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。     

Enzyme-linked immunosorbent assay for the detection of antibodies to infectious bronchitis virus

An enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis (IB) virus is described. The immune response of chickens following vaccination with IB virus was monitored using this test, and the titers were compared with those obtained by serum neutralization. The ELISA appears to be suitable for IB serology.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

Protection of laying hens against infectious bronchitis with inactivated emulsion vaccines

Commercially-reared laying chickens were challenged at 31 weeks of age with a virulent infectious bronchitis (IB) virus. They showed a sharp drop in egg production, despite having been vaccinated at four and eight weeks old with live attenuated IB vaccines to a recommended schedule. In contrast, similar birds that had been further immunised at point-of-lay with inactivated oil emulsion IB vaccine, or with a combined IB/Newcastle disease (ND) emulsion vaccine, showed no detectable fall in egg production after the same challenge. Unvaccinated susceptible specific pathogen-free birds challenged at the same time stopped laying almost completely. In the birds revaccinated with emulsion vaccine, measurement of haemagglutination inhibition antibody levels to IB showed their geometric mean titres to be raised from less than 5 log2 at the time of vaccination to over 10 log2 four weeks later. Their antibody levels did not rise further followining the IB challenge whereas in the birds that had not been revaccinated antibody rises to nearly 10 log2 were detected after the same challenge. For pullets vaccinated earlier with live IB vaccine, revaccination with inactivated IB or IB/ND oil emulsion vaccine at point-of-lay provides a safe and effective way of protecting their egg production against IB infection.     

Serological differentiation of avian infectious bronchitis field isolates using an enzyme immunoassay: presence of Dutch strains in West Germany

Six field virus isolates were identified as the etiologic agent of avian infectious bronchitis (IB) in West Germany. A serological comparison was carried out using the M-41 strain from the Massachusetts serotype and the Dutch variant strains D-207, D-1466, D-3128, and D-3896 in a cross indirect immunoperoxidase test. Three isolates demonstrated similarity to the M-41 strain; the remainder showed a closer antigenic relationship to the Dutch variant strains. These results correspond to differences observed in a hemagglutination-inhibition test. The enzyme immunoassay is proposed as another possible method for differentiating IB virus strains.     

Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.     

Some field observations on the antibody response to avian infectious bronchitis on Auckland layer farms

Layer flocks on four Auckland poultry farms were monitored monthly for Infectious Bronchitis (IB) antibody levels, using the haemagglutination inhibition test. The same birds were bled each month and antibody levels compared with egg production. The results showed that IB vaccination at 4(1/2) and 14(1/2) weeks using the live, attenuated, New Zealand A strain virus, protected layers from IB infection on a farm with good management techniques but vaccination on another commercial farm gave less then ideal protection due possibly to intercurrent disease. Also antibody levels in naturally infected layers responded more vigorously when exposed to the field strain, compared with the response in vaccinated birds.     

鸡传染性支气管炎病毒免疫机制和免疫预防研究进展

由鸡传染性支气管炎病毒（Infectious bronchitis virus,IBV）引起的鸡传染性支气管炎（Infectious bronchitis,IB）是高度传染的全球性鸡病之一,严重危害养鸡业。IBV众多的血清型及其基因组的不断变异,给IB的免疫防控带来很大的困难。IBV主要侵害鸡的呼吸系统、泌尿生殖系统和消化系统,病鸡出现呼吸困难、产蛋下降、肾炎和腺胃炎等症状和病变。IBV的特点是变异频繁,血清型复杂,所致疾病的临床表现差异很大。因此,IB已成为养禽业最难控制的疫病之一。鸡对IBV的免疫机制是国内外研究的热点之一。传统疫苗已不能完全保护免疫鸡群,开发IBV基因工程疫苗,从主要免疫原性蛋白的良好表达到免疫策略的不断完善,已成为未来预防IB的趋势。     

IgM responses in chicken serum to live and inactivated infectious bronchitis virus vaccines.

Intramuscular (i.m.) administration of infectious bronchitis virus (IBV) oil-emulsion vaccine (OEV) to IBV-primed or unprimed chickens resulted in the production of zero or minimal concentrations of IBV-specific IgM in the serum, as measured by enzyme-linked immunosorbent assay of gel chromatography fractions. Live-attenuated infectious bronchitis (IB) vaccine given i.m. or by eyedrop stimulated the production of IBV-specific IgM in similar amounts following inoculation by both routes. These levels were comparable to those found in earlier studies following intranasal inoculation with a virulent strain of IBV and confirm that the detection of IBV-specific IgM is a valuable aid to the diagnosis of recent infection. As expected, administration of live-attenuated IB vaccines i.m. or by eyedrop protected the respiratory tract against challenge with virulent virus 24 days later; however, OEV given i.m. did not.     

Infectious bronchitis agar-gel precipitin test--use of infected allantoic fluid as antigen

The use of infected allantoic fluid (AF) as precipitating infectious bronchitis (IB) antigen was investigated. The results show that unconcentrated AF harvested at the right time can be a very satisfactory precipitating IB antigen. With the majority of the virus strains used, unconcentrated AF, harvested 68 hours postinoculation (PI), showed more precipitating activity than that harvested at 20 or 120 hours PI. If further antigen concentration is required, dialysis with polyethyleneglycol MW 20,000, freeze-drying, and precipitation with polyethyleneglycol 6,000 are satisfactory methods of concentration.     

鸡产蛋下降综合症油乳剂灭活苗的免疫及攻毒保护试验

对应用当地分离病毒株研制的鸡产蛋下降综合症系列油乳剂灭活苗进行了免疫试验。结果表明,免疫后,鸡产蛋下降综合症（ＥＤＳ７６）油乳剂灭活苗、鸡新城疫（ＮＤ）－产蛋下降综合症二联油乳剂灭活苗及鸡新城疫－鸡传染性支气管炎（ＩＢ）－产蛋下降综合症三联油乳剂灭活苗免疫组的血清ＥＤＳ７６ＨＩ抗体迅速上升,维持４个月后仍在６．８－８（ｌｏｇ２）以上,并且能够抵抗强毒的攻击。证明所研制的ＥＤＳ７６油苗、ＮＤ－ＥＤＳ７６二联油苗、ＮＤ－ＩＢ－ＥＤＳ７６三联油苗对ＥＤＳ７６具有良好的免疫作用,能够抵抗ＥＤＳ７６强毒的攻击并产生高而持久的血清ＨＩ抗体。此外,对ＮＤ－ＥＤＳ７６二联苗、ＮＤ－ＩＢ－ＥＤＳ７６三联苗免疫组的血清ＮＤＨＩ抗体测定结果表明,上述二联苗、三联苗能产生与接种ＮＤ油苗一样良好的ＮＤ免疫效果,在免疫后１３０天时,其血清ＨＩ抗体仍高达９－１１（ｌｏｇ２）以上,与对照组有极显著的差异     

The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989

The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Bam HI fingerprints the commonest virus identified in our study was EHV1.IP (P is for prototype strain). There was a single notable exception in that the Bam HI fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.IB that was identified as a cause of abortion in Central Kentucky in 1970 to 1974. We present evidence that EHV1.IB caused abortion in California in 1964 and has remained unaltered in its Bam HI restriction pattern. No antigenic differences were found among 4 distantly related EHV1 isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHV1 isolates is further evidence for a recent origin for EHV1 and may help to explain the natural history of this virus in the horse in which it seems to be a cause of serious epidemics of abortion and perinatal mortality, and less commonly of encephalitis.     

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.     

Immunogenicity and efficacy of a bivalent vaccine against infectious bronchitis virus

Infectious bronchitis (IB) is a highly contagious viral disease and is responsible for considerable economic losses in the poultry industry, worldwide. To mitigate the IB-associated losses, multiple vaccines are being applied in the sector with variable successes and thus necessitating the development of a potent vaccine to protect against the IB in the poultry. In the present study, we investigated a bivalent live attenuated vaccine consisting of IB virus (IBV) strain H120 (GI-1 lineage) and D274 (GI-12 lineage) to evaluate its protection against heterologous variant of IBV (GI-23 lineage) in chicken. Protection efficacy was evaluated based on the serology, clinical signs, survival rates, tracheal and kidney histopathology and the viral shedding. Results demonstrated that administering live H120 and D274 (named here Classivar®) vaccine in one day-old and 14 days-old provided 100 % protection. We observed a significant increase in the mean antibody titers, reduced virus shedding, and ameliorated histopathology lesions compared to routinely used vaccination regimes. These results revealed that usage of different IBV vaccines combination can successfully ameliorate the clinical outcome and pathology in vaccinated chicks especially after booster vaccination regime using Classivar®. In conclusions, our data indicate that Classivar® vaccine is safe in chicks and may serve as an effective vaccine against the threat posed by commonly circulating IBV strains in the poultry industry.