In vitro bactericidal efficacy of equine polymorphonuclear leukocytes against Corynebacterium equi

Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%) was greater than that for C equi (85% +/- 6%) (P less than 0.001), but the bactericidal efficacy was equivalent. More than 90% of the ingested or attached bacteria were destroyed during the 3-hour incubation period (mean percentage of C equi killed = 96 +/- 2%; mean percentage of S aureus killed = 91 +/- 8%). These results indicated that a failure of bacterial killing by neutrophils is unlikely to be important in the pathogenesis of C equi pneumonia in the horse.     

In vitro activity of tiamulin against porcine mycoplasmas

The activity of tiamulin against Mycoplasma hyopneumoniae, M flocculare, M hyorhinis and M hyosynoviae grown in liquid medium was assessed in vitro. With the first three of these mycoplasmas, the activity of tylosin and oxytetracycline was observed in parallel. Tiamulin was more active against M hyopneumoniae and M flocculare, but there was less disparity between the three antibiotics with the strain of M hyorhinis tested. Tiamulin was notably more active against M hyosynoviae than against M hyopneumoniae. It was more difficult to suppress M hyopneumoniae than the other mycoplasmas with tiamulin. This persistence of M hyopneumoniae was more striking when M hyopneumoniae and M hyosynoviae were tested in parallel.     

Determination of the activity of ponazuril against Sarcocystis neurona in cell cultures

The present study examined the efficacy of ponazuril in inhibiting merozoite production of Sarcocystis neurona in cell cultures. Ponazuril inhibited merozoite production by more that 90% in cultures of S. neurona treated with 1.0 microg/ml ponazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 5.0 microg/ml ponazuril. Ponazuril may have promise as a therapeutic agent in the treatment of S. neurona induced equine protozoal myeloencephalitis (EPM) in horses.     

In vitro activity of difloxacin against canine bacterial isolates.

The in vitro activity of difloxacin against canine bacterial isolates from clinical cases was studied in the United States and The Netherlands. Minimal inhibitory concentrations (MIC), the postantibiotic effect, the effect of pH on antimicrobial activity, and the bacterial killing rate tests were determined according to standard techniques. The MICs of American and Dutch isolates agreed in general. The MICs of the American gram-negative isolates ranged from 0.06 to 2.0 microg/ml, and the MICs of the Dutch gram-negative isolates ranged from 0.016 to 8.0 microg/ml. A few European strains of Proteus mirabilis and Klebsiella pneumoniae had relatively high MICs. Bordetella bronchiseptica also was less susceptible to difloxacin. The MICs of the American gram-positive cocci ranged from 0.125 to 4.0 microg/ml, and the MICs of Dutch isolates ranged from 0.125 to 2.0 microg/ ml. Difloxacin induced a concentration-dependent postantibiotic effect that lasted 0.2-3 hours in cultures with Escherichia coli, Staphylococcus intermedius, Streptococcus canis, Proteus spp., and Klebsiella pneumoniae. There was no postantibiotic effect observed against canine Pseudomonas aeruginosa. Decreasing the pH of the medium increased the MIC of Proteus mirabilis for difloxacin. The MICs of Escherichia coli and Klebsiella pneumoniae were lowest at neutral pH and were slightly increased in acid or alkaline media. At a neutral pH, most tested bacterial species were killed at a difloxacin concentration of 4 times the MIC. Similar results were obtained when these same bacteria were tested against enrofloxacin. A Klebsiella pneumoniae strain in an acidic environment was readily killed at difloxacin or enrofloxacin MIC, but at neutral pH the drug concentration had to be raised to 4 times the MIC for a bactericidal effect. After 24 hours of incubation at pH 7.1, difloxacin and enrofloxacin had similar bactericidal activity for all bacteria tested except Staphylococcus intermedius. Against S. intermedius, difloxacin was more bactericidal than enrofloxacin.     

In vitro antimicrobial activity of sulfonamides against some porcine pathogens

The minimal inhibitory concentrations (MIC) of sulfonamides were determined against Bordetella bronchiseptica (n = 10), Pasteurella multocida (n = 10), Haemophilus pleuropneumoniae (n = 20), and Streptococcus suis (n = 10) strains isolated from pigs with atrophic rhinitis, pneumonia, or meningitis. Sulfonamides tested in an agar dilution method were sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfadoxine, sulfisoxazole, sulfamerazine, sulfamethoxazole, sulfamethoxypyridazine, sulfanilamide, sulfatroxazole, and sulfisomidine. Results indicated that monotherapy of S suis infections with sulfonamides should not be encouraged because the MIC50 of all sulfonamides investigated was greater than 32 micrograms/ml. The MIC50 of the sulfonamides against B bronchiseptica ranged from 0.5 to 8 micrograms/ml, against P multocida from 2 to 32 micrograms/ml, and against H pleuropneumoniae from 8 to 64 micrograms/ml. The MIC50 of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamerazine, and sulfamethoxazole for the gram-negative bacteria did not exceed 16 micrograms/ml. Among these compounds, sulfamethoxazole had the highest activity. The frequently prescribed sulfamethazine had an overall low antimicrobial activity.     

马泰勒虫不同PCR检测方法的比较

为寻求一种快速、有效的马泰勒虫PCR检测方法，用Bec-UF2、Equi-R；EMA-1F、EMA-1R两对引物对56份马血液样本中马泰勒虫的核蛋白体基因和表面蛋白基因进行了常规PCR方法检测，用EMA-5、EMA-6；EMA-7、EMA-8两对引物对56份马血液样本中马泰勒虫的表面蛋白基因进行了PCR和套式PCR方法检测。结果显示，以Bec-UF2、Equi-R为引物的PCR方法的阳性检出率为57．1％（32／56），以EMA-5、EMA-6；EMA-7、EMA-8为引物的套式PCR方法的阳性检出率为51．8％（29／56），以EMA-1F、EMA-1R为引物的PCR方法的阳性检出率为17．9％（10／56）。结果表明，以Bec-UF2、Equi-R为引物的常规PCR方法为检测马泰勒虫的最佳方法。     

马泰勒虫病PCR检测方法的建立和应用

为寻求一种快速、有效的马泰勒虫 (Theileria equi,T.equi) PCR检测方法。基于马泰勒虫18S rRNA基因序列,在其V4高变区设计特异性引物Te-18F、Te-18R,通过PCR技术获得了531 bp的核酸片段。用该引物对马泰勒虫、马泰勒虫和驽巴贝斯虫混合样本、驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫基因组模板进行特异性试验。同时对马泰勒虫基因组模板进行不同浓度稀释后扩增,以便于确定试验的敏感性。用本试验建立的方法与常规显微镜镜检方法对45份马属动物血样进行检测。特异性试验显示,在被检测的9个样本中,只有马泰勒虫及马泰勒虫和驽巴贝斯虫混合模板中扩增出了符合大小的特异核酸片段。驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫的扩增结果均为阴性。灵敏度试验结果表明,PCR对马泰勒虫的扩增效率可达到10^-13。对本试验建立的PCR检测马泰勒虫方法评估结果显示,PCR对马泰勒虫的检出率为17.78%(8/45),显微镜镜检结果只有8.89%(4/45)两者的符合率为100%。本试验建立的马泰勒虫PCR检测方法不失,为一种好的检测方法。     

马泰勒虫感染新西兰兔的病理组织学观察

旨在研究马泰勒虫感染家兔后器官组织病理变化。以家兔为动物模型,去脾后人工感染马泰勒虫、在其体内进行扩繁,PCR检测是否成功感染,每日进行血液涂片和血常规检查,记录感染率,同时用1只健康兔作为对照;达感染高峰时,试验兔和对照兔进行剖检、制作组织切片,观察其主要器官病理变化。结果显示,感染兔心肌发生颗粒变性及水泡变性,心肌细胞间质增宽;肝脏小叶间静脉淤血,肝细胞呈现水泡变性,甚至气球样变;肺脏呈现典型间质性肺炎的病理变化;肾脏主要表现为急性肾小球肾炎;健康兔的各器官未见明显异常。血常规检测结果显示,感染兔有炎性血象、贫血和轻度脱水;感染兔临死前血液涂片检查发现红细胞中含大量马泰勒虫裂殖子,而健康兔未见异常。PCR检测得到512bp的目的条带,与预期目的片段相符。综上表明,感染兔心脏、肝脏、肺脏、肾脏均有明显的病理变化,与健康兔对比差异明显。     

3个马群感染马泰勒虫的分子病原学调查

试验采用显微镜观察、PCR和竞争性酶联免疫吸附试验(competitive enzyme-linked immunosorbentassay,cELISA)等方法对贵州矮马、西南马和伊犁马的马泰勒虫病的感染状况进行研究。结果显示,从32份新鲜的血液涂片中,观察到形态完整的马泰勒虫(Theileria equi)虫体,检出率12.5%。从贵州矮马、西南马及伊犁马3个马群血液总DNA中都检测到马泰勒虫的18S rRNA基因片段,与已知序列的同源性为97%~100%;相比之下,贵州矮马与伊犁马的阳性率相近,分别为76.62%和73.81%,西南马较低,仅为33.33%。另外,经cELISA检测,与伊犁马(31.71%)相比,贵州矮马和西南马血液中抗马泰勒虫抗体的阳性率较低,分别为24.68%和12.12%,并与两个马群的血液理化指标存在一定的联系:与中性细胞数量、γ-谷氨酰转移酶和肌酸激酶的含量呈弱正相关;与红细胞、白细胞、血小板、淋巴细胞数量及血红蛋白含量呈弱负相关。这些研究结果提示3个马群中均存在较高比例的马泰勒虫感染。     

Investigation of Molecular Etiology of Theileria equi Infection in Three Herds

To explore the prevalence of Theileria equi (T.equi) infection in horse in Guizhou province, the antibody level and 18S rRNA gene were detected from blood samples of Guizhou pony, Southwest horse and Yili horse using competitive ELISA and PCR methods.Giemsa-stained blood smear was prepared to observe T.equi in red blood cells.Intact protozoans of T.equi were observed in red blood cells of horses at Giemsa-stained slide smears with a detection rate of 12.5%.The 18S rRNA gene fragment of T.equi was detected in Guizhou pony, Southwest horse and Yili horse, and the consistent rates with the known nucleotide sequence were 97% to 100%.The PCR result indicated that the positive rates of T.equi in Guizhou pony (76.62%) and Yili horse (73.81%) were similar, which were higher than that in Southwest horse (33.33%).Furthermore, the antibody levels against T.equi in Guizhou pony (24.68%) and Southwest horse (12.12%) were lower than that in Yili horse (31.71%).A weak correlation between the antibody level and the blood physicochemical indexes was calculated from Guizhou pony and Southwest horse, including weak positive correlations with neutrophils numbers, gamma-glutamyl transferase and creatine kinase levels, and weak negative correlations with the numbers of red blood cell, white blood cell, platelet and lymphocyte and contents of hemoglobin.It suggested that a higher proportion of T.equi infection present in three herds.     

In vitro proliferative and cytotoxic responses of PBL from Theileria annulata-immune cattle

Peripheral blood lymphocytes were isolated from healthy calves and were subsequently infected with sporozoites of Theileria annulata in vitro. The infected cells were passaged for 50 times and thereafter inoculated into animals from which they were previously isolated. Within 4-5 days, schizont-containing cells were demonstrable in the lymph nodes of all animals. Few days later, merozoites were detected in erythrocytes. A slight decrease in the counts of lymphocytes and leucocytes was also found. After 2 months these animals and a group of uninfected calves were heavily infected by tick-infestation and showed severe symptoms of theileriosis with 60% schizont-containing cells in the lymph nodes and a parasitaemia of about 35%. Because of the severity of the infection, all control calves were treated with Halofuginone. In contrast, the initially immunized cattle (by inoculation of culture cells), survived the infection without chemotherapy. Less than 10% of their lymph node cells contained schizonts, whereas less than 1% of their erythrocytes were found to be infected with merozoites. In all immunized animals, specific cytotoxic PBL, with the capacity to lyse autologous but not allogeneic infected cells, were demonstrated. In addition, a population of PBL were found to be able to inhibit the growth of T.annulata-infected culture cells in vitro. However, in comparison to PBL of immune animals, PBL of acute infected calves were superior in their capacity to inhibit the proliferation of schizont-containing cells. In mixed lymphocyte reactions, T. annulata-infected cells could induce a more pronounced proliferative response in PBL from immune than in PBL of uninfected animals.     

Pulmonary disposition of tilmicosin in foals and in vitro activity against Rhodococcus equi and other common equine bacterial pathogens

The objectives of this study were to determine the serum and pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against Rhodococcus equi and other common bacterial pathogens of horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of body weight) was administered to seven healthy 5- to 8-week-old foals by the intramuscular route. Concentrations of tilmicosin were measured in serum, lung tissue, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils. Mean peak tilmicosin concentrations were significantly different between sampling sites with highest concentrations measured in blood neutrophils (66.01+/-15.97 microg/mL) followed by BAL cells (20.1+/-5.1 microg/mL), PELF (2.91+/-1.15 microg/mL), lung tissue (1.90+/-0.65 microg/mL), and serum (0.19+/-0.09 microg/mL). Harmonic mean terminal half-life in lung tissue (193.3 h) was significantly longer than that of PELF (73.3 h), bronchoalveolar cells (62.2 h), neutrophils (47.9 h), and serum (18.4 h). The MIC90 of 56 R. equi isolates was 32 microg/mL. Tilmicosin was active in vitro against most streptococci, Staphylococcus spp., Actinobacillus spp., and Pasteurella spp. The drug was not active against Enterococcus spp., Pseudomonas spp., and Enterobacteriaceae.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

In vitro antimycoplasmal activity of six Jordanian medicinal plants against three Mycoplasma species

The in vitro effect of six Jordanian traditional medicine plant methanolic extracts were tested against 32 isolates of Mycoplasma species; Mycoplasma mycoides subsp. mycoides LC (6), Mycoplasma capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions in Jordan. All Mycoplasma species showed susceptibility to Artemisia herba-alba and Artemisia arborescens with MIC ranges from 3.125–12.5 mg/ml. Allium sativum and Punica grantum showed limited activity against some Mycoplasma isolates. Olea europea and Citrullus colocynthis showed no in vitro activity against any of the Mycoplasma species tested. Artemisia herba-alba and Artemisia arborescens may therefore be useful for the treatment of mycoplasma infections. The authors are British Civil Servants and as such their work is subject to British Crown Copyright. This means the exclusive copyright for the article cannot be transferred.     

豫北地区临床分离鸡大肠杆菌的体外抑菌作用测定

为了有效地治疗鸡大肠杆菌病,采用微量倍比稀释方法测定了17种抗菌药物对临床分离的豫北地区15株鸡大肠杆菌的体外最小抑菌浓度（MIC）,并根据其MIC及MIC范围（MICRange）使用SPSS 13.0中Probit过程计算出17种抗菌药物的MIC50和MIC90。结果表明：多粘菌素B的抑菌作用最强,MIC50、MIC90分别为0.11、0.87μg/mL;加替沙星的抑菌作用次之,MIC50、MIC90分别为2.53、3.88μg/mL,其它3种药物恩诺沙星、左氧氟沙星、环丙沙星的抑菌作用相当,但不及加替沙星,MIC50、MIC90分别为10.11-11.79μg/mL、15.16-21.13μg/mL;多西环素和阿莫西林等12种抗菌药物的抑菌作用较小,MIC50、MIC90分别为18.53-388.50μg/mL和30.59-713.42μg/mL。