应用琼脂扩散试验检测新城疫抗原

应用ＰＥＧ平板进行琼脂扩散试验检测新城疫抗原,提高了对新城疫抗原的检出率。采用多器官统一判定方法,即脾、胸腺、盲肠扁桃体,肠淋巴结和法氏囊等器官检出阳性时即可判为新城疫,对新城疫死鸡的检出率为１００％,解决非典型新城疫诊断困难这一难题,方法简单准确,快速易行,值得推广和应用。     

应用琼脂扩散试验检测新城疫抗原

应用PEG平板进行琼脂扩散试验检测病禽组织器官中的新城疫抗原，采用抗原与兔抗ND高兔血清的点阵分析。确定了兔抗ND高兔血清诊断抗体的最佳稀释度为1：2，采用多器官检查和统一判定方法，即心、肝，脾、肺、肾，脑，盲肠扁桃体，法氏囊等器官检出阳性时即可判为新城疫。     

间接免疫荧光染色检测鸭源副黏病毒及在鸭体内的抗原定位

以抗新城疫病毒F蛋白的单克隆抗体为一抗,建立了间接免疫荧光染色法(IFA)检测石蜡切片中鸭副黏病毒(DPMV)的方法。以建立的IFA对DPMV人工感染鸭的各组织器官进行检测,结果显示:各组织器官均能检测到DPMV,但不同时间取样,阳性信号分布的器官不同。脾脏、胸腺、法氏囊、肠道、肝脏、肺脏DPMV的阳性检出率较高,表明这些器官为DPMV的主要靶器官。在所检测的阳性组织中,病毒抗原分布在细胞浆中。IFA检测石蜡切片中的DPMV具有直观、特异性强的优点,是对DPMV进行检测和抗原定位的良好方法。     

间接法DOT—ELISA检测新城疫病毒的研究

本文首次成功地建立了间接法Dot-ELISA检测新城疫病毒,对新城疫发病鸡群口咽、泄殖腔拭子的NDV抗原阳性检出率分别为58.33％(91/156)、62.50％(90/144),两者没有显著差异(P>0.05),结果表明,间接法Dot-ELISA对新城疫的早期诊断具有简单、快速、经济、敏感性高、特异性强和重复性好等优点,是疫病早期诊断与流行病学调查的有效手段。     

复方金银花对鸡体内NDV分布的影响

选用1日龄雏鸡,无污染环境饲养21 d后,随机分成感染组、中药治疗组、空白组。中药治疗组饮水给予复方金银花制剂。以单克隆抗体介导的免疫组化（IHC）法检测攻毒鸡体内NDV抗原的分布及定位,以确定复方金银花对新城疫病毒感染鸡的组织嗜性的影响。结果显示,感染组和中药治疗组鸡的16个组织器官中均能检测到NDV抗原,病毒以气管、肺脏、十二指肠、空肠等器官中分布为最高。复方金银花对新城疫的组织损伤有一定的保护作用,可缩短组织修复的时间。     

不同基因型新城疫病毒强毒株对鸡的致病性

就不同基因型新城疫病毒强毒株对鸡的致病性进行了比较。用3种基因型（Ⅵg、Ⅶd亚型、Ⅸ型）新城疫病毒强毒株分别人工感染25日龄雏鸡，结果各株病毒接种鸡均100％发病、100％死亡，表现出嗜内脏型新城疫病毒感染引起的典型的新城疫症状和病理变化。免疫组化检测发现．攻毒鸡多种组织器官中能检测到病毒抗原，在能检测到病毒抗原的器官中，病毒抗原主要定位于淋巴细胞、网状细胞、巨噬细胞、肝细胞及各种上皮细胞的胞浆内；新城疫病毒对肠相关淋巴组织的亲嗜性优先于附近的上皮。本试验结果表明．基因Ⅵg、Ⅶd亚型、Ⅸ型新城疫病毒强毒株对鸡均具有高度致病性。     

新城疫浓缩灭活疫苗的初步研究

以10^3.0、10^4.0、10^5.0 EID503个不同剂量接种非免疫鸡胚,收获鸡胚液,病毒含量测定结果显示,以10^3.0-10^4.0～EID50剂量接种的鸡胚收获的抗原含量高。用该优化方法繁殖了一批新城疫抗原,采用截留分子量为100kD的浓缩滤膜对新城疫抗原进行浓缩,对不同浓缩倍数的新城疫抗原制备疫苗进行了免疫效力试验。结果表明,以新城疫3倍浓缩抗原制备疫苗,以5μL剂量（现有相关产品效力检验的1／4剂量）免疫SPF鸡,即可达到现有同类产品的效力检验质量标准,高抗体水平至少可以维持35d。本研究为进一步研制新城疫灭活浓缩疫苗用于低剂量免疫肉鸡提供了基础。     

对β-微量法监测新城疫抗体的几点改进

目前，尽管养鸡场对新城疫进行了多次免疫，但以不拉稀、无呼吸道症状、不死亡，仅产蛋率下降为特征的非典型新城疫仍不断发生。究其原因，主要有以下几点：一、免疫程序不合理；二、使用质量低劣的疫苗；三、野毒的毒性增强；四、鸡群的局部（气管）免疫水平较低。监测抗体的方法：β－微量法β－微量法在基层大量应用的制约因素是抗原和阳性血清价格太高且不易购到。自制抗原，方法简单易行，并可将每次测新城疫抗体的成本由3．5元降为0．10元（按每次测100份计）。制备抗原：将新城疫Insota系疫苗，用灭菌生理盐水稀释10倍，加人青霉素…     

应用荧光RT-PCR方法检测H7N9亚型禽流感病毒

为了验证荧光RT-PCR方法检测H7N9亚型禽流感病毒的可行性,应用建立的荧光RT-PCR方法检测H7N9亚型禽流感抗原、H9亚型禽流感抗原、H5亚型禽流感抗原、新城疫抗原评估检测方法的特异性,检测不同稀释度的H7N9亚型禽流感抗原,并用建立的方法检测采自三鸟批发市场100份泄殖腔、喉拭子。结果是建立的荧光RT-PCR方法,H7体系能检测出1:10-10抗原稀释度,N9体系能检测1:10-9抗原稀释度,对H9亚型禽流感抗原、H5亚型禽流感抗原、新城疫抗原无交叉反应;检出100份泄殖腔、喉拭子结果均为阴性。结果表明,荧光RT-PCR方法H7N9亚型禽流感病毒具有快速、灵敏、特异的特点,适合对禽类的大规模监测。     

应用ELISA双抗体夹心法检测传染性法氏囊病免疫器官病毒抗原分布动态

利用ＩＢＤＶ高免血清ＩｇＧ作为包被抗体，成功地建立了双抗体夹心法ＥＬＩＳＡ，对人工感染雏鸡免疫器官抗原动态分布进行了检测。结果表明，不同毒株感染雏鸡免疫器官病毒抗原含量不一致，持续时间也有差异。采用本方法共检测ＩＢＤ阳性样品２７５份，检出率为１００％，检测对照阴性样品２５份，结果均为阴性，比ＡＧＰ法检出率高４０％左右，灵敏度高１００～２００倍。试验证明双抗体夹心法ＥＬＩＳＡ可用于组织抗原分布的检测，并且该方法具有特异性、高度敏感性，稳定性、快速性等优点，是一种实用的组织病毒检测方法。     

Salmonella typhimurium infection in domesticated fowl in a children's zoo.

Salmonella typhimurium infection occurred in a children's zoo where 11 fowl and 85 mammals were kept. Initially, the guinea pigs were infected and transmitted the infection to the fowl and rabbits. These mammals responded to medication and cleared of the infection; however, the birds were judged to contain excreters despite four regimens of treatment with antibiotics. Cloacal swabs were taken from all the birds. One turkey was positive for Salmonella and was destroyed. Pooled fecal samples from the birds were again positive. All the birds were tested serologically, and two birds, a goose and a turkey, were positive with Salmonella pullorum-gallinarum antigen, which was assumed to be a cross reaction with S. typhimurium antigen. The two birds were destroyed and the goose yielded Salmonella. The infection was finally eradicated, and the serologic examination was considered to be the most useful procedure for detection of the excreters.     

Newcastle disease--seroepidemiologic study of a highly contagious epizootic in poultry and in wild birds in Switzerland

Newcastle disease (ND) is a highly contagious viral disease particularly of domestic poultry. Switzerland is currently declared free from ND. A serosurvey using an ELISA was performed to investigate infections with ND-Virus (NDV) in 260 Swiss laying hen flocks, 169 backyard poultry flocks and 1576 wild birds. For laying hen flocks, a stochastic model was applied to analyse the results from serological testing. Four laying hen flocks were identified as NDV-seropositive, and the true NDV seroprevalence in this population was most likely between 1.3 and 1.5%. NDV antibodies were also detected in five of the 169 backyard poultry-flocks. ND-antibody positive birds were found in 10% of all wild birds examined, with the highest proportions among cormorants, grebes, birds of prey, owls, and swifts. The study indicated that positive flocks must have been in contact with NDV strains causing sub-clinical infection, since no clinical signs had been observed. Moreover, trade of poultry or poultry eggs was considered to be an important factor associated with seropositivity in backyard poultry flocks. Contact to wild birds did not seem to be of major importance.     

Determination of the distribution of lentogenic vaccine and virulent Newcastle disease virus antigen in the oviduct of SPF and commercial hen using immunohistochemistry

The control of Newcastle disease (ND) in South Africa has proved difficult since 2002 following the introduction of lineage 5d/VIId Newcastle disease virus (NDV) strain ("goose paramyxovirus" - GPMV) to which commercially available ND vaccines appeared less effective. Most of the ND infections, even in fully vaccinated hens were characterized consistently by a drop in egg production. In this study, commercial and SPF hens-in-lay were vaccinated with La Sota vaccine and challenged with a GPMV isolate. Immunohistochemical labeling was used to determine the distribution of viral antigen in the oviduct of the hens. Following reports that cloacal vaccination offered better protection against egg production losses than the oro-nasal route, the efficacy of cloacal and ocular routes of vaccination against challenge were compared. Results showed that La Sota vaccine offered birds 100% protection against the virulent ND (GPMV) virus challenge from clinical disease and death, but not against infection and replication of the GPMV, as birds showed varying degrees of macropathology. Histopathology of the oviduct of infected birds revealed multifocal lymphocytic inflammation in the interstitium as well as mild glandular ectasia and mild edema. Finely granular NDV-specific immunolabeling was demonstrated in the cytoplasm of epithelial cells and mononuclear (lymphohistiocytic) cells in the interstitium of the oviduct. Both vaccine and virulent GPMV showed greatest tropism for the uterus (versus the magnum and isthmus). There was no clear difference in the protection of the oviduct and in the distribution of oviductal GPMV antigens between the two routes of vaccination.     

Prevalence and incidence of Newcastle disease and prevalence of Avian Influenza infection of scavenging village chickens in Timor-Lesté

A longitudinal study to monitor prevalence and incidence of antibodies against Newcastle disease (ND) virus and prevalence of antibodies against Avian Influenza (AI) virus in scavenging village chickens was conducted in 20 villages within 4 districts of Timor-Lesté. A total of 3600 blood samples was collected from 1674 individual birds in 300 household chicken flocks during three sampling periods (December 2008-February 2009, March-May 2009, and June-August 2009). The mean interval between household visits was 101.6±1.9 days. None of the birds enrolled in the study was vaccinated against ND or AI. A haemagglutination inhibition (HI) test was used to determine antibody titres against ND virus and a competitive ELISA and HI tests were used to detect antibody against AI virus. The bird-level ND seroprevalence pooled across all samplings (adjusted for clustering by households) was 4.4% (95% CI 3.5-5.2). The bird-level ND seroprevalence in each of the three sampling periods (adjusted for clustering by household) was 3.0% (95% CI 2.0-4.0), 6.6% (95% CI 5.1-8.0) and 3.6 (95% CI 2.5-4.6), respectively. A total of 12.6% individual birds tested ND seropositive at least once over the total study period (95% CI 10.5-14.7). The flock-level ND seroprevalence (at least one bird tested had antibodies against ND virus) pooled across all samplings was 15.9% (95% CI 13.5-18.3). A total of 35.3% flocks had a minimum of one bird being ND seropositive at least once over the study period. The bird-level incidence rate for the period between the first and the second sampling and between the second and the third sampling was 5.6 (95% CI 4.1-7.5) and 0.5 (95% CI 0.5-3.8) per 10,000 bird-years-at-risk, respectively. A total of 1134 serum samples from the last sampling period between June and August 2009 was tested for antibodies against AI virus. Only 4 samples tested Influenza A positive, indicating a bird-level seroprevalence level for Influenza A of 0.4% (CI 0.0-0.7%). These Influenza A positive samples were further tested for HI antibodies against AI virus subtypes of H5N1, H5N3, H7N3 and H9N2, but all tested negative, suggesting that the influenza antibodies in those four birds resulted from exposure to low pathogenic AI viruses of different H subtypes. Our results indicate that village chickens in Timor-Lesté are exposed to ND virus; there was a higher risk of infection during the early months of 2009 than either immediately prior or subsequent to this. No evidence of infection of village chickens with H5, H7 or H9 AI viruses was detected in this study.     

A survey of Newcastle disease in Swiss laying-hen flocks using serological testing and simulation modelling

Newcastle disease (ND) is a highly contagious viral disease of birds particularly domestic poultry. Switzerland is currently declared free from ND; since vaccination is prohibited, the detection of antibodies against ND virus (NDV) results in the destruction of the respective flock (stamping-out policy). However, in 1995 and 1996, antibody-positive flocks were detected and sporadic ND outbreaks even occurred in Switzerland. Therefore, a serosurvey was done to look for evidence of NDV infections in Swiss laying-hen flocks. The survey was designed to provide 95% confidence of detecting at least one seropositive flock if the flock prevalence were 1%. Thirty blood samples from each of 260 commercial laying-hen flocks were collected during 1996 in a central poultry slaughterhouse. Sera were screened for NDV antibodies with a commercial blocking enzyme-linked immunosorbent assay (ELISA). Samples with a questionable or positive test result were retested with the same ELISA. A stochastic computer model was applied to define a cut-off number of test-positive samples to help to differentiate between true- and false-positive flocks and to estimate the true flock prevalence of infection. Four flocks were identified as NDV-seropositive and the NDV true seroprevalence among commercial laying-hen flocks in Switzerland was most likely between 1.35 and 1.55%. This indicates that Swiss laying-hen and parental flocks with more than 150 animals have been in contact with strains of NDV that cause subclinical infection in chicken, because no clinical symptoms have been observed. In this context, computer simulation was a useful technique to interpret survey results.     

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.     

Immunogenicity of a Locally Produced Newcastle Disease I-2 Thermostable Vaccine in Chickens in Uganda

A locally-produced Newcastle disease (ND) I-2 thermostable vaccine of embryo-infective dose (EID50) 10(8.5) per ml was administered to 100 laboratory chickens in four test groups, each of 25 birds. It was given by the eye-drop method, in drinking water, in drinking water freshly medicated with levamisole, or using millet grains as a vaccine carrier. A fifth control group consisting of 25 birds received the heat-sensitive La Sota vaccine (EID50 10(9) per ml) by the eye-drop method. The immunological responses were monitored by the enzyme-linked immunosorbent assay (ELISA) ND antibody technique using serum samples collected from 18 birds in each group at 3-week intervals for 3 months. The overall mean ND antibody log(10) titres and percentage positivities were 3.1, 88%; 2.9, 70%; 3.0, 83%; 3.2, 87% and 3.3, 87%, respectively. The use of water alone or medicated with levamisole for vaccine administration produced significantly lower ND antibody titres only in the first 3 weeks. The immunogenicity shown by the I-2 vaccine as a potential vaccine is discussed in relation to free-range poultry management conditions in Uganda.