鸡产蛋下降综合症病毒快速纯化及其抗血清制备

为研制鸡产蛋下降综合症病毒（ EDS-76）检测用标准抗原及标准多抗血清,以EDS-76国际通用毒株AV-127株为抗原,进行了EDS-76病毒抗原纯化及多抗血清制备。研究表明,经氯仿抽提,并经PEG浓缩处理病毒的尿囊液可获得良好的纯化效果,纯化病毒背景干净,血凝效价达19Log2。以此研制出的鸡抗 EDS-76多抗血清血凝抑制效价达13Log2,间接免疫荧光效价达1∶2000,Western-blot试验分析表明该多抗血清可以识别两个分子大小在50 kD至85 kD之间的蛋白抗原,HI及IFA 试验发现所制备的特异性,多抗血清与12种常见禽病病原（ NDV、AIV-A、ALV-J、ALV、GPV、REV、MDV、IBV、CAV、IBDV、TMUV、REOV）均不反应。本研究建立的快速简便的EDS-76病毒纯化方法以及获得的抗EDS-76多抗血清,为进一步研制EDS-76标准抗原以及标准多抗血清提供了有效的方法与材料。     

鸡新城疫强毒株的分离与鉴定

从山东某鸡场的产蛋下降而无其他典型ND症状的高抗体蛋鸡群中分离到一株病毒。经过试验鉴定．该分离株具有血凝性，且可被ND标准阳性血清所抑制和中和，不能被AI（H5亚型与H9亚型）标准阳性血清抑制；该病毒的最小致死量病毒致死鸡胚的平均时间（MDT）为50．4，1日龄SPF鸡脑内接种分离病毒致病指数（ICPI）为1．85，6周龄SPF鸡静脉接种致病指数（WPI）为2．42，回归试验鸡出现了新城疫症状和病变，该病毒株确定为新城疫病毒强毒型，并暂命名为ShD-dzh04。     

鸡新城疫病毒的分离与鉴定

从山东某鸡场的产蛋下降而无其他典型新城疫(Newcastle Disease)症状的高抗体蛋鸡群中分离到一株病毒。经过试验鉴定,该分离株具有血凝性,且可被ND标准阳性血清所抑制和中和,不能被AI(H5亚型与H9亚型)标准阳性血清抑制;该病毒的最小致死量病毒致死鸡胚的平均时间(h)为50.4 h,1日龄SPF鸡脑内接种分离病毒致病指数(ICPI)为1.85,6周龄SPF鸡静脉接种致病指数(IVPI)为2.42,回归试验鸡出现了新城疫症状和病变,经外源病毒检测及回归试验该病毒株被确定为新城疫病毒,并属强毒型,命名为ShD-5-04。     

Serologic survey of slaughter-age ostriches (Struthio camelus) for antibodies to selected avian pathogens

Serum samples from 163 slaughter-age ostriches (Struthio camelus) in Ohio and Indiana were tested for antibodies to avian influenza virus (AIV), Newcastle disease virus (NDV), paramyxovirus (PMV) 2, PMV3, PMV7, infectious bursal disease virus (IBDV), Bordetella avium, Mycoplasma synoviae, Mycoplasma gallisepticum, Ornithobacterium rhinotracheale, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium. One ostrich had antibodies to AIV H5N9, 57% of the ostriches had antibodies to NDV, four ostriches had antibodies to both NDV and PMV2, and one ostrich had antibodies to NDV, PMV2, PMV3, and PMV7. None of the ostriches had antibodies to IBDV, B. avium, M. synoviae, M. gallisepticum, O. rhinotracheale, S. pullorum, S. gallinarum, and S. typhimurium. This is the first report of antibodies to avian influenza and PMV7 in ostriches in the United States.     

Development and application of a multiplex polymerase chain reaction for avian respiratory agents

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.     

Strategies of newcastle disease vaccination for commercial ostrich farms in Japan

Strategies of Newcastle disease (ND) vaccination were demonstrated in a commercial ostrich farm in Japan. Three of 13 seven-month-old ostriches kept in a pen were vaccinated with a live ND vaccine by eye dropping for the 1st and 2nd vaccinations and spraying for the 3rd to 5th vaccinations. Antibodies against ND virus (NDV) were detected in all of the unvaccinated ostriches by virus neutralization test. At 2.5 months post final vaccination, 2 ostriches introduced into the pen raised antibodies against NDV. These data indicate that NDV may be transmitted from vaccinated to unvaccinated ostriches in the flock and that the virus may be sustained for a certain period in the flock. These data may be helpful for ND vaccination management in ostrich farms.     

A survey of disease in five commercial flocks of meat breeder chickens

Causes of sickness and death in approximately 30,000 chickens in 5 meat breeder flocks were investigated between May 1979 and April 1980. Approximately 23% of disease was due to neoplasms; 81% of these were Marek's disease despite vaccination against this infection. Other frequent diagnoses included cellulitis (15%), respiratory disease (14%), lesions of the reproductive tract (11%) and tenosynovitis/arthritis (9%). Antibodies to Mycoplasma gallisepticium, avian adenovirus, infectious bursal disease virus and reticuloendotheliosis virus were present in all flocks. Antibody to Newcastle disease virus (NDV) was found in 2 flocks but titres were not considered protective against a virulent NDV challenge. Antibody to egg drop syndrome 1976 virus was found in 2 flocks comprised of the same breed of bird.     

Serological and virological studies of Newcastle disease and avian influenza in slaughter-age ostriches (Struthio camelus) in Japan

Serum samples from 191 ostriches (Struthio camelus) in Japan were tested for antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV). Twenty-two (12%) contained NDV-specific neutralizing antibodies by a virus-neutralization (VN) test without vaccination. Antibodies to AIV were not detected in the any sera by an agar gel precipitation test. Seven serum samples that had vaccinated with live NDV by eye drop were all positive by the VN test at 1 month post vaccination. A haemagglutination inhibition (HI) test for NDV seemed not to be suitable for ostriches because of non-specific agglutination of chicken red blood cells. No haemagglutinating viruses were isolated. This is the first report on detection of antibodies against NDV in ostriches in Japan.     

免疫层析试纸条(ICS)检测禽流感抗体

以纯化的禽流感病毒（avian influenza virus,AIV）核蛋白作为捕捉抗原，金标兔抗鸡抗体作为金标二抗，建立检测禽流感血清抗体的胶体金免疫层析试纸条（immunochromatographic strip, ICS）。对标准阳性血清不同滴度进行检测，ICS试验的敏感性高于琼脂扩散试验（AGP）。特异性试验证实，禽流感的ICS试验与新城疫（ND）、传染性支气管炎（IBV）、鸡传染性法氏囊病（IBD）及减蛋综合症（EDS-76）均无交叉反应。同时，用ICS试验和HI试验检测105份鸡血清，两者具有较好的符合性。结果认为：禽流感ICS具有快速简便、特异、灵敏等优点。     

Prevalence of infectious diseases in ring-necked pheasant flocks in Poland

The health status of ring-necked pheasants in view of the prevalence of infectious diseases was estimated in Polish pheasantries in the years 1997-2000. Anatomicopathological, microbiological and serological examinations were carried out on birds derived from 26 pheasantries, including birds randomly selected from 18 flocks and sick or dead birds sent from 8 pheasantries. Antibodies specific to the following viruses were detected in serum blood samples: HE, AE, AP, REO, AI, Adeno group 1, MD, ND, as well as Mycoplasma gallisepticum specific antibodies. However, in none of the examined flocks was the presence of antibodies against reticuloendoteliosis virus found. Marble spleen disease and salmonellosis proved to be the most frequent cause of death during the growing period.     

Serosurvey of five viruses in chickens on smallholdings in Bangladesh

A serologic survey was undertaken in chickens in smallholdings in Bangladesh for avian influenza A virus (AIV), egg drop syndrome '76 virus (EDS'76V), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and reovirus (RV) in three phases: January 2002-May 2003, September 2003-August 2004, and August 2005-March 2006. Four hundred thirty-six sera collected in the 2nd phase, 295 in the first phase, 755 in the 1st plus 2nd phases and 295 in the 1st phase were investigated for AIV, EDS'76V, IBV and RV, respectively, using enzyme linked immunosorbent assays. All 854 sera collected in the three phases were screened for NDV using hemagglutination inhibition test. In chickens 20% were seropositive to AIV, 3% to EDS'76V, 74% to IBV, 88% to NDV, and 47% to RV. The seroprevalence in flocks was 23% to AIV, 6% to EDS'76V, 79% to IBV, 89% to NDV and 56% to RV. Twenty-five percent chickens had > or = 10log(2)HI titers to NDV.     

Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus

The hypothesis that an effective protection of progeny chickens against inclusion body hepatitis/hydropericardium syndrome (IBH/HP) can be achieved by dual vaccination of breeders with fowl adenovirus (FAV) serotype 4 and chicken anemia virus (CAV) was tested. Thus, 17-wk-old brown leghorn pullet groups were vaccinated by different schemes including single FAV (inactivated), single CAV (attenuated), FAV and CAV dually, or were not vaccinated (controls). Subsequent progenies of these breeders were challenged with the virulent strains FAV-341 and CAV-10343 following three strategies: 1) FAV-341 intramuscularly (i.m.) at day 10 of age (only FAV-vaccinated and control progenies); 2) FAV + CAV i.m. simultaneously at day 10 of age (all progenies); 3) CAV i.m. at day 1 and FAV orally at day 10 of age (all progenies). The induction of IBH/HP in these progenies was evaluated throughout a 10-day period. Both breeder groups vaccinated against FAV and those vaccinated against CAV increased virus neutralizing specific antibodies. Challenge strategy 1 showed 26.6% mortality in control progeny chickens and 13.3% in the progeny of FAV-vaccinated breeders. Presence of lesions in the liver of these groups showed no significant differences (P > 0.05), suggesting a discreet protective effect of the vaccine. Challenge strategy 2 showed 29.4% mortality in controls and 94% of chickens showed hepatic inclusion bodies (HIB). Single CAV vaccination of breeders did not demonstrate a beneficial effect, with both mortality and liver lesions resembling the nonvaccinated controls. FAV vaccination of breeders significantly reduced both mortality (7.4%) and liver lesions (26% HIB) (P < 0.05), providing protection against this challenge strategy. Dual vaccination of breeders with FAV and CAV proved to be necessary to achieve maximum protection of the progeny (no mortality and 7% HIB). Challenge strategy 3 produced no mortality but consistent liver damage in controls (96% HIB). In this case, both CAV and FAV + CAV-vaccinated breeders showed best protection results in terms of liver histopathology (8% and 0% HIB, respectively). FAV vaccination alone produced 24% HIB, similar to challenge strategy 2, demonstrating a lower protective effect.     

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.     

Establishment and Application of a Nested PCR Assay for Detection of Fowl Adenovirus Group Ⅰ

According to the sequence of hexon gene of fowl adenovirus groupⅠ(FAVⅠ) strain published in GenBank,two pairs of primers were designed and synthesized.The outer primers amplified a fragment of 475 bp in length, and the inner primers amplification fragment was 237 bp in length. A nested PCR assay for rapid detection of FAVⅠ was established.A specific 237 bp fragment was amplified from DNA templates of FAVⅠstrain,but no bands were amplified with templates extracted respectively from avian influenza virus (AIV) subtype H9,Newcastle disease virus (NDV), infectious bursal disease virus (IBDV),duck plague virus (DPV), reticuloendotheliosis (REV), avian reovirus (ARV), Marek's disease virus (MDV). Sensitivity of the 1st and 2nd amplifications by the nested PCR assay were 100 pg and 1 fg,respectively.The sensitivite of the 2nd amplifications increased by 10⁵ times.The results showed that the nested PCR was specific,sensitive,rapid,accurate,and could be used as a routine assay for the detection of FAVⅠ.This method had good reproducibility, specificity and sensitivity, and might detect low content FAVⅠ accurately and rapidly. This method could be used as a method for the diagnosis and detection of clinical cases,and molecular epidemiological investigation of FAVⅠ.     

禽Ⅰ型副粘病毒各种禽源分离株的免疫原性

分别制备了源于鸡、鹅、鸽、鹌鹑、孔雀、画眉鸟、珍珠鸡的禽型副粘病毒(APMV-1)7个强毒分离毒株和商品弱毒疫苗株克隆30(C30)的灭活油乳剂苗,并用这8种灭活油乳剂苗和C30株的活疫苗分别在鸡、鹌鹑、鹅和鸽进行了免疫及交叉攻毒保护试验。免疫后(PI)分别测定试验禽血清的新城疫病毒(NDV)血凝抑制(HI)抗体的滴度,并于PI 5周用强毒株进行攻毒。结果表明,鸽的HI抗体几何平均滴度(MAT)为9.00~10.0 log2,鸡的为7.13~7.63 log2,鹌鹑的为5.00~5.13 log2,鹅对灭活油乳剂苗的为5.63~6.38 log2、而对C30株活疫苗的仅为3.38log2;除了C30株活疫苗免疫鹅提供的保护率比较低(20%)外,8种油乳剂苗都能对同源或者异源强毒株的攻毒提供比较高的保护率(66.75%~100%)。研究结果表明,经典疫苗株C30与近年来从各种不同禽类分离的致病性APMV-1野毒株之间、不同禽源分离株之间的抗原性,以及各种不同禽源分离株的之间的免疫原性差异均不大。     

Protective immunity against Newcastle disease: the role of antibodies specific to Newcastle disease virus polypeptides

Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea.     

鸽新城疫病毒的分离及其生物学特性测定

用ＳＰＦ鸡胚从疑似鸽新城疫（鸽ＮＤ）病鸽群中分离到一株病毒ＱＬ株,该病毒株能凝集鸡红细胞（ＲＢＣ）,这种凝集作用能被抗新城疫病毒（ＮＤＶ）阳性血清抑制;用抗ＮＤＶ单抗ＰＥＧ夹心ＥＬＩＳＡ测定分离株为阳性。分离株经肌肉注射能使鸽发病和死亡,出现与自然发病鸽一致的症状和病变,但肌注ＳＰＦ鸡只感染,不见临床症状。对该分离株作进一步生物学特性鉴定,按照国际上规定的ＮＤＶ毒力判定标准,测定了该毒株最低致死量致死鸡胚的平均死亡时间（ＭＤＴ）、１日龄雏鸡脑内接种致病指数（ＩＣＰＩ）和６周龄雏鸡静脉接种致病指数（ＩＶＰＩ）,结果ＭＤＴ为１０５小时、ＩＣＰＩ为１．３３、ＩＶＰＩ为１．０。试验结果表明本分离株为鸽新城疫病毒。     

A survey for paramyxoviruses in caged birds, wild birds, and poultry in New Zealand

AIMS: To determine the presence of avian paramyxovirus (APMV) types 1, 2, and 3 in caged and wild birds, and APMV-2 and -3 in poultry in New Zealand. METHODS: Blood samples collected from caged (231) and wild birds (522) from various regions of New Zealand in 1997-99 were tested by haemagglutination inhibition (HI) test for antibodies to APMV types 1, 2, and 3. Blood samples collected from 1778 commercial poultry in 1996-99 were tested for APMV-2 and APMV-3 antibodies and the samples that reacted with APMV-3 antigen were tested for antibodies to APMV-1. Isolation of APMV was attempted from cloacal swabs collected from 116 of the caged birds and 175 of the wild birds sampled. RESULTS: Antibodies to APMV types 1, 2, and 3 were detected in 4.8, 1.7, and 2.6%, respectively, of caged bird samples. The majority of these caged birds were 'exotic' or 'fancy' poultry breeds. Amongst wild birds, 4.2% had titres to APMV-2 and over half of these were passerine birds; 1.7% of the samples had titres to APMV-1 and 0.8% to APMV-3 antigen. No virus was isolated from any of the cloacal swabs tested. Of the 1778 poultry serum samples tested, only 5 reacted with APMV-3 antigen and these were later found to be cross-reactions to APMV-1. No reactions were detected with APMV-2 antigen. CONCLUSIONS: APMV-1 is present in caged birds, wild birds, and poultry of New Zealand. There is no conclusive evidence of the presence of APMV-2 and APMV-3 in poultry or APMV-3 in wild birds. The results do not provide conclusive evidence for the presence of APMV-2 in wild birds in New Zealand.