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Avian influenza virus (AIV) variants emerge frequently, which challenges rapid diagnosis. Appropriate diagnosis reaching the sub- and pathotype level is the basis of combatting notifiable AIV infections. Real-time RT-PCR (RT-qPCR) has become a standard diagnostic tool. Here, a total of 24 arrayed RT-qPCRs is introduced for full subtyping of 16 hemagglutinin and nine neuraminidase subtypes of AIV. This array, designated Riems Influenza A Typing Array version 2 (RITA-2), represents an updated and economized version of the RITA-1 array previously published by Hoffmann et al. RITA-2 provides improved integration of assays (24 instead of 32 parallel reactions) and reduced assay volume (12.5 µL). The technique also adds RT-qPCRs to detect Newcastle Disease (NDV) and Infectious Bronchitis viruses (IBV). In addition, it maximizes inclusivity (all sequences within one subtype) and exclusivity (no intersubtypic cross-reactions) as shown in validation runs using a panel of 428 AIV reference isolates, 15 reference samples each of NDV and IBV, and 122 clinical samples. The open format of RITA-2 is particularly tailored to subtyping influenza A virus of avian hosts and Eurasian geographic origin. Decoupling and re-arranging selected RT-qPCRs to detect specific AIV variants causing epizootic outbreaks with a temporal and/or geographic restriction is possible.

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 2 4 and 2 1 for AIV and NDV, respectively, and to an IDEXX antibody titer of 10 3 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

Background: Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks., Results: The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 10 1 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won't cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%., Conclusion: The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

Chickens in live bird markets (LBMs) from six different regions of Tanzania were surveyed for Newcastle disease (ND) virus (NDV) and avian influenza virus in 2012. ELISA-based serology, virus isolation, and characterization, including pathotyping was conducted. Virulent NDV was isolated from almost 10% of the tested samples, with two distinct genotypes being detected. One genotype was similar to recent viruses circulating in Kenya and Uganda, which share a northern border with Tanzania. Several viruses of this genotype were also isolated from Tanzania in 1995, the last time surveillance for NDV was conducted in the country. The second genotype of virus from Tanzania was closely related to viruses from Mozambique, a southern neighbor, and more distantly to viruses from South Africa, Botswana, and several European countries. Partial fusion gene sequence from the isolated viruses showed identical fusion cleavage sites that were compatible with virulent viruses. Selected viruses were tested by the intracerebral pathogenicity index, and all viruses tested had scores of >1.78, indicating highly virulent viruses. Serology showed only a third of the chickens had detectable antibody to NDV, suggesting that vaccination is not being commonly used in the country, despite the availability of vaccines in agricultural-related markets. All samples were taken from clinically healthy birds, and it is believed that the birds were sold or slaughtered before showing ND clinical signs. LBMs remain a biosecurity risk for farmers through the return of live infected birds to the farm or village or the movement of virus on fomites, such as uncleaned wooden cages.

A one-step multiplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and quantification of four avian respiratory viruses: avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV). In comparison with the singleplex rRT-PCR, the specificity, the sensitivity and the reproducibility of the new assay were evaluated and validated using 70 clinical samples. The optimal cutoff point, the corresponding limit of quantification (LoQ) and the limit of detection (LoD) were statistical established based on receiver operating characteristic (ROC) curve analysis. The results showed that the multiplex assay presents higher sensitivity and specificity. Correlation coefficients (R 2 ) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95% LoDs of multiplex assay were in the range [3-19] copies genomic/ µl, and its corresponding cutoff cycles were in the range [34.16-36.59]. No competitive inhibition for the detection of the four targets and no specific amplification or cross reactivity with other tested viruses was observed. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. All identified samples by the multiplex rRT-PCR assay proved to be 100% concordant with the results of the singleplex assays. The results achieved showed that the multiplex assay is very suitable as a routine laboratory test for rapid and specific detection and quantification of co-infections in field samples., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Acute respiratory tract infections are of paramount importance in the poultry industry. We developed an xTAG bead assay for the simultaneous detection and discrimination of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The assay lacked nonspecific reactions with other common avian viruses and the limit of detection was 6.75 × 10 2 - 3.52 × 10 3 copies/μL. We examined 60 clinical specimens and found 18 positive for respiratory viruses. Our result demonstrated that xTAG-multiplex PCR method is a high-throughput, rapid, specific and sensitive assay for use in epidemiological studies and clinical detection of avian respiratory pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combination with MiSeq platform was applied to target negative- and positive-sense single-stranded RNA viral sequences. This method allowed successful sequence assembly of full or near full length avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) viral genome. Moreover, SISPA analysis applied to unknown clinical cases of mixed viral infections produced genome assemblies comprising 98% NDV and 99% of IBV genomes. Complete or near complete virus genome sequence was obtained with titers at or above 10 4.5 EID 50 /ml (50% embryo infectious dose), and virus identification could be detected with titers at or above 10 3 EID 50 /ml. Taken together, these studies demonstrate a simple template enrichment protocol for rapid detection and accurate characterization of avian RNA viruses., (Published by Elsevier Inc.)

Urbanization is intensifying worldwide, and affects the epidemiology of infectious diseases. However, the effect of urbanization on natural host-pathogen systems remains poorly understood. Urban ducks occupy an interesting niche in that they directly interact with both humans and wild migratory birds, and either directly or indirectly with food production birds. Here we have collected samples from Mallards (Anas platyrhynchos) residing in a pond in central Uppsala, Sweden, from January 2013 to January 2014. This artificial pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. Nine hundred and seventy seven (977) fecal samples were screened for RNA viruses including: influenza A virus (IAV), avian paramyxovirus 1, avian coronavirus (CoV), and avian astrovirus (AstroV). This intra-annual dataset illustrates that these RNA viruses exhibit similar annual patterns to IAV, suggesting similar ecological factors are at play. Furthermore, in comparison to wild ducks, autumnal prevalence of IAV and CoV are lower in this urban population. We also demonstrate that AstroV might be a larger burden to urban ducks than IAV, and should be better assessed to demonstrate the degree to which wild birds contribute to the epidemiology of these viruses. The presence of economically relevant viruses in urban Mallards highlights the importance of elucidating the ecology of wildlife pathogens in urban environments, which will become increasingly important for managing disease risks to wildlife, food production animals, and humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis μ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

Newcastle disease (ND) is a contagious and often fatal disease that affects over 250 bird species worldwide, and is caused by infection with virulent strains of avian paramyxovirus-1 (APMV-1) of the family Paramyxoviridae, genus Avulavirus. Infections of poultry with virulent strains of APMV-1 (Newcastle disease virus) are reportable to the World Organization for Animal Health (OIE). Vaccination of poultry species is a key measure in the control of ND. Other APMV-1 viruses of low virulence, which are not used as vaccines, are also often isolated from wild bird species. The APMV-1 virus, like avian influenza virus (AIV), is a hemagglutinating virus (HA) and able to agglutinate chicken red blood cells (RBC). Because the clinical presentation of ND can be difficult to distinguish from disease caused by AIV, techniques for differential diagnosis are essential, as well as the ability to detect mixed infections. When an HA positive virus is detected from virus isolation, additional assays can be performed to determine which virus is present. Both antigenic and molecular methods are necessary as some virulent ND viruses from cormorants in the USA after 2002 have lost their ability to hemagglutinate chicken RBC and molecular methods are needed for identification.

Although it is well established that wild birds, such as cormorants, carry virulent avian paramyxovirus serotype 1 (APMV-1; causative agent of Newcastle disease) and avian influenza virus (AIV), the prevalence of these viruses among Double-crested Cormorants (Phalacrocorax auritus) in the Great Lakes region of North America has not been rigorously studied. We determined the prevalences of APMV-1 and AIV in Double-crested Cormorants from the interior population of eastern North America. From 2009 to 2011, oropharyngeal and cloacal swabs and serum samples were collected from 1,957 individual Double-crested Cormorants, ranging from chicks to breeding adults, on breeding colony sites in Michigan, Wisconsin, and Mississippi, USA, and Ontario, Canada, as well as on the wintering grounds of migratory populations in Mississippi, USA. Prevalence of antibodies to APMV-1 in after-hatch year birds was consistently high across all three years, ranging from 86.3% to 91.6%. Antibody prevalences in chicks were much lower: 1.7, 15.3, and 16.4% in 2009, 2010, and 2011, respectively. Virulent APMV-1 was detected in six chicks sampled in 2010 in Ontario, Canada. Only one adult was positive for AIV-specific antibodies and five individuals were positive for AIV matrix protein, but the latter were negative for H5 and H7 AIV subtypes. We provide further evidence that Double-crested Cormorants play an important role in the maintenance and circulation of APMV-1 in the wild, but are unlikely to be involved in the circulation of AIV.

A new technique that uses gold immunochromatographic strips enhances the detection sensitivity by inducing the clustering of additional gold nanoparticles (AuNPs) around the immunogold particles immobilized on nitrocellulose strips. The additional AuNPs provide an intense signal that can be detected by the naked eye. The AuNPs were synthesized and conjugated to monoclonal antibodies using self-assembly. Other antibodies were immobilized in a defined detection zone on the nitrocellulose membrane. The detection principle is based on a "sandwich" immunoreaction, where gold-labeled antibodies serve as signal vehicles. To improve the sensitivity of the strips, we use a mixture of 1% HAuCl4 and 10 mmol L(-1) NH2OH·HCl to "enlarge" the gold nanoparticles. The detecting limits of Avian influenza virus (AIV) and Newcastle disease virus (NDV) are significantly increased. Compared with commercial test strips, this method is 100-fold more sensitive. This method is easy to perform and can be carried out on-site in test laboratories., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Background: Active and passive surveillance for avian influenza virus (AIV) and Newcastle disease virus (NDV) is widespread in commercial poultry worldwide, therefore optimization of sample collection and transport would be valuable to achieve the best sensitivity and specificity possible, and to develop the most accurate and efficient testing programs. A H7N2 low pathogenicity (LP) AIV strain was selected and used as an indicator virus because it is present in lower concentrations in swabbings and thus requires greater sensitivity for detection compared to highly pathogenic (HP) AIV. For similar reasons a mesogenic strain of NDV was selected. Using oro-pharyngeal and cloacal swabs collected from chickens experimentally exposed to the viruses we evaluated the effects of numerous aspects of sample collection and transport: 1) swab construction material (flocked nylon, non-flocked Dacron, or urethane foam), 2) transport media (brain heart infusion broth [BHI] or phosphate buffered saline [PBS]), 3) media volume (2 ml or 3.5 ml), 4) transporting the swab wet in the vial or removing the swab prior to transport, or transporting the swab dry with no media, and 5) single swabs versus pooling 5 or 11 swabs per vial., Results: Using real-time RT-PCR (rRT-PCR), virus isolation (VI) and commercial antigen detection immunoassays for AIV we observed statistically significant differences and consistent trends with some elements of sample collection and transport; media, dry transport and swab construction. Conversely, the number of swabs pooled (1, 5 or 11) and whether the swab was removed prior to transport did not impact virus detection. Similarly, with NDV detection by both VI and rRT-PCR was not affected by the numbers of swabs collected in a single vial (1, 5 or 11)., Conclusions: We observed that flocked and foam swabs were superior to non-flocked swabs, BHI media was better than PBS, and transporting swabs wet was better for virus recovery and detection than transporting them dry. There was no observable difference in detection whether the swab was removed prior to transport or left in the vial. Also, with both AIV and NDV, there was no observed difference in virus detection between pools of 1, 5 or 11 swabs.

Newcastle disease virus (NDV), member of the Paramyxoviridae family and avian influenza virus (AIV), member of the Orthomyxoviridae family, are two main avian pathogens causing serious economic problems in poultry health. Both are enveloped, single-stranded, negative-sense RNA viruses and cause similar symptoms, ranging from sub-clinical infections to severe diseases, including decrease in egg production, acute respiratory syndrome, and high mortality. Similar symptoms hinder the differentiation of infection with the two viruses by standard veterinary procedures like clinical examination or necropsy. To overcome this problem, we have developed a new duplex real-time PCR assay for the detection and differentiation of these two viruses. Eighteen NDV strains, fourteen AIV strains, and twelve other (negative control) strains viruses were isolated from allantoic fluids of specific pathogen-free (SPF), embryonated eggs. Four-weeks-old SPF chickens were co-infected with both viruses (NDV - LaSota and AIV - H7N1). Swabs from cloaca and trachea were collected and examined. The results obtained in this study show that by using duplex real-time PCR, it was possible to detect and distinguish both viruses within less than three hours and with high sensitivity, even in case a bird was co-infected. Additionally, the results show the applicability of the real-time PCR assay in laboratory practice for the identification and differentiation of Newcastle disease and influenza A viruses in birds.

Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Avian influenza viruses (AIVs) and Newcastle disease viruses (NDVs) co-circulate in the poultry population in China. These viruses cause repeated disease outbreaks that exhibit similar clinical symptoms and epidemiological patterns. H5 and H9 influenza viruses are the major pathogens infecting poultry stocks. Recently, H3 AIV (one of the main subtypes in waterfowl) has become endemic in chickens. A multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was designed for simultaneous detection and differentiation of avian H3, H5, H9 subtype AIVs and NDVs. Four primer sets were evaluated, three of which specifically targeted the hemagglutinin genes of H3, H5 and H9 AIVs, while the other targeted the NDV fusion gene. The sensitivity and specificity of the mRT-PCR assay was determined. The assay detected the major clades or genotypes of all of the reference AIVs and NDVs currently circulating in China. In addition, the mRT-PCR results obtained from screening 380 clinical swabs and 12 experimental tracheal samples were consistent with those obtained using conventional virus isolation methods. The mRT-PCR assay was established successfully for the detection and differentiation of avian H3, H5, and H9 subtype AIVs and NDVs. The method should, therefore, provide a valuable diagnostic tool for these infections., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Virus isolation rates for influenza A virus (FLUAV) and Avian paramyxovirus serotype 1 (APMV-1) from wild bird surveillance samples are lower than molecular detection rates for the specific viral genomes. The current study was conducted to examine the possibility of increased virus isolation rates from real-time reverse transcription polymerase chain reaction (real-time RT-PCR) using alternative virus isolation substrates such as embryonating duck eggs (EDEs), embryonating turkey eggs (ETEs), Madin-Darby canine kidney (MDCK) cell cultures, and African green monkey kidney (Vero) cell cultures. Rectal swabs of birds in the orders Anseriformes and Charadriiformes were tested by real-time RT-PCR for the presence of FLUAV and APMV-1 genomes, and virus isolation (VI) was attempted on all real-time RT-PCR-positive samples. Samples with threshold cycle (Ct) ≤ 37 had VI rates for FLUAV of 62.5%, 50%, 43.8%, 31.5%, and 31.5% in embryonating chicken eggs (ECEs), ETEs, EDEs, MDCK cells, and Vero cells, respectively. A higher isolation rate was seen with ECEs compared to either cell culture method, but similar isolation rates were identified between the different embryonating avian eggs. Virus isolation rates for APMV-1 on samples with real-time RT-PCR Ct ≤ 37 were 75%, 100%, 100%, 0%, and 37.5% in ECEs, ETEs, EDEs, MDCK cells, and Vero cells, respectively. Significantly higher VI rates were seen with ECEs as compared to either cell culture method for all real-time RT-PCR-positive samples. Because of the limited availability and high cost of ETEs and EDEs, the data support the continuing usage of ECEs for primary isolation of both FLUAV and APMV-1 from real-time RT-PCR-positive wild bird surveillance samples.

An avian influenza (AI) surveillance was undertaken in Maharashtra state, India during the period 2010-2011. There are no reports of AI surveillance in emus from India. A total of 202 blood samples and 467 tracheal and cloacal swabs were collected from eight emu farms. A hemagglutination inhibition (HI) assay was performed for detection of antibodies against AI H5N1, H7N1, H9N2, and avian paramyxovirus type 1 (APMV-1) viruses. A microneutralization (MN) assay was performed to confirm the presence of neutralizing antibodies against AI H9N2 and to compare with HI assays. A total of 28.2% and 28.7% of samples were positive for antibodies against AI H9N2 by HI and MN assays, respectively, using > or = 1:40 as a cut-off titer; 15.3% samples were positive for APMV-1 by HI assay using a > or = 1:10 cut-off titer. Seropositivity of AI H9N2 was nil in the grower (<1 yr) age group and highest (78%) in the breeder (2-3 yr) age group, whereas seropositivity against APMV-1 was observed in all age groups. Performance of both HI and MN assays was similar, suggesting the utility of using the MN assay along with HI assay for surveillance studies. This is the first report of the seroprevalence of AI H9N2 and APMV-1 in emus in India.

Herpesvirus isolations from peripheral white blood cells of 253 White Storks (Ciconia ciconia) were obtained during a long-term study (1983 to 2001). The storks lived for a few months to 20 years at four rehabilitation centres. Isolates were obtained from 83 of 253 storks. This herpesvirus is indigenous for storks and unrelated to any other avian herpesvirus. Significantly more herpesvirus isolates were obtained during spring than in autumn samplings. The intervals between the first and last virus isolation ranged from 1 to 15 years. Herpesvirus isolates were simultaneously obtained from white blood cells and from pharyngeal swabs of four of 34 storks but not from cloacal swabs. Neutralizing antibodies to stork herpesvirus were detected in 178 of 191 examined blood plasma samples. Neutralizing antibodies against stork herpesvirus did not correlate with herpesvirus viraemia. The results further substantiate the persistence of herpesvirus in White Storks and underline the previously unrecorded long periods of virus and antibody presence. Virulent avian paramyxovirus type 1 (APMV-1; Newcastle disease virus) was isolated from white blood cells during 1992 and 1993 from four healthy migrating storks, and possessed virulence markers on the cleavage site of the H and F genes. These properties resemble the NE type of APMV-1. Haemagglutination inhibition antibodies against APMV-1 were detected in 16 of 191 blood plasma samples. Avian influenza A virus was not isolated and antibodies against subtypes H5 and H7 were not detected.

Tracheal and cloacal swabs as well as blood samples from 408 feral urban (Columba livia forma domestica) and 170 free-ranging wood pigeons (Columba palumbus) in Germany were tested for infection with avian influenza viruses (AIVs). Neither influenza A virus was isolated in the swab samples nor influenza A virus RNA detected using real-time reverse transcriptase-PCR (RT-qPCR). In three urban feral pigeons, avian paramyxovirus (APMV) serotype 1 was isolated. Two of 123 serum samples from hunted free-ranging wood pigeons contained specific antibodies against influenza A virus but not against the subtypes H5 and H7. In conclusion, our study indicates that even after the occurrence of zoonotic highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the area of investigation in Germany, pigeons do not play a major part in the transmission of influenza viruses. The risk of AIV infection for humans from urban and free-ranging wood pigeons is, if at all, of minimal importance.

Waterfowl are the natural reservoirs of avian influenza viruses (AIVs), from which the virus can spread to other species including humans, poultry, and swine. For the surveillance of AIV in their natural reservoir, most laboratories initially screen the samples using real-time reverse-transcriptase-polymerase chain reaction because of its high speed and sensitivity. Thereafter, virus isolation is used to isolate viruses from positive samples. Although many studies point to the need of testing both cloacal and oropharyngeal (OP) samples in AIV surveillance programs, most laboratories focus only on cloacal samples. This study was undertaken to determine the utility of OP samples as target samples in AIV surveillance programs under a strict cold chain of samples from the field to the laboratory. A total of 16 AIV (15.1%) were isolated from the 106 OP samples examined. Upon subtyping, four hemagglutinin subtypes (H1, H3, H4, and H6) and three neuraminidase subtypes (N1, N2, and N8) were detected in nine different combinations. Mixed infection with two different subtypes was found in four samples. No AIVs were isolated from the corresponding cloacal samples. These results highlight the fact that testing of properly frozen OP samples could add value to the understanding of the epidemiology and ecology of AIV in waterfowl populations.

Several studies have shown that migratory birds play an important role in the ecology, circulation and dissemination of pathogenic organisms. In October 2006, a health status evaluation was performed on a large population of migratory birds passing through the territory of Ustica (Italy), an island located on the migration route of many species of birds to Africa, and various laboratory tests were conducted. In total, 218 faecal swabs and the internal organs of 21 subjects found dead in nets were collected for bacteriological and virological examination, including avian influenza and Newcastle disease. In addition, 19 pooled fresh faecal samples were collected for mycological examination. The bacteriological analysis produced 183 strains belonging to 28 different species of the Enterobacteriaceae family. In particular, Salmonella bongori, Yersinia enterocolitica and Klebsiella pneumonia strains were isolated. Almost all of the isolates were susceptible to sulphamethoxazole/trimethoprime (99.4%), cefotaxime (98.9%), nalidixic acid (96.7%), chloramphenicol (95.6%), and tetracycline (93.4%). Alternatively, many strains were resistant to ampicillin (42.6%), amoxicillin-clavulanic acid (42.6%), and streptomycin (43.7%). According to reverse transcriptase-polymerase chain reaction analysis, all of the samples were negative for the M gene of avian influenza virus. Moreover, isolation tests conducted on specific pathogen free eggs were negative for avian influenza and Newcastle disease. Several hyphomycetes and yeasts belonging to different genera were present in the specimens, and Cryptococcus neoformans was observed in a pooled faecal sample. Antibiotic resistance in wildlife can be monitored to evaluate the impact of anthropic pressure. Furthermore, migratory birds are potential reservoirs of pathogenic agents; thus, they can be regarded as sentinel species and used as environmental health indicators.

The persistence of 3 low-pathogenicity avian influenza viruses (LPAIV) (H4N6, H5N1, and H6N8) and one human influenza virus (H1N1) as well as Newcastle disease virus (NDV) and enteric cytopathogenic bovine orphan (ECBO) virus was investigated in lake sediment, duck feces, and duck meat at 30, 20, 10, and 0°C using a germ carrier technique. Virus-loaded germ carriers were incubated in each substrate, and residual infectivity of the eluted virus was quantified on cell culture after regular intervals for a maximum of 24 weeks. Data were analyzed by a linear regression model to calculate T(90) values (time required for 90% loss of virus infectivity) and estimated persistence of the viruses. In general, the persistence of all of the viruses was highest in lake sediment, followed by feces, and was the lowest in duck meat at all temperatures. For the avian influenza virus subtypes, T(90) values in sediment ranged from 5 to 11, 13 to 18, 43 to 54, and 66 to 394 days at 30, 20, 10, and 0°C, respectively, which were 2 to 5 times higher than the T(90) values of the viruses in the feces and meat. Although the individual viruses vary in tenacity, the survival time of influenza viruses was shorter than that of NDV and ECBO virus in all substrates. The results of this study suggest that lake sediment may act as a long-term source of influenza viruses in the aquatic habitat, while the viruses may remain infectious for extended periods of time in duck feces and meat at low temperatures, allowing persistence of the viruses in the environment over winter.

Avian influenza viruses (AIVs) are important zoonotic pathogens whose natural reservoir is waterfowl. In addition to AIV, waterfowl are often coinfected with other viruses, such as the paramyxoviruses, of which Newcastle disease virus (NDV) is of particular importance because of the highly virulent nature of certain strains of this virus for domestic poultry. In routine surveillance of waterfowl for AIV, a number of cloacal samples were encountered that were positive for AIV by real-time reverse transcription polymerase chain reaction (RT-PCR), but did not yield AIV by inoculation in embryonated chicken eggs. On further testing, these samples were also positive for NDV by conventional RT-PCR. It was hypothesized that if both NDV and AIV are present in a sample, the former may overgrow AIV yielding false-negative AIV results. Such samples were treated with chicken anti-NDV polyclonal antiserum and then inoculated in embryonated chicken eggs. Several samples were found to be positive for different subtypes of AIV, indicating that, in the presence of mixed infection with NDV and AIV, it is imperative to remove the influence of NDV, so a true picture of AIV prevalence emerges. An additional benefit is that information on the circulation of NDV in these birds sheds light on their epidemiologic and ecologic significance.

In-solution enzymatic and nonenzymatic digestion methods have been successfully implemented in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based virus identification, extending to typing/subtyping of deadly influenza viruses. However, these methods are inefficient in obtaining more precise information on surface proteins of myxovirus particles, not only the hemagglutinin and neuraminidase of influenza virus but also the hemagglutinin-neuraminidase of Newcastle disease virus (NDV). Imbalances in viral protein composition cause ion suppression of tryptic fragments from low-abundant target proteins (surface proteins), adversely affecting reproducibility of mass spectra. Additionally, the coexistence of tryptic peptides from several proteins requires sophisticated statistical solutions for precise result interpretations. To circumvent these, we apply detergent-based (gel-free) partitioning of whole viruses into soluble surface proteins and insoluble virus materials, using differential centrifugation. MALDI-TOF or MALDI-TOF/TOF MS was applied to analyze tryptic peptides from separated viral proteins. In this study, we achieved type/subtype of avian influenza virus (AIV) within 5 h, based on 4 major proteins, by significantly reducing ion suppression and signal overlap from various protein sources. Hence, our approach can both yield dependable results and allow Web-based search engines to be directly employed, obviating the need for additional statistical strategy. Additionally, we demonstrate the utility of the method using NDV.

A germ carrier technique was adapted for the determination of the persistence of influenza viruses in moist environments. The technique was employed with 3 low pathogenic avian influenza viruses (H4N6, H5N1, and H6N8), one human influenza virus (H1N1), and two model viruses (NDV and ECBO) in lake water at five different temperatures (30, 20, 10, 0, and -10°C). Viral quantitation was carried out at regular intervals on cell culture for a maximum duration of 16 weeks. Serial data were analyzed by linear regression model to calculate T-90 values (time required for one log reduction in the virus titer). Persistence of all of the viruses was highest at -10°C followed by 0, 10, 20, and 30°C. At -10°C, the single freeze-thaw cycle resulted in an abrupt decline in the virus titer, followed by long term persistence. Generally, influenza viruses persisted shorter than model viruses while ECBO has the highest survival time in lake water. Individual influenza viruses differed in their persistence at all temperatures. The findings of the present study suggest that AIV can remain infectious in lake water for extended periods of time at low temperatures., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Since wild ducks constitute a vital element in the epizootiology of avian influenza viruses (AIVs) as well as avian paramyxoviruses (APMVs) and play a key role in the ecology and inter-species transmission of these viruses, it is crucial to elucidate the diversity and prevalence of these viruses within these bird populations. This report shows the presence, antigenic diversity, and inter-annual prevalence variations of AIVs in apparently healthy northern pintail (Anas acta) wintering in Japan. We also provide evidence that this host carries APMV-1: Newcastle disease virus (NDV) and other haemagglutinating viruses. Composite samples (n=2381) of fresh fecal materials were collected from northern pintail during November 2007-March 2008 at different locations of Tohoku district, main Island, Japan. We isolated 47 haemagglutinating viruses, out of which 25 were identified as AIVs, representing 9 combinations of 5 different haemagglutinin (HA) and 6 neuraminidase (NA) subtypes. Both H5 and H7 subtypes were identified and found to be low pathogenic. A further 11 viruses were grouped into APMV-1 (NDV). The rest of the viruses (n=11) remained to be identified. Some of the HA subtypes and NA subtypes detected during the first season reoccurred in the second season, as well as some of their combinations; yet, several new subtypes and combinations appeared during the second season. These findings indicate that different subtypes of AIVs, NDV and other haemagglutinating viruses circulate subclinically in the pintail populations sampled. Pintails should be regarded, potentially, as important spreaders of AIVs and NDVs, particularly due to their extensively ramified flyways, which include various inter-continental routes.

Objective: In order to monitor the present situation of Avian influenza virus (AIV) and Newcastle disease virus (NDV) in migratory waterfowls effectively, 158 tracheal and cloacal swab samples for wild birds were collected from Sanjiang natural reserve during migratory seasons in October 2005, April 2006 and October 2006., Methods: Serial passages in specific pathogen free embryonated chicken eggs, haemagglutination activity (HA) text, hemagglutination inhibition (HI) text and RT-PCR detection were used to isolate and identify AIV and NDV., Results: Twenty AIV isolates and 13 NDV isolates were collected in the test. Twenty AIV isolates were all from aquatic birds in October 2006, and among these isolates, 12 AIV subtypes were identified definitely, 11 subtypes were found in mallards-H2N2 (2/20), H2N6 (2/20), H3N4 (1/20), H3N6 (2/20), H3N7 (2/20), H3N8 (2/20), H6N2 (2/20), H11N2 (1/20), H11N3 (1/20), H11N5 (2/20), H11N6 (1/20), and 1 subtype was found in garganey-H5N2 (1/20). Thirteen NDV isolates were collected in all three migratory seasons from 5 different species of waterfowls, including mallard (8/13), bean goose (1/13), white-fronted goose (1/13), common teal (1/13) and mandarin duck (2/13)., Conclusion: The results indicated that mallard, which possesses huge population size and world wide distribution, could be considered one of the most important natural carrier of AIV and NDV and may have more important ecological significance on viruses transmission than other species of wild birds.

Composting has been used for disposal of poultry carcasses and manure following outbreaks caused by avian influenza virus (AIV) and Newcastle disease virus (NDV), but methods are needed to test for survival of the viruses in compost to ensure biosecurity. Methods developed in the present study include extracting viruses from compost and purifying viral RNA. The extracted viruses were detected by virus isolation using embryonated chicken eggs, and the purified RNA was detected by real-time reverse transcription PCR (RRT-PCR). The virus isolation and the RRT-PCR methods were evaluated with 3 compost preparations that were produced from chicken manure mixed with corn silage, wood shavings, or wheat straw. The detection limits of both methods were 1,700 and 1,000 embryo lethal doses of AIV and NDV per gram of compost, respectively. The copy number of viral RNA quantified by RRT-PCR was highly correlated with the amount of virus in compost. The results suggested that the RRT-PCR method may be used as an alternative to the virus isolation method for rapid detection and accurate quantification of AIV and NDV in compost.

The paper presents the results of the 2003 and 2006 environmental virological monitoring surveys on the Malyi Zhemchuzhnyi Island where a large breeding colony of sea gull (Laridae) is located. In the past several years, expansion of cormorants (Phalacrocorax carbo) has enhanced the intensity of populational interactions. The investigators isolated 13 strains of influenza A virus (Orthomyxoviridae, Influenza A virus) subtype H13N1 (from sea gulls (n = 4), cormorants (n = 9) 1 strain of Dhori virus (Orthomyxoviridae, Thogotovirus) from a cormorantwith clinical symptoms of the disease, 3 strains of Newcastle disease virus (Paramyxoviridae, Avulavirus) from cormorants. RT-PCR revealed influenza A virus subtype H5 in 3.1% of the cloacal lavages from cormorants. Neutralization test indicated that sera from cormorants contained specific antibodies against West Nile (Flaviviridae, Flavivirus) (15.0%), Sindbis (Togaviridae, Alphavirus) (5.0%), Dhori (10.0%), and Tahini (Bunyaviridae, Orthobunyavirus) (5.0%); sera from herring gulls had antibodies against Dhori virus (16.7%); there were no specific antibodies to Inco (Bunyaviridae, Orthobunyavirus) and mountain hare (Lepus timidus) (Bunyaviridae, Orthobunyavirus) virus.

Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

The paper presents the results of monitoring of viruses of Western Nile (WN), Japanese encephalitis (JE), tick-borne encephalitis (TBE), Geta, Influenza A, as well as avian paramicroviruses type I (virus of Newcastle disease (ND)) and type 6 (APMV-6) in the Primorye Territory in 2003-2006. Totally throughout the period, specific antibodies to the viruses were detected by neutralization test in wild birds (7.3%, WN; 8.0%, Geta; 0.7% Batai; 2.8%, Alpine hare (Lepus timidus); by hemagglutination-inhibition test in cattle (11.4% WN; 5.9%, JE; j 3.0%, TBE; 11.6%, Geta), horses (6.1, 6.8, 0, and 25.3%, respectively), and pigs (5.4, 1.5, 0, and 5.9%, respectively) by enzyme immunoassay (IgG) in human beings (0.8, 0.5, 6.8, and 3.2%, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was used to reveal RNA of the NP segment of influenza A virus in 57.9 and 65% of the cloacal swabs from wild and domestic birds, respectively; and the HA-segment of subtype HH was not detected in 2005. HA/H5 RNA was recorded in 5.5 and 6.7% of the swabs from wild and domestic birds, respectively; 6% of the specimens from domestic birds were M-segment positive in 2006. RNA of influenza A virus NA/H7 and RNA was not detected throughout the years. In 2004, the cloacal swabs 8 isolated influenza A strains: two H3N8 and two H4N8 strains from European teals (Anas crecca), two (H3N8 and H6N2) strains from Baikal teals (A. formosa), one (H10N4) strain from shovelers (A. clypeata), and one (H4N8) from garganeys (A. querquedula). In 2004, one ND virus strain was isolated from the cloacal swabs from European teals (A. crecca). RT-PCR revealed RNA of this virus in some 8 more cloacal swabs from black ducks (A. poecilorhyncha) (3 positive specimens), pheasants (Phasianus colchicus) (n = 2), garganeys (A. querquedula) (n = 1), gadwalls (A. strepera) (n = 1), and geese (Anser anser domesticus) (n = 1). Sequencing of the 374-member fragment of the ND virus F gene, which included a proteolytic cleavage site, could assign two samples to the weakly pathogenetic variants of genotype 1, one sample to highly pathogenic variants of genotype 3a, five to highly pathogenic ones of genotype 5b. Isolation of APMV-6 (2003) from common egrets (Egretta alba) and geese (Ans. anser domesticus) is first described.

A duplex reverse transcription-polymerase chain reaction (dRT-PCR) assay has been developed for the simultaneous, rapid and specific detection/discrimination of avian influenza virus (AIV) and Newcastle disease virus (NDV). Primers targeting the matrix protein gene (M) of AIV and the fusion protein gene (F) of NDV were evaluated experimentally with 13 AIV and 19 NDV strains. PCR products of the expected size of 144 bp and 316 bp were amplified from AIV/NDV samples, respectively, while no cross-reaction was observed with negative controls or with 16 other avian pathogens. The endpoint of detection was defined as approximately 10(+0.5) 50% egg infectious dose (EID(50))/0.2 ml for AIV and 10(+2.2) EID(50)/0.2 ml for NDV. The assay was able to detect AIV/NDV with similar sensitivity in spiked stool samples and in specimens from vaccinated birds. The developed dRT-PCR assay is a rapid, cost-effective tool, which provides powerful novel means for the early diagnosis of avian influenza and Newcastle disease.

A total of 543 migrating passerines were captured during their stopover on the island of Helgoland (North Sea) in spring and autumn 2001. They were sampled for the detection of avian influenza A viruses (AIV) subtypes H5 and H7, and for avian paramyxoviruses serotype 1 (APMV-1). The goal of the study was to examine the role of migrating birds as potential vectors for these zoonotic viral diseases. For virus detection samples were taken from a) short-distance migrants such as chaffinches (Fringilla coelebs, n = 131) and song trushes (Turdus philomelos, n = 169), and b) long-distance migrants such as garden warbler (Sylvia borin, n = 142) and common redstarts (Phoenicurus phoenicurus, n = 101). Virus detection was done on conjunctival, choanal cleft and cloacal swabs. Embryonated SPF chicken eggs were used to isolate and propagate virus followed by virus identification in a hemagglutination test, hemagglutination inhibition test and in an agar gel diffusion test. In none of the tested samples AIV was detected. Therefore, we conclude that the tested four species of passerines were infected by these pathogens. Six out of 543 birds (1.1 %) were found to carry non-pathogenic and lentogenic strains of APMV-1. This indicates that the passerine species examined in this study may play only a minor role as potential vectors of APMV-1.

Two hundred thirty specimens of wild birds were collected from some areas in Heilongjiang Province during the period of 2003-2004, including two batches of specimens collected randomly from a same flock of mallards in Zhalong Natural Reserve in August and December, 2004, respectively. Primary virus isolation and identification for avian influenza virus (AIV) and Newcastle disease virus (NDV) were performed. The results showed that only two specimens of young mallards collected from Zhalong Natural Reserve in August, 2004 were positive to AIV (isolation rate 0.9%), and one strain (D57) of these two virus isolates was identified to be H9 subtype by hemagglutination inhibition test. Meanwhile, the two batches of blood serum samples of mallards from Zhalong were also examined for antibodies against AIV and NDV. Among 38 blood serum samples collected in August, antibodies against the hemagglutinin of H1, H3, H5, H6 and H9 subtypes of AIV were found in 1, 0, 2, 0 and 8 samples, respectively; and 11 samples were found with antibody against NDV. Whereas the NDV isolation in both two batches of specimens of mallard was negative, all of the 32 blood serum samples collected in December were negative for antibodies against AIV and NDV.

Influenza A and Newcastle disease viruses are pathogens of social and economical importance known to be disseminated throughout the world by migratory birds. Many efforts have been made to control the introduction of these viruses into new environments, and complete world surveillance has yet to be achieved. Virus isolation and immunofluorescence techniques are time consuming, have inherent limited sensitivity and present a lack of host cells permissive universally to all Influenza A viruses. In this paper, the use of a duplex RT-PCR is described capable of sorting out any NDV and Influenza A virus strain simultaneously in oral and cloacal swab specimens. This method includes fluorescent detection of amplicons that provide accurate analysis of many DNA fragments within one base discrimination. Reference viruses were used for standardization of the assay and samples from wild-type viruses were screened, with four positive results for Influenza A detected in migratory birds captured in the state of Sao Paulo. This screening test can be considered a first step for further studies of these viruses circulating in avian species in Brazil, and hopefully will contribute to broaden the sample spectrum from wild birds, leading to a better understanding of these viruses and their participation in the southeastern region of the country.

Inland great cormorants (Phalacrocorax carbo) culled in France were examined in the winter of 1997-98 and 1998-99 for antibodies to Newcastle disease (ND) and influenza A strains H5 and H7 by the hemagglutination inhibition test. Antibodies to influenza A group antigen were tested by agar gel precipitin test. Ten of 53 adult individuals were seropositive for ND virus. All sera were negative for influenza A antibodies. It is speculated that ND occurred in the sampled population.

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.

Regular surveillance of domestic poultry from Hong Kong and southern China for influenza A viruses was conducted in Hong Kong from November 1977 to October 1978. Tracheal or cloacal swabs taken from 2844 ducks, geese and fowls yielded 141 influenza viruses of which 135 were derived from ducks. The isolation rate from ducks was twice as high from the cloaca (12.1 per cent) as from the trachea (5.8 per cent). The overall frequency of isolation was 10.3 per cent and 2.3 per cent for poultry originating from southern China and Hong Kong, respectively. Fourteen of the influenza antigenic combinations recorded in this period of surveillance had been obtained in the previous two years while five were new--H2N2, H2Nav6, Hav5N2, Hav6Nav1 and Hav7Nav4. The number of combinations recorded over three years was 39, the most frequent being Hav4Nav1 which comprised one fifth of the total. All the combinations found in geese and fowl isolates, apart from Hav7Nav6 found in a fowl, have been recorded in duck isolates. Faecal/oral transmission of virus from infected pond water was thought to be an important factor in the high incidence of influenza in ducks. The existence of a large number of combinations of haemagglutinin and neuraminidase antigens and the isolation of two viruses from the cloaca suggest that recombination is occurring in these birds. Reasons for the limited isolation of some antigenic combinations are considered. The paramyxoviruses, Newcastle disease virus and the Hong Kong avian paramyxovirus, were also isolated. Over the three year period the isolation rate of influenza and paramyxoviruses from ducks appeared to be cyclical and seasonal. Influenza was found mainly in the summer and paramyxoviruses in the winter.

Twenty-seven chicken red blood cell agglutinating agents were isolated from 187 tracheal swabbings of apparently healthy migratory mallard ducks (Anas platyrhynchos) in the Mississippi flyway. Twenty-four of the isolants were type A influenza virus; 3 lentogenic Newcastle disease viruses were isolated. Isolations were not made from either 65 giant Canada geese (Branta canadensis) or 60 Franklins' gulls (Larus pipixcan).

One strain of influenza A virus ( H4N6 ), three strains of Newcastle disease virus (NDV) and one strain of paramyxovirus (PMV) type 4 were isolated from tracheal and cloacal swabs of sentinel domestic ducks by inoculation of tested samples into the amniotic and allantoic cavities of chick embryos (CE).

Forty-four haemagglutinating viruses were isolated from the pooled tracheal/cloacal swabs of the dead birds from 170 consignments of caged birds arriving at Heathrow Airport over a period of 6 months. Two isolates were identified as Newcastle disease virus but the remaining 42 were all identified as influenza viruses with Hav 7 Neq 2 antigens. All the consignments from which influenza viruses were isolated originated in India but had widespread destinations. The NDV isolates were from birds originating in central America and destined for Japan.

As part of the World Health Organization's international programme on the ecology of influenza, cloacal swabs were collected from 3,736 birds belonging to 67 species over a 3-year period in Western Australia for the isolation of ortho- and paramyxoviruses. A total of 24 influenza A viruses were isolated from various species of ducks, shearwaters , noddies , terns and a coot , and were subtyped as H1N9 , H3N8 , H4N4 , H4N6 , H6N2 , H6N4 , H?N2, H?N6 and H? N9 . The H? haemagglutinins did not react in tests with reference antisera. Whether they represent a novel haemagglutinin subtype or atypical members of an established subtype remains to be determined, although preliminary results indicate that they may be atypical members of the H7 subtype. The H1N9 isolate is the first reported isolate of this particular antigenic combination. A total of 17 Newcastle disease viruses was isolated from ducks, noddies , terns and a black- fronted plover : preliminary results suggest that they are avirulent for domestic chickens. This study indicates that ortho- and paramyxoviruses are present in a variety of wild birds in Australia.

Lectins of different specificities do not interfere with the maturation of myxo-viruses; their inhibitory effect on virus replication is mainly due to prevention of the detachment of infectious virus particles from the host cell. In chick embryo fibroblasts infected with an influenza virus and treated with concanavalin A, budding occurs into intracytoplasmic vacuoles, but this phenomenon is not observed with a parainfluenza virus and with different cells.