某猪场3种重大疫病的抗体检测分析

为评估某猪场猪瘟、蓝耳病及伪狂犬病三大疫病的免疫状况及感染状态,及时发现潜在风险,为重大疫病的预警防控提供理论依据,应用中国兽医药品监察所猪瘟病毒抗体间接ELISA检测试剂盒、IDEXX与法国LSI蓝耳病抗体检测试剂盒以及SVANOVIR伪狂犬野毒抗体检测试剂盒,对采自某猪场各阶段猪只的50份血清进行了抗体检测。结果显示,该猪场猪瘟、蓝耳病、伪狂犬gE抗体总体阳性率分别为92.0%、38.0%(IDEXX)、90.0%(LSI)和42.0%;猪瘟抗体阳性率很高但整体抗体滴度较低且离散度较大,免疫效果一般;2种蓝耳病抗体检测试剂盒的检测结果在一定程度上具有一致性,指示生长育肥猪蓝耳病病毒的感染压力较大;种猪群伪狂犬gE抗体的阳性率偏高,说明该场伪狂犬野毒感染情况严重。通过对抗体的监测及其结果的科学分析,能够及时发现猪场存在的问题,以便针对性地采取防控措施,指导养猪生产。     

闽北地区猪伪狂犬病抗体检测分析

为了解猪伪狂犬病在闽北地区的流行情况,整理并分析2015年度血清抗体检测数据。采用ELISA检测方法对闽北地区45个规模化猪场进行检测分析,通过检测样品血清中伪狂犬病gE抗体和gB抗体的水平进行结果判定。结果显示：送检的45个规模化猪场,伪狂犬病gE抗体阳性场达25个,猪场阳性率达55.6%;共检测1304份血清,其中伪狂犬病gE抗体阳性213份、可疑24份、阴性1067份,抗体阳性率达20.6%。从伪狂犬病gE抗体阴性猪场抽检猪伪狂犬gB抗体380份,gB抗体阳性258份,猪群伪狂犬病抗体保护率仅有67.9%。     

某规模种猪场猪伪狂犬病的净化与防控

为了检查出北京市郊区某规模种猪场隐性伪狂犬病感染猪并加以淘汰,达到净化猪场的目的,应用伪狂犬病病毒(PRV)gE蛋白抗体ELISA检测试剂盒对不同阶段猪群血清进行抽检。结果表明:该种猪场不同阶段猪均存在PRV gE抗体阳性猪,采取普检种猪并淘汰PRV gE抗体阳性猪、调整免疫程序、抓好综合性防控措施、采用PRV gB蛋白抗体ELISA检测试剂盒检测免疫抗体等方法,使该种猪场猪的伪狂犬病得到了净化。说明用PRV gE蛋白抗体ELISA检测试剂盒对血清样本进行检测,发现并淘汰PRV gE抗体阳性猪、调整免疫程序可以净化猪伪狂犬病。     

泉州市洛江区规模化猪场伪狂犬野毒感染情况调查

为掌握泉州市洛江区规模化猪场伪狂犬野毒感染情况,采集15个规模化猪场745份血清样品,运用ELISA方法检测伪狂犬野毒感染抗体(gE抗体)。结果显示,洛江区规模化猪场伪狂犬野毒感染gE抗体阳性率为17.32%、场阳性率为80%,经产母猪、后备母猪、哺乳仔猪、保育仔猪和育肥猪gE抗体阳性率在6.67%~20.6%,各乡镇规模化猪场gE抗体阳性率在5%~22.29%,表明洛江区规模化猪场不同程度存在伪狂犬野毒感染,伪狂犬病防控和净化形势仍然严峻。     

2017年苏州市规模猪场猪伪狂犬病血清学调查

为了解我苏州地区猪伪狂犬病的野毒感染情况,对苏州市3个市(区)的18家自繁自养规模猪场共971份猪血清样品进行了猪伪狂犬病gB抗体和gE抗体的监测与分析,结果显示:971份样品中,伪狂犬gE抗体阳性497份,gE抗体阳性率为51.18%,其中生产母猪样品394份,伪狂犬gE抗体阳性率为63.2%;公猪样品130份,伪狂犬gE抗体阳性率为46.92%.结果显示,苏州地区中存在猪伪狂犬病痛毒感染,且某些猪场猪伪狂犬病病毒的感染率较高.     

规模化猪场猪伪狂犬病野毒感染的调查

为了解北疆部分规模化猪场伪狂犬病野毒感染情况,本试验于2016年6月-2017年7月共采集北疆5个不同规模的规模化猪场567份血清,采用gE-ELISA和全病毒ELISA方法进行伪狂犬野毒及免疫抗体的检测。结果表明,北疆地区猪伪狂犬病全病毒抗体总阳性率为99.32%(290/292),gE抗体总阳性率为52.74%(154/292);其中B、D、E三场全病毒抗体阳性率都达到了100%,C场阳性率最低,为98.15%(53/54),D场gE抗体阳性率最高为83.33%(20/24),B场gE抗体阳性率最低为35%(14/40);不同猪群调查结果显示,怀孕母猪gE抗体猪阳性率最高为68.18%(30/44),公猪较高为59.38%(19/32),保育猪阳性率最低为35.14%(26/74);A场在2016年6月-2017年7月3次检测中,gE抗体阳性率总体呈下降趋势,其中生产母猪和怀孕母猪下降明显,其他猪群阳性率波动较大。检测结果揭示,北疆地区伪狂犬野毒感染净化工作的压力依然很大,需要进行严格的检疫、免疫以及净化。     

云南省某规模化猪场不同日龄猪伪狂犬病病毒抗体的检测

为了评估云南省某猪场猪群的猪伪狂犬病毒野毒感染及免疫状况,试验随机采集该猪场的不同日龄猪的血清80份,采用猪伪狂犬病病毒gE(PRV-gE)抗体检测试剂盒和猪伪狂犬病病毒gB(PRV-gB)抗体检测试剂盒同时进行猪的狂犬病抗体检测,以掌握不同日龄猪伪狂犬病毒野毒的感染情况并制订适合该猪场的猪伪狂犬病免疫程序。结果表明:该场不同日龄猪PRV-gE抗体全部为阴性,无野毒感染; PRV-gB抗体阳性率均为100%,PRV-gB抗体水平相对较稳定。说明该猪场的免疫程序较为合理。     

规模猪场应用伪狂犬gE基因缺失疫苗免疫及淘汰措施净化猪伪狂犬病技术

正伪狂犬病(PR)病毒主要毒力基因为gE和gI,诱导产生保护性免疫基因为gB、gC、gD等,通过给猪接种伪狂犬gE基因缺失疫苗,检测PR-gB和PR-gE抗体,区分免疫动物和感染动物,淘汰感染动物,从而为伪狂犬病的净化提供可操作的技术手段。本课题通过在一个规模猪场应用伪狂犬gE     

Kappa检验比较2种猪伪狂犬病gE抗体ELISA检测试剂盒

采用2种猪伪狂犬病gE抗体ELISA检测试剂盒分别检测1100份猪血清,应用Kappa检验比较试验数据的一致性。结果显示:2种猪伪狂犬病抗体检测试剂盒都具有很好的重复性,检测定性结果一致性强度为极好,Kappa值=0.929(95%置信区间:0.899~0.959),其符合率达98.2%,说明2种猪伪狂犬病抗体检测试剂盒都适用于PRV gE抗体检测。     

比较两种ELISA试剂盒在猪伪狂犬病血清抗体检测中的一致性

比较两种ELISA试剂盒检测结果的一致性,为流行病学调查和临床诊断筛选适合的商品化试剂盒。采用来自两个厂家的野毒抗体(gE)和免疫抗体(gB)ELISA检测试剂盒分别检测362份和392份猪血清,应用Kappa检验比较试验数据的一致性。结果显示:两种猪伪狂犬病抗体(g E)检测试剂盒联合检测出95份抗体阳性和256份阴性,符合率97.24%,结果一致性为极强(Kappa值0.932);两种猪伪狂犬病抗体(gB)检测试剂盒联合检测出351份抗体阳性和9份阴性,符合率91.84%,一致性为弱(Kappa值0.347)。以上结果说明,两种猪伪狂犬病抗体(gE)检测试剂盒都适用于PRV gE抗体检测,而两种猪伪狂犬病抗体(gB)检测试剂金差异较大,需慎重选择。     

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.     

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.     

A serological survey of selected pathogens in wild boar in Slovenia

Serum samples collected from 178 shot wild boars (Sus scrofa) were tested for the presence of antibodies against classical swine fever virus, Aujeszky's disease virus (ADV), porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, swine influenza virus, porcine parvovirus (PPV), swine vesicular disease virus, Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella spp., Brucella spp. and Haemophilus parasuis (HPS) throughout Slovenia during the hunting season 2003/2004. The number of samples corresponds to 3% of the total hunting bag. By enzyme-linked immunosorbent assay (ELISA) antibodies against ADV were detected in 55 sera (31%), against PRCV in five sera (3%), PPV in 87 sera (49%), APP in 93 sera (52%), M. hyopneumoniae in 38 sera (21%), Salmonella spp. in 85 sera (47%) and HPS in 33 sera (18%).     

Intranasal vaccination of pigs against Aujeszky's disease: comparison with one or two doses of attenuated vaccines in pigs with high maternal antibody titres

Ten-week-old pigs with high levels of maternally derived antibody (MDA) against Aujeszky's disease virus (ADV) were given either a single intranasal vaccination or one or two doses (with an interval of three weeks) of commercially available attenuated ADV vaccines intramuscularly. The pigs did not produce a clear neutralising antibody response to ADV. However, pigs vaccinated intranasally and pigs given two doses of attenuated ADV vaccines were protected against intranasal challenge with virulent ADV two months after the first vaccination. Pigs given one parenteral dose of attenuated ADV vaccine were insufficiently protected. Protection was shown by shorter periods of growth arrest and fever and a greater reduction of virulent virus shedding after challenge in vaccinated pigs than in unvaccinated control pigs. Although intranasal vaccination conferred protection comparable to two parenteral doses of attenuated vaccines, it reduced shedding of virulent virus much more effectively. These results, together with those of other studies, show that intranasal vaccination confers better protection against Aujeszky's disease in pigs with MDA than parenteral vaccination. However, the efficacy of intranasal vaccination also decreases with increasing levels of MDA at the time of vaccination.     

Microbiological studies of wild rodents in farms as carriers of pig infectious agents

In 15 breeding and fattening pig herds, 85 mice (Mus musculus) and 40 rats (Rattus norvegicus) were captured and bacteria and viruses looked for. Bordetella bronchiseptica, Pasteurella sp., E. coli, Campylobacter jejuni and Treponema sp. were isolated from different samples. Rota-virus was also identified and neutralizing transmissible gastroenteritis antibodies were detected in the serum of one rat and mice from three different farms. Wild rats were also orally infected with Aujeszky's disease virus (ADV) and classical swine fever (CSF) virus. All the rats survived the ADV experimental infection and some of them showed ADV neutralizing antibodies in their sera. No multiplication of the SF virus was obtained.     

Studies on immunisation of pigs with the Bartha strain of Aujeszky's disease virus.

The K strain of Aujeszky's disease virus (ADV) grown in Vero cells was used to vaccinate pigs. Following intramuscular inoculation, the pigs remained healthy, no vaccine virus was excreted and virus could be detected only at the inoculation site. One inoculation gave good protection against challenge with a virulent strain of ADV, and the amount of virulent ADV excreted was geatly curtailed. Following vaccination only low leads of serum neutralizing antibody were detected (geometric mean titre 1/2), but three weeks after challenge very high levels were found (GMT 1/1773). Intranasal vaccination gave similar results. There was minimal excretion of vaccine virus. The clinical reaction on challenge was less severe than in the intramuscularly challenged group, although lower antibody levels were detected three wekks following challenge (GMT 1/483). A field trial, using this strain given subcutaneously, indicated that one inoculation of this vaccine is effective.     

Sero-epidemiological screening of pig sera collected at the slaughterhouse to detect herds infected with Aujeszky's disease virus, porcine influenza virus and Actinobacillus (Haemophilus) pleuropneumoniae in the framework of an integrated quality control (IQC) system

Over a period of six months, approximately 4700 blood samples were collected from 97 pig-finishing farms in the provinces of Noord-Brabant and Gelderland and screened for antibodies with respect to Aujeszky's disease virus (ADV), porcine influenza virus (PI) and Actinobacillus (Haemophilus) pleuropneumoniae (App). There were significant differences in the percentages of seropositive pigs between the two provinces, which may be related to the difference in the density of the pig population in the two provinces. In practice, it was possible to perform a reliable sera collecting procedure at the slaughterhouse. No farms remained seronegative with respect to most of the disease agents during the sampling period. There was a high degree of variation in the percentages of seropositive pigs per farm as to most of the disease agents. Evidence was found that animals that were seropositive with respect to ADV were significantly more susceptible to becoming seropositive with respect to App. serotype 2, and vice versa. The same connection was observed for PI serotype H1N1 and PI serotype H3N2. Furthermore, evidence was found that pigs seropositive with respect to PI serotype H1Ni only, or to PI serotype H1N1 and ADV or PI serotype H3N2 show a significant decrease in average daily weight gain compared to pigs that were seronegative.     

Electron microscopy of Aujeszky's disease virus in explants of Gasser's ganglion from pigs with a latent infection

Different developmental stages of the Aujeszky's disease virus were demonstrated by electron microscopy in the ultra-thin slices by the cultivated fragments of the Gasserian ganglion (G. g.) of two pigs latently infected with the Aujeszky's disease virus (ADV). In a pig vaccinated with the inactivated vaccine against the disease, the virus was detected in the G. g. cells 186 days after virus challenge, the reactivation of latency being obtained after immunosuppression with dexamethasone. In the non-vaccinated pig the virus was detected in G. g. cells after three months from experimental infection. In the ultra-thin slices the largest amount of virus was located in the nuclei and cytoplasm of satellite and Schwann's cells, in the connective-tissue cells and in the extracellular space. In the ganglion cells the virus was present in the cytoplasm and sporadically in the myelinized axons.     

Horizontal transmission of Aujeszky's disease virus from sheep to pigs

Eight 2-month-old merino lambs were inoculated intranasally with different (10(2.0)-10(5.0)TCID50) amounts of Aujeszky's disease virus (ADV). Electron microscopic studies indicated that ADV replicated in extra-neural sites, in the epithelial cells of the mucosa of the upper and lower respiratory tract. Although the virus was excreted continuously in nasal discharges, horizontal transmission to contact lambs failed. The surviving exposed and contact lambs had no demonstrable antibodies against ADV and they were susceptible when challenged by ADV. However, the virus was transmitted to susceptible pigs in contact with the exposed lambs. One of the five contact pigs showed characteristic clinical signs of Aujeszky's disease, developed a nonsuppurative meningoencephalomyelitis and ADV was recovered from the brain, nasal discharge and other organs. Restriction enzyme analysis of DNA from this virus confirmed the sheep origin of the isolate. The other 4 pigs seroconverted. ADV infection in sheep is therefore a possible source of infection for pigs, but the lack of horizontal transmission in sheep was confirmed.     

Induction of immune responses to glycoprotein gD of Aujeszky's disease virus with DNA immunization.

In an attempt to produce a DNA vaccine to prevent Aujeszky's disease, the induction of immune responses against Aujeszky's disease virus (ADV) gD was investigated in mice. The plasmid was constructed by placing ADV gD gene downstream of murine cytomegalovirus immediate early promoter of expression vector pMYK, which was injected twice on the skin of mice by using a gene-gun. All mice showed neutralizing antibodies against ADV gD at 4 weeks after immunization. The induction of cytotoxic T lymphocytes and splenic natural killer cells was also observed at 6 weeks post immunization. These results indicate that ADV gD gene in the form of DNA vaccine may induce specific as well as non-specific immune responses in vivo.