Determination of the antigenic relationships among six serotypes of infectious bronchitis virus using the enzyme-linked immunosorbent assay and serum-neutralization test

The indirect enzyme-linked immunosorbent assay (ELISA) was applied to detect antibody to infectious bronchitis (IB) virus in chickens. A cross-reactivity study was performed to determine the antigenic relationships among six strains: Massachusetts, Connecticut, Beaudette, and three Ontario field isolates. For comparison, the cross-reactivity study was performed in eggs using the serum-neutralization (SN) test for IB. The ELISA proved to be more broadly cross-reactive than the IB-SN test. This suggests that the ELISA for IB will be useful as a rapid diagnostic test for serum profiling in unvaccinated flocks, regardless of the IB serotype involved.     

A sensitive dot immunobinding assay for serodiagnosis of African swine fever virus with application in field conditions.

The present work describes a simple dot immunobinding assay (DIA) for African swine fever virus (ASFV) antibody detection that can be used under field conditions. The assay uses nitrocellulose strips dotted with a cytoplasmic soluble antigen (CS-P) of ASFV. The nitrocellulose strips are adhered to a plastic handle. The test serum samples react with the CS-P, and antibodies are detected using a protein A-peroxidase conjugate. Both incubations are carried out at 20 C. The efficacy of the DIA as a screening test for ASFV was compared to an enzyme-linked immunosorbent assay (ELISA) and an immunoblotting (IB) test using 343 sera collected from natural African swine fever epizootics and from inapparent ASFV carriers. The DIA had comparable sensitivity to both reference techniques, and all samples positive in the ELISA and IB test were also positive in the DIA. False-positive reactions were not detected when whole blood or poorly preserved serum samples were tested by DIA. Some poorly preserved sera that were positive initially by the ELISA were no longer ELISA positive in a later run, although they were positive in IB and DIA. These positive DIA and IB test results could be caused by the differences in antibody epitope binding.     

A solid-phase fluorescent immunoassay for detecting canine or mink enteritis parvoviruses in faecal samples

Mink enteritis virus (MEV) and canine parvovirus (CPV) were detected in faecal samples from experimentally or naturally infected minks and dogs, respectively, using antibody-coated polyacrylamide beads (immunobeads, IB) as the solid phase for immunofluorescence (IF) tests. The specificity and sensitivity of the immunobead assay (IBA) were studied by comparing it with an enzyme-linked immunoassay (ELISA), a haemagglutination (HA) test and an IF test using tissue cultures. The IBA was as sensitive as ELISA, but more sensitive than the HA test and the IF test. Furthermore, the use of IB as the matrix for the immunological reactions allows FITC- or enzyme-conjugated antibodies to be used as indicators of the reactions and a simultaneous investigation of several pathogenic agents.     

Immunization of day-old chicks having maternally derived antibodies against infectious bronchitis: degree of protection as monitored by ciliary activity after intratracheal challenge.

One-day-old chicks with maternally derived antibodies were vaccinated against infectious bronchitis (IB) with 3000 EID50 of the IB vaccine virus designated H120. The degree of protection induced by intranasal-eye drop (IE) vaccination was compared to that achieved by spray (S) vaccination. The protection afforded by vaccination was monitored by intratracheal challenge with IBV strain M-41 (clinical signs, ciliary activity in tracheal explants, virus isolation) and by serological tests (ovoneutralization, microneutralization in cell culture, haemagglutination inhibition (HI) test, ELISA). Intranasal-eye drop vaccination provided protection against intratracheal challenge. Immunity developed around 31 days of age. Spray vaccination failed to give protection against challenge by the same route. No difference was demonstrable in effectiveness between the two routes of vaccination by serological tests. No elevation of the antibody level occurred in either group. The level of maternally derived antibodies declined with age.     

鸡传染性支气管炎M41毒株S1基因的克隆与真核表达研究

根据Genbank中公布的IBVS1基因序列 ,设计了一对引物 ,克隆出IBV强毒株M41的S1基因 ,基因大小为 1 .62kb ,导入真核表达载体系统pIRS1neo中 ,表达蛋白分子量为 61ku ,并具有中和活性。为今后开发鸡传染性支气管炎基因工程疫苗奠定了基础。     

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.Abbreviations AGID agar gel immunodiffusion - bp base pair - ELISA enzyme-linked immunosorbent assay - FBS fetal bovine serum - IB immunoblot - kD kilodalton - OLV ovine lentivirus - MEM minimal essential medium - p.i. post-infection - r recombinant - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TM transmembrane - WV whole virus     

Application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens

Objective A real-time polymerase chain reaction (PCR)/high-resolution melt (HRM) curve analysis protocol was developed in our laboratory to differentiate infectious bronchitis (IB) virus reference strains. In the current study, this method was used to detect and classify IB viruses in field submissions. Procedure Over an 11-month period samples from 40 cases of suspected IB virus were received and 17 submissions were positive for IB virus by polymerase chain reaction. HRM curve analysis classified each strain as subgroup 1, 2 or 3 strain (12 submissions) or a strain that was unable to be classified (5 submissions). The 3′ untranslated region (UTR) and partial S1 gene nucleotide sequences for the 17 IB virus strains were determined and their identity with those of the relative reference strains compared to confirm the classifications generated using the HRM curve analysis. Results Of the 12 IB field viruses classified as subgroup 1, 2, or 3 using HRM curve analysis, the 3′UTR and S1 gene nucleotide sequences had identities ≥99% with the respective subgroup reference strain. Analysis of the 3′ UTR and S1 gene nucleotide sequences for the five IB virus strains that could not be classified indicated that four belonged to one of the subgroups, and one was a potential recombinant strain (between strains from subgroups 2 and 3). A novel recombinant strain was also detected. Conclusion HRM curve analysis can rapidly assign the majority of IB viruses present in field submissions to known subgroups. Importantly, HRM curve analysis also identified variant genotypes that require further investigation.     

肾型鸡传染性支气管炎病毒新疆株的生物学特性研究

通过致病力试验和免疫原性检测对肾型鸡传染性支气管炎病毒新疆株(IBVXJ-2株)进行生物学特性的研究。致病力检测结果证实,IBVXJ-2株可以致SPF鸡胚卷缩、僵化,EID50达10-8.3/0.1 mL;鸡胚肾细胞在接毒后96 h出现皱缩、脱落等明显的细胞病变;对20日龄非免疫鸡攻毒后第2天,试验鸡出现甩鼻、伸颈张口呼吸等症状。将IBVXJ-2株抗原经乳化后接种1月龄非免疫鸡,在接种后22 d和44 d用ELISA和HI检测到了IBV特异性抗体。     

鸡传染性支气管炎实验室诊断方法研究进展

传染性支气管炎是鸡的一种重大传染病,临床上无典型的病变特征,诊断较为困难。现有的实验室检测方法有血清学、病原学和分子生物学方法,但该病病原变异复杂,血清型众多,交叉免疫性低,临床上又多采用活毒进行免疫,以至实验室无法对疫苗毒和野毒进行准确区分。为了更好地控制本病,有必要根据临床特点对目前各种检测鸡传染性支气管炎的方法及其优缺点进行分析,为临床寻找一种适合的快速准确的诊断方法、制定合理的免疫程序和发展新型的诊断方法提供参考。     

Protective immunity to infectious bronchitis in broilers vaccinated against Marek's disease either in ovo or at hatch and against infectious bronchitis at hatch

Two experiments were conducted using commercial broiler chickens to determine if Marek's disease (MD) vaccines HVT/SB-1 and HVT plus CVI-988 given either in ovo or at hatch adversely affected the efficacy of infectious bronchitis (IB) vaccines (Ark and Mass serotypes) given by eyedrop on the day of hatch. Nonvaccinated negative controls and controls that received only IB vaccines were included in each study. Birds were challenged with either infectious bronchitis virus (IBV) Mass-41 or IBV Ark-99 on either day 26 or 27 of age. Protection was assessed 5 days post-IBV challenged by virus isolation from the trachea. The day of hatch mean antibody titer to IBV was 12,668 +/- 4704 and 2503 +/- 3243 by enzyme-linked immunosorbent assay in experiments 1 and 2, respectively. In each study, nonvaccinated controls had a significantly higher (P < or = 0.05) incidence (88%-100%) of IBV challenge virus isolation than did controls vaccinated for IB but not for MD. Analysis of data from both studies showed that protection to IB in groups that received only IB vaccines at hatch ranged from 55.0% to 77.3%, whereas protection to IB in groups receiving both MD and IB vaccines ranged from 50.0% to 95.5%. In both experiments and within IBV challenge serotype, broilers given MD vaccines (in ovo or at hatch) and IB vaccines at hatch had protection rates to IBV challenges that were not significantly less (P < or = 0.05) than IB protection rates of groups that received only IB vaccines at hatch. Analysis of these data shows that administration of high-titered MD vaccines either in ovo or at hatch did not affect the efficacy of an IB vaccination (serotypes Ark and Mass) given by eyedrop at hatch.     

Detection of antibodies to infectious bronchitis of fowl using the ELISA, hemagglutination-inhibition test and agar-gel precipitation test]

Chicks of a conventional poultry flock, Shaver Starcross 288 hybrid, were vaccinated with infectious bronchitis (IB) virus H 120 at the age of 21 days. Three weeks later, the chicks were divided into three groups and separate groups were infected with infectious bronchitis viruses M 41 and D 274 or revaccinated with virus H 120. The content of specific antibodies to antigens prepared from homologous and heterologous viruses of infectious bronchitis used for chick vaccination and infection was investigated at regular intervals in the separate groups of chicks by means of an ELISA technique and haemagglutination-inhibition test (HIT). Serotype specificity of haemagglutination-inhibition test was documented by the results; the specificity was obvious mainly after the first vaccination and two weeks after infection, or after chick revaccination (Fig. 1). The dynamics of postinfective or postvaccinal antibodies, recorded by the ELISA technique, had analogical patterns in the separate groups of chicks, and there were no larger differences in the values determined on the basis of different antigens during the investigation (Fig. 2). A total of 52 group samples of fowl serum was examined by the ELISA technique and agar-gel precipitin test (AGPT) in another part of this study. Ten serums of identical origin represented the separate groups. The result of this examination was evaluated from the percentage of samples with precipitin activity in the group, or from the average value of ELISA. Mutual comparison of the mentioned values indicated that the precipitin activity was limited by the positivity degree of ELISA reaction (Fig. 3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune responses of breeding chickens to trivalent oil emulsion vaccines: responses to infectious bronchitis

Three similar flocks of broiler breeder parent chickens that had been given live infections bronchitis (IB) vaccines during rearing were injected at 20 weeks of age with three different oil emulsion vaccines: a commercial monovalent Newcastle disease (ND) vaccine (flock A); an experimental bivalent vaccine containing ND and infectious bursal disease (IBD) components (flock B); and an experimental trivalent vaccine containing ND, IBD and IB components (flock C). One week after vaccination 40 hens from flock A and 40 from flock C were taken to the laboratory and their egg yields individually recorded. At 37 weeks of age they were challenged by aerosol exposure to virulent IB virus. The egg production dropped significantly in the hens from flock A but not in the hens from flock C. On the farm, flock C showed a higher mean IB virus antibody titre four weeks after vaccination but titres rose in all three flocks indicating the presence of active IB virus infection. No differences in egg yields were found between the three farm flocks.     

Letter to the editor

Extract

Sir, — In the course of research on infectious bronchitis (IB) in chickens in New Zealand, one strain of IB virus was seleted and attenuated as a local vaccine virus and produced in limited quantities at Massey University for trial purposes.     

Establishment of Indirect ELISA Method for Detecting Antibodies against Avain Infectious Bronchitis Virus

The purpose of this research was to establish a rapid detection serological method against avain infectious bronchitis virus (IBV).In this study,an indirect ELISA method was established using IBV as the detected antigen and a variety of testing conditions were optimized.The results showed that the optimal antigen coating concentration was 19.2 μg/mL and the optimal condition for coating was incubated at 37 ℃ for 1 h and then 4 ℃ overnight.The optimal dilutions of serum and enzyme labeled antibody were 1:800 and 1:7 000 incubated at 37 ℃ for 60 min,respectively.The optimal condition of chromogenic substrate was incubated at 37 ℃ for 10 min in the dark.The specificity,repeatability and sensitivity tests proved that the indirect ELISA did not cross-react with positive antiviral sera of other chicken diseases,had good repeatability and could detect IBV antibody when serum was diluted 1:12 800.We concluded that the established indirect ELISA was specific,repeatable and sensitive.     

4种中药多糖对免疫雏鸡黏膜免疫功能的影响

为了探讨4种多糖增强雏鸡黏膜免疫的作用和机理.将450羽罗曼公鸡随机分成9组,14日龄用新支二联弱毒苗点眼滴鼻,同时,8个试验组分别肌肉注射高、低剂量的黄芪多糖(APS)、板蓝根多糖(IRPS)、牛膝多糖(AIRPS)和山药多糖(CYPS)溶液,对照组注射生理盐水,连续注射3 d,1次/d.于免疫后第10 d、20 d、30 d、40 d、50 d和60 d每组随机抽取5羽迫杀,刮取空肠黏液,用ELISA方法测定SIgA的含量;采盲肠扁桃体和十二指肠.用免疫组织化学法测定盲肠扁桃体和肠绒毛SIgA细胞阳性面积.实验结果表明:4种多糖均能显著提高空肠黏液中SIgA的含量和盲肠扁桃体、小肠绒毛的SIgA阳性细胞数,低剂量的APS、IRPS的效果优于高剂量的ARPS、CYPS.提示4种多糖均可以有效地增强黏膜的屏障作用,从而提高机体抗感染能力.     

鸡传染性支气管炎病毒抗体间接ELISA检测方法的建立

为建立快速检测鸡传染性支气管炎病毒(IBV)的血清学方法,本试验以IBV为检测抗原,建立了一种检测IBV抗体的间接ELISA方法,并对各种检测条件进行了优化.研究结果显示,抗原的最佳包被浓度为19.2 μg/mL,最佳包被条件为37 ℃ 1 h后4 ℃过夜;血清的最佳稀释度为1:800,37 ℃孵育60 min;酶标二抗稀释度为1:7 000,37 ℃孵育60 min;底物显色为37 ℃避光作用10 min.经特异性、重复性、敏感性试验证明,该方法与鸡常见病原的阳性血清均无交叉反应,重复性较好及血清稀释至1:12 800时仍可检测到IBV抗体.结果表明,本试验所建立的间接ELISA方法具有良好的特异性、重复性和敏感性.     

鸡传染性支气管炎病毒致病机理的研究进展

鸡传染性支气管是由鸡传染性支气管炎病毒引起鸡的一种急性高度接触性呼吸道传染病,由于病毒血清型较多,易于发生变异而难以免疫预防,成为养鸡业发展的重大阻力。文章就该病毒的致病机理方面的研究情况做一综述,为防制鸡传染性支气管炎提供科学依据。     

Research Progress on Molecular Biological Characteristics and Attenuated Vaccine of Infectious Bronchitis Virus

Infectious bronchitis (IB) is an acute and susceptible infectious disease,which has been classified as B loemia and causes a grave threat to the poultry industry.Now,the primary prevention measures of IB are vaccine inoculation.With the diversity of infectious bronchitis virus (IBV) serotype and easy variation,and the weakness of cross protection,the prevention and control of IB were a major problem in poultry industry.In this ariticle,it summarized the molecular biological characteristics, and the development and immunization strategy of the attenuated vaccine of IB to provide scientific references for the research and application of IB attenuated vaccine.     

传染性支气管炎病毒的分子生物学特性及其弱毒疫苗的研究进展

传染性支气管炎是一种急性、高度接触性传染病,被世界动物卫生组织(OIE)列为B类疫病,对世界养禽业的发展造成了严重威胁.目前,传染性支气管炎的主要预防措施仍然是接种疫苗,但由于传染性支气管炎病毒具有血清型众多、变异速度快且交叉保护性弱等特点,使得传染性支气管炎的防制一直是养禽业面临的重大难题.文章主要对传染性支气管炎病毒的分子生物学特征及其弱毒疫苗的发展历程和免疫策略等方面进行了阐述,以期为新型传染性支气管炎弱毒疫苗的开发及应用提供科学参考.     

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.