Diagnosis of duck plague in waterfowl by polymerase chain reaction

A recently developed polymerase chain reaction (PCR) assay was used for diagnosis of duck plague in waterfowl tissues from past and current cases of waterfowl mortality and to identify duck plague virus in combined cloacal/oral-pharyngeal swab samples from healthy mallards (Anas platyrhynchos) after a disease outbreak. The PCR was able to detect viral DNA from all the individual or pooled tissues assayed from 10 waterfowl, including liver and spleen samples from three Muscovy ducks (Cairina moschata domesticus) that did not yield virus isolates. The strong staining intensity of the PCR products from the waterfowl tissues indicated that large amounts of virus were present, even when virus was not isolated. Duck plague DNA was also detected in a cloacal swab sample from a wood duck (Aix sponsa) carcass submitted for diagnosis. The PCR assay identified duck plague DNA in 13 swab samples that produced virus isolates from carrier mallards sampled in 1981 after a duck plague die-off. The duck plague PCR clearly demonstrated the ability to quickly diagnose duck plague in suspect mortality cases and to detect virus shed by carrier waterfowl.     

Outbreak of duck plague (duck herpesvirus enteritis) in numerous species of captive ducks and geese in temporal conjunction with enforced biosecurity (in-house keeping) due to the threat of avian influenza A virus of the subtype Asia H5N1

The continuing westward spread of avian influenza A virus of the subtype H5N1 in free-living and domestic birds forced the European Union and the German federal government to enhance all biosecurity measures including in-house keeping of all captive birds from October 20 to December 15, 2005. Movement of captive ducks and geese of many different species from a free-range system to tight enclosures and maintenance for prolonged times in such overcrowded sheds resulted in pronounced disturbance of natural behaviour, interruption of mating and breeding activities and possibly additional stress. Under these conditions the birds developed signs of severe disease and enhanced mortality twentyfour days later. A total of 17 out of 124 (14%) adult birds and 149 out of 184 year-old birds (81 %) died during the outbreak. A herpesvirus was isolated from many organs of succumbed ducks and geese that was identified as a duck plague herpesvirus by cross neutralization test using known antisera against duck plague virus. The published host range of duck plague comprises 34 species within the order Anseriformes. We report here on additional 14 species of this order that were found to be susceptible to duck plague virus. The exact source of the herpesvirus could not identified. However, low antibody titres in some ducks at day of vaccination indicate that at least some of the birds were latently infected with a duck plague herpesvirus. The remaining healthy appearing birds were subcutaneously vaccinated with a modified live duck plague vaccine (Intervet, Boxmeer, NL) that stopped losses and resulted in seroconversion in most of the vaccinated birds.     

Latency sites and reactivation of duck enteritis virus

Duck virus enteritis (DVE) is a contagious disease caused by herpesvirus in waterfowl populations. Recovered birds become carriers and shed the virus periodically. Reactivation of latent duck enteritis virus (DEV) has been implicated in outbreaks of DVE in domestic and migrating waterfowl populations. In this study, the sites for virus latency were determined in white Pekin ducks infected with the DEV-97 strain. At 3 wk postinfection, infectious virus was not detectable in tissues or cloacal swabs (CSs). At 7 and 9 weeks postinfection, the viral DNA was detected by polymerase chain reaction in the trigeminal ganglia (TG), suggesting that the virus is latent. Viral DNA was detected in the peripheral blood lymphocytes (PBL), spleen, thymus, bursa, and CSs only after in vitro cocultivation. In vivo virus reactivation was demonstrated when dexamethasone or a combination of dexamethasone and cyclophosphamide was inoculated in latently infected ducks. The reactivation of DEV occurred without any clinical evidence of the disease, but the virus was detected in PBL and CSs. We conclude from this study that DEV establishes latency in TG and lymphoid tissues including PBL.     

Laboratory confirmed outbreaks of duck virus enteritis (duck plague) in the United Kingdom from 1977 to 1982

From 1977 to 1983 the Central Veterinary Laboratory, Weybridge confirmed 19 outbreaks of duck virus enteritis in the United Kingdom. All the outbreaks involved collections of captive waterfowl and there were no reported cases in commercial ducks. In many instances the disease was associated with contact with migrating waterfowl, particularly male mallards (Anas platyrhynchos). Muscovy ducks (Cairina moschata) and related species appeared to be particularly susceptible. The most sensitive system for isolating the virus was muscovy duck embryo tissue cultures. The duckling inoculation test was found to be the most reliable method of confirming the disease.     

The occurrence of virus enteritis (duck plague) in wild birds]

296 samples from wild birds of 15 species were incorporated into long term exploration of duck plague on ducks in farms. By using virological and serological standard methods 9 virus carriers and 20 serum samples showing positive antibody titers could be detected. The epidemiology as well as the relation of the incidence of duck plague in wild birds and farm poultry is discussed.     

Superinfection in ducks persistently infected with duck plague virus

Superinfections with homologous or heterologous strains of duck plague virus resulted in the deaths of birds persistently infected with duck plague virus. Not all birds that were superinfected died. Protection against mortality depended on the route of exposure, strain of the initial duck plague virus, and strain of the superinfecting virus.     

Assessment of the immune response to duck plague vaccinations

An experiment was conducted to assess the immune responses of ducks to duck plague (DP) vaccinations employing one commercial and one laboratory-adapted (LA) DP vaccines. Virus neutralisation and leucocyte migration-inhibition tests were conducted at regular intervals before and after vaccinations. Similarly, ducks in vaccinated and control groups were subjected to challenge infection with virulent DP virus.The commercial vaccine yielded a poor immune response and partial protection on challenge whereas satisfactory responses were obtained in ducks receiving two doses of LA vaccine. The humoral as well as cellular factors were stimulated indicating possible involvement of both the immune responses in the protection from duck plague.     

Pathological and immunological study of goose embryos experimentally infected with duck plague virus

A total of 240 embryonated goose eggs obtained from two susceptible flocks were used. Half of the eggs were inoculated into the allantoic cavity with a virulent strain (7593) of duck plague virus isolated from an acute outbreak, and the other half were inoculated with the attenuated vaccine virus (KAPEVAC). Ten, 100 or 1000 CPU/0.1 ml virus were given on days 12 and 20 of incubation. Embryos that died and surviving embryos killed at 5-day intervals were examined by light and electron microscopy. The yolk and the serum of embryos that survived until hatching were assayed for antibody content. Lymphocytes separated from the blood were used for the immuno-rosette formation and lymphocyte stimulation tests. Pathomorphological changes indicative of virus replication occurred in the liver, kidney, myocardium, gizzard muscle and chorioallantoic membrane (CAM) of the embryos in the case of both virus strains. The time of onset and severity of these changes and the time and rate of embryonic mortality depended on the virulence of the strain used for inoculation, the virus dose and the time of inoculation. Virus-neutralizing (VN) antibodies were demonstrable neither in the yolk nor in the serum of goslings exsanguinated after hatching. The lymphocytes recognized the virus antigen in the in vitro cellular tests and responded to it with blastogenic transformation. As opposed to adult birds, in the embryos duck plague virus infection did not cause damage to the digestive tract mucosa and the lymphoid organs.     

Identification of duck plague virus by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.     

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.     

Isolation from ducks of a hypervirulent strain of duck plague virus and an avian type 6 paramyxovirus

This paper describes the isolation and identification of a duck plague virus (DP) and a paramyxovirus (PMV₆), from the livers and intestines collected in 4-month old mule ducks, under fattening, exhibiting 75% mortality and necrotic-haemorrhagic gross lesions. These viruses were isolated in specific pathogen free (SPF) muscovy duck eggs and SPF chicken eggs respectively. Then the DP virus was adapted to duck and chicken fibroblasts. The disease was reproduced in 2-week old SPF muscovy ducklings, intramuscularly inoculated with the previous organs, as well as in contact ducks. From them, only the DP virus was isolated again. Experimentally the intramuscular inoculation of the duck plague French vaccinal strain, 4 h post contact, did not prevent the disease and did not decrease its severity.

Regarding the DP virus, the typical signs and lesions observed in experimentally infected muscovy ducks as well as the presence of intranuclear inclusions of the epithelial cells of their oesophagus, intestines, bursa of Fabricus and liver on the one hand, and on the other hand, of the epithelial cells of the duck egg chorio-allantoïc membrane and fibroblasts inoculated with the samples first defined, allowed the characterization of the virus. Direct electron microscopy, as well as the results of seroneutralization tests with different specific avian Herpes virus antisera confirmed the DP virus identification. Moreover the DP isolate was not antigenically different from the serotype actually known.

The haemagglutinating virus (PMV₆) was characterized by direct electron microscopy as well as with 18 specific avian Myxovirus antisera; its identification was confirmed too by the specific seroconversion observed 4 weeks post-inoculation of this virus, in 11 weeks old SPF muscovy ducklings.

Finally an assay was carried out to appreciate the pathogenicity of theses viruses inoculated either separately or associated. It showed the high pathogenicity of the DP strain. The PMV₆ was apathogenic and no synergic effect with the DP virus was demonstrated. It appears to be the first isolation of PMV₆ in France, to our knowledge. The epidemiological circumstances related to theses isolations are discussed. The failure of the emergency vaccination in contact ducks, might be attributed to the high virulence of the DP strain.     

Vertical transmission of duck plague virus (DPV) by apparently healthy DPV carrier waterfowl

Duck plague virus (DPV) was transmitted vertically in muscovy, pekin, and mallard ducks that were persistently infected with the LA-SD-73, MSN-WI-77, or CO-WI-73 isolates of DPV. The effects of vertical transmission on the fertility and hatchability of eggs laid by DPV carrier ducks varied with the DPV isolate and duck species. Fertility was reduced significantly only in eggs laid by MSN-WI-77 virus carrier pekin and muscovy ducks. The hatchability of eggs laid by DPV carrier mallards and muscovies was significantly reduced from that of uninfected control ducks. All ducklings tested that hatched from eggs laid by DPV carrier waterfowl shed DPV in the feces. The DPV carrier ducklings shed DPV in small amounts. Vertical transmission of DPV in domestic flocks can lower fertility and hatchability. In wild waterfowl, vertical transmission may be a means of virus perpetuation from generation to generation.     

Association between outbreaks of highly pathogenic avian influenza subtype H5N1 and migratory waterfowl (family Anatidae) populations

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 threatens poultry production and human health. Understanding the role that migratory waterfowl play in introducing and maintaining this infection is critical to control the outbreaks. A study was conducted to determine if the occurrence of HPAI subtype H5N1 outbreaks in village poultry in Romania, 2005-2006, was associated with proximity to populations of migratory waterfowl. Reported outbreaks--which could be grouped into three epidemic phases--and migratory waterfowl sites were mapped. The migratory waterfowl site closest to each outbreak was identified. The distances between outbreaks occurring in phase 1 and 2 of the epidemic and the closest migratory waterfowl site were significantly (P<0.001) less than in phase 3, but these distances were only useful in predicting when outbreaks occurred during phase 1 (October-December, 2005) of the epidemic. A spatial lag (rho=0.408, P=0.041) model best fit the data, using distance and [distance]*[distance] as predictors (R2=0.425). The correlation between when outbreaks were predicted to occur and when they were observed to occur was 0.55 (P=0.006). Results support the hypothesis that HPAI virus subtype H5N1 infections of village poultry in Romania during the autumn of 2005 might have occurred via exposure to migratory populations of waterfowl.     

Rapid diagnosis of duck plagues virus infection by loop-mediated isothermal amplification

Duck virus enteritis is a serious disease among farmed and free-living ducks (Anatidae) and a constant threat to the commercial duck industry in China. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to rapidly detect and diagnose duck plague virus (DPV) in both farmed and wild waterfowl, and compared with polymerase chain reaction (PCR) method and real-time PCR method in accuracy, sensitivity and specificity. A set of four specific primers was successfully designed to recognize six distinct genomic sequences of UL6 protein from DPV, including one forward inner primer, one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 61.5 °C and 60 min, respectively. Comparative experiments showed that LAMP assay was a simple, rapid, accurate, sensitive and specific method for detecting DPV, and was superior to PCR assay in sensitivity and specificity for DNA amplification. In addition, challenge tests indicated the newly developed LAMP method was more sensitive for the diagnosis of DPV infection than virus isolation and PCR. LAMP assay would be a good alternative method for on-farm disease diagnosis.     

应用ELISA检测鸭瘟抗体

应用ＥＬＩＳＡ间接法检测鸭瘟抗体,具有简单、快捷、特异的优点。小鸭免疫鸭瘟弱毒疫苗２周后抗体水平明显上升,４周达最高峰。用１０００个致死量鸭瘟强毒攻击,结果,抗体效价与免疫保护无明显的相关性。     

商品肉鸭鸭瘟病毒的分离与鉴定

采用鸭胚成纤维细胞培养从山东和北京两地暴发的鸭瘟临床病例中分离到两株鸭肠炎病毒（DEV），分别命名为SD和BJ。以单抗介导的间接免疫荧光（IFA）检测方法，对两个分离株感染细胞滴片进行间接IFA检测，可见感染细胞内有明显的蓝绿色荧光。试验感染7日龄北京鸭可引起鸭瘟的典型临床症状．死亡率为100％（3／3），取试验感染死亡鸭肝脏、法氏囊和脑组织等制备石蜡包埋切片，利用单抗进行免疫组化检验，除脑组织外均检测到病毒抗原。根据在GenBank上已发表的DEV两段序列设计两对引物，采用聚合酶链式反应（PCR）对野毒sD株人工感染鸭肝脏和BJ珠自然发病鸭肝脏病科提取核酸为模板进行扩增，得到预期大小为765bp和1954bp的目的片段，对长片段进行测序，与发表序列进行比较，毒株间的碱基序列同源性达到99．73％。     

番鸭小鹅瘟病直接荧光诊断试剂的研制

将抗番鸭GPV单抗腹水采用透析法标记异硫氰酸荧光素（FITC）,制备成抗GPV荧光抗体,研制检测GPV抗原的直接免疫荧光诊断方法。结果显示GPV荧光抗体仅与GPV阳性的组织切片或细胞呈现特异性荧光,与番鸭细小病毒（MPV）、番鸭呼肠孤病毒（MDRV）、鸭副粘病毒（DPMV）、鸭病毒性肝炎病毒（DHV）和正常番鸭组织切片不反应;与间接荧光方法的符合率为92．9％。表明GPV荧光抗体具有较好的特异性、敏感性和准确性,可用于临床快速诊断番鸭小鹅瘟病。     

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.