Effect of IBV-H120 vaccination in broilers on colibacillosis susceptibility after infection with a virulent Massachusetts-type IBV strain

Vaccination against infectious bronchitis (IB) is aimed to protect against clinical IB. The question is, however, whether vaccinated birds are also protected against predisposure for colibacillosis after a subsequent IBV infection. We examined this research question in four experiments. One-day-old commercial broilers, housed in isolators, were vaccinated with IB vaccine strain H120 by coarse spray or ocularly. Twenty-eight days after vaccination, broilers were challenged with the virulent IBV strain M41. Five days later, broilers were inoculated with Escherichia coli strain 506. Body weight uniformity, severity of E. coli airsacculitis, and systemic E. coli infection at 7 days following E. coli inoculation were used as parameters for colibacillosis. IBV vaccination reduced both the number of broilers with E. coli airsacculitis as well as the severity of airsacculitis significantly after challenge with IBV-M41 and E. coli 506. However, in spray-vaccinated groups, no significant reduction of the number of birds with systemic colibacillosis or the severity of this infection was obtained, and body weight uniformity was not significantly improved compared with nonvaccinated, IBV-M41, and E. coli 506-challenged groups. Eye-drop vaccination resulted in conflicting results.     

Infectious bronchitis virus nucleoprotein specific CTL response is generated prior to serum IgG

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.     

Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. The aim of this study was to investigate how IBV affects the course of events upon infection with E. coli. Broilers were inoculated with IBV H120 vaccine virus or virulent M41 and challenged 5 days later with E. coli 506. A PBS and E. coli group without previous virus inoculation were included. Sections of trachea, lung and airsacs were stained for CD4, CD8, gammadelta-TCR, alphabeta1-TCR, and for macrophages (KUL-01) and both pathogens. Changes in the mucociliary barrier of trachea, lung and airsacs did not predispose for bacterial superinfection. The disease in the lungs of the E. coli group and both IBV/E. coli groups was similar. Lesions in the airsacs were more pronounced and of longer duration in the IBV/E. coli groups. The immunocytological changes differed substantially between the E. coli group and both IBV/E. coli groups. In trachea, lungs and airsacs the CD4+ and CD8+ populations were significantly larger than in the E. coli and PBS groups. In the lungs and the airsacs the macrophages were more numerous in the IBV/E. coli and the E. coli groups than in the PBS group. The presence of high numbers of T cells and macrophages in IBV infected birds most likely induced an altered immune response, which is responsible for the enhanced clinical signs of colibacillosis.     

The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4dpi, the number of KUL-01(+) PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1beta, IL-6, IL-8, IL-10, IL-18 and IFN-gamma mRNA was found 1dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.     

鸡传染性支气管炎强毒分离株遗传进化分析和免疫保护试验

从山东省发病鸡群中分离鉴定了一株鸡传染性支气管炎病毒（Infectious bronchitis virus,IBV）强毒株SDIB821/2012,对其进行S1基因序列测定分析和免疫保护试验。S1基因遗传进化分析结果显示,SDIB821/2012属于以QXIBV为代表的基因型,与同属一个基因型的IBV参考株氨基酸同源性为91.6%～98.5%,与疫苗株491同源性为77.6%,与H120和MA5同源性均为74.8%。免疫保护试验结果显示,根据试验鸡临床症状和发病死亡情况,弱毒活疫苗491对SDIB821/2012的保护率为90%,而H120和MA5对SDIB821/2012的保护率分别仅为40%和33%。攻毒后各免疫组喉头、泄殖腔棉拭样品以及气管、肺脏和肾脏组织均可检测到病毒,表明3种IB疫苗均不能对SDIB821/2012提供完全的免疫保护。     

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活苗(La Sota株+M41株+Re-9株)应用效果研究

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活苗(La Sota株+M41株+Re-9株)(三联苗(Re-9株))为国内首个采用基因重组H9亚型禽流感疫苗株研制的新支流三联灭活疫苗。为研究疫苗上市后的实际应用效果,使用7日龄AA肉鸡和260日龄产蛋期蛋鸡评价三联苗(Re-9株)有效性。结果显示:肉鸡免疫后14 d即可激发抗体,免疫后21 d,新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、H9亚型禽流感病毒(H9 AIV)的血凝抑制(HI)抗体分别达8.6log2、6.2log2、8.3log2,出栏时仍维持在较高水平;于免疫后14 d抽样进行NDV、H9 AIV攻毒,对照组均100%发病或排毒,免疫鸡均可获得100%保护;在蛋鸡上,免疫三联苗(Re-9株)后28 d,NDV、IBV、H9 AIV的HI抗体效价分别达12.0log2、7.8log2、12.2log2,免疫后6个月仍可以维持在较高水平。结果表明:三联苗(Re-9株)在实际应用中免疫效果较好,本研究将为鸡新城疫、传染性支气管炎、H9亚型禽流感的防控提供有力支撑。     

2006年至2010年山东省鸡传染性支气管炎病毒分子变异的研究

为研究山东省鸡传染性支气管炎病毒(IBV)的遗传变异规律,本研究2006年～2010年从山东省发病的商品鸡中分离鉴定了17株IBV,并对其S1基因、N基因和M基因分别进行RT-PCR扩增、测序及遗传进化分析.序列分析结果表明:与疫苗株H120相比,17个分离株S1蛋白的变异程度较大,存在广泛的基因突变和氨基酸替代,多数病毒株还存在氨基酸的插入;N蛋白无碱基的缺失和插入,仅存在核苷酸的突变和氨基酸的替代;M蛋白除病毒株CK/CH/SD09/005插入3个碱基外,其它16个分离株仅存在少数的碱基突变和氨基酸替代.S1基因、N基因和M基因的系统进化分析结果表明多数分离株的3个基因在进化上相对平行,与国内分离株LX4同属一个进化分支,同源性较高;分离株SDYT0605的3个基因与疫苗株H120同源性较高,可能是免疫压力下变异的疫苗株;分离株SDTA06111、SDWF0608和CK/CH/SD09/005的S1基因、N基因和M基因分属于不同的进化分支,可能发生了基因重组.本研究结果显示基因突变、插入和不同基因之间的重组是免疫压力下IBV变异的主要方式.     

Chicken embryonal vaccination with avian infectious bronchitis virus

A commercial infectious bronchitis virus (IBV) vaccine of the Massachusetts 41 strain was injected in embryonating chicken eggs on embryonation day (ED) 18. The IBV vaccine was pathogenic for embryos, and it was passaged in chicken kidney tissue culture to reduce the pathogenicity. At the 40th tissue culture passage (P40-IBV), the virus became apathogenic for the embryos. Maternal antibody-positive or -negative chicks hatching from eggs injected with P40-IBV developed antibody to IBV and were protected against challenge exposure at 4 weeks of age with virulent Massachusetts 41 IBV. Although P40-IBV protected chicks when administered on ED 18, this virus did not protect chicks well if given at hatch. When combined with the turkey herpesvirus (HVT), P40-IBV given on ED 18 did not interfere with the protection against challenge exposure with virulent Marek's disease virus, nor did the presence of HVT interfere with protection by P40-IBV. Thus, under laboratory conditions, IBV vaccine could be combined with HVT to form a bivalent embryonal vaccine.     

Study of infection with an Iranian field-isolated H9N2 avian influenza virus in vaccinated and unvaccinated Japanese quail

In the present study, we examined the mortality rate, egg production, and clinical signs of quail experimentally infected with a field isolate of A/Chicken/Iran/339/02 (H9N2) avian influenza virus obtained from an infected commercial layer farm with severe morbidity and mortality. A total of 120 quail at 14 days old were randomly divided into four groups of vaccinated (B and C) and unvaccinated (A and D) birds. Vaccination was done on days 20 and 32, and viral inoculation of birds in groups C and D was then carried out on day 43. For evaluation of viral transmission, at 24 hr postinoculation additional unvaccinated birds were placed in direct contact with challenged birds. All the birds were evaluated for clinical signs, egg production, antibody production, viral titration in lung homogenates, and viral transmission following inoculation. All unvaccinated-challenged birds were infected and showed clinical signs, whereas the infection rate along with clinical signs of vaccinated-challenged birds reached 30%-40%. Although vaccination induced high antibody titers, reduction in food and water consumption was evident in this vaccinated-challenged group compared with the unchallenged control group. These results could indicate that inactivated vaccine did not fully prevent the infection, although it was capable of protecting birds against clinical signs and significantly decreased viral titers in lungs after intranasal challenge.     

Evaluation of the antigen relatedness and efficacy of a single vaccination with different infectious bronchitis virus strains against a challenge with Malaysian variant and QX-like IBV strains

BackgroundThe predominant infectious bronchitis virus (IBV) strains detected in chickens in Malaysia are the Malaysian variant (MV) and QX-like, which are associated with respiratory distress, nephropathy, and high mortality. On the other hand, the antigenic relatedness and efficacy of IBV vaccines against these 2 field IBV strains are not well characterized.ObjectivesThis study aimed to determine the antigen relatedness and efficacy of different IB vaccine strains against a challenge with MV and QX-like strains.MethodsThe antigen relatedness and the ability of different IB vaccine strains in conferring protection against MV and QX-like were assessed based on the clinical signs, macroscopic lesions, and ciliary activity.ResultsThe MV strain IBS037A/2014 showed minor antigenic subtype differences with the vaccine virus Mass H120 and 4/91 strains but showed major antigenic subtype differences with the K2 strain. The Malaysian QX-like strain IBS130/2015 showed major antigenic subtype differences with the MV strain IBS037A/2014 and the vaccine strains except for K2. Chickens vaccinated once with Mass (H120) or with non-Mass (4/91 and K2) developed antibody responses with the highest antibody titer detected in the groups vaccinated with H120 and 4/91. The mean ciliary activities of the vaccinated chickens were between 56 to 59% and 48 to 52% in chickens challenged with IBS037A/2014 and IBS130/2015, respectively. The vaccinated and challenged birds showed mild to severe lesions in the lungs and kidneys.ConclusionsDespite the minor antigenic subtype differences, a single inoculation with Mass or non-Mass vaccines could not protect against the MV IBS037A/2014 and QX-like IBS130/2015.     

几种病毒与禽病原性大肠杆菌的人工联合感染

以2种剂量的低致病性禽流感病毒(lowly pathogenic avian influenza virus,LPAIV)，传染性支气管炎病毒（infectious bronchitis virus,IBV)疫苗株H120和H52，新城疫病毒（Newcastle disese viurs,NDV)Lasota株分别于气管内注射10日龄易感鸡，2d后，气管注射禽病原性大肠杆菌O37株（O78），连续观察5d，结果，除LPAIV单独感染组有6．25％的死亡率外，其余各病毒单独接种组均健活；大肠杆菌O37株单独接种组的死亡率为62．50％，较高剂量的LPAIV，IBV H120和H52，NDV Lasota株与大肠杆菌O37株有效强的协同致病作用，死亡率分别达到81.25%,100.00%,93.75%和87.50%，而较低剂量的上述病毒则无明显的协同作用，IBV，NDV疫苗株与大肠杆菌联合接种组的多数死亡鸡病程推迟。     

国产IBV疫苗株核衣壳蛋白基因的亲缘关系

利用自行设计的引物Cx和Cs，通过RT－PCR方法分别扩增出鸡传染性支气管炎病毒（IBV）D41株，H120GD株、H120SH株、H52GD株等4个国产疫苗株和标准强毒M41－E4株完整的N基因cDNA,然后将其分别克隆到pGEM T-Easy或pMD 18-T载体中并测序。测序结果表明，这5个IBV毒株可分2组，其中D41株，H120GD株和H120SH株为一组，它们的核苷酸和推导氨基酸序列同源性为99．7％－99．8％和99．0％－99．5％，而H52GD株与M41－E4株构成另一组，其核苷酸和推导氨基酸序列同源性为99．7％和99．5％；而2组之间的最大同源性仅为91．0％和93．2％。在系统发生进化树上，这2组分别位于不同的分支簇上。值得注意的是，国内的H52GD株与国外报道的H52株不在同一分支簇上，相反却与国内强毒M41－E4株以及国外报道的M41株在同一分支簇上。这一结果表明，国内的H52GD疫苗株与国外报道的H52疫苗株不同，它们在亲缘关系上更靠近M41－E4株和M41株。     

Evaluation of the hemagglutination-inhibition test for measuring the response of chickens to avian infectious bronchitis virus vaccination

An infectious bronchitis virus (IBV) hemagglutination-inhibition (HI) test was used to assay serum-antibody titers after IBV vaccination of IBV-susceptible specific-pathogen-free broilers and commercial layers. Three-week-old broilers were vaccinated via eye-drop with IBV strains that represent the antigenic spectrum of commercial vaccines--Holland, Massachusetts 41 (41 Ms), Connecticut 46, Florida 18288, or JMK strain--and revaccinated 3 weeks later with either the same or a heterologous strain. Weekly serum samples were tested by IBV HI with homologous and heterologous antigens. Vaccinates, except for those vaccinated with the Holland strain, were HI-positive with homologous but not heterologous antigens by 1 to 2 weeks postvaccination. Sixteen-week-old IBV-vaccinated commercial layers were revaccinated with IBV Holland 52 (H 52) strain and subsequently infected with Arkansas 99 (Ark 99) and SE 17 strains. In contrast to the limited HI cross-reactivity of serum from IBV-vaccinated broilers, there were extensive cross-reactions in HI tests with 41 Ms, H 52, Ark 99, and SE 17 antigens of revaccinated layers. These results demonstrate that the IBV HI test is more strain-specific than previous reports indicate, especially when the test samples are from early postvaccination.     

鸡传染支气管炎病毒H52、H120和M41株的PCR鉴别方法研究

根据GenBank发表的序列,对鸡传染性支气管炎病毒（IBV）H52、H120和M41株全基因组序列进行比较,设计并合成了两对特异性引物,其中一对引物能以M41株为模板,特异性扩增出1791bp的目的片断,从而特异鉴定IBVM41株;另一对引物能以H52株和H120株为模板,特异性扩增1700bp的目的片断,再用限制性内切酶NaeI对PCR产物进行酶切,H120株能够被切成1000bp和700bp两个片段,而H52株的PCR产物不存在该酶切位点,从而能区分H52株和H120株。本研究建立的PCR方法和技术具有快速、简单、特异性强等优点,可用于IBVH52、H120和M41株活疫苗的分子鉴别。     

鸡传染性支气管炎病毒SX01株的分离鉴定及M基因序列分析

本试验通过鸡胚矮小试验、血凝试验和RT-PCR等方法,从华北地区某鸡场发生疑似肾型传染性支气管炎的发病鸡群分离了1株鸡传染性支气管炎病毒,命名为SX01株.结果显示该分离株能引起鸡胚典型的临床症状;经1%胰酶处理后的尿囊液可凝集鸡红细胞,而未处理的则无血凝活性;利用RT-PCR对分离株M基因序列测定分析结果显示,SX01株的M基因核苷酸序列与GX-GL分离株同源性高达99.4%,与QX基因型毒株SDZB0808核苷酸同源性高达99.1%,与H120株同源性仅为90.0%,与BJ/00/02株M基因核苷酸同源性较低,为90.6%.遗传进化分析结果显示,该分离株与中国地方型分离株(GX-GL、SDZB0808、CK/CH/LJL/110302、DY07、IBVSX7)亲缘关系较近,位于同一进化分支;与H120代表的Mass型疫苗株的亲缘关系较远,是一株肾型传染性支气管炎病毒毒株.     

4株IBV中国地方分离株部分结构基因变异的研究

本研究通过RT-PCR分别获得了4个国内IBV分离株的S1、M和N基因，并进行了克隆及测序。序列分析结果表明：HaN2-95株的S1、M和N基因核苷酸序列均与IBV H120疫苗株的同源性最高；尽管HaN1-95株的S1基因与IBV H52疫苗株的亲缘关系最近，但是该毒株的M基因和N基因却与IBV Gray株的同源性最高；GX1-98株的S1和M基因均与IBV H52疫苗株的亲缘关系最近，但其N基因却与IBV Gray株和Ark99株有高度的同源性；GX2-98株的S1基因却与IBV Holte株的亲缘关系最近。上述结果提示国内有些IBV分离株的出现可能与疫苗株的使用有关。     

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.     

Determination of the antigenic relationships within the Massachusetts (M41) type of infectious bronchitis virus using the hemagglutination-inhibition test

The antigenic relationships, antigenic spectrum, and immunogenicity of seven IBV-Massachusetts-41 isolates were investigated using the hemagglutination-inhibition (HI) test. HI titers equal to 32 are considered suspicious, titers lower than 32 are considered negative, and titers higher than 32 are considered positive immune responses to infectious bronchitis virus (IBV). Some isolates of Massachusetts-41 ( M41 ) were capable of inducing large quantities of antibodies in chickens following inoculation and demonstrated a wider antigenic spectrum than others. Variations in antigenic spectrum observed within M41 isolates in this study are in agreement with previous reports. This variation is of importance in selecting a proper vaccine strain for a successful immunization program for IBV.