Pseudomonas fluorescens contamination of a feline packed red blood cell unit and studies of canine units

Background: While screening programs have reduced the risk of infectious disease transmission by donors in human and veterinary blood banking, bacterial contamination of blood products has emerged as a major complication in human medicine. Objectives: To describe a Pseudomonas fluorescens (Pf)‐contaminated feline packed RBC (pRBC) unit and experimentally investigate Pf‐contaminated canine pRBCs. Methods: Canine pRBCs were inoculated with Pf‐rich pRBCs from the sentinel feline unit and stored at 4°C or 20°C for 72 hours. Aliquots from the pRBCs were serially evaluated by microscopy, culture, and a eubacterial 16S rRNA real‐time PCR assay. Results: One Pf‐contaminated feline unit turned black after 22 days of storage and was removed from the blood bank; a source was not found, and no other contaminated units were identified. Canine pRBCs spiked with 5 or 25 μL of the sentinel unit became culture‐ and/or 16S PCR‐positive at ≥8 hours at 20°C and 48 hours at 4°C and developed a color change at ≥24 hours. Sensitivity studies indicated that without incubation, inoculation of ≥100 μL Pf‐rich pRBCs was necessary for a positive 16S PCR test result. Conclusions: P. fluorescens grows in stored pRBCs slowly at 4°C and rapidly at 20°C. Screening of blood products for color change, estimating bacterial concentration with microscopy, and 16S PCR testing are simple and fast ways to detect bacteria in stored blood. Aseptic collection, temperature‐controlled storage, and regular visual monitoring of stored units is recommended. Discolored units should not be transfused, but examined for bacterial contamination or other blood product quality problems.     

Risk factors for fecal shedding of Salmonella from horses in a veterinary teaching hospital

Identification of risk factors for horses shedding Salmonella in their feces helps identify patients at-risk of infection and protect the overall population through heightened biosecurity. Fecal samples from 230 hospitalized horses were cultured for Salmonella spp. Historical data were collected on 21 putative risk factors and assessed for association with the risk of a horse being culture positive using forwards stepwise logistic regression. Salmonella was isolated from 13 horses—most commonly from either the first (n=5) or second (n=4) sample collected. Only presenting complaint (confounded by age, breed and gender) was significantly (P≤0.05) associated with positive Salmonella-culture results. Analysis of residuals showed that the model was robust, but individual risk-factor estimates were changed by removal of outliers. Overall, presenting complaint (for example, lower-respiratory-tract disease) was the most important indicator of culture status.     

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Detection of Salmonella organisms and assessment of a protocol for removal of contamination in horse stalls at a veterinary teaching hospital

OBJECTIVES: To assess methods of detecting environmental contamination with Salmonella organisms and evaluate a cleaning and disinfection protocol for horse stalls in a veterinary teaching hospital. DESIGN: Original study. SAMPLE POPULATION: 37 horses with diarrhea likely to be caused by Salmonella infection and their stall environments. PROCEDURES: Fecal samples were collected from horses daily during hospitalization; samples were obtained from stall sites after cleaning and application of disinfectants. Fecal and environmental samples were cultured for Salmonella spp and tested via polymerase chain reaction (PCR) assay to detect Salmonella DNA. RESULTS: 1 horse died and 2 were discharged prior to sample collection. Fecal samples from 9 of 34 horses yielded growth of Salmonella organisms on bacteriologic culture, and 23 yielded positive results via PCR assay on > or = 1 occasion. Among environmental samples from 21 stalls, salmonellae were detected at > or = 1 stall site on 6 of 78 occasions, and > or = 1 stall site yielded positive results via PCR assay on 69 of 77 occasions. Salmonella DNA was detected more frequently in samples of stall drains, cracks, and corners. Salmonella spp were cultured from samples of 3 stalls after both initial and second cleaning and disinfection cycles, but no organisms were detected in samples obtained after use of a peroxygen disinfectant. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that stalls in which horses with salmonellosis were housed should only be used to accommodate newly hospitalized horses after samples (collected after 2 cycles of cleaning and disinfection) from drains, cracks, and corners yield negative results on bacteriologic culture.     

四川省松潘县牦牛体表蜱、高原鼠兔携带巴尔通体和无形体的PCR检测与进化分析

旨在了解四川省松潘县牦牛体表蜱、高原鼠兔巴尔通体和无形体感染情况。采集牦牛体表的蜱和捕获高原鼠兔，对蜱进行形态学初步鉴定后，提取蜱和高原鼠兔脾总DNA，PCR扩增蜱16S rRNA、巴尔通体rpoB和无形体16S rRNA基因，对PCR产物阳性产物测序、比对及构建系统进化树，从而确定蜱种类及蜱和高原鼠兔感染巴尔通体和无形体的种类及感染率。结果显示：在松潘县进安乡、山巴乡、下八寨乡各采集到蜱102、97和131只，共计330只，经鉴定均为青海血蜱。蜱巴尔通体仅检出1种巴尔通体，与B.melophagi亲缘关系最近，进安乡、山巴乡和下八寨乡检出率分别为16.7%、8.2%和18.3%，其中下八寨乡检出率显著高于进安乡（P<0.05）；蜱源无形体进安乡、山巴乡和下八寨乡检出率分别为9.8%、12.4%和26.7%，下八寨乡检出率显著高于进安乡（P<0.01），检出的无形体均为1种，与牛无形体（A.bovis）亲缘关系最近；下八寨乡检出的鼠兔源巴尔通体与B.queenslandens亲缘关系最近，感染率为6.7%；进安乡、山巴乡和下八寨乡检出的鼠兔源巴尔通体与B.grahamii亲缘关系最近，感染率分别为8.7%、17.9%和13.3%，3个点检出率无显著差异；未定种Bartonella sp.（MN296294）和Bartonella sp.（MN296293）仅分别在进安乡和下巴乡检出；与蜱均检出无形体不同，高原鼠兔均未检出无形体。此外，蜱和高原鼠兔均未发现2种及2种以上病原混合感染。松潘县青海血蜱携带巴尔通体和无形体，高原鼠兔感染巴尔通体，且首次在高原鼠兔体内检测到疑似B.queenslandens的病原体，提示当地居民有感染这两类病原风险。     

Detection of bacteriuria and bacteremia in newborn calves by a catalase-based urine test

Background: Bacteremia occurs frequently in newborn calves. The predictive value of clinical signs is low, suggesting the use of calf‐side diagnostic tests. Objectives: To investigate testing of urine catalase activity (Uriscreen test) for bacteriuria and bacteremia detection. Animals: Five colostrum‐free calves and 3 colostrum‐fed control calves. Methods: Controlled experimental trial. Colostrum‐free calves were inoculated PO with Escherichia coli O78+. A clinical score was established to define the onset of the illness. Blood and urine (cystocentesis) samplings and cultures, and Uriscreen tests, were performed 4–6 times from inoculation to death. Three control calves received the same management as 3 inoculated calves, but with colostrum and without inoculation. Results: Bacteremia was demonstrated in all of the inoculated colostrum‐free calves and in none of the control calves. The E. coli O78+ strain, E. coli, and Klebsiella spp. were recovered from 4/5, 5/5, and 2/5 inoculated colostrum‐free calves, respectively. Urine cultures were negative for the 2 groups at the start of the experiment; 5/5 colostrum‐deprived inoculated calves were positive for E. coli thereafter whereas 3/3 controls remained negative. Concordance of Uriscreen tests with bacteremia and bacteriuria was 0.86 and 0.88, respectively. Kappa value of agreement between Uriscreen and bacteremia and bacteriuria was 0.73 and 0.76, respectively. Sensitivity of Uriscreen for bacteremia and bacteriuria was 80.0 and 86.6%, respectively, and specificity was 92.8 and 88.8%, respectively. Conclusions and Clinical Relevance: The results suggest that Uriscreen can be used for detection of bacteremia in neonatal calves in connection with a constant bacteriuria.     

Detection of Salmonella in Finishing Pigs on Farm and at Slaughter in Piedmont,Italy

Salmonella is one of the most common causes of human gastroenteritis often associated with pork consumption. The aims of this cross‐sectional study were to collect preliminary data on the presence of Salmonella enterica in pigs in Piedmont (Italy), through sampling on farm and at slaughter and to gather pilot data on serotypes and phagetypes present in the sampled area and distribution of anti‐microbial resistance among isolated strains. Salmonella was detected through culture and identified with Salmonella spp. and Salmonella Typhimurium PCR; positive samples were serotyped, phagetyped and tested for antibiotic susceptibility. Positive samples (from 9% of faeces up to 29% of tonsils) were found in 64% of the herds. Salmonella spp. was retrieved also from scalding water. Most of the isolates were Salmonella Derby, Salmonella Typhimurium and Salmonella 4,5,12:i:‐. The results of Salmonella Typhimurium specific PCR suggested that Salmonella 4,5,12:i:‐ might be unrecognized by serotyping. Anti‐microbial resistance was recorded in 75–100% of the isolates. Phagetyping allowed the identification of DT104B and DT46A strains. These results set the bases for further research studies that would aim to estimate the real herd prevalence in Piedmont and the diffusion of serotypes and anti‐microbial resistant strains within the same region.     

长春市市售猪肉鸡肉中金黄色葡萄菌和沙门氏菌分离及耐药性分析

对肉品中金黄色葡萄球菌及沙门氏菌进行耐药分析,以期为其风险评估提供依据。采集吉林省长春市市售猪肉、鸡肉样品,按照国标方法分离金黄色葡萄球菌、沙门氏菌,利用K-B法药敏试验检测金黄色葡萄球菌和沙门氏菌对头孢噻肟钠、左旋氧氟沙星、庆大霉素、四环素4种抗生素的耐药性。结果显示：共分离出金黄色葡萄球菌46株,检出率为46％;沙门氏菌48株,检出率为16．84％。金黄色葡萄球菌对4种抗生素的耐药率在0～56．52％之间,共产生6种耐药谱。沙门氏菌对4种抗生素的耐药率在16．67％～87．5％之间,共产生9种耐药谱。长春市肉品中金黄色葡萄球菌、沙门氏菌耐药现象普遍。