Newcastle disease and avian influenza A virus in wild waterfowl in South Africa

In an intensive ostrich farming area in South Africa with a history of ostrich influenza outbreaks, we conducted a survey of avian influenza virus (AIV) and Newcastle disease virus (NDV) in wild aquatic birds. During late autumn and winter 1998, the time of year when outbreaks in ostriches typically start to occur, 262 aquatic birds comprising 14 species were sampled and tested for both virus infections. From eight samples, AIV, serotype H10N9, could be isolated. All isolates were apathogenic as determined by the intravenous pathogenicity index (0.00). Conversely, none of 33 sera of these wild birds showed antibodies against H10. However, one bird was found serologically positive for H6 AIV. This AIV serotype was later isolated from ostriches during an avian influenza outbreak in this area. No NDV was isolated although 34 of 46 serum samples contained NDV-specific antibodies. This is the first H10N9 isolate to be reported from Africa. In addition, our data support the notion that wild aquatic birds may function as a reservoir for AIV and NDV in South Africa.     

CAV与REV共感染SPF鸡对疫苗免疫反应的抑制作用

用1日龄SPF鸡人工感染鸡贫血病毒(CAV)和禽网状内皮增生病病毒(REV),探讨病毒感染对鸡体疫苗免疫反应的影响。结果表明,在用禽流感病毒(AIV,H5和H9)疫苗免疫后,CAV与REV单独感染均显著抑制了鸡体对H5和H9亚型禽流感病毒灭活疫苗的HI抗体反应,在CAV与REV共感染后,这种抑制作用更为明显。CAV单独感染后鸡体对新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)疫苗的免疫反应受到抑制,但与对照组在统计学上的差异不显著,然而,CAV可以显著加重REV感染对鸡体在NDV和IBDV疫苗免疫后抗体反应的抑制作用。从而证实CAV与REV共感染在疫苗免疫抑制上有协同作用。     

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.     

禽肉产品中禽流感与新城疫多重PCR-DHPLC检测方法的建立

应用多重RT-PCR反应(mRT-PCR)结合变性高效液相色谱(DHPLC)技术建立禽产品中禽流感病毒和新城疫病毒的快速检测方法。以禽流感病毒NP基因和新城疫病毒F基因各设计1对引物,经优化单相RT-PCR(sRT-PCR)反应体系,建立多重RT-PCR(mRT-PCR)同时检测禽流感和新城疫,其PCR扩增产物经DHPLC技术进行快速检测分析后,检测极限分别达10-0.5ELD50/0.1mL和10-1.5ELD50/0.1mL。与普通凝胶电泳比较,敏感性高2个数量级。特异性试验表明,mRT-PCR-DHPLC仅检测到禽流感病毒与新城疫病毒,而对其他5种禽类病原体和SPF鸡胚尿囊液均未检测到阳性吸收峰。所建立的方法检测不同亚型禽流感病毒29株、不同来源及不同毒力的新城疫病毒16株,结果与实时荧光定量PCR检测结果完全一致;与病原分离法同时检测人工感染鸡的组织脏器,两种方法检测禽流感的符合率为94.4%(34/36),检测新城疫的符合率为95.4%(21/22)。病原分离法的检出率虽高于DHPLC方法,但经统计学分析两者差异不显著,两种方法同时对27份进口鸡胗样品与90份棉拭子样品进行检测,阴性符合率为100%,可适用于进出口禽肉产品的检测。     

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.     

一步法多重RT-PCR检测新城疫、禽流感、传染性支气管炎病毒试验的研究

为了提高检测鸡肉中新城疫病毒（Newcastle disease virus,NDV)，禽流感病毒(Avian influenza virus,AIV)，传染性支气管炎病毒（Avian infectious bronchitis virus,IBV)的效率和敏感性，利用引物设计软件Primer Primer5.0设计了NDV的F基因片段，AIV的M基因片段，IBV的M基因片段的引物，在以前一步法RT-PCR扩增各种病毒RNA的基础上，进行了两种病毒和三种病毒的一步法多重RT-PCR的试验。结果确定了两种病毒的一步法多种RT-PCR的试验条件。三种病毒的一步法多重RT-PCR的试验条件基本确立，本试验初步建立了检测NDV，AIV，IBV一步法多重RT-PCR技术。     

Immuno-PCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers

Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step.     

禽流感病毒和新城疫病毒二联RT-PCR检测方法的建立

参照国内外已发表的禽流感病毒(AIV)和新城疫病毒(NDV)的基因序列及其相关的RT-PCR检测方法,根据禽流感病毒M蛋白基因和新城疫病毒NP基因各设计一套特异性通用引物,扩增目的带分别为600bp和340 bp。通过对相关病毒检测,建立了AIV和NDV通用型二联RT-PCR检测方法。该方法具有快速、敏感、特异等优点,可为AIV和NDV的检测、流行病学调查及疫苗使用等奠定基础。     

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.     

Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum

Viruses conveyed in shipments of eggs, viral diagnostic reagents, or avian serum samples are a potential hazard for susceptible poultry. Different methods of treatment of those materials to eliminate the hazard of virulent and avirulent strains of Newcastle disease virus (NDV) or avian influenza virus (AIV) were evaluated. The NDV strains tested were more thermostable than the AIV strains. The results suggest that standard pasteurization methods would not reliably inactivate the concentrations of NDV used. beta-Propiolactone (BPL) (greater than or equal to 0.025%) inactivated NDV or AIV in allantoic fluid, but higher concentrations were needed to inactivate virus diluted in serum. Hemagglutination (HA) of NDV and AIV and hemolysis (HL) activity of NDV were reduced or eliminated by 0.4% BPL. Formalin (greater than or equal to 0.04%) inactivated either virus but adversely affected HA and HL activity. NDV or AIV was inactivated by binary ethylenimine (BEI) (0.01 M) with no adverse effect on HA or HL. Heat (56 C) or BEI (0.01 M) had no apparent effect on hemagglutination-inhibition (HI) titers of NDV and AIV antisera, the effect of formalin (0.1%) was variable, and BPL (greater than or equal to 0.25%) depressed the HI titers of both antisera. The optimum method should achieve virus inactivation without harming the treated material.     

Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak

During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures.     

Influenza virus infections in migrating waterfowl: results of a two year study in Germany

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in Germany, extensive surveillance studies were carried out between March 2003 and January 2005. More than 3.000 samples of 79 different species of wild birds (migratory and resident birds) were taken and 1.151 established pools investigated. Samples came from 80 different regions of Germany. Forty AIV isolates representing 14 combinations of eight different hemagglutinin and eight neuraminidase subtypes, among them H5 and H7, were identified. All H5 and H7 isolates were found to be of low pathogenicity. The overall incidence of the investigated pools based on virus isolation was 3,5 % for AIV, with considerable variability noted among species, season and location. All AIV were isolated from birds sampled in autumn. Most of the AIV isolates came from the resting or wintering areas of mallards breeding far north. This study adds to the understanding of the ecology of influenza viruses in wild birds and empahsizes the constant need for surveillance in times of an ongoing and expanding epidemic of highly pathogenic AI.     

Highly pathogenic avian influenza virus subtype H5N1 in Mute swans in the Czech Republic

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in the Czech Republic extensive surveillance was carried out between January and April 2006. A total of 2101 samples representing 61 bird species were examined for the presence of influenza A by using PCR, sequencing and cultivation on chicken embryos. AIV subtype H5N1 was detected in 12 Mute swans (Cygnus olor). The viruses were determined as HPAI (highly pathogenic avian influenza) and the hemagglutinin sequence was closely similar to A/mallard/Italy/835/06 and A/turkey/Turkey/1194/05. Following the first H5N1 case, about 300 wild birds representing 33 species were collected from the outbreak region and tested for the presence of AIV without any positive result. This is the first report of highly pathogenic avian influenza subtype H5N1 in the Czech Republic. The potential role of swan as an effective vector of avian influenza virus is also discussed.     

禽四种病毒多重PCR诊断技术的建立和应用

根据禽呼肠孤病毒（ARV）、禽流感病毒（AIV）、新城疫病毒（NDV）和鸡传染性支气管炎病毒（IBV）的基因保守区设计了4对特异性引物,建立了针对四种病毒的多重PCR检测体系,并对该体系进行了条件优化及特异性、敏感性试验。该体系扩增的四种病毒基因片断大小分别为199bp（ARV）、264bp（AIV）、362bp（NDV）和459bp（IBV）,且特异性、敏感性良好,能够检出1pgAIV和10PgARV、NDV、IBV的RNA。临床应用结果证明,该体系具有很高的实用价值和应用前景。