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Abstract

OBJECTIVE To culture Lactobacillus spp from veterinary probiotics and measure their in vitro oxalate-degrading capacity.

SAMPLE 2 commercial veterinary probiotics containing Lactobacillus spp.

PROCEDURES Lactobacillus spp were cultured anaerobically on selective deMan, Rogosa, Sharpe agar medium and subcultured for speciation by 16S rDNA gene sequencing. Isolates were inoculated into broth containing sodium oxalate (5 mg/L) and incubated anaerobically for 72 hours. An oxalate-degrading isolate of Lactobacillus acidophilus (American Type Culture Collection [ATCC] 53544) was the positive control sample; sterile broth containing a known quantity of sodium oxalate was the negative control sample. Oxalate concentrations were detected with ion chromatography. Oxalate degradation was assessed with Dunnett tests to detect differences in mean oxalate concentration for each isolate, compared with results for the negative control.

RESULTS Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei or Lactobacillus zeae (too closely related to differentiate) were isolated from probiotic 1, and L plantarum was isolated from probiotic 2. Sequencing of the 16S rDNA gene confirmed 100% homology to type species. Lactobacillus acidophilus (ATCC 53544) and L acidophilus from probiotic 1 significantly decreased oxalate concentrations by 85.3 and 161.9 mg/L, respectively. Lactobacillus plantarum from probiotics 1 and 2 significantly increased oxalate concentrations by 56.1 and 36.1 mg/L, respectively. Lactobacillus casei did not alter oxalate concentrations.

CONCLUSIONS AND CLINICAL RELEVANCE Lactobacillus acidophilus isolates significantly reduced oxalate concentrations. In vivo studies are needed to determine whether probiotics containing L acidophilus decrease urine oxalate concentrations and reduce risk of urolith recurrence in dogs with a history of calcium oxalate urolithiasis.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate whether the application of steam to a variety of surface types in a veterinary hospital would effectively reduce the number of bacteria.

Sample—5 surface types.

Procedures—Steam was applied as a surface treatment for disinfection to 18 test sites of 5 surface types in a veterinary hospital. A pretreatment sample was obtained by collection of a swab specimen from the left side of each defined test surface. Steam disinfection was performed on the right side of each test surface, and a posttreatment sample was then collected in the same manner from the treated (right) side of each test surface. Total bacteria for pretreatment and posttreatment samples were quantified by heterotrophic plate counts and for Staphylococcus aureus, Pseudomonas spp, and total coliforms by counts on selective media.

Results—Significant reductions were observed in heterotrophic plate counts after steam application to dog runs and dog kennel floors. A significant reduction in counts of Pseudomonas spp was observed after steam application to tub sinks. Bacterial counts were reduced, but not significantly, on most other test surfaces that had adequate pretreatment counts for quantification.

Conclusions and Clinical Relevance—Development of health-care–associated infections is of increasing concern in human and veterinary medicine. The application of steam significantly reduced bacterial numbers on a variety of surfaces within a veterinary facility. Steam disinfection may prove to be an alternative or adjunct to chemical disinfection within veterinary practices.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To assess the status of antimicrobial resistance (AMR), identify extraintestinal virulence factors (VFs) and phylogenetic origins, and analyze relationships among these traits in extraintestinal pathogenic Escherichia coli (ExPEC) isolates from companion animals.

Sample—104 E coli isolates obtained from urine or genital swab samples collected between 2003 and 2010 from 85 dogs and 19 cats with urogenital infections in Japan.

Procedures—Antimicrobial susceptibility of isolates was determined by use of the agar dilution method; a multiplex PCR assay was used for VF gene detection and phylogenetic group assessment. Genetic diversity was evaluated via randomly amplified polymorphic DNA analysis.

Results—Of the 104 isolates, 45 (43.3%) were resistant to > 2 antimicrobials. Phylogenetically, 64 (61.5%), 22 (21.2%), 13 (12.5%), and 5 (4.8%) isolates belonged to groups B2, D, B1, and A, respectively. Compared with other groups, group B2 isolates were less resistant to all tested antimicrobials and carried the pap, hly, and cnf genes with higher frequency and the aer gene with lower frequency. The aer gene was directly associated and the pap, sfa, hly, and cnf genes were inversely associated with AMR. Randomly amplified polymorphic DNA analysis revealed 3 major clusters, comprised mainly of group B1, B2, and D isolates; 2 subclusters of group B2 isolates had different VF and AMR status.

Conclusions and Clinical Relevance—Prevalences of multidrug resistance and human-like phylogenetic origins among ExPEC isolates from companion animals in Japan were high. It is suggested that VFs, phylogenetic origins, and genetic diversity are significantly associated with AMR in ExPEC.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To measure the relationship between gross lesions in swine carcasses observed at a processing plant and Salmonella contamination and to determine whether nonexpert assessments of lesion status would correspond with swine pathologists' judgments.

Animals—Carcasses of 202 conventionally raised and 156 antimicrobial-free pigs in a Midwestern US processing plant examined from December 2005 to January 2006.

Procedures—4 replicates were conducted. For each, freshly eviscerated carcasses were identified as having or lacking visceral adhesions by a nonexpert evaluator and digital carcass photographs were obtained. Swab specimens were obtained from carcasses before the final rinse stage of processing, and bacterial culture for Salmonella spp and Enterococcus spp was performed. Subsequently, carcass photographs were numerically scored for lesion severity by 3 veterinary pathologists. Results were used to test the ability of lesion detection to predict bacterial contamination of carcasses and the agreement between judgments of the inexperienced and experienced assessors.

Results—The probability of Salmonella contamination in carcasses with lesions identified at the abattoir was 90% higher than that in carcasses lacking lesions, after controlling for replicate identity and antimicrobial use. The receiver operating characteristic curve and Cohen κ indicated close agreement between lesion detection at the abattoir and by the 3 pathologists.

Conclusions and Clinical Relevance—Findings indicated the presence of lesions could be used to predict Salmonella contamination of swine carcasses and that a nonexpert processing-line assessment of lesions could be used to discriminate between healthy and chronically ill swine before their entry into the human food supply.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To compare results for 3 commercially available microbiological media plates with those for standard bacteriologic testing of bovine milk.

Sample—Milk samples from postpartum cows and cows with a high somatic cell count (SCC) or clinical mastitis (CM).

Procedures—Sample-ready Staphylococcus culture medium (SRSC) plates were used to detect Staphylococcus aureus in milk samples obtained from postpartum cows and cows with a high SCC or CM. Rapid coliform count (RCC) plates were used to detect coliforms in milk samples obtained from cows with CM. Aerobic count (AC) plates were used to detect streptococci in CM samples. Fresh mastitic milk samples were frozen and then thawed to evaluate the effects of freezing for the SRSC and RCC plates. The effects of dilution (1:10) of samples were determined. Agreement of results between the commercially available plates and standard bacteriologic testing was evaluated.

Results—The ability of SRSC plates to detect S aureus in milk samples was highest with diluted samples from postpartum cows and cows with a high SCC or CM. Sensitivity of the RCC plate for detection of coliforms was highest with diluted mastitic milk samples. The AC plates had a poor positive predictive value for detection of streptococci in mastitic milk samples. Freezing increased S aureus detection.

Conclusions and Clinical Relevance—Overall, the SRSC and RCC plates were accurate, were easy to use, and yielded results comparable to those of standard bacteriologic testing for the detection of S aureus and coliforms in bovine milk.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate whether an equine-derived canine H3N8 influenza A virus was capable of infecting and transmitting disease to ponies.

Animals—20 influenza virus-seronegative 12- to 24-month-old ponies.

Procedures—5 ponies were inoculated via aerosol exposure with 107 TCID50 of A/Canine/Wyoming/86033/07 virus (Ca/WY)/pony. A second group of 5 ponies (positive control group) was inoculated via aerosol exposure with a contemporary A/Eq/Colorado/10/07 virus (Eq/CO), and 4 sham-inoculated ponies served as a negative control group. To evaluate the potential for virus transmission, ponies (3/inoculation group) were introduced 2 days after aerosol exposure and housed with Ca/WY- and Eq/CO-inoculated ponies to serve as sentinel animals. Clinical signs, nasal virus shedding, and serologic responses to inoculation were monitored in all ponies for up to 21 days after viral inoculation. Growth and infection characteristics of viruses were examined by use of Madin-Darby canine kidney cells and primary equine and canine respiratory epithelial cells.

Results—Ponies inoculated with Ca/WY had mild changes in clinical appearance, compared with results for Eq/CO-inoculated ponies. Additionally, Ca/WY inoculation induced significantly lower numbers for copies of the matrix gene in nasal secretions and lower systemic antibody responses in ponies than did Eq/CO inoculation. The Ca/WY isolate was not transmitted to sentinel ponies.

Conclusions and Clinical Relevance—Inoculation of ponies with the canine H3N8 isolate resulted in mild clinical disease, minimal nasal virus shedding, and weak systemic antibody responses, compared with responses after inoculation with the equine H3N8 influenza isolate. These results suggested that Ca/WY has not maintained infectivity for ponies.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To isolate and characterize bacteriophages with strong in vitro lytic activity against various pathogenic Pseudomonas aeruginosa strains isolated from dogs with ocular infections.

Sample—26 genetically distinct P aeruginosa isolates.

ProceduresP aeruginosa strains were derived from dogs with naturally acquired ulcerative keratitis. From a large-scale screening for bacteriophages with potential therapeutic benefit against canine ocular infections, 2 bacteriophages (P2S2 and P5U5) were selected; host ranges were determined, and phage nucleic acid type and genetic profile were identified via enzymatic digestion. Electron microscopy was used to characterize bacteriophage ultrastructure. Bacteriophage temperature and pH stabilities were assessed by use of double-layer agar overlay titration. A cocultivation assay was used to evaluate the effect of the bacteriophages on bacterial host growth.

Results—P5U5 was active against all P aeruginosa isolates, whereas P2S2 formed lytic plaques on plates of 21 (80.8%) isolates. For each bacteriophage, the genomic nucleic acid was DNA; each was genetically distinct. Ultrastructurally, P2S2 and P5U5 appeared likely to belong to the Podoviridae and Siphoviridae families, respectively. The bacteriophages were stable within a pH range of 4 to 12; however, titers of both bacteriophages decreased following heating for 10 to 50 minutes at 45° or 60°C. Growth of each P aeruginosa isolate was significantly inhibited in coculture with P2S2 or P5U5; the dose response was related to the plaque-forming unit-to-CFU ratios.

Conclusions and Clinical Relevance—Bacteriophages P2S2 and P5U5 appear to be good candidates for phage treatment of infection caused by pathogenic P aeruginosa in dogs.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To assess the degree of biological similarity (on the basis of genotype determined via pulsed-field gel electrophoresis [PFGE]) between isolates of 2 Staphylococcus schleiferi subspecies (S schleiferi subsp coagulans and S schleiferi subsp schleiferi) in clinical samples obtained from dogs.

Sample Population—161 S schleiferi isolates from 160 canine patients.

Procedures—A commercial microbiology identification system was used to identify each isolate as S schleiferi. Isolates underwent slide and tube coagulase testing and antimicrobial susceptibility testing. A mecA PCR assay and a latex agglutination test for penicillin-binding protein 2a (PBP2a) were also performed on each isolate. Clonal clusters with a similarity cutoff value of 80% were identified via PFGE.

Results—Of the 161 isolates, 61 (38%), 79 (49%), and 21 (13%) were obtained from cutaneous sites, ears, and other sites, respectively; 110 (68%) were coagulase negative, and 51 (32%) were coagulase positive. Among the coagulase-negative and coagulase-positive isolates, 65% (71/110) and 39% (20/51) were oxacillin resistant, respectively. All oxacillin-resistant isolates yielded positive results via mecA PCR assay and PBP2a latex agglutination testing. Via PFGE, 15 major clusters and 108 individual pulsed-field profiles were identified. Oxacillin-resistant and oxacillin-susceptible isolates clustered separately. Clonal clusters were heterogeneous and contained representatives of both subspecies.

Conclusions and Clinical Relevance—Coagulase-positive and coagulase-negative isolates were not genotypically distinct and may represent a single S schleiferi sp with variable coagulase production, rather than 2 biologically distinct subspecies. Further studies are needed to characterize clinical or epidemiological differences associated with infections with coagulase-positive and coagulase-negative S schleiferi in dogs.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs.

Sample Population—Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri.

Procedures—The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28)

Results—A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs.

Conclusions and Clinical Relevance—The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs. [Am J Vet Res 2010;71:1195-1200)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To estimate prevalence and determine association between antimicrobia resistance and toxin gene profile of Clostridium difficile in commercial pigs at the preharvest food-safety level.

Animals—68 sows and 251 young pigs from 5 farms in North Carolina and 3 in Ohio.

Procedures—Fecal samples were collected from sows (8/farm) and matched young pigs (32/farm) at farrowing and again at the nursery and finishing stages. Clostridium difficile isolates were tested for susceptibility to 6 antimicrobials. A PCR assay was used to detect genes coding for enterotoxin A (tcdA), cytotoxin B (tcdB), and binary toxin (cdtB).

Results—C difficile prevalence in young pigs at farrowing was 73% (n = 183) with significantly higher prevalence in Ohio (87.5%) than in North Carolina (64%). Clostridium difficile was isolated from 32 (47%) sows with no significant difference between the 2 regions. A single pig had a positive test result at the nursery, and no isolate was recovered at the finishing farms. Resistance to ciprofloxacin was predominant in young pigs (91.3% of isolates) and sows (94%). The antimicrobial resistance profile ciprofloxacin-erythromycin-tetracycline was detected in 21.4% and 11.7% of isolates from young pigs and sows, respectively. Most isolates had positive results for tcdA (65%), tcdB (84%), and the binary toxin cdtB (77%) genes. Erythromycin resistance and tetracycline resistance were significantly associated with toxin gene profiles.

Conclusions and Clinical Relevance—The common occurrence of antimicrobial-resistant C difficile and the significant association of toxigenic strains with antimicrobial resistance could contribute to high morbidity in farms with farrowing pigs. (Am J Vet Res 2010;71:1189—1194)

Full access
in American Journal of Veterinary Research