of different cleaning and disinfection strategies on this persistence. Two broiler houses were followed on each of five farms over 3-4 flocks. A total of 353 E. coli isolates from cellulitis lesions were analyzed in this study, and 314 of these isolates (89%) were DNA fingerprinted by PFGE. In each ranch, there were several DNA fingerprint patterns that were present over successive flocks, regardless of the cleaning and disinfection strategy utilized. Isolates persisted as long as 191 days, implying that these E. coli are capable of persisting in the broiler house environment for long periods of time. In addition, these E. coli isolates were associated with cellulitis lesions in successive flocks. Thus, the isolates of E. coli that are associated with cellulitis in broiler chickens appear to be endemic in the litter environment of the broiler house.

Suitability of the broth-disk elution test for evaluating susceptibility of obligate anaerobes to trimethoprim-sulfonamides.

Species of anaerobic bacteria isolated from clinical veterinary specimens were tested for susceptibility to trimethoprim-sulfonamides by the broth-disk elution technique. Three different media were used for each organism: prereduced anaerobically sterilized (PRAS) brain-heart infusion broth (BHI), thioglycollate broth, and a semidefined PRAS medium. Susceptibility results from these media were compared with those determined by interpreting the minimal inhibitory concentration obtained using an agar dilution technique. Results from broth-disk testing in semidefined medium agreed in 68.7% of the cases, in 53.7% for thioglycollate broth, and in 36.9% for BHI. The greatest deviation between techniques occurred with isolates belonging to the genus Bacteroides, followed by those of the genus Clostridium and those of the genus Fusobacterium. This deviation was directly proportional to increasing concentrations of thymidine in the BHI and thioglycollate broths but not with the semidefined medium. We conclude that the broth-disk elution method for measuring susceptibility of obligate anaerobes to trimethoprim-sulfonamides is unsuitable.

Distribution of indole-producing urease-negative pasteurellas in animals.

Three hundred fifty-six animal isolates of indole-positive urease-negative cultures of Pasteurella, which would formerly have been classified as P. multocida, were examined with respect to their relationship to the recently described P. multocida subspecies (ssp.) multocida, septica, and gallicida and P. canis, P. stomatis/Taxon 16, and Pasteurella sp. B. Two hundred sixty-three (73.9%) of the cultures could be identified with one of these taxa, and 93 isolates (26.1%), representing 17 different biotypes, were unassignable. Pasteurella multocida ssp. multocida was the predominant taxon throughout and in most of the 25 animal species from which isolations were made. In dogs, P. canis was the most frequent. Different degrees of host predilection were observed also in P. multocida ssp. septica for cats, P. canis for sheep, and 2 of the unassignable biotypes for cattle and dogs, respectively. Overall, the respiratory tract was the most frequent source of isolates, but a propensity of P. multocida ssp. septica for localization in the central nervous system of cats was noted.

Multiplex polymerase chain reaction for distinguishing Taylorella equigenitalis from Taylorella equigenitalis-like organisms.

It is difficult to distinguish isolates of Taylorella equigenitalis, the cause of contagious equine metritis, from a T. equigenitalis-like organism isolated from asymptomatic donkeys and horses. Although T. equigenitalis is responsible for a severe, contagious disease of the reproductive tract of equids, the T. equigenitalis-like organism, although contagious, does not appear to produce disease. Because of the economic consequences of correctly distinguishing isolates of these 2 microorganisms, a polymerase chain reaction (PCR)-based assay was developed that will distinguish isolates of T. equigenitalis from the T. equigenitalis-like microorganism. The primers used in the PCR assay were designed to amplify unique regions of the gene encoding the 16S ribosomal RNA.

Apparent outbreaks of Clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital.

Intestinal colonization with toxigenic strains of Clostridium difficile was documented in 9 of 10 horses with acute onset diarrhea in a veterinary medical teaching hospital, whereas a similar isolate was detected in only 1 of 23 other horses without diarrhea in the hospital. One horse with diarrhea was infected simultaneously with both C. difficile and Salmonella krefeld. Clostridium difficile was detected by fecal culture on selective medium, confirmed with a latex particle agglutination test, and identified as toxigenic by polymerase chain reaction amplification of toxin A and toxin B gene sequences. Using an arbitrarily-primed polymerase chain reaction, 6 distinct C. difficile isolates were detected in the feces of the 9 affected horses at the time of the outbreak of diarrhea.

A statistical model for assessing sample size for bacterial colony selection: a case study of Escherichia coli and avian cellulitis.

A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined.

Association of complement sensitivity with virulence of Pasteurella multocida isolated from turkeys.

Two strains of Pasteurella multocida, both derivatives of strain P1059, were compared for virulence for 14-week-old turkeys and sensitivity to turkey plasma. Strain P1059-1, a nalidixic-acid-resistant mutant of P1059 with an LD50 of approximately 10(3) colony-forming units (CFU), was more resistant to the bactericidal effects of fresh turkey plasma at 37 C than avirulent strain P1059-1A. P1059-1A, with an LD50 of approximately 10(8) CFU, is an acapsular variant of P1059-1 that spontaneously arose after prolonged passage on artificial medium. The bactericidal effect on P1059-1A was removed when turkey plasma was treated with heat or with zymosan, maneuvers that removed hemolytic complement activity from turkey plasma.

Fate of Pasteurella multocida in the blood vascular system of turkeys following intravenous inoculation: comparison of an encapsulated, virulent strain with its avirulent, acapsular variant.

A virulent, encapsulated strain of Pasteurella multocida was compared with a spontaneously arising, avirulent acapsular variant following injection into the bloodstream of 14-week-old turkeys. Neither strain was detectable in the blood by 1 hour, but they reappeared 4 hours postinoculation in approximately equal numbers. The concentration of both strains increased with time, but the virulent strain reached concentrations 100,000-fold higher than the avirulent strain 15-24 hours after inoculation. In the liver and spleen the virulent strain reached higher concentrations than the avirulent strain, particularly 15 hours postinoculation. However, histopathological examination indicated that the difference between concentrations of the two strains was more likely due to an increased propensity for extracellular multiplication of the virulent strain rather than to greater efficiency in phagocytosis of the avirulent strain. In vitro, the two strains became associated minimally, though equally, with the mononuclear phagocytes and were destroyed. We conclude that humoral bactericidal defenses are primarily responsible for the differences in behavior between these two strains of P. multocida in vivo.

Serologic associations between fowl cholera and other diseases.

As part of a case-control study designed to identify fowl cholera risk factors, 2087 blood samples were collected from 71 California meat-turkey flocks. Samples were tested for antibodies to three mycoplasmas and four viruses pathogenic for turkeys. Flocks that had antibodies to Newcastle disease virus and/or Mycoplasma meleagridis had an increased risk of having an outbreak of fowl cholera. This information should prove useful for fowl cholera control programs in meat turkeys.

A descriptive study of fowl cholera in California meat turkeys: August 1985-July 1986.

One hundred sixty meat-turkey premises in California were monitored for outbreaks of fowl cholera from August 1985 through July 1986. Nearly 27 million turkeys in 720 flocks were at risk during the year. Fifty-three flocks of approximately 3 million turkeys on 34 different premises experienced confirmed fowl cholera outbreaks. The epidemic curve for the year indicated that the majority of outbreaks occurred in the late summer and fall, particularly in October. The incidence of outbreaks during this time was not significantly associated with seasonal variation in the size of the turkey population. The mean flock age at outbreak was 10.8 weeks, with a range of 5-18 weeks.

Pasteurella multocida in psittacines: prevalence, pathology, and characterization of isolates.

Although the pathogenicity of Pasteurella multocida for psittacines (parrots and their relatives) has been documented in several case reports, the associated pathologic syndromes have not been well defined nor have the isolates been characterized. In addition, the prevalence of P. multocida in psittacines has not been determined. Three hundred twenty-eight psittacines (253 clinically healthy and 75 clinically ill) were cultured for P. multocida. Pasteurella multocida was not isolated from the pharynx, choana, or cloaca of psittacines. However, in five dead psittacines submitted for necropsy, P. multocida was isolated. These isolates were characterized, and all belonged to either somatic serotype 3 or 4,7. Pasteurella multocida somatic serotype 3 was isolated from psittacines with septicemia, whereas P. multocida somatic serotype 4,7 was isolated from psittacines with cutaneous lesions. The majority (four out of five) of the P. multocida isolates belonged to the subspecies multocida, and all isolates were susceptible to penicillin G, sulfisoxazole, gentamicin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole but resistant to streptomycin. DNA fingerprints demonstrated that isolates belonging to the same somatic serotype were genetically related. The isolate from a cockatiel that had been caught by a cat belonged to somatic serotype 3 and was not genetically related to the other two isolates belonging to this somatic serotype.

Pasteurella multocida in raptors: prevalence and characterization.

Several cases dealing with Pasteurella multocida infection have been documented in raptors. However, the isolates have not been fully characterized nor has the prevalence of P. multocida in raptors been determined. Three hundred ninety-eight raptors were cultured for P. multocida. Results indicated that P. multocida was not normally carried in the pharyngeal, choanal, or cloacal regions. However, P. multocida was isolated from raptors with avian cholera. Isolates from eight cases were characterized by biotype, somatic serotype, and antibiogram. Most (six of eight) of the P. multocida isolates belonged to somatic serotype 1. The remaining two P. multocida isolates belonged to somatic serotypes 3 and 3,4. The majority of the isolates belonged to the subspecies multocida. All isolates were susceptible to penicillin G, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole. Various restriction site heterogeneities of P. multocida chromosomal DNA were found among the raptor isolates. Results indicated that isolates of P. multocida somatic serotype 1 from diurnal raptors were genetically related.