Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

Influence of acute exposure to a low dose of systemic insecticide fipronil on locomotor activity and emotional behavior in adult male mice

Fipronil (FPN) is a systemic insecticide that antagonizes the gamma-aminobutyric acid type A (GABA_A) receptors in insects. Recently, adverse effects of FPN on mammals have been reported, but most of those were caused by high doses of FPN and additives in the products. We investigated the effects of low-dose pure FPN on the emotional behavior of mice. Nine-week-old male mice conducted behavioral tests 24 hr after FPN administration by gavage at doses of 0.05 or 5 mg/kg based on the no-observed-effect level (NOEL), showed a significant increase in locomotor activity and dose-dependent responses on the time they spent in the central zone in the open field test. Pure FPN below the NOEL dose may affect the emotional behavior of mice.     

不同氨基酸对泰万菌素发酵影响的研究

通过单因素试验考察了泰万菌素发酵培养基中不同氨基酸种类对其发酵过程的影响,并利用正交试验对发酵培养基中3种主要氨基酸的用量配比进行了优化,结果表明发酵培养基加入0.2%亮氨酸、0.1%色氨酸、0.1%甘氨酸对泰万菌素发酵转化率影响显著,发酵摇瓶效价从14000μ/mL提高到18000μ/mL,效价提高了28.5%,完成了对泰万菌素提高发酵转化率和发酵单位的目的,并为大规模商业化生产泰万菌素奠定了良好的基础。     

我国和欧盟兽药质量监督抽检工作机制对比研究

为更好地完善我国兽药质量监督抽检计划,通过对欧盟药品抽检管理工作机制的研究,本文分析了欧盟和我国兽药监督抽检管理方式的不同,从抽检责任部门、抽检目的、抽检计划内容、抽检计划制定流程、抽检品种的遴选和检测参数的确定等6方面进行了对比分析,探讨我国兽药质量监督抽检工作的管理中可以借鉴的方面。     

A proposal to leverage high-quality veterinary diagnostic laboratory large data streams for animal health,public health,and One Health

Test data generated by ~60 accredited member laboratories of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) is of exceptional quality. These data are captured by 1 of 13 laboratory information management systems (LIMSs) developed specifically for veterinary diagnostic laboratories (VDLs). Beginning ~2000, the National Animal Health Laboratory Network (NAHLN) developed an electronic messaging system for LIMS to automatically send standardized data streams for 14 select agents to a national repository. This messaging enables the U.S. Department of Agriculture to track and respond to high-consequence animal disease outbreaks such as highly pathogenic avian influenza. Because of the lack of standardized data collection in the LIMSs used at VDLs, there is, to date, no means of summarizing VDL large data streams for multi-state and national animal health studies or for providing near-real-time tracking for hundreds of other important animal diseases in the United States that are detected routinely by VDLs. Further, VDLs are the only state and federal resources that can provide early detection and identification of endemic and emerging zoonotic diseases. Zoonotic diseases are estimated to be responsible for 2.5 billion cases of human illness and 2.7 million deaths worldwide every year. The economic and health impact of the SARS-CoV-2 pandemic is self-evident. We review here the history and progress of data management in VDLs and discuss ways of seizing unexplored opportunities to advance data leveraging to better serve animal health, public health, and One Health.     

鹅大肠埃希菌的分离鉴定及系统进化树分析

为对发病鹅感染致病性大肠埃希菌的临床治疗提供指导并防控致病性大肠埃希菌感染,本研究无菌采集腹泻鹅及病死鹅脾脏、肝脏等组织器官进行病原菌分离培养、染色观察、生化鉴定、致病性试验,对菌株部分基因进行测序、构建遗传进化树并进行药敏试验。结果显示,普通琼脂培养基上生长出灰白色、圆形、凸起、边缘整齐菌落,麦康凯琼脂培养基上长出玫红色、光滑圆润菌落;分离菌株经革兰氏染色,镜下可见革兰氏阴性菌,两端钝圆、大小中等,多呈单个存在的杆状菌;致病性试验表明,该分离菌株对小鼠、雏鹅均有较高致死率;遗传进化树结果显示,分离菌株与患者粪便分离株（GenBank登录号：CP024992.1）同源性最高,达99.5%;体外抑菌试验发现菌株耐药情况较严重,对大多数抗菌药均有较强抗性,仅头孢噻呋对该分离株有较强的抑菌作用,合理用药后病情得到有效控制。本研究为有效防治鹅大肠埃希菌病提供了理论依据,对规模化鹅场防控大肠埃希菌等其他细菌性疾病具有重要价值。     

牦牛源溶血致病性表皮葡萄球菌的分离鉴定及药敏分析

对1株分离自西藏那曲地区养殖场有出血性症状牦牛的致病性菌进行分子鉴定及药敏分析,为西藏地区牦牛出血性疾病提供治疗依据。通过对病死牦牛肺脏、肝脏进行细菌分离纯化获得疑似菌株,对所得疑似菌株进行形态学观察与哥伦比亚血平板试验筛选出疑似致病菌株,再对疑似致病菌株进行生理生化鉴定试验、16S rDNA通用引物检测及测序、同源性比对,后经药敏试验得到该疑似致病菌株的敏感药物,最后通过动物回归试验验证其敏感药物的治疗效果。结果表明,通过对病死牦牛肺脏、肝脏分离纯化获得6株疑似菌株,经形态学观察试验、哥伦比亚血平板试验筛选出1株具有溶血性的革兰氏阳性球菌S-4。经生理生化鉴定试验、16S rDNA测序、同源性比对,鉴定S-4菌株为表皮葡萄球菌;药敏试验结果显示,S-4菌株对恩诺沙星、新霉素、多黏菌素B、卡那霉素、环丙沙星敏感,对氟苯尼考、多西环素中介敏感,对链霉素、红霉素、四环素、青霉素、头孢氨苄耐受;动物回归试验显示,该菌株具有致病性,且恩诺沙星、新霉素、卡那霉素3种药物治疗效果良好,多黏菌素B、环丙沙星治疗效果差。本试验获得1株具有致病性的牦牛源表皮葡萄球菌,该致病菌在养殖过程中可使用恩诺沙星、新霉素、卡那霉素3种药物进行治疗。     

气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因,用纯化的重组蛋白建立其间接ELISA检测方法,a。利用大肠杆菌密码子的偏爱性优化CctA基因序列,克隆至原核表达载体pET-28a （+）,双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌,经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化,SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗,用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法,以复性后的重组CctA蛋白作为检测抗原,摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清,同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示,得到大小约853 bp的条带,与预期相符,且测序结果正确。SDS-PAGE结果表明,成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示,豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL,于4 ℃包被过夜;封闭液选择10%胎牛血清,37 ℃孵育2 h;二抗的稀释度为1：8 000;室温避光显色10 min后终止反应,测定D_{450 nm}值。当P/N>4.6时,间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白,建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法,有望成为气肿疽灭活疫苗免疫效果验证的替代方法。