Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.     

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.     

鸭疫里默氏杆菌基因组文库的构建及免疫原性基因初步筛选

以血清1型鸭疫里默氏杆菌(RA)CH-1株为材料提取基因组DNA,采用限制性内切酶Sau3A进行部分酶切消化,纯化回收1.0~6.5 kb片段,用T4DNA连接酶将回收片段与经BamH酶切并去磷酸化的ZAP Express载体进行连接,用噬菌体包装蛋白包装。经测定包装滴度为5.23×106pfu/mL,蓝白斑筛选重组率为96.8%,表明已成功构建血清1型RA CH-1株部分基因组文库。在此基础上以RA抗血清为抗体探针进行免疫筛选,获得3个阳性克隆,经多次重复筛选后再体内删除,得到含有插入片段的质粒并测序。生物信息学分析结果显示,该序列(GenBank登录号:DQ151838)含有1个编码369个氨基酸的完整开放阅读框架,是尚未见报道的RA基因序列,并且存在多个预测的抗原表位。     

柔嫩艾美耳球虫表面抗原SAG2基因的克隆与表达

根据生物信息学预测的基因序列设计引物,应用RT-PCR方法从柔嫩艾美耳球虫第二代裂殖子总RNA扩增获得了鸡柔嫩艾美耳球虫表面抗原2(surface antigen 2,SAG2)基因序列,将其与pGEM-T easy载体连接后转化E.coliDH5α,筛选阳性克隆,以带有限制酶切位点的特异性引物用PCR方法扩增不含SAG2 N端信号肽序列的ORF序列后克隆至表达载体pET-32 a(+),构建了重组表达质粒pET-32 a(+)-SAG2,并将其转化至E.coliBL21(DE3)。经IPTG诱导,获得了SAG2重组抗原在大肠杆菌的高效表达,重组蛋白的表达量约占菌体总蛋白的35%,融合蛋白的分子量约为47 ku。菌体经超声处理后进行SDS-PAGE分析表明,表达蛋白以包涵体的形式存在。     

不同佐剂和免疫途径对柔嫩艾美耳球虫SO7抗原免疫保护效果的影响

利用PGEX-6P-1融合表达系统将SO7基因在大肠杆菌中进行融合表达,对表达产物进行初步纯化和复性,制备免疫原。分别使用不同剂量的人参总皂甙和重组γ-干扰素以及弗氏完全佐剂作为免疫佐剂,于5日龄、12日龄和19日龄对雏鸡进行3次免疫,同时设立蛋白口服免疫组、蛋白皮下注射免疫组、卵囊口服免疫组、未免疫未攻毒组和未免疫攻毒组作对照,于26日龄用105个柔嫩艾美耳球虫卵囊进行攻毒,8d后扑杀,对各组的存活率、相对增重率、病变减少率、相对卵囊产量和抗球虫指数ACI等指标进行统计分析,比较免疫保护效果。与非免疫攻毒组以及不加佐剂的免疫攻毒组相比,佐剂使用后各组的增重、病变记分、相对卵囊产量、ACI等指标均有一定的改善,对柔嫩艾美耳球虫的人工感染可以提供部分保护。3种佐剂中,γ-干扰素和人参总皂甙能增强重组蛋白的免疫保护效果,且佐剂的剂量对免疫保护的效果有一定的影响,以5000Uγ-干扰素效果最佳,效果与E.tenella活卵囊免疫相当,弗氏完全佐剂的免疫增强效果不明显。单独使用重组蛋白皮下注射或口服免疫,虽有一定的保护作用,但不明显。一定剂量的γ-干扰素对SO7抗原的免疫保护效果起明显的增强作用,是一个具有很好临床应用前景的新型佐剂。     

Prevention of isinglass on the chronic atrophic gastritis in rats and its mechanism

AIM: To evaluate the effect of isinglass on chronic atrophic gastritis(CAG) in rats and its mechanism. METHODS: An animal model of CAG in accordance with the previous experience of combined administration of 60% ethanol, 20 mmol/L sodium deoxycholate and 0.1% ammonia water was established in SD rats. Isinglass was used as preventive therapy while we were establishing CAG rat model. Finally all the rats were executed and pathologic changes of the gastric mucosa were studied by gross appearance and microscopy and serum epidermal growth factor (EFG) and growth hormone(GH) contents were tested. RESULTS: In each isinglass prevention group, inflammation grade of gastric antrum was less than that in model group (P<0.01) while the mean ratio of the thickness of gastric mucosal gland and muscularis mucosa (L1/L2), the number of gastric glands in 1 mm lengths of mucosal layer in longitudinal sections were much better than those in model group (P<0.01).They were very close to normal control group (P>0.05). The expression of proliferating cell nuclear antigen (PCNA) in gastric mucosa and serum EFG level were higher than those in model group (P<0.01, P<0.05), but serum GH content showed no different between isinglass prevention group and model group. CONCLUSION: Isinglass preventes the gastric mucosal atrophy in the CAG model. Its mechanism may be related to the effects of decreasing the gastric mucosal damage， promoting the cell proliferation and increasing of internal EFG secretion.     

Antiproliferation effects of 188Re labeled monoclonal antibody on prostate cancer cells in vitro

AIM: To investigate the effect of 188Re labeled monoclonal antibody on prostatic specific membrane antigen 7E11C5.3,radioimmunotherapy for the treatment of human prostate cancer cell line LNCaP in vitro.METHODS: 188Re-7E11C5.3 was prepared by direct 2-mercaptoethanol reduction method.Labeling efficiency and radiochemical purity was measured by paper chromatography.Immunoreactive fraction was determined by linear extrapolation.Cytotoxicity to LNCaP cells was determined by MTT assay.RESULTS: The labeling yield of 188Re-7E11C5.3 was (93.16±2.18)%,the radiochemical purity was (95.62±0.48)%,and the immunoreactive fraction was (74.86±1.86)%.The inhibitory effect of 188Re-7E11C5.3 on cell proliferation of LNCaP cells was significantly higher than that of 188Re-mIgG or 188ReO-4.The 50% inhibitory doses (IC50) of 188Re-7E11C5.3,188Re-mIgG,and 188ReO-4 were (23.38±3.73)×107 Bq/L,(59.21±8.02)×107 Bq/L and (68.89±10.91)×107 Bq/L,respectively.CONCLUSION: 188Re-7E11C5.3 can effectively inhibit the growth of in vitro cultured prostate cancer cells and shows much potential for prostate cancer radioimmunotherapy.     

猪繁殖与呼吸综合征病毒感染对猪肺泡巨噬细胞外源性抗原加工递呈相关分子和共刺激分子的影响

采用定量竞争PCR技术对猪繁殖与呼吸综合征病毒（PRRSV）感染后不同时相猪肺泡巨噬细胞（PAM）中外源性抗原加工递呈相关分子SLA-DR、Ii链和SLA-DM分子及共刺激分子CD40、CD80、CD86的mRNA转录动态进行了定量检测。结果表明,PRRSV感染后PAM的SLA-DR mRNA转录水平在3、7和14 d下调,低于对照组;感染后3 d,Ii链、SLA-DM和CD40的mRNA转录水平极显著下调（P〈0.01）;CD80和CD86mRNA转录水平也在感染后3 d明显下调（P〈0.05）。用流式细胞术检测PAM表面的SLA-DR抗原的结果表明,在感染后3 d短暂上升,7 d之后一直低于对照组。由此说明,PRRSV感染早期对猪肺泡巨噬细胞的外源性抗原加工和递呈功能产生显著影响,导致其外源性抗原递呈功能下降,进而影响机体的体液免疫应答。     

Pesticide use and residues on Queensland wool

Objective To determine practices for control of louse infestation and blowfly strike in Queensland sheep flocks that are associated with organophosphorous and synthetic pyrethroid residues on wool.
Design Information on residues was obtained from a survey of Queensland wool clips. Information on pesticide use was obtained from a trace-back postal survey. The association between pesticide use and residues was assessed using generalised linear models, controlling for potential confounding by flock location.
Procedure Between 1995 and 1997 Queensland wool clips were randomly sampled. Samples were tested for the presence and amount (mg per kg of greasy wool) of organophosphorous and synthetic pyrethroid pesticides. A questionnaire seeking information on flock characteristics and pesticide use was sent to the manager of each flock from which a wool sample was tested.
Results The median amount of OP and SP residue was 0.8 and 0.25 mg/kg, respectively, and 91 and 95% of wool samples contained < 8 mg/kg of OP and SP residues, respectively. The frequency of OP pesticide use for louse control was significantly (P = 0.005) associated with mean OP residue amount, and the timing of SP use for louse control, in relation to shearing, was significantly (P < 0.001) associated with mean SP residue amount.
Conclusion Most Queensland wool clips have acceptable amounts of residues after the use of OP and SP pesticides, but wool growers can further reduce residues by effectively controlling louse infestation with pesticide applications early after shearing and the use of non-chemical methods of ectoparasite control.