猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立

采用特异性好的鼠源抗猪流行性腹泻病毒(PEDV)单克隆抗体为捕获抗体,兔源多克隆抗体为检测抗体,建立PEDV双抗体夹心ELISA检测方法。结果显示,该方法的最佳反应条件为:抗PEDV单克隆抗体E1包被质量浓度4.40μg·mL~(-1),37℃包被2h,采用5%BSA封闭液封闭1h,兔抗PEDV抗体工作质量浓度为5.91μg·mL~(-1),酶标二抗稀释度为1∶2000,以OD450nm≥0.381作为阳性判定标准。该ELISA方法对猪轮状病毒和猪传染性胃肠炎病毒无交叉反应。敏感度可达30μg·mL~(-1)(5×103.12);重复性变异系数小于10%。采用该方法和RT-PCR方法同时检测临床样品42份,阳性样品符合率为92.30%,表明建立的PEDV双抗体夹心ELISA检测方法具有特异性好、敏感性高和方便快捷等优点,可用于PEDV快速检测。     

牛病毒性腹泻-粘膜病病毒地方株动物回归试验

【目的】自流产奶牛粪便及血液中分离得到的非细胞病变型牛病毒性腹泻-粘膜病病毒(BVDV),为了解分离毒株的特性,进行回归动物试验;【方法】将此分离毒回归2月龄健康犊牛,接毒25天后扑杀剖检,观察临床症状及病理变化。自犊牛体内采取脾、骨髓、肠淋巴结及血液接种MDBK细胞进行培养,分离病毒,进行细胞传代,将此细胞毒进行RT-PCR检测;【结果】犊牛表现为典型的急性病毒性腹泻症状和病变。自病料分离病毒经MDBK细胞盲传至第9代,均未出现细胞病变。将此细胞毒进行RT-PCR检测,扩增出相应的665bp的片段;【结论】将分离株接种易感犊牛以复制出BVD-MD病症,并从试验牛体内分离得到BVDV,从而进一步确证分离到的毒株属BVDV。     

Comparison of 2 methods for establishing rat model of chronic osmotic diarrhea

AIM: To compare 2 different rat models of chronic osmotic diarrhea with the lactose enrichment and magnesium citrate methods. METHODS: The SD rats (n=31) were randomly divided into lactose enrichment diarrhea group (Lac group, 12 rats), magnesium citrate diarrhea group (Mg group, 12 rats) and normal control group (Con group, 7 rats). The rats in Lac group were fed with a lactose enrichment diet and freely drank water. The rats in Mg group were fed with a standard diet and drank magnesium citrate solution. The Con group was fed with a standard diet and normal water. The general conditions and the characteristics of stool were observed every day. The grip strength and motor activity of the rats were measured after diarrhea for 14 d. The trans-epithelial resistance of the colonic mucosa was detected by Ussing chamber. The pathological changes of villus height and villus surface area in the intestine were observed with hemato-xylin-eosin staining. RESULTS: The rats in both diarrhea groups displayed the clinical manifestations of slow increase in body weight, spare and dull hair, lacking in strength and physical inactivity. The grip strength and motor activity of the rats in both diarrhea groups decreased significantly compared with Con group. The diarrhea rate and diarrhea index of the rats in the 2 diarrhea groups were both significantly higher than those in Con group (P<0.05). Moreover, the diarrhea index in Lac group was significantly increased compared with Mg group. The thickness of intestinal mucosa in Lac group was thinner than that in Mg group at the same time. However, the damage of intestinal villi in Mg group was more serious than that in Lac group.CONCLUSION: Both methods of lactose enrichment-induced diarrhea and magnesium citrate-induced diarrhea meet the requirement of chronic osmotic diarrhea, but there are different advantages between the diarrhea models. Therefore, we should select the correct diarrhea model to meet different aims of the experiments.     

女贞枸杞散预防断奶仔猪腹泻效果和对血液白细胞数的影响

为探讨以女贞子、枸杞子、穿心莲、大青叶等多味中药组成的"女贞枸杞散"对断奶仔猪腹泻的预防效果和对外周血各类白细胞数量的影响。根据体重相近、公母各半的原则选用80头体重为10kg左右的"杜长大"三元杂交断奶仔猪,随机分4组,分别为试验Ⅰ组(日粮中加0.4%"女贞枸杞散"),Ⅱ组(添加0.6%),Ⅲ组(添加0.8%)和对照组(普通日粮)。详细记录仔猪腹泻情况,计算腹泻率、腹泻频率、腹泻指数等。试验结束后采血,测定各类白细胞数量,结果表明,"女贞枸杞散"能有效地降低断奶仔猪的腹泻率,改善仔猪的生长性能,促进血液中的白细胞尤其是淋巴细胞的的生成,提高机体免疫力。     

低聚木糖和酸化剂对断奶仔猪生产性能的影响

本试验研究了低聚木糖及其与酸化剂合用对断奶仔猪生产性能的影响。选择72头35±1日龄、平均断奶体重(10.33±0.34)kg的健康三元(杜×长×大)杂交仔猪,按体重和性别分成4个处理组,每处理组3个重复。第1组为对照组,饲喂玉米-豆粕型基础日粮;第2组为低聚木糖(200mg/kg)组;第3组为酸化剂(8g/kg)组;第4组为合用组,添加低聚木糖(200mg/kg)和酸化剂(8g/kg)。研究结果表明,各试验组日增重、料重比得到显著改善(P<0.05),腹泻率显著下降(P<0.01)。     

牛病毒性腹泻病毒持续感染的免疫学研究进展

牛病毒性腹泻病毒(BVDV)是危害养牛业的重要病原之一。BVDV感染能够引起广泛的临床症状,包括牛病毒性腹泻-黏膜病、持续感染、免疫耐受、繁殖障碍、血小板减少与出血综合征等。由于其病原的复杂性,给该病的控制及疫苗研制带来了一定的困难。近年来,对其免疫学的研究取得了一定进展,特别是在病原的基本免疫学特征、体液免疫、细胞免疫、免疫耐受和免疫调节方面做了深入研究,同时也在参与免疫应答的细胞因子方面做了研究,这些都为牛病毒性腹泻-黏膜病的预防和控制研究提供了新的思路。     

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.     

Isolation and Identification of a Variant Porcine Epidemic Diarrhea Virus and the Immune Efficacy of Its Inactivated Vaccine

This study was aimed to isolate a mutant strain of porcine epidemic diarrhea virus and prepare a PEDV inactivated vaccine with high valence by suspension culture process for immunizing against PEDV effectively in China.200 small intestines and theirs contents of diarrhea piglets died of diarrhea,collected from many large-scale pig farms in China,were detected by RT-PCR and sequenced,a mutant strain of porcine epidemic diarrhea virus was selected and put on the suspension-cultured Vero cells in a 2 L reactor for virus isolation and continuous cell culture,the harvested virus suspension,which was identified and determined TCID₅₀,was inactivated by formaldehyde and mixed with aluminum hydroxide gel adjuvant to prepare PEDV inactivated vaccine.After its physical behavior,stability viscosity,sterility test were checked out,the safety and immune efficacy were studied by immunizing the pregnant pigs and theirs piglets.The results were as follows:86 samples were detected positive in 200 samples,cytopathy occurred after the mutant strain samples screened were passaged to 5th generation,the virus suspension was harvested in 10th generation and identified as a mutant strain of PEDV,named PEDV-GF10 strain.The virus titer of harvested virus suspension was measured up to 1×10^8.0 TCID₅₀/mL after concentrated.After the vaccine was checked out,the sows,40 and 25 days before delivery in experimental groups,were injected into Xuehai acupoint with 4 mL vaccine and the pigs in blank group were free of immunifications.The results showed that there were no obvious differences in the production status of the sows in experimental groups and blank group and the temperature of theirs 3-day-old healthy piglets injected different doses of vaccine,and the vaccine was safe to the sows and piglets.Forty 3-day-old piglets producted by pregnant sows in experimental groups and blank group were randomly selected and taken 4 mL 1×10^8.0TCID₅₀/mL F10 virus culture.The PEDV morbidity of piglets in blank group was 100% after injection and the antibodies were negative;10% piglets in blank group had mild diarrhea symptoms,the protection rate was up to 90%,antibody of passive immunity in piglets lasted for more than 35 days.Virus titer of mutant strain of PEDV-GF10 improved a lot by suspension cell culture,the PEDV-GF10 inactivated vaccine was safe,and could effectively prevent and control the variation strain of PEDV in China.     

牛病毒性腹泻病毒及其进化研究进展

牛病毒性腹泻病毒（Bovine viral diarrhoea virus，BVDV）在我国发现有20多年，而在世界范珏来讲已有近100年的历史。由于牛病毒性腹泻病毒造成奶牛生产性能下降、繁殖障碍、持续感染等，是导劐集约化奶牛场严重亏损的重要原因。且牛病毒性腹泻导致的黏膜病致死率几乎1000A，已经严重阻碍养斗业的发展，但目前我国对牛病毒性腹泻还没有好的防控措施。文章对牛病毒性腹泻的发生历史总结，便于石牛病毒性腹泻病毒的诊断和预防中参考。     

Acute cortisol responses of calves to scoop dehorning using local anaesthesia and/or cautery of the wound

Objective To measure plasma cortisol responses in calves dehorned using a scoop after administration of local anaesthesia and/or cautery of the wounds.
Design A physiological study with controls.
Procedure There were six treatments: control handling with and without local anaesthesia, dehorning, dehorning after local anaesthesia, dehorning followed by wound cautery, and dehorning after local anaesthesia followed by wound cautery. Blood samples were taken before and after dehorning.
Results Dehorning caused an increase in plasma cortisol concentrations, which decreased a little to plateau values and then declined to pretreatment values 3 to 4 h after dehorning. The peak was smaller after local anaesthesia was administered but when its effects wore off, cortisol concentrations increased and thereafter were similar to those in the dehorned animals. The combination of local anaesthesia and cautery resulted in a plasma cortisol response similar to those in control calves with or without local anaesthesia.
Conclusions If plasma cortisol concentrations reflect the distress being experienced by the calves, then local anaesthesia reduces the acute distress for about 3 h after dehorning but not during the subsequent 3 to 4 h. Combining local anaesthetic and cautery prevented the significant increase in plasma cortisol following dehorning and may eliminate the acute distress caused by scoop dehorning.