Optimization of hydrophile-lipophile balance for improved efficacy of Newcastle disease and avian influenza oil-emulsion vaccines

Preparations of inactivated Newcastle disease (ND) and avian influenza (AI) oil-emulsion vaccines with surfactant hydrophile-lipophile-balance (HLB) values between 4.3 and 9.5 were evaluated for their efficacy in broiler-type white rock chickens. Chickens were vaccinated at 3-4 weeks of age and bled at 2-week intervals over 8 weeks. Post-vaccinal hemagglutination-inhibition (HI) geometric mean titers (reciprocals) ranged from 197 to 485 for ND vaccines and from 184 to 1040 for AI vaccines. Based on the HI response, an HLB value of 7.0 induced the greatest stimulation of antibody titers. Ten percent surfactant in the oil phase of the vaccines induced maximum titers at this HLB. The oil:aqueous ratios of the vaccines did not greatly influence the overall serologic response when the vaccines had an HLB of 7.0. These results indicate that manipulating surfactant HLB values of OE vaccine may maximize the HI response in broilers.     

Serological response in broiler chicks to different commercial Newcastle disease and infectious bronchitis vaccines.

Broiler chicks were administered vaccines against Newcastle disease and infectious bronchitis (both Arkansas and Massachusetts strains) at 2 weeks of age as either primary or secondary vaccinations. The vaccine was administered as a spray at 2 weeks of age to chicks that had received Newcastle disease vaccine alone, bronchitis vaccine alone, both vaccines in combination, or no vaccine at day 1 in the hatchery. The Newcastle disease hemagglutination-inhibition response was significantly lower in chicks receiving Newcastle disease vaccine as a secondary vaccine at 2 weeks than in those receiving the vaccine as a primary vaccination at that age. In contrast, the bronchitis hemagglutination-inhibition response was significantly higher in chicks receiving bronchitis vaccine as a secondary vaccination at 2 weeks than in those receiving the vaccine as a primary vaccination at that age.     

Protection induced by infectious laryngotracheitis virus vaccines alone and combined with Newcastle disease virus and/or infectious bronchitis virus vaccines

Two types of live attenuated vaccines have been used worldwide for the control of infectious laryngotracheitis virus (ILTV): 1) chicken embryo origin (CEO) vaccines; and 2) tissue culture origin vaccines (TCO). However, the disease persists in spite of extensive use of vaccination, particularly in areas of intense broiler production. Among the factors that may influence the efficiency of ILTV live attenuated vaccines is a possible interference of Newcastle Disease virus (NDV) and infectious bronchitis virus (IBV) vaccines with the protection induced by ILTV vaccines. The protection induced by CEO and TCO vaccines was evaluated when administered at 14 days of age alone or in combination with the B1 type strain of NDV (B1) and/or the Arkansas (ARK) and Massachusetts (MASS) serotypes of IBV vaccines. Two weeks after vaccination (28 days of age), the chickens were challenged with a virulent ILTV field strain (63140 isolate, group V genotype). Protection was evaluated at 5 and 7 days postchallenge by scoring clinical signs and quantifying the challenge virus load in the trachea using real-time PCR (qPCR). In addition, the viral load of the vaccine viruses (ILTV, NDV, and IBV) was quantified 3 and 5 days postvaccination also using qPCR. The results of this study indicate that the NDV (B1) and IBV (ARK) vaccines and a multivalent vaccine constituted by NDV (B1) and IBV (ARK and MASS) did not interfere with the protection induced by the CEO ILTV vaccine. However, the NDV (BI) and the multivalent (B1/MASS/ARK) vaccines interfered with the protection induced by the TCO vaccine (P < 0.05). Either in combination or by themselves, the NDV and IBV vaccines decreased the tracheal replication of the TCO vaccine and the protection induced by this vaccine, since the ILTV-vaccinated and -challenged chickens displayed significantly more severe clinical signs and ILTV load (P < 0.05) than chickens vaccinated with the TCO vaccine alone. Although NDV and IBV challenges were not performed, the antibody responses elicited by NDV and/or the IBV vaccinations were significantly reduced (P < 0.05) when applied in combination with the CEO vaccine.     

The efficacy of an avian metapneumovirus vaccine applied simultaneously with infectious bronchitis and Newcastle disease virus vaccines to specific-pathogen-free chickens

Avian metapneumovirus (aMPV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) are important respiratory pathogens of chickens. To achieve early posthatch protection against all three diseases it would be helpful to deliver live aMPV, IBV, and NDV vaccines simultaneously at 1 day of age. However, previous work has indicated that the efficacy of aMPV vaccines may be affected when codelivered with IBV or NDV vaccines. The efficacy of an aMPV vaccine when codelivered to chickens in a trivalent combination with an NDV and an IBV vaccine was examined. The serological antibody response to the aMPV vaccine given with the IBV and NDV vaccine was significantly lower than when the aMPV vaccine was given alone. However, the aMPV vaccine did not affect the serological response to the IBV and NDV vaccines. Irrespective, the efficacy of the aMPV vaccine was not affected based on clinical signs postchallenge. This is the first report showing aMPV, IBV, and NDV vaccines can be codelivered without affecting the efficacy of the aMPV vaccine.     

鸡新城疫、传染性支气管炎、减蛋综合征、禽流感四联浓缩油乳剂灭活疫苗的研制

利用PALL滤器分别对鸡新城疫、传染性支气管炎、减蛋综合征和禽流感4种病毒进行10倍浓缩,然后按照一定比例将抗原浓缩液与油佐剂配比,制备了四联油乳剂灭活疫苗。通过了实验室检查,田间试验表明:开产前种鸡免疫后第4周,新城疫和禽流感的HI抗体滴度在10.2log2以上、传染性支气管炎和减蛋综合征均大于9.5log2,且抗体高峰维持时间长;SPF鸡免疫后4周进行攻毒保护试验,结果免疫攻毒组保护率100%,对照组攻毒发病率100%。试验结果表明:采用抗原浓缩技术制备的四联苗抗体水平高,维持时间长,能够有效地保护鸡群。     

Efficacy of avian influenza oil-emulsion vaccines in chickens of various ages

An experimental avian influenza (AI) oil-emulsion vaccine was formulated with 1 part inactivated A/turkey/Wisconsin/68 (H5N9) AI virus emulsified in 4 parts oil. Broilers were vaccinated subcutaneously (SC) either at 1 or 3 days old or at 4 or 5 wks old. Commercial white leghorn (WL) layers were vaccinated SC at 12 and 20 wks old or at only 20 wks old. Maximum geometric mean hemagglutination-inhibition titers postvaccination (PV) were 1:86-1:320 for broilers, 1:597 for twice-vaccinated layers, and 1:422 for once-vaccinated layers. Ninety to 100% of vaccinated broilers were protected against death and morbidity when challenged with highly pathogenic A/chicken/Penn/83 (H5N2) AI virus 4 weeks PV, and all were protected when challenged 8 wks PV. All controls and most vaccinates were infected by challenge virus, and 90-100% of controls died or exhibited clinical signs. Vaccinated commercial pullets were protected against morbidity, death, and egg-production decline at either peak of lay (25 wks old) or at 55 wks old. All unvaccinated controls became morbid or died, and egg production ceased 72 hours after challenge. The 0.5-ml vaccine dose was determined to contain 251 and 528 mean protective doses (PD50S) in 4-wk-old and 1-year-old SPF WL chickens, respectively, challenged 4 wks PV.     

Effect of Newcastle disease and infectious bronchitis live vaccines on the immune system and production parameters of experimentally infected broiler chickens with H9N2 avian influenza

H9N2 Avian influenza (AI) is an infectious disease which considered to have low pathogenic virulence, but in the case of coinfection with other pathogens it has the potential to become a major threat to the poultry industry. Infectious bronchitis (IB) and Newcastle diseases (ND) are other common problems to the poultry industry, which there are an extensive vaccination program against these viral pathogens. To investigate the effects of administration of infectious bronchitis and Newcastle disease live vaccines (IBLVs and NDLVs) in the presence of H9N2 AI infection on the immune system and some production parameters, 180 one-day-old broiler chicks were randomly allocated into six groups with different vaccination programs including H120 IBLV, 4/91 IBLV, B1 NDLV and LaSota NDLV. At the age of 20 days, all birds of the experimental groups except the negative control group, were inoculated intra-nasally (at dose of 10⁶ EID₅₀) with H9N2 AIV. After the inoculation, gross and microscopic lesions of the immune organs, serological changes and some production parameters were examined. The findings of this study showed that coinfection of H9N2 AI with NDLVs exacerbated the gross and microscopic injuries in the immune organs; especially the bursa of Fabricius. LaSota + AIV group had the most severe lesion in the bursa of Fabricius, spleen and thymus. Furthermore, the birds of LaSota + AIV group consumed the least amount of feed and water and their final body weight were significantly (P ≤ 0.05) lower in comparison with the other groups. Interestingly, in the context of this experiment both 4/91 and H120 IB live vaccines enhanced the HI antibody titers against H9N2 AIV, but the 4/91 showed the most significant (P ≤ 0.05) increase compared to the other experimental groups.     

The preparation and efficacy of manually emulsified Newcastle disease oil-emulsion vaccines

Experimental Newcastle disease oil-emulsion vaccines were prepared by manually shaking small quantities of antigen, surfactant, and mineral oil and compared with oil-emulsion vaccines made by the more conventional mechanical methods. Emulsion stability (prolonged emulsification of aqueous antigen) of the manually prepared vaccines was achieved by four different processes and tested for efficacy in broilers. White rock broilers were vaccinated at 3-5 weeks of age and bled at 1- to 2 week intervals thereafter for 8 weeks. Cumulative 8-week hemagglutination-inhibition (HI) mean titers (reciprocals) ranged from 15 to 250 for manually emulsified vaccines and from 18 to 240 for mechanically emulsified vaccines. Eight-week cumulative mean HI responses induced by manually emulsified vaccines were never significantly (P less than 0.05) lower than their specific mechanically emulsified comparison vaccines and were occasionally significantly higher. Highest HI titers were induced when hydrophile-lipophile balances of 7, 9, and 6 were used for the preparation of manually emulsified vaccines. In general, most of the vaccine emulsions were stable for more than 30 days. Vaccine efficacy, however, was not always diminished by poor emulsion stability. These results indicate that manual emulsification can be used for the production of oil-emulsion vaccines of high efficacy that can provide benefits over existing mechanical methods.     

AA肉鸡免疫禽流感油乳剂灭活疫苗细胞免疫应答的变化

为了解肉鸡生长期内免疫前后的细胞免疫反应,本实验采用CD3/CD4/CD8三色流式细胞术、MTT法和Griess法依次检测了AA肉鸡免疫禽流感灭活疫苗和非免疫状态下外周血、胸腺和脾脏中T淋巴细胞及其亚群的相对含量、外周血中T淋巴细胞对PHA-P的增殖反应能力和诱导巨噬细胞NO分泌量的变化。结果表明,非免疫状态下,AA肉鸡外周血CD4+、CD8+、CD3+T淋巴细胞相对百分含量在7日龄时均为最低,脾脏中CD4+T淋巴细胞相对百分含量在整个试验期一直呈下降趋势,免疫后外周血、脾脏和胸腺中CD4+、CD8+、CD3+T淋巴细胞相对百分含量均明显高于非免疫组,且脾脏CD4+T淋巴细胞呈阶段性上升状态;各时期淋巴细胞刺激指数(SI)值和巨噬细胞NO分泌量检测结果表明,免疫组均高于同期对照组(p<0.05)。研究结果还表明:肉鸡在7日龄时细胞免疫水平最低,灭活疫苗可以激发肉鸡产生较强的细胞免疫应答。     

Effect of infectious bursal disease on the response of chickens to Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus.

At 35 days of age, chickens which as 1-day-old chicks were inoculated with the infectious bursal disease virus (IBDV) had significantly lower antibody titers against Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus than did those never inoculated with IBDV. The IBDV also had a marked effect on the development of air-sac lesions. Birds infected with IBDV that were later inoculated with M synoviae (day 14), Newcastle disease virus (days 14 and 28) experienced an increased incidence and greater seversity of airsacculitis than did chicks which were not exposed to IBDV.     

Cell mediated and humoral immune response of chickens to inactivated oil-emulsion infectious bronchitis vaccine

A lymphocyte transformation microassay was used to study cell mediated immunity (CMI) in chickens following primary and secondary vaccination with inactivated oil emulsion infectious bronchitis (IB) vaccine and subsequent challenge with Massachusetts-41 (M-41). Humoral immunity was monitored for comparison, using the haemagglutination inhibition (HI) microassay. Positive stimulation indices (2 to 2.7 after primary and 2 to 4.8 after secondary vaccination) were lower and HI titres were higher than those previously reported following primary and secondary vaccination with live IB vaccines. The highest HI titres appeared in birds which had received the inactivated vaccine as a secondary vaccination. Challenge of vaccinated and revaccinated birds resulted in strong HI and weak CMI secondary responses. There was no correlation between CMI and HI antibody production. Monitoring egg production and clinical signs showed that a high level of protection against challenge resulted from revaccination with an inactivated oil adjuvant vaccine.     

Immune responses of breeding chickens to trivalent oil emulsion vaccines: responses to Newcastle disease and infectious bursal disease

Bivalent Newcastle disease (ND)/infectious bursal disease (IBD) and trivalent ND/IBD/infectious bronchitis (IB) inactivated oil emulsion vaccines were prepared in the laboratory and evaluated under field conditions. Broiler breeder parent chickens previously vaccinated with live vaccines were inoculated with commercial monovalent ND and experimental bivalent or trivalent oil emulsion vaccines. The commercial vaccine induced a higher initial ND haemagglutination inhibition (HI) response than the experimental vaccines but, by 34 weeks after vaccination, the mean ND HI levels were not significantly different in any of the three flocks. All three vaccines provided sufficient ND immunity to protect against the clinical disease and egg production losses. The IBD responses of both flocks vaccinated with oil emulsion vaccine were similar to each other and only slightly lower than those flocks vaccinated with monovalent IBD oil emulsion vaccine in earlier experiments. Six weeks after vaccination, sufficient immunity was transferred to protect all the progeny against IBD challenge up to 33 days of age and some of them up to 45 days of age. Thirty-four weeks after vaccination of the parents with oil emulsion vaccine, the progeny were totally immune up to 27 days of age and some of them were immune until 37 days. Application of oil emulsion vaccines in bivalent or trivalent form did not impair the responses of the chickens to the monovalent components.     

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗对禽流感H9亚型流行株攻毒的保护作用

为了监测鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活疫苗(LaSota株+M41株+SS/94株)对H9亚型禽流感病毒流行毒株的免疫保护效果,采用H9亚型禽流感病毒SS/94株及2009—2010年现地分离的3株H9亚型禽流感病毒对已免疫上述三联灭活苗的SPF鸡进行攻毒试验。结果显示,试验鸡以0.3 mL/只的剂量免疫三联灭活苗后21 d,其H9亚型禽流感病毒的HI抗体效价可达8～11log2,此抗体水平可抵抗2×106EID50的H9亚型禽流感病毒SS/94株、BLCN09株、WDZ09株、YT10株的攻击,攻毒保护率均达90%(9/10)以上。可见,以SS/94株作为禽流感疫苗抗原制备的三联灭活苗具有良好的免疫原性,能使免疫鸡抵抗2009—2010年期间现地分离的多株H9亚型禽流感病毒的攻击。