Identification of a common conserved neutralizing linear B-cell epitope in the VP3 protein of waterfowl parvoviruses

ABSTRACT

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of ⁴³⁸LHNPPP⁴⁴³ in VP3 protein of GPV, NGPV and MDPV. To the authors’ best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.     

Wood ducks (Aix sponsa) as potential reservoirs for avian influenza and avian paramyxoviruses

Influenza A viruses (IAVs) and avian paramyxoviruses (APMVs) are important pathogens of poultry worldwide, and both commonly occur in wild waterfowl, especially ducks in the family Anatidae. Although wood ducks (Aix sponsa) are members of the Anatidae, their behaviour differs from most other species in this family, which could affect the transmission of IAVs and APMVs. We collected cloacal and oropharyngeal swab and blood samples from more than 700 wood ducks across nine states in the eastern United States of America. No IAVs were isolated, and based on blocking enzyme-linked immunoassay ELISA results, antibodies to IAVs were only detected in 0.2% of samples. In contrast, 23 (3%) APMVs were isolated (22 Newcastle disease virus and 1 APMV-6), and antibodies to multiple serotypes of APMVs were detected in more than 60% of the samples. After-hatch-year birds were more likely to be antibody positive for APMV-4 and APMV-6 compared to hatch-year birds. Female birds were more likely to be antibody positive for APMV-4 than were male birds. Our results indicate that wood ducks are probably not an important host for IAV but are frequently infected with APMVs.     

Avian host range of Chlamydophila spp. based on isolation,antigen detection and serology

Published reports and our own diagnostic data on the avian host range of avian Chlamydophila spp. are presented in an attempt to provide evidence for the large number of bird species that have been naturally infectedwith chlamydia. The term 'chlamydia-positive' is based on either isolation of the organism and antigen detection or on serological detection of circulating antibodies. The list of chlamydia-positive birds contains the six majordomestic species (chicken, turkey, Pekin duck, Muscovy duck, goose, and pigeon), the three minor domestic species (Japanese quail, bobwhite quail, and peafowl) and a total of 460 free-living or pet bird species in 30 orders. The order Psittaciformes contains by far the most (153 of 342; 45%) chlamydia-positive bird species. More than 20% of all species per order are positive for chlamydia in the orders Lariformes (gulls, 26 of 92species; 28%), Alciformes (alks, six of 23 species; 26%), Sphenisciformes (penguins, four of 16 species; 25%), and Anseriformes (ducks and geese, 33 of 157 species; 21%). Only 5% of all bird species (14 of 259 species) inthe order Phasianiformes (gallinaceus birds) are chlamydia-positive. The different percentages of chlamydiapositive bird species reflect: (i) a high rate of investigations (e.g. of domestic birds) compared with infrequenttesting (e.g. of Charadriiformes or Cuculiformes), (ii) frequent zoonotic implications (e.g. psittacine and columbiform birds), and (iii) an assumed high susceptibility to infection and subsequent seroconversion (e.g.waterfowl).     

Genetic diversity of avian paramyxovirus type 4 isolates from wild ducks in Korea from 2006 to 2011

Thirteen isolates of avian paramyxovirus type 4 (APMV-4) isolated from wild ducks in Korea from 2006 to 2011 were genetically characterized by sequence analysis of the N-terminal region of the APMV-4 fusion (F) protein gene. The results revealed that the amino acid sequence homology within Korean isolates was 97.5 % or greater. The homologies of the Korean isolates with the APMV-4/duck/HK/D3/75 and APMV-4/duck/BE/15129/07 strains were 86.9–88.0 and 95.5–96.1 %, respectively. All Korean isolates had sequence motifs of ¹¹⁶DIQPR↓F¹²¹ at the F0 cleavage site. Phylogenetic analysis based on the N-terminal region of the F protein gene of APMV-4 isolates revealed that all 2006–2011 Korean isolates formed a single genotypic cluster that was phylogenetically different from APMV-4/duck/HK/D3/75 or APMV-4/duck/BE/15129/07 strains. Korean APMV-4 isolates were more closely related to APMV-4/goose/ZA/N1468/10 (isolated in South Africa) than to the Belgium APMV-4 virus. Korean APMV-4 isolates were further divided into at least two subgroups (A and B) based on phylogenetic analysis. Subgroup A viruses were isolated throughout Korea, whereas subgroup B viruses were detected only in isolates from Cheju island in 2011, suggesting that Korean APMV-4 exhibits marked genetic diversity and differs from viruses currently circulating in Europe and other locations.     

Vaccination of domestic ducks against H5N1 HPAI: A review

Domestic ducks play an important role in the epidemiology of H5N1 highly pathogenic avian influenza (HPAI) viruses. Consequently, successful control of H5N1 HPAI in ducks is important for the eradication of the disease in poultry and in preventing infections in humans. Domestic ducks, however, include different species and breeds, and the susceptibility to infection, disease and response to vaccination can vary depending on the species and age of the bird. Most domestic duck species are descendants of mallard ducks (Anas platyrhynchos), but in Asian countries Muscovy ducks (Cairina moschata) are also commonly farmed. Current vaccines and vaccination practices are insufficient for the control of H5N1 HPAI virus infections in domestic waterfowl and new vaccination strategies are needed. Although vaccination has proven effective in protecting ducks against disease, shedding of the virus still occurs in clinically healthy vaccinated populations. To improve protection of ducks against H5N1 HPAI, vaccination programs must take into account the susceptibility of ducks to circulating viruses and the particular production systems and husbandry practices of the country. Vaccination needs to be implemented as part of a comprehensive control strategy that also includes biosecurity, surveillance, education and elimination of infected poultry.     

Susceptibility of wild passerines to subtype H5N1 highly pathogenic avian influenza viruses

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have spread throughout many areas of Asia, Europe and Africa, and numerous cases of HPAI outbreaks in domestic and wild birds have been reported. Although recent studies suggest that the dissemination of H5N1 viruses is closely linked to the migration of wild birds, information on the potential for viral infection in species other than poultry and waterfowl is relatively limited. To investigate the susceptibility of terrestrial wild birds to infection with H5N1 HPAI viruses, common reed buntings (Emberiza schoeniclus), pale thrushes (Turdus pallidus) and brown-eared bulbuls (Hypsipetes amaurotis) were infected with A/mountain hawk-eagle/Kumamoto/1/07(H5N1) and A/whooper swan/Aomori/1/08(H5N1). The results showed that common reed buntings and brown-eared bulbuls were severely affected by both virus strains (100% mortality). While pale thrushes did not exhibit any clinical signs, seroconversion was confirmed. In common reed buntings, intraspecies-transmission of A/whooper swan/Aomori/1/08 to contact birds was also confirmed. The findings show that three passerine species; common reed buntings, brown-eared bulbuls and pale thrushes are susceptible to infection by H5N1 HPAI viruses, which emphasizes that continued surveillance of species other than waterfowl is crucial for effective monitoring of H5N1 HPAI virus outbreaks.     

Expression of capsid proteins and non-structural proteins of waterfowl parvoviruses in Escherichia coli and their use in serological assays.

While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and non-structural proteins. To enable this, the capsid and non-structural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in Escherichia coli. These proteins were purified and used as antigens in western blotting assays of antibodies against GPV and MDPV. The results showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the non-structural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and non-structural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the non-structural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine.     

Genomic sequence of an avian paramyxovirus type 1 strain isolated from Muscovy duck (Cairina moschata) in China

The complete sequence of an avian paramyxovirus type 1 (APMV-1) strain, FP1/02, isolated from Muscovy duck in China, was determined. Sequence analysis indicated that the complete genome of strain FP1/02 contained 15,192 nucleotides (nt), following the rule of six. The genome contained an extra 6-nt insertion in the non-coding region of the NP gene when compared with other APMV-1 strains, such as strains La Sota and Beaudette C. The cleavage site of the F protein was (112)R-R-Q-K-R↓F(117), indicating that the FP1/02 strain was virulent, but the morbidity and mortality varied with the species of duck. Genotypic analysis based on the F gene revealed that APMV-1 FP1/02 was a member of genotype VII. Phylogenetic analysis showed that the FP1/02 strain shared high identity with other APMV-1 strains such as ZJ1, SF02 and NA-1 isolated from geese.     

The mallard duck (Anas platyrhynchos) as intermediate host for Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis)

Morphometric and DNA investigation results of Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis) and Sarcocystis sp. (cyst type IV) from the mallard duck (Anas platyrhynchos) are presented. No significant morphometric differences between the investigated Sarcocystis species were found. ITS-1, 18S rRNA, and 28S rRNA gene sequences of these species showed 100% identity. The conclusion is drawn that it is one and the same Sarcocystis species in different intermediate hosts.     

Genomic analysis of Newcastle disease virus strain NA-1 isolated from geese in China

Newcastle disease virus (NDV) has been thought to infect only domestic avian species, with waterfowl such as geese either not being infected, even by virulent strains, or developing only in apparent infection. In 1997, a new infectious disease producing high morbidity and mortality among geese broke out in many provinces of China, which was caused by a serotype I avian paramyxovirus (APMV-1)--NDV. To investigate how NDV spreads between chickens and geese, the complete genome of one NDV strain isolated from a goose was cloned and analyzed. The results indicate that there is conservation in NDV structural genetic evolution but that there are also considerable differences between goose and chicken NDV strains. Separate patterns of NDV evolution exist among wild bird species. Meanwhile, there is evidence indicating that the goose NDV may have evolved from chicken NDV strain Herts/33. In addition, the possibility was investigated that this new strain of NDV may bind to different sialic acid receptor binding sites than the normal NDV strains that have been investigated so far. This might provide clues to the evolution of the goose NDV.     

Complete genome sequence of goose parvovirus Y strain isolated from Muscovy ducks in China

A goose parvovirus (GPV) Y strain was isolated from Muscovy ducks in Anhui Province of China. By polymerase chain reaction method, its complete genomic sequence was found to be 5,106 bp in length, consisting of 444-bp inverted terminal repeat, 1,844-bp non-structural protein and 2,199-bp capsid protein (VP) regions. Then its sequence was aligned with the sequences of GPV and Muscovy duck parvovirus published in the GenBank using the neighbor-joining method. The phylogenetic analyses based on the VP3 gene sequences revealed that the GPV Y strain along with those from Taiwan belonged to the subgroup IIb, while other GPV strains from Muscovy ducks belonged to the subgroup Ib and most of other GPV strains isolated in China mainland were clustered in the subgroup IIa. The absence of the deduced 703–705NRT glycosylation site in VP region may explain the host specificity of the GPV Y strain. The complete genomic sequence of the GPV Y strain from Muscovy ducks will help to understand the molecular and evolutionary characteristics of GPV.     

Quantitative double antibody sandwich ELISA for the determination of gliadin

A sensitive double antibody sandwich ELISA (DAS-ELISA) based on chicken anti-gliadin IgY and biotinylated monoclonal antibody (mAb) was developed for the quantification of gliadin in foods. The anti-gliadin IgY and mAb specifically detected gliadin in wheat, barley, and rye by indirect ELISA and Western-blot assay. Using anti-gliadin IgY as capture antibody and biotinylated mAb as detecting antibody, the sensitivity of DAS-ELISA has a linear standard range of 4-40?ng/mL, showing that the limit of detection (LOD) corresponds to 4?ng/mL gliadin in assay buffer, equivalent to 0.8?ppm in foods. The intra-assay expressed as percentage of coefficients of variation (%CV) was 7.25% average of six food samples. The interassay precision was 9.51% in food samples. The combination of anti-gliadin IgY and biotinylated mAb in the DAS-ELISA provides a reliable, sensitive, and inexpensive tool for the detection of gliadin in gluten-free and gluten-containing food products.     

Serological survey for the prevalence of antibodies to egg drop syndrome 1976 virus in domesticated and wild birds in Israel

A serological survey of chicken, turkey, goose and duck flocks for the presence of antibodies to egg drop syndrome virus 1976 (EDS 76) has been carried out in Israel. In most of the chicken flocks sampled egg production was not normal, but no antibodies against this virus were detected. Likewise, turkey breeding flocks were similarly negative. All Pekin duck flocks tested showed some serological activity. Muscovy ducks that were reared in direct or indirect contact with Pekin ducks had haemagglutination-inhibition antibodies to EDS 76 virus. Seven cattle egrets trapped on a duck farm were serologically positive. No antibodies were detected in a collection of water fowl raised in a zoological garden.     

ELISA-based detection of mislabeled albacore (Thunnus alalunga) fresh and frozen fish fillets

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of albacore (Thunnus alalunga) and its differentiation from other less-valued scombrid species such as yellowfin tuna (Thunnus albacares), bullet tuna (Auxis rochei), atlantic bonito (Sarda sarda), bigeye tuna (Thunnus obesus), little tunny (Euthynnus alleteratus) and skipjack tuna (Katsuwonus pelamis). The assay uses polyclonal antibodies raised in rabbits against soluble muscle protein extract from fresh albacore. These polyclonal antibodies were rendered species-specific by blocking them with the heterologous soluble muscle proteins, allowing discrimination between fresh albacore and the rest of the scombrid species, except for yellowfin tuna. A total of 40 commercial albacore fresh and frozen fillets were analysed, revealing an incorrect labelling in 32.5% of the albacore samples. However, positive samples (67.5%) could be albacore or yellowfin tuna and should require a DNA assay as discriminatory technique.     

Anti‐idiotype and anti‐anti‐idiotype antibodies for aflatoxin from laying hens

Anti‐idiotype (anti‐id) antibodies (IgY2)for aflatoxin (AF) were obtained from the egg yolks of laying hens immunized with affinity‐purified rabbit polyclonal anti‐aflatoxin B₁ (AFB₁) carboxymethyloxime‐bovine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity purified and then subjected to various analyses. Inhibition of the binding of pAbl to the solid‐phase AFB₁‐BSA by IgY2 and the binding of pAb1 to the solid‐phase IgY2 by free AFB₁ were demonstrated in a biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) system. The concentration of IgY2 causing 50% inhibition (ID₅₀) of the binding of pAb1 to AFB₁‐BSA was found to be 2.45 μg/assay. The ID₅₀ concentration of the binding of pAb1 to IgY2 by free AFB₁ was found to be 0.30 μg/assay. Inhibition of the binding of AFB₁‐horseradish peroxidase (HRP) to the solid‐phase pAbl by IgY2 (ID50 = 9.65 μg/assay) was also demonstrated in the direct ELISA. Egg yolk anti‐anti‐id antibodies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti‐AFB₃‐hemisuccinate‐BSA monoclonal antibody. IgY3 was subjected to affinity chro‐matography purification with Sepharose gel armed with AFB₂‐carboxymethytoxime, and then subjected to various analyses. ELISA analysis revealed that IgY3 has characteristics similar to other anti‐AFB antibodies induced in various experimental animals. In the direct ELISA, the ID₅₀ of the binding of AFB₁‐HRP to solid‐phase lgY3 by AFB₁ was found to be 0.12 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of IgY3 to solid‐phase AFB₁‐BSA by AFB₁ was found to be 2.2 ng ml^‐1. The IgY3‐based ELISA analysis showed higher sensitivity than that of the egg yolk antibodies directly against AFB‐protein conjugates (IgY1). A good correlation was found for the data obtained from IgY3‐based and pAb1‐based ELISAs in the analysis of AFB in the fungal culture filtrates.     

Propagation of viruses infecting waterfowl on continuous cell lines of Muscovy duck (Cairina moschata) origin

Duck circovirus, duck hepatitis A virus 1, goose parvovirus and goose haemorrhagic polyomavirus are economically damaging pathogens of waterfowl, and replicate poorly or not at all in established cell lines. AGE1.CR, AGE1.CR.pIX and AGE1.CS cell lines, originating from the Muscovy duck, were tested for their suitability to isolate and identify these viruses. Immunofluorescence (IF) and quantitative polymerase chain reaction investigations verified that all cell lines are permissive for all four viruses; however, AGE1.CR.pIX proved to be the most productive and most sensitive for viral infection. IF experiments revealed that the time of one infectious cycle is approximately 12 to 14 h in the AGE1.CR.pIX cells in the case of the three DNA viruses, while it is 10 to 12 h for DHAV-1. Specific viral infectivity and the limit of detection by IF varied between 55 and 1484 copies, depending on the viruses and cell lines. Despite the high sensitivity of the cell lines for viruses, their viral productivity remained relatively low for the investigated field isolates. However, optimization of virus infection and/or the adaptation of the viruses to the cells can raise viral productivity and can make these cell lines suitable for vaccine development and production.     

Description of <Emphasis Type="Italic">Sarcocystis anasi</Emphasis> sp. nov. and <Emphasis Type="Italic">Sarcocystis albifronsi</Emphasis> sp. nov. in birds of the order Anseriformes

On the basis of the already published morphological, 18S rDNA, 28S rDNA data (Kutkienė et al., Parasitol Res 99:562–565, 2006; Parasitol Res 102:691–696, 2008; Parasitol Res 104:329–336, 2009), and ITS-1 region investigation results of sarcocysts presented in this paper, Sarcocystis albifronsi sp. nov. from the white-fronted goose (Anser albifrons) and Sarcocystis anasi sp. nov. from the mallard duck (Anas platyrhynchos) are described.     

Experimental infections of herring gulls (Larus argentatus) with H5N1 highly pathogenic avian influenza viruses by intranasal inoculation of virus and ingestion of virus-infected chicken meat

The present study investigated the susceptibility of herring gulls (Larus argentatus) exposed to two strains of Asian lineage H5N1 highly pathogenic avian influenza (HPAI) virus by evenly separating six gulls into two groups and inoculating them intranasally with 10⁶ median embryo infectious doses of either A/whooper swan/Mongolia/244/05 (H5N1) or A/duck meat/Anyang/AVL-1/01 (H5N1). Two additional gulls were fed 5.0 g meat from a specific pathogen free chicken that died after experimental infection with A/whooper swan/Mongolia/244/05. Morbidity and mortality were observed in the gulls infected with A/whooper swan/Mongolia/244/05 by both routes of exposure. Gulls infected with A/duck meat/Anyang/AVL-1/01 exhibited high morbidity, but no mortality. The concentration and duration of viral shedding were similar between gulls infected with either strain of H5N1 HPAI virus by intranasal inoculation and gulls exposed to A/whooper swan/Mongolia/244/05 through ingestion of virus-infected chicken meat. The susceptibility of herring gulls in this study varied between the two strains of Asian lineage H5N1 HPAI virus. These results also provide preliminary data to support that ingestion of virus-infected raw or uncooked chicken meat is a viable route of exposure to some H5N1 HPAI viruses in herring gulls. Additional studies are necessary to further evaluate the efficiency of this route of exposure to a variety of H5N1 HPAI virus strains in herring gulls and other avian species in order to better understand the potential role of scavenging species in the epidemiology of H5N1 HPAI virus.