Brucella abortus infection in indigenous Korean dogs.

Three dogs reared on a dairy farm with a high incidence for Brucella abortus were serologically positive for B. abortus and no other Brucella spp. The identity of the organism was confirmed to be B. abortus by AMOS (abortus melitensis ovis suis)-polymerase chain reaction with specific primers for B. canis. One hundred percent homology of the canine isolate and the bovine pathogen isolated from the farm was demonstrated. The only possible source of infection was infected cattle on the same farm. It is suggested that dogs be routinely included in brucellosis surveillance and eradication programs.

Characterization of the protective response against a homologous challenge infection with Strongyloides venezuelensis in rats.

The protective response in rats against a homologous challenge infection with Strongyloides venezuelensis was characterized. In an initial infection with 1000 filariform larvae and migrating larvae (L(3)) of S. venezuelensis, the population of L(3) in the lungs on day 3 postinfection (PI), and that of adult worms in the small intestine on day 7 PI, were 180.8+/-14.5 and 336.8+/-70.7, respectively. The latter were gradually expelled towards day 42 PI. After the initial infection, the rats developed strong immunity against a homologous challenge infection as manifested by a marked reduction in worm populations, stunted body length and width, damage to reproductive organs, impaired egg production and rapid expulsion of the worms by day 14 after challenge. Expulsion of the worms was preceded by a significantly elevated (P<0.05) peripheral blood eosinophil (PBE) count, both in the initial (200.0+/-26.5 x 10(3)ml) and the challenge infection (400.9+/-165.4 x 10(3)ml). These findings suggest that rats acquire strong homologous immunity following initial exposure to S. venezuelensis. It is suggested that PBEs are involved in worm expulsion. A major target of these effector mechanisms is the reproductive system of S. venezuelensis.

Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk.

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.

Nonenteric Escherichia coli isolates from dogs: 674 cases (1990-1998).

OBJECTIVE: To determine nonenteric sites associated with Escherichia coli isolates in dogs and the antimicrobial susceptibilities of the isolates. DESIGN: Retrospective study. SAMPLE POPULATION: 17,000 canine specimens. PROCEDURE: Medical records of 17,000 canine specimens submitted for bacteriologic culture were examined and the number of isolations of E coli was determined. For these cases, records were further examined with respect to body system involvement, sex, concurrent infection with other species of bacteria, and antimicrobial susceptibility. RESULTS: 674 E coli isolates (424 from urine, 62 from the skin, 52 from the respiratory tract, 45 from the ear, 43 from the female reproductive tract, 25 from the male reproductive tract, and 23 from other organ systems) were identified. There was a significantly higher proportion of isolates from urine specimens from spayed females than from sexually intact females or males. Escherichia coli was isolated in pure culture from 65.9% of the specimens. Most E coli isolates were susceptible to norfloxacin (90%), enrofloxacin (87.5%), gentamicin (90.7%), and amikacin (85.9%). CONCLUSIONS AND CLINICAL RELEVANCE: Most nonenteric E coli infections in dogs involve the urinary tract. Amikacin, gentamicin, norfloxacin, and enrofloxacin have the highest efficacy against canine E coli isolates. For E coli isolates from dogs, in vitro susceptibility to commonly used antimicrobial agents has remained fairly stable during the past decade.

Standardization of the diagnostic criteria for canine ehrlichiosis. Towards a universal case definition.

Canine ehrlichiosis is a highly variable syndrome presenting a significant differential diagnostic difficulty. It imitates many metabolic and infectious diseases and lacks standardized diagnostic criteria, common reagents, and database resources. A clinical diagnosis of canine ehrlichiosis may be based on the manifestation of fever, thrombocytopenia, anorexia, nasolacrimal discharge, epistaxis, and exclusion of autoimmune and common canine vector borne diseases. These parameters are not invariably observed especially in the atypical form of the disease often caused by species other than Ehrlichia canis. A definitive diagnosis is based on the presence of specific antibodies to ehrlichial agent(s), the demonstration of the etiologic agent(s) itself, or specific amplicons by a strigently quality controlled PCR protocol. The relationship of the various clinical and laboratory parameters, the status of the currently available tests, and their real or presumed predictive value are discussed in the context of stimulating an effort to formulate an international standard for the diagnosis of this and related diseases of man and animals.

Serologic cross-reactivity of antibodies against Borrelia theileri, Borrelia burgdorferi, and Borrelia coriaceae in cattle.

OBJECTIVE: To evaluate the immune response induced by Borrelia theileri infection and to determine whether B theileri induces cross-reacting antibodies to other bovine borreliae. ANIMALS: Two 3-month-old calves, 1 of which was splenectomized. PROCEDURE: Calves were exposed to Boophilus microplus infected with B theileri. Rectal temperature, PCV, bacteremia, and clinical signs of infection were monitored. Serum was obtained weekly and used to evaluate the humoral response to homologous antigen and B burgdorferi and B coriaceae, using an indirect fluorescent antibody (IFA) test, and to B burgdorferi, using a commercially available ELISA. The identity of cross-reacting antigens was explored, using monoclonal antibodies to genus- and species-specific antigens in an IFA test. RESULTS: B theileri-infected calves produced antibodies that cross-reacted with B burgdorferi and B coriaceae whole-cell antigens. Borrelia theileri whole-cell antigen was recognized by genus-specific monoclonal antibody H9724 but not by species-specific antibody H5332. False-positive reactions were not observed when serum from B theileri-infected calves was tested by use of the ELISA for B burgdorferi. CONCLUSIONS: B theileri induces humoral responses in infected cattle that can be confused with those of other borrelial infections. Care must be taken to definitively distinguish between the various borreliae that may cause disease in cattle. CLINICAL RELEVANCE: Serologic cross-reactivity must be taken into account when making a serodiagnosis of Lyme borreliosis or epizootic abortion in epidemiologic studies involving cattle.

Evaluation of activity of selected ophthalmic antimicrobial agents in combination against common ocular microorganisms.

OBJECTIVE: To determine in vitro efficacy of gentamicin, tobramycin, and miconazole when used in combination, with or without atropine, against Pseudomonas or Aspergillus sp. PROCEDURE: Selected ophthalmic agents were combined for predetermined times. Sterile disks impregnated with the combined solutions were prepared and placed on Mueller-Hinton plates that were seeded with Pseudomonas or Aspergillus sp. Zones of growth inhibition were measured at postincubation hours 24 and 48. RESULTS: Tobramycin alone inhibited growth of Pseudomonas sp, whereas miconazole inhibited growth of Aspergillus sp. Significant differences in zones of growth inhibition when atropine was combined with tobramycin, when gentamicin was combined with miconazole, or when atropine was combined with miconazole and gentamicin, were not detected. CLINICAL RELEVANCE: Combining selected ophthalmic therapeutic agents for as long as 6 hours does not appear to alter the in vitro efficacy of the agents against microorganisms used in this study.

Methicillin resistance among staphylococci isolated from dogs.

OBJECTIVE: To determine whether methicillin-resistant staphylococci from dogs expressed the mecA gene and to determine what proportion of canine staphylococcal isolates positive for the mecA gene were resistant to oxacillin and other antibiotics. SAMPLE POPULATION: 25 methicillin-resistant (10 coagulase-positive and 15 coagulase-negative) and 15 methicillin-susceptible (8 coagulase-positive and 7 coagulase-negative) staphylococci isolated from dogs. PROCEDURE: All strains were tested for methicillin resistance by use of oxacillin agar screening and identified by use of standard techniques. Minimum inhibitory concentrations of 16 antibiotics were determined for all 40 isolates. A polymerase chain reaction method targeting a 533-basepair fragment of the mecA gene was used to detect mecA gene expression. RESULTS: 23 of the 25 methicillin-resistant isolates and none of the methicillin-susceptible isolates possessed the mecA gene. For 10 of 16 antibiotics, the proportion of mecA-positive isolates that were resistant or of intermediate susceptibility was significantly higher than the proportion of mecA-negative isolates that were resistant or of intermediate susceptibility. Only 1 methicillin-resistant coagulase-positive isolate was identified as Staphylococcus intermedius; the other 9 were identified as S. aureus. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirm that staphylococci isolated from dogs may have methicillin resistance mediated by the mecA gene. Isolates positive for the mecA gene were more likely to be resistant to various antibiotics than were isolates negative for the mecA gene. Results suggest that in dogs, infections caused by staphylococci that have the mecA gene may be difficult to treat because of resistance to antibiotics.

Cutaneous antimicrobial preparation prior to intravenous catheterization in healthy dogs: clinical, microbiological, and histopathological evaluation.

The purpose of this study was to determine the effects of a one-minute chlorhexidine gluconate skin preparation protocol prior to cephalic vein catheterization. Twenty-three healthy beagle dogs had one leg aseptically prepared and the opposite leg served as a control. Twenty-six- and 77-hour time groups were studied. Chlorhexidine-treated legs had significantly lower cutaneous bacterial counts than the control legs prior to catheter insertion and prior to catheter withdrawal for both time groups. Control legs developed significantly more dermatitis than the treated legs after 77 h. A one-minute preparation with 4% chlorhexidine gluconate was an effective method for sustained reduction of cutaneous bacterial counts at peripheral intravenous catheter insertion points in dogs. Increased cutaneous bacterial counts were associated with significantly more microscopic dermatitis in untreated legs after 77 h of catheterization.

Detection of Staphylococcus aureus in milk by use of polymerase chain reaction analysis.

OBJECTIVE: To evaluate polymerase chain reaction (PCR) analysis for detection of Staphylococcus aureus (nuc gene) in fresh and formalin-preserved milk. SAMPLE POPULATION: Samples from 80 lactating sheep and 100 lactating dairy cows. PROCEDURE: 4 lactating sheep were inoculated with S aureus by intramammary infusion. A set of primers specific for the nuc gene of S aureus was used to develop a PCR technique, and modification of the rapid boil method was used to isolate bacterial DNA. Milk was obtained from experimentally infected sheep before and after infusion with S aureus, and from the 100 cows and remaining 76 sheep. Samples were screened by bacteriologic culture and PCR. To validate the PCR assay, S aureus or other pathogens were added to distilled water and "normal" sheep milk samples, with and without formalin. RESULTS: The PCR assay was 100% specific for S aureus when known negative and positive samples were tested. Sensitivity was 100% for samples with added S aureus or other pathogens. Sensitivity was lower for samples obtained from experimentally infected sheep, but increased from 53% to 90% with increased washing of target DNA. CONCLUSIONS: The PCR technique based on the nuc gene is able to detect S aureus in sheep milk yields results faster than does traditional culturing, is highly specific, and is able to detect S aureus in formalin-fixed milk samples. CLINICAL RELEVANCE: The assay is particularly suitable for analysis of samples shipped or stored without refrigeration. Although antibiotics in milk may inhibit growth in culture, they should not affect the results of the PCR assay.

Effects of mastitis on the volume and composition of colostrum produced by Holstein cows.

The effects of mastitis during the late nonlactating period on colostral volume and concentrations and total yields of immunoglobulin (Ig) G1, fat, and protein in colostrum were investigated using matched pairs of mammary glands from multiparous Holstein cows. Samples of mammary secretions were collected at approximately 14 and 7 d prepartum and within 3 h after calving. At each sampling time, the glands and secretions were examined for gross abnormalities, and the California Mastitis Test was performed. Duplicate secretion samples from each gland were cultured, and somatic cell count, pH, and fat and protein concentrations were determined. The volume of colostrum obtained at the first milking of each gland was quantified using a quarter milking device, and its IgG1 concentration was measured. Colostral volume from persistently infected mammary glands was lower than that from matched uninfected glands, as was the total mass of IgG1. However, infection did not alter IgG1 concentration in colostrum. Fat and protein percentages were lower in prepartum secretions but not in colostrum from infected glands. Persistent infection was associated with increased somatic cell count and pH of secretions at all sampling times, and California Mastitis Test scores were higher for colostrum from infected glands. The appearance of secretions was extremely variable, but the presence of flakes or clots in colostrum was associated with infection. We concluded that mastitis during the late nonlactating period alters mammary gland function but is unlikely to be an important contributor to the high rate of failure of passive transfer of immunoglobulins in calves.

Bronchopulmonary disease in the cat: historical, physical, radiographic, clinicopathologic, and pulmonary functional evaluation of 24 affected and 15 healthy cats.

The results of clinical and pulmonary functional evaluation of 24 cats with bronchopulmonary disease and 15 healthy cats are presented. Affected cats had historical evidence of excessive reflexes (coughing, sneezing); physical evidence of airway secretions (crackles), obstruction (wheezing), and increased tracheal sensitivity; radiographic evidence of bronchial and interstitial lung disease; and cytological evidence of airway inflammation or mucous secretions. Bacterial isolates from healthy and affected cats were predominantly Gram-negative rods, indicating that bronchi of cats are not always sterile and that normal flora should be considered in interpreting cultures from cats with suspected bronchopulmonary disease. Cats were grouped according to relative disease severity based on scored historical, physical, and radiographic abnormalities. The mean (+/- standard deviation) baseline lung resistance measurement in healthy cats was 28.9 cm H2O/L/s (+/- 6.2 cm H2O/L/s), whereas in mildly, moderately, and severely affected cats it was 38.3 cm H2O/L/s (+/- 21.5 cm H2O/L/s), 44.8 cmH2O/L/s (+/- 7.7 cm H2O/L/s), and 105.2 cm H2O/L/s (+/- 66.9 cm H2O/L/s), respectively. In healthy cats, dynamic lung compliance was 19.8 (+/- 7.4), whereas in mildly, moderately, and severely affected cats it was 14.7 mL/cm H2O (+/- 3.8 mL/cm H2O), 17.7 mL/cm H2O (+/- 6.9 mL/cm H2O), and 13.0 mL/cm H2O (+/- 7.9 mL/cm H2O), respectively. Thus, airway obstruction was present in many of the affected cats. Based on acute response to the bronchodilator, terbutaline, airway obstruction was partially reversible in many affected cats, although the degree of reversibility varied. Furthermore, based on bronchoprovocation testing, 6 (of 7) affected cats evaluated also had increased airway responsiveness to aerosolized methacholine.

Evaluation of udder health and mastitis in llamas.

OBJECTIVE: To investigate intramammary infections in llamas, identify the pathogens responsible, and determine whether effects of intramammary infection could be detected by use of mastitis indicator tests commonly used for cows. DESIGN: Observational study. ANIMALS: 100 llamas on 10 farms. PROCEDURE: Milk samples were evaluated by bacterial culturing and by determination of somatic cell count (SCC), using direct microscopic and automated counting methods, California Mastitis Test score, pH, and N-acetyl-beta-d-glucosaminidase activity. Correlation coefficients were determined among the various mastitis indicator tests, and test results were determined for milk from infected and uninfected glands. RESULTS: Evidence of intramammary infection was evident in 76 of 369 (21%) milk samples, with 54 of 94 (57%) llamas having at least 1 infected gland. Staphylococcus sp other than Staphylococcus aureus were the predominant pathogens. None of the llamas had clinical signs of mastitis, and significant differences were not detected in SCC, California Mastitis Test score, pH, or N-acetyl-beta-d-glucosaminidase activity between infected and uninfected samples. California Mastitis Test scores were negative or trace for 307 of 313 (98%) samples, and SCC were low. In contrast, pH and N-acetyl-beta-d-glucosaminidase activity of milk from uninfected glands were higher than values reported for milk from uninfected cows, and neither variable was significantly correlated with the number of somatic cells in samples of llama milk. CLINICAL IMPLICATIONS: Although intramammary infections develop in llamas, inflammation (mastitis) appears to be rare. Values for mastitis indicator tests used for cows cannot be directly extrapolated to llamas. Subclinical mastitis is apparently not an important problem in llamas in the United States.

Evaluation of fetal infection and abortion in pregnant ponies experimentally infected with Ehrlichia risticii.

Fetal infectivity of Ehrlichia risticii was investigated in 19 ponies that were E risticii negative on the basis of results of an indirect fluorescent antibody (IFA) test. Thirteen pregnant ponies were infected by IV administration of E risticii between 90 and 180 days of gestation. Six pregnant ponies served as noninfected controls. Each infected pony had clinical signs of equine monocytic ehrlichiosis, was confirmed to be ehrlichemic, and developed an IFA titer to E risticii. Two infected ponies became recumbent, were unresponsive to supportive care, and were euthanatized. After recovery from clinical illness, the remaining ponies were observed throughout gestation for reproductive abnormalities. On abortion, each fetus was necropsied and tissue specimens from the liver, bone marrow, spleen, colon, and mesenteric lymph nodes were inoculated into canine monocyte cell cultures. Six infected ponies aborted at a mean 217 days of gestation, which was between postinoculation days 65 and 111. Five fetuses were recovered for evaluation, and E risticii was isolated from 4 of them. All 5 fetuses recovered had similar histologic finding, including enterocolitis, periportal hepatitis, and lymphoid hyperplasia with necrosis of the mesenteric lymph nodes and spleen. All 5 fetuses tested negative for IgG to E risticii, although 3 had low IgM titer to E risticii. The remaining 5 infected ponies had normal parturition. Presuckle IFA titer to E risticii was measured in 4 of the term foals, and results for 3 were positive. Two foals from infected ponies were monitored for 6 months and daily gain in body weight was comparable to that of a control foal. None of the control ponies became ill or seroconverted during the clinical illness phase, and none aborted throughout gestation Two control ponies seroconverted to E risticii 6 weeks before parturition. Results of this study indicate that E ristcii is a primary abortifacient under experimental conditions.

Identification of Ehrlichia risticii as the causative agent of two equine abortions following natural maternal infection.

Two pregnant mares diagnosed as having equine monocytic ehrlichiosis based on history, clinical signs, and high serum antibody titers to Ehrlichia risticii aborted subsequent to recovery from illness. Mare 1 and mare 2 experienced clinical illness at 120 and 143 days of gestation and aborted at 203 and 226 days of gestation, respectively. The fetuses were expelled in fresh condition, and both mares retained their placentas upon abortion. Gross findings for the fetuses included meconium staining and petechiation of external surfaces. Internally, there was increased volume of feces within the small and large intestines and liver discoloration with enlargement. Microscopic findings included lymphohistiocytic enterocolitis, hepatitis, and myocarditis. Lymphoid hyperplasia and depletion were present in spleen, thymus, and lymph nodes. Ehrlichia risticii was recovered from bone marrow, spleen, lymph node, colon, and liver of the first fetus and bone marrow and colon of the second fetus. Electron microscopic evaluation of the organism isolated in cell culture revealed morphology consistent with E. risticii. The isolated organism was inoculated into a naive pony, and this pony developed high levels of antibody against E. risticii, became ehrlichemic, and developed clinical signs of depression, anorexia, and mild diarrhea. These findings confirm that E. risticii is an abortifacient under conditions of natural infection and should be considered as a differential diagnosis of equine abortions.

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immunogens. III. Characterization of immunodominant peptides.

Immunoblot analysis utilizing bovine sera from naturally or experimentally infected with Theileria sergenti were used to determine the immunodominant polypeptides of T. sergenti (Korean isolate). The previously recognized major bands, 18 kDa, 29 kDa, 34 kDa and 45 kDa, were excised after electrophoresis and transfer to PVDF membrane. The individual bands were sequenced. The 34 kDa polypeptide which was the most antigenic and immunogenic peptide was observed in the Western blot. However, Chou-Fasman prediction sites (antigenic site) for antigen determinants of the 45 kDa, 24 kDa, 29 kDa and 18 kDa polypeptide were 6, 4, 2 and 0, respectively. However, the 45 kDa polypeptide showed no reaction with anti-T, sergenti hyperimmune serum.

Cultural, molecular, and immunological characterization of the etiologic agent for atypical canine ehrlichiosis.

More than 100 cases of canine ehrlichiosis, with three fatalities, were serologically negative by the indirect immunofluorescent antibody (IFA) test with Ehrlichia canis or E. sennetsu antigen but were reactive at titers of 10 to 640 with E. risticii. Ehrlichia-like agents were isolated from three such cases. The agents isolated from those cases were morphologically indistinguishable from each other and from a prototype, E. risticii, the etiologic agent of equine monocytic ehrlichiosis, in terms of growth characteristics and by light or electron microscopy. The patterns of and products from PCR were identical to those of E. risticii. The 16S rRNA sequences were distinct from those of E. canis and E. ewingii but were identical to those of E. risticii. A PCR product corresponding to the 5' half of the 16S rRNA gene was obtained from amplification of DNA from E. risticii and both sources of the atypical canine ehrlichiosis agent but was not obtained from uninfected host cells. The entire sequence of 719 nucleotides was identical for all three sources. The percentages of relatedness of the partial 16S rRNA gene of the atypical canine ehrlichiosis agent to E. risticii, E. sennetsu, E. platys, E. equi, E. phagocytophila, E. canis, E. chaffeensis, and E. ewingii were 100.0, 98.9, 83.7, 83.0, 83.0, 82.2, 81.8, and 81.5, respectively. These data are consistent with the identity of these isolates as E. risticii. The caninotropic characteristics of naturally acquired infections due to E. risticii are herein described for the first time, and the epizootiological implications are discussed in relation to the host range of E. risticii, which may include dogs as reservoirs.

Leptospirosis serology in Korean wild animals.

A study was undertaken on wild animals to determine the seroprevalence of animal leptospirosis in Korea. Using the serum microagglutination test for 19 serogroups, it was shown that two of 26 rats (Rattus rattus) had antibodies to Leptospira canicola. When data for domestic animals were included, the most prevalent (nearly 50%) serogroup was Leptospira canicola.

Human serum for in vitro cultivation of Babesia bovis.

Various combinations of human serum (from blood of groups A and Rhesus positive) with bovine serum, i.e. 20% + 20% (Medium I), 30% + 10% (II), 40% + 0% (III) and 0% + 40% (IV) and Medium-199 (60%) were used in the propagation of Babesia bovis. Babesia bovis stabilate revived by inoculation in a bovine calf was used at a level of 6% parasitized erythrocytes (PPE). The medium was replenished every 24 h. The medium changed from bright red to dark-coffee color every 24 h. The increase in PPE was maximal between 24 and 48 h. It was also observed that the increase in PPE was significantly higher in a 1:2 dilution compared with a 1:1 dilution. The increase in PPE was highest in Medium I with a 50% replacement of bovine with human serum. However, the parasites could not be subcultured and maintained continuously. Fifty percent replacement was thus optimal for the human-bovine serum combination in a microaerophilous stationary phase (MASP) system. The results indicate that B. bovis can also multiply in a human/bovine serum MASP culture system at least for a period of 48 h, and this is consistent with the zoonotic nature of Babesia species.