Pulsed-field gel electrophoresis of Staphylococcus hyicus and Staphylococcus chromogenes genomic DNA and its taxonomic, epidemiologic and ecologic applications in veterinary medicine.

One hundred and thirty-eight strains of Staphylococcus hyicus and 21 strains of S. chromogenes isolated from animals were analyzed by pulsed-field gel electrophoresis (PFGE) after restriction endonuclease Smal digestion of chromosomal DNA. Eighty-eight strains of S. hyicus from pigs with or without exudative epidermitis (EE) generated 16 to 26 fragments in the size range of < 1 to 485 kb, and yielded 39 different patterns. With regard to the strains from pigs with EE, PFGE patterns differed according to the country of origin. Outbreaks of EE occurring on four separate pig farms in Japan involved S. hyicus with different PFGE patterns. The PFGE patterns shown by S. hyicus strains from 4 kinds of animals were compared. Strains from pigs differed from those isolated from chickens (n = 45; 18 to 24 fragments of < 1 to 425 kb), cows (n = 3; 17 to 19 fragments of < 1 to 475 kb), and goats (n = 2; 16 or 17 fragments of < 1 to 1,125 kb). Also, each of the chicken, cow and goat strains had a host-specific fragment. The results suggest that PFGE analysis might be a useful marker for distinguishing ecovars within S. hyicus. In contrast, strains of S. chromogenes from pigs and cows generated 17 to 24 fragments ranging from < 1 to 545 kb. The PFGE patterns of S. chromogenes strains were more highly conserved than those of S. hyicus. S. chromogenes strains could be distinguished from S. hyicus strains by fragments within the range of 305 to 545 kb. The results indicate that PFGE analysis could be used to distinguish between S. hyicus and S. chromogenes. We conclude that PFGE analysis is a useful tool not only for species or strain identification but also for epidemiologic or ecologic studies of S. hyicus and S. chromogenes.

Genomic DNA fingerprinting, using pulsed-field gel electrophoresis, of Staphylococcus intermedius isolated from dogs.

OBJECTIVES: To investigate the degree of polymorphism in the pulsed-field gel electrophoresis (PFGE) patterns of Staphylococcus intermedius and to assess the value of this typing method for discriminating strains. SAMPLE POPULATION: 52 S intermedius isolates from diseased and healthy dogs. PROCEDURE: Chromosomal DNA of S intermedius was digested with restriction endonuclease Sma I, and the fragments were separated by PFGE in a 1% agarose gel. RESULTS: Sma I cut the chromosomal DNA into 15 to 23 fragments ranging from about < 1 to 679 kb, and most of the detectable fragments were < 155 kb. Nine fragments, 115, 48, 33, 26, 16, 13, 10, 4, and < 1 kb, were shared by all or almost all (> 71%) of the strains examined. Of the 52 strains, each had a different pattern. S intermedius had a high degree of restriction fragment length polymorphism. The PFGE patterns obtained for S intermedius were stable and reproducible when the strains were tested in the different experiments. CONCLUSIONS: Genomic DNA fingerprinting by PFGE is an effective technique for discriminating S intermedius strains. The PFGE method appears to be a useful molecular marker for epidemiologic or ecologic studies of S intermedius.

Prolonged Bartonella bacteremia in cats associated with cat-scratch disease patients.

Recent evidence supports a causal relationship between Bartonella (Rochalimaea) henselae, cat-scratch disease (CSD), and bacillary angiomatosis. Cats appear to be the primary reservoir. Blood from 19 cats owned by 14 patients diagnosed with CSD was cultured. Blood samples from cats owned by veterinary students (n = 25) having no association with CSD or bacillary angiomatosis were cultured as controls. Eighty-nine percent (17 of 19) of cats associated with CSD patients and 28% (7 of 25) of controls were bacteremic with Bartonella species (chi-square = 16.47; P < 0.001). Twenty-three isolates were characterized as B. henselae, while one isolate from the cat of a CSD patient appeared to be a new Bartonella species. Thirteen cats remained culture positive during the ensuing 12-month period. Our results support the conclusion that B. henselae is the predominant species involved in CSD and is transmitted by cats. The incidence of Bartonella bacteremia in control cats suggests that B. henselae bacteremia is prevalent among the domestic cat population in the United States.

Influence of estrogen on antibacterial and immunoglobulin secretory activities of uterine fluids from ovariectomized mares.

Effect of estrogen (E2) and progesterone (P4) on uterine antibacterial activity and immunoglobulin concentrations in mares was studied. In 2 in vitro experiments, 6 mixed-breed mares were ovariectomized, and uterine fluid and blood serum were analyzed. Antibacterial assay methods were used to determine inhibitory effects on Streptococcus zooepidemicus of uterine fluid samples collected on days 3, 5, and 8, and serum obtained on day 8 of treatment. Single radial immunodiffusion methods were used to quantify amounts of IgA and IgG in uterine fluid and serum on days 3, 5, 8, and 14 of treatment. Neither E2 nor P4 increased activity of serum and uterine fluid against S zooepidemicus. Numbers of colony-forming units per milliliter of bacteria were significantly (P < 0.01) lower in control Hanks' balanced salt solution with 1.0% gelatin (HBSSG) than in uterine fluids. Bacterial numbers were significantly (50%) greater in uterine fluids and serum than in HBSSG controls for both treatments. Both fluids, especially serum, supported significantly (P < 0.01) more growth of S zooepidemicus than did HBSSG when incubated for 0, 2, and 4 hours. These findings are in contrast to previous reports of antibacterial activity in the uterus of sexually intact mares undergoing an estrous cycle: great reduction of bacterial count in uterine fluid from mares in diestrus, and significant increases in bacterial numbers in uterine fluid or serum from mares in estrus. Treatment comparisons between serum and uterine fluid IgA and IgG concentrations were not significantly different, although overall IgA concentration in the uterus was higher than concentration in serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiological studies of congo red Escherichia coli in broiler chickens.

This prospective cohort study was designed to confirm the association between Congo red binding Escherichia coli (CREC) and E. coli air sacculitis in commercial broilers. It was also designed to evaluate CREC as an air sacculitis risk factor and to explore the CREC relationship to other air sacculitis risk factors (poultry house temperature, air-ammonia levels, and presence of other diseases). In addition, this study was used to assess a possible role of the broiler-breeder flocks and hatchers in the spread of CREC air sacculitis. Congo red E. coli-associated airsacculitis risk was based on CREC exposure of the chicks in the hatchers. Breeder flocks with greater than 30 CREC colonies/plate from hatcher air sampling tests were placed in the high risk group; flocks with less than five CREC colonies/plate were placed in the low risk group. Increased risks of death due to air sacculitis (RR = 2.26), and increased death rates due to CREC air sacculitis (RR = 9.45) in high-risk flocks, identified CREC as an important air sacculitis risk factor. The attributable risk percent of CREC airsacculitis from hatcher exposure of CREC was 89.4%, pointing to the hatcher as the source of CREC infection. The association of specific broiler-breeder flocks to high levels of CREC in the hatchers, and subsequent air sacculitis, suggests that the broiler-breeders are the ultimate source of CREC.

Experimental reproduction of airsacculitis and septicemia by aerosol exposure of 1-day-old chicks using Congo red-positive Escherichia coli.

To further demonstrate the association of phenotype and virulence of Congo red Escherichia coli (CREC), experiments were designed to reproduce airsacculitis and colisepticemia in 1-day-old chicks via aerosol exposure. In eight separate experiments in which a total of 462 chicks were exposed to CREC, the mortality rate was 4.11% and the morbidity rate was 13.4%, with both the dead and diseased chicks showing lesions of fibrinous airsacculitis, pericarditis, and perihepatitis. In contrast, when the corresponding non-CREC derivatives (the same E. coli strains, but not expressing the CR phenotype) were used as the aerosol inoculum in five additional experiments using 284 chicks, no chicks died and no lesions were observed. These results clearly show a strong correlation between the expression of the CR phenotype and virulence in avian E. coli.

Clinical and serologic evaluations of induced Borrelia burgdorferi infection in dogs.

Adult Beagles were used to evaluate clinical signs and serologic response after inoculation with, or exposure to, Borrelia burgdorferi. An indirect immunofluorescent assay (IFA) and 2 ELISA were used to monitor the serologic response to B burgdorferi. Feeding infected ticks on 4 dogs (group 1) failed to cause seroconversion, and SC inoculation with 500 organisms caused minimal seroconversion in 2 of 4 dogs (group 2). At 56 days, approximately 3.01 X 10(8) B burgdorferi organisms were injected IV into group-1 dogs, and intraperitoneally into group-2 dogs. A control group of 4 dogs (group 3) had noninfected ticks feed on them, and then were given IV injection of physiologic saline solution. Increases in immunoglobulin M (IgM) titers were detected in 2 of 4 group-2 dogs approximately 7 days after the initial exposure. These titers returned to negligible values 20 days later. Immunoglobulin G titers increased approximately 10 days after the initial exposure and were mildly increased 56 days later, when dogs were exposed a second time. Both the IV and intraperitoneal injections (second exposures) resulted in increased IgM titers, which in both groups eventually returned to preexposure values after approximately 2 months. Immunoglobulin G titers increased within a week after the second exposure, and in 3 dogs monitored for 8 months, returned to negligible values after the 8-month period. One control dog had a slightly increased IgG titer 24 days after the second inoculation. The possibility of urine transmission is suggested. Clinical status, hemograms, serum biochemical profiles, ECG and results of urinalyses remained normal throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to Borrelia burgdorferi in dogs in North Carolina.

An indirect immunofluorescence assay was used to detect antibodies against Borrelia burgdorferi in sera from 600 dogs in 1983 and 402 dogs in 1985. In 1983, the overall prevalence rate of dogs with B burgdorferi titers greater than or equal to 1:64 was 3.6%, whereas in 1985, the prevalence rate was 2.7%. An unexplainable higher seroprevalence was detected in 1 group of dogs tested in 1983. These dogs were from the southern coastal plains of North Carolina. In the dogs tested in 1985, this regional difference in sero-prevalence was not noticed. Statistical differences were not noticed (P greater than 0.05) between dogs from 2 sources or when gender was considered. Seemingly, the prevalence of anti-B burgdorferi antibodies in dogs in North Carolina was low.

Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs.

Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs. Adsorption studies confirmed that the antibodies were specific for B. burgdorferi. Experimentally exposed dogs were asymptomatic. Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease. Naturally exposed dogs were from four geographic regions of the country. No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country. The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity to fewer and different protein bands. Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not. Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for the observed differences.

Epidemiological study of the relationship between Congo red binding Escherichia coli and avian colisepticemia.

An epidemiological prospective (longitudinal) study design was used to evaluate the association of Congo red positive Escherichia coli and avian colisepticemia. High and low risk exposure groups of chickens were identified at hatching, and placed in separate identical houses on the same farm. Approximately 14,000 birds were placed in each house for the seven week grow-out period, during which all birds which died were necropsied and cultured, together with a representative sample of birds which were culled weekly. The findings implicated Escherichia coli as the etiological agent of avian colisepticemia. A relative risk of 6.5 and attributable risk of 73.5% supported the hypothesis that the Congo red medium identifies a virulent form of Escherichia coli which causes airsacculitis-colisepticemia in poultry.

Congo red medium to distinguish between invasive and non-invasive Escherichia coli pathogenic for poultry.

In the course of our molecular studies of virulence factors associated with invasive avian Escherichia coli infections, it was first necessary to distinguish between common E. coli and those that cause septicemia in poultry. We found a direct correlation between the ability of clinical isolates of E. coli to bind Congo red dye (CR) and their ability to cause septicemic infection in chickens. This finding was supported by bacteriological studies of 30 broiler flocks (26 sick and 4 healthy) and by virulence studies in chickens and mice. All 144 isolates of E. coli from internal tissues of diseased birds were determined to be CR-positive (red colonies). Congo-red-positive E. coli colonies were isolated from air sacs, pericardium, liver, lung, joint fluid, and heart blood of chickens with lesions of colisepticemia. In contrast, of 170 E. coli isolates from the poultry house environment and from the trachea and cloaca of healthy birds, more than half were CR-negative (white colonies). No CR-negative (white) E. coli colonies were found in internal organs from birds with typical lesions of colisepticemia. We feel that these preliminary findings suggest that the CR dye binding could be used as a phenotypic marker to distinguish between invasive and noninvasive isolates.

Prevalence of Alcaligenes faecalis in North Carolina broiler flocks and its relationship to respiratory disease.

In order to assess the role of Alcaligenes faecalis in respiratory disease of broilers, a study was conducted to determine the prevalence of this bacterium in North Carolina broilers and to determine the relationship of A. faecalis infection to clinical disease. Our studies showed that A. faecalis is prevalent in North Carolina commercial broilers during the winter months. Bacteriological examination of turbinates and tracheas revealed that almost 40% of individual birds between 35 and 45 days of age yielded positive cultures; 62% of tested flocks were infected. When present, A. faecalis was usually the predominant bacterium isolated. Furthermore, because of a higher frequency of A. faecalis isolation in broiler flocks with respiratory disease (75% vs. 29% in flocks without respiratory diseases), these studies suggest a causal relationship between this bacterium and clinical respiratory disease.

Differentiation of Alcaligenes-like bacteria of avian origin and comparison with Alcaligenes spp. reference strains.

Although standard biochemical tests used for the identification of Alcaligenes spp. revealed only minor differences, the oxidative low-peptone technique clearly differentiated between Alcaligenes-like bacteria of avian origin and Alcaligenes spp. reference strains. Based on their colonial morphology, biochemical profiles, and hemagglutination, the Alcaligenes-like bacteria of avian origin were further divided into two subgroups, C1-T1 and C2-T2. Colonies of subgroup C1-T1 were nondescript, round, raised, glistening, translucent, greyish, and about 2 mm in diameter. Colonies of subgroup C2-T2 were off-white, flat, dry and wrinkled, generally round, and resembled tiny lily pads. Biochemical profiles by the oxidative low-peptone method showed the C1-T1 subgroup alkalinizing only three substrates (citrate, acetate, and succinate), whereas the C2-T2 subgroup alkalinized eight substrates (citrate, acetate, butyrate, itaconate, malonate, saccharate, succinate, and M-tartrate). Subgroup C1-T1 agglutinated human, chicken, and turkey erythrocytes, whereas subgroup C2-T2 did not. The recognition of these two subgroups within the Alcaligenes-like bacteria of avian origin is important, since it may explain the differences seen in pathogenicity among isolates.

Prevalence of Clostridium botulinum type C in substrates of phosphate-mine settling ponds and implications for epizootics of avian botulism.

Prevalence and conditions for occurrence of Clostridium botulinum type C were examined on phosphate-mine settling ponds and a natural wetland in northern Florida between April 1981 and March 1982. Substrate samples were collected monthly (winter) and semi-monthly (summer) from 16 locations on seven ponds. Selected environmental parameters were measured at each location at the time of sampling. Mouse inoculation tests and toxin neutralization tests using enrichment culture filtrates were conducted to identify C. botulinum type C in the samples. The bacteria were identified in 26 (5.6%) of 467 sediment samples. Occurrences were distributed over four of the seven ponds and included nine of the 16 sample locations, but were restricted to the months April through October. The organism occurred over a wide range of ecological conditions found on the ponds during these months. If the presence of C. botulinum type C in the substrate is a prerequisite for botulism to occur, the prevalence and fairly wide distribution of this organism on settling ponds makes it difficult to predict where future outbreaks may occur.

Pathogenicity of various isolates of Alcaligenes faecalis for broilers.

Day-old broilers or specific-pathogen-free chickens were inoculated intranasally with approximately 1 X 10(8) organisms of eight different field isolates of Alcaligenes faecalis. Major differences in the pathogenicity of isolates and their ability to colonize the trachea were found. Only two isolates (Wilson and Lockamy) produced mild clinical signs of respiratory disease ("snicking," dyspnea). The same two also colonized the respiratory tract, especially the trachea, in large numbers; they persisted for 31 days. Of the remaining six isolates, five were also able to colonize the respiratory tract but did so to a lesser degree and less persistently, without causing clinical signs. Only one isolate (CS) was incapable of becoming established in the respiratory tract of chicks after intranasal inoculation.

Differential microbiological diagnosis of protothecosis from non-human sources.

Primary isolation of Prototheca was accomplished on MacConkey's, sheep blood agar, and Sabouraud dextrose agar. Prototheca was differentiated from similar organisms by its ability to grow on MacConkey's agar, and by its colonial morphology. Further differentiation was based on staining procedures to reveal the characteristic microscopic morphology. In the process of identification of several isolates of Prototheca zophii obtained from animal sources, a simple guideline for isolation and identification of these organisms was developed.