Serological analysis in humans in Malaysian Borneo suggests prior exposure to H5 avian influenza near migratory shorebird habitats.

Cases of H5 highly pathogenic avian influenzas (HPAI) are on the rise. Although mammalian spillover events are rare, H5N1 viruses have an estimated mortality rate in humans of 60%. No human cases of H5 infection have been reported in Malaysian Borneo, but HPAI has circulated in poultry and migratory avian species transiting through the region. Recent deforestation in coastal habitats in Malaysian Borneo may increase the proximity between humans and migratory birds. We hypothesise that higher rates of human-animal contact, caused by this habitat destruction, will increase the likelihood of potential zoonotic spillover events. In 2015, an environmentally stratified cross-sectional survey was conducted collecting geolocated questionnaire data in 10,100 individuals. A serological survey of these individuals reveals evidence of H5 neutralisation that persisted following depletion of seasonal H1/H3 HA binding antibodies from the plasma. The presence of these antibodies suggests that some individuals living near migratory sites may have been exposed to H5 HA. There is a spatial and environmental overlap between individuals displaying high H5 HA binding and the distribution of migratory birds. We have developed a novel surveillance approach including both spatial and serological data to detect potential spillover events, highlighting the urgent need to study cross-species pathogen transmission in migratory zones.

Subclinical Infection and Transmission of Clade 2.3.4.4 H5N6 Highly Pathogenic Avian Influenza Virus in Mandarin Duck (Aix galericulata) and Domestic Pigeon (Columbia livia domestica).

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aixgalericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.

Wild Bird Surveillance for Avian Influenza Virus.

Avian influenza (AI) viruses have been routinely isolated from a wide diversity of free-living avian species, representing numerous taxonomic orders. Birds in orders Anseriformes and Charadriiformes are considered the natural reservoirs for all AI viruses; it is from these orders that AI viruses have been most frequently isolated. Since first recognized in the late 1800s, AI viruses have been an important cause of disease in poultry and, occasionally, in non-gallinaceous birds and mammals. While AI viruses tend to be of low pathogenicity (LP) in wild birds, the 2014-2015 incursion of highly pathogenic avian influenza (HPAI) clade 2.3.4.4 H5Nx viruses into North America and the recent circulation of HPAI H5 viruses in European wild birds highlight the need for targeted, thorough, and continuous surveillance programs in the wild bird reservoir. Such programs are crucial to understanding the potential risk for the incursion of AI into human and domestic animal populations. The aim of this chapter is to provide general concepts and guidelines for the planning and implementation of surveillance plans for AI viruses in wild birds.

Avian Influenza Virus Sample Types, Collection, and Handling.

Successful detection of avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel immunodiffusion tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; oropharyngeal swabs from gallinaceous poultry and cloacal swabs from waterfowl are the preferred specimens for most diagnostic tests, although it is optimal to collect swabs from both locations, if possible. In addition to collecting the appropriate sample for the tests to be performed, selecting the right materials for sample collection (i.e., type of swab) is very important. This chapter will outline the collection of different specimen types and procedures for proper specimen handling.

Avian influenza at animal-human interface: One-health challenge in live poultry retail stalls of Chakwal, Pakistan.

BACKGROUND: Live poultry retail stalls (LPRSs) are believed to be the source of human infection with avian influenza viruses (AIVs); however, little is known about epidemiology of these viruses in LPRSs of Pakistan. OBJECTIVES: The current study was conducted to estimate the virological and serological prevalence of AIVs in humans and poultry and associated risk factors among seropositive butchers. METHODS: A field survey of LPRSs of Chakwal District was conducted between December 2015 and March 2016. In total, 322 samples (sera = 161 and throat swab = 161) from butchers and 130 pooled oropharyngeal swabs and 100 sera from birds were collected. Baseline sera (n = 100) from general population were also tested. Data were collected by structured questionnaires. Sera were tested by hemagglutination inhibition (HI) test further confirmed by micro-neutralization test (MN). Swabs were processed by real-time RT-PCR. Logistic regression analyses were conducted to identify risk factors. RESULTS: In butchers, 15.5% sera were positive for antibodies against H9 virus using a cutoff of ≥40 in HI titer; 6% sera from general population were positive for H9. Seroprevalence in poultry was 89%, and only 2.30% swabs were positive for H9. Presence of another LPRS nearby and the number of cages in the stall were risk factors (OR > 1) for H9 seroprevalence in butchers. CONCLUSIONS: This study provides evidence of co-circulation of H9 virus in poultry and exposure of butchers in the LPRSs, which poses a continued threat to public health. We suggest regular surveillance of AIVs in occupationally exposed butchers and birds in LPRSs.

Modulation of lethal HPAIV H5N8 clade 2.3.4.4B infection in AIV pre-exposed mallards.

In 2016/2017, a severe epidemic of HPAIV H5N8 clade 2.3.4.4 group B (H5N8B) affected Europe. To analyse the role of mallards in the spatiotemporal dynamics of global HPAIV H5N8B dispersal, mallards (Anas platyrhynchos), naturally exposed to various AIV and therefore seropositive, were challenged with H5N8B. All experiments were controlled by infection and co-housing of seronegative juvenile Pekin ducklings. All ducks that survived the first infection were re-challenged 21 dpi with the homologous H5N8B strain. After the first H5N8B infection, seropositive mallards showed only mild clinical symptoms. Moderate to low viral shedding, occurring particularly from the oropharynx and lasting for 7 days maximum, led to severe clinical disease of all contact ducklings. All challenged seronegative Pekin ducks and contact ducklings died or had to be euthanized. H5-specific antibodies were detected in surviving birds within 2 weeks. Virus and viral RNA could be isolated from several water samples until 6 and 9 dpi, respectively. Conversely, upon re-infection with homologous H5N8B neither inoculated nor contact ducklings showed any clinical symptoms, nor was an antibody titer increase of seropositive mallards or any seroconversion of contact ducklings observed. Mallard ducks naturally pre-exposed to LPAIV can play a role as a clinically unsuspicious virus reservoir for H5N8B effective in virus transmission. Mallards with homologous immunity did not contribute to virus transmission.

EXPERIMENTAL INFECTIONS AND SEROLOGY INDICATE THAT AMERICAN WHITE IBIS (EUDOCIUMUS ALBUS) ARE COMPETENT RESERVOIRS FOR TYPE A INFLUENZA VIRUS.

The American White Ibis (Eudocimus albus) is a nomadic wading bird common to wetland habitats in the southeastern US. In south Florida, US, habitat depletion has driven many ibis to become highly urbanized. Although they forage in neighborhood parks, artificial wetlands, backyards, and golf courses, the majority continue to nest in natural wetlands, often in dense, mixed species colonies. Adults and juveniles commonly disperse thousands of kilometers to other breeding colonies along the Gulf and southeast Atlantic coasts, presenting the potential for close contact with humans, domestic animals, and other wild bird species. Historically, wading birds were not considered to be significant hosts for influenza A virus (IAV), yet as ibis regularly move among various human, domestic animal, and wildlife interfaces, their potential to be exposed to or infected with IAV deserves attention. We experimentally challenged wild-caught, captive-reared White Ibis (n=20) with IAV, tested wild White Ibis for IAV, and serologically tested wild White Ibis for antibodies to IAV. White Ibis were highly susceptible to experimental challenge with H6N1 and H11N9 IAVs, with cloacal shedding lasting an average of 6 d. All 13 infected birds seroconverted by 14 d postinfection as determined by microneutralization. In contrast, no birds challenged with H3N8 were infected. We tested 118 swabs and 578 serum samples from White Ibis captured in southeastern Florida for IAV infection and antibodies to IAV, respectively. Although no IAVs were isolated, 70.4% serum samples were antibody positive by blocking enzyme-linked immunosorbent assay (bELISA). Neutralizing antibodies to H1-H12 were detected in 96.0% of a subset of bELISA positive birds (n=196) and 81.0% tested antibody positive to two or more hemagglutinin subtypes, indicating that exposure to multiple IAVs is common. These results provide evidence that White Ibis are susceptible and naturally infected with IAV and may represent a component of the IAV natural reservoir system.

Dual-mode detection of avian influenza virions (H9N2) by ICP-MS and fluorescence after quantum dot labeling with immuno-rolling circle amplification.

Avian influenza virus (AIVs), hosted in poultry, are the pathogens of many poultry diseases and human infections, which bring huge losses to the poultry breeding industry and huge panic to society. Therefore, it is of great significance to establish accurate and sensitive detection methods for AIVs. In this work, a dual-mode detection method based on immuno-rolling circle amplification (immuno-RCA) and quantum dots (QDs) labeling for inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence detection of H9N2 AIV was developed. The dual-mode detection of the QDs by ICP-MS and fluorescence is used to achieve mutual verification within the analysis results, thus improving the accuracy of the method. With the immuno-RCA, the sensitivity of the method was increased by two orders of magnitude. The limit of detection of the proposed method is 17 ng L-1 and 61 ng L-1, and the linear range of the proposed method is 0.05-5 ng mL-1 and 0.1-5 ng mL-1 with ICP-MS and fluorescence detection, respectively. The relative standard deviation (n = 7) is 4.9% with ICP-MS detection and 3.1% with fluorescence detection. Furthermore, the proposed method was applied to the analysis of chicken serum samples, no significant different was found for two modes detection and the recoveries of the spiking experiments are acceptable, indicating that the method has good practical potential for real sample analysis.

Label-free localized surface plasmon resonance biosensor composed of multi-functional DNA 3 way junction on hollow Au spike-like nanoparticles (HAuSN) for avian influenza virus detection.

In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily.

Serologic Evidence for Influenza A Virus Exposure in Three Loon Species Breeding in Alaska, USA.

Limited information exists about exposure to influenza A viruses (IAVs) in many wild waterbird species, including loons. We analyzed serum samples from breeding adult Pacific (Gavia pacifica), Red-throated (Gavia stellata), and Yellow-billed (Gavia adamsii) loons sampled at three locations along the coast of Alaska, US from 2008 to 2017 to gain a better understanding of the potential role loons play in IAV ecology. We screened loon sera for IAV antibodies using three tests-blocking enzyme-linked immunosorbent assay (bELISA), agar gel immunodiffusion (AGID), and hemagglutination inhibition (HI)-and examined patterns in seroprevalence among species and sampling locations. We found evidence of IAV infection in all loon species and at all breeding locations, although concordance was imperfect among serological tests. Diagnostic tests yielded seroprevalence estimates of 24% (42/172) with bELISA, 8% (5/60) with AGID, and 6% (4/70) with HI. The IAV subtypes to which loon sera reacted using HI were consistent with those detected in waterfowl and gulls at other locations in Alaska, suggesting that loons may be exposed to IAV maintained in sympatric waterbirds. Our study provided evidence that loons inhabiting Alaska were exposed to IAV. However, given imperfect concordance among serologic tests, and relatively low seroprevalence as compared to other avian taxa exposed to IAV in Alaska, they make poor IAV surveillance targets.

The development of a multiplex serological assay for avian influenza based on Luminex technology.

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.

Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged in China in 2017.

H7N9 low pathogenic influenza viruses emerged in China in 2013 and mutated to highly pathogenic strains in 2017, resulting in human infections and disease in chickens. To control spread, a bivalent H5/H7 inactivated vaccine was introduced in poultry in September 2017. To monitor virus evolution and vaccine efficacy, we collected 53,884 poultry samples across China from February 2017 to January 2018. We isolated 252 H7N9 low pathogenic viruses, 69 H7N9 highly pathogenic viruses, and one H7N2 highly pathogenic virus, of which two low pathogenic and 14 highly pathogenic strains were collected after vaccine introduction. Genetic analysis of highly pathogenic strains revealed nine genotypes, one of which is predominant and widespread and contains strains exhibiting high virulence in mice. Additionally, some H7N9 and H7N2 viruses carrying duck virus genes are lethal in ducks. Thus, although vaccination reduced H7N9 infections, the increased virulence and expanded host range to ducks pose new challenges.

A Comparison of Toll-Like Receptor 5 and 21 Ligands as Adjuvants for a Formaldehyde Inactivated H9N2 Avian Influenza Virus Vaccine in Chickens.

Low pathogenic avian influenza virus (AIV) infection in chickens can result in economic losses and has impacts on human health. Poultry vaccination is a tool that can be used to decrease infection and transmission of AIVs. Prior research has demonstrated that Toll-like receptor (TLR) ligands can act as vaccine adjuvants and their addition to inactivated AIV vaccines can enhance immune responses elicited in chickens. The objective of this study was to compare the adjuvant capabilities of TLR5 ligand (flagellin) and TLR21 ligand (CpG ODN 2007) administered either alone or in combination with an intramuscular formaldehyde inactivated H9N2 whole virus vaccine in chickens. Along with the inactivated virus, chickens were administered either a single dose of CpG ODN 2007 (2 or 10 µg), flagellin (0.4 or 2 µg), or a combination of both ligands. An additional group received AddaVax™, an oil emulsion style adjuvant. Chickens were vaccinated twice and serum and lachrymal samples were collected weekly following the primary vaccination, and antibody-mediated immune responses were quantified. Results showed that vaccines containing CpG ODN 2007 induce significantly greater systemic and lachrymal antibody responses than vaccines containing flagellin or AddaVax. Combinations of flagellin and CpG ODN 2007 did not demonstrate inhibitory, additive, or synergistic effects on systemic or lachrymal antibody-mediated immune responses. Additionally, for both flagellin and CpG ODN 2007, a fivefold higher dose of each did not induce significantly higher antibody-mediated immune responses compared with the lesser dose. Future studies should examine the induction of cell-mediated immune responses when flagellin, CpG ODN 2007, or other TLR ligands are administered either alone or combined as adjuvants for inactivated H9N2 AIV vaccines.

Persistence of maternal antibodies to influenza A virus among captive mallards (Anas platyrhynchos).

Wild waterfowl are maintenance hosts of most influenza A virus (IAV) subtypes and are often the subjects of IAV surveillance and transmission models. While maternal antibodies have been detected in yolks and in nestlings for a variety of wild bird species and pathogens, the persistence of maternal antibodies to IAVs in mallard ducklings (Anas platyrhynchos) has not been previously investigated. Nonetheless, this information is important for a full understanding of IAV transmission dynamics because ducklings protected by maternal antibodies may not be susceptible to infection. In this study, we examined the transfer of IAV-specific maternal antibodies to ducklings. Blood samples were collected approximately every five days from ducklings hatched from hens previously infected with an H6 strain of IAV. Serum samples were tested for antibodies to IAV by an enzyme-linked immunosorbent assay. The median persistence of maternal antibodies in ducklings was 12.5 days (range: 4-33 days) post-hatch. The majority of ducklings (71%) had detectable maternal antibodies from 4 to 17 days post-hatch, while a small subset of individuals (29%) had detectable maternal antibodies for up to 21-33 days post-hatch. Antibody concentrations in hens near the time of egg laying were correlated with maternal antibody concentrations in the initial blood sample collected from ducklings (0-4 days post-hatch). Knowledge of the duration of maternal antibodies in ducklings will aid in the interpretation of IAV serological surveillance results and in the modeling of IAV transmission dynamics in waterfowl.

Antigenic characterization of highly pathogenic avian influenza A(H5N1) viruses with chicken and ferret antisera reveals clade-dependent variation in hemagglutination inhibition profiles.

Highly pathogenic avian influenza (HPAI) A(H5N1) viruses pose a significant economic burden to the poultry industry worldwide and have pandemic potential. Poultry vaccination against HPAI A(H5N1) viruses has been an important component of HPAI control measures and has been performed in Vietnam since 2005. To systematically assess antigenic matching of current vaccines to circulating field variants, we produced a panel of chicken and ferret antisera raised against historical and contemporary Vietnamese reference viruses representing clade variants that were detected between 2001 and 2014. The antisera were used for hemagglutination inhibition (HI) assays to generate data sets for analysis by antigenic cartography, allowing for a direct comparison of results from chicken or ferret antisera. HI antigenic maps, developed with antisera from both hosts, revealed varying patterns of antigenic relationships and clustering of viruses that were dependent on the clade of viruses analyzed. Antigenic relationships between existing poultry vaccines and circulating field viruses were also aligned with in vivo protection profiles determined by previously reported vaccine challenge studies. Our results establish the feasibility and utility of HPAI A(H5N1) antigenic characterization using chicken antisera and support further experimental and modeling studies to investigate quantitative relationships between genetic variation, antigenic drift and correlates of poultry vaccine protection in vivo.

Serological evidence of H5-subtype influenza A virus infection in indigenous avian and mammalian species in Korea.

In Korea, H5-subtype highly pathogenic avian influenza (HPAI) has caused huge economic losses in poultry farms through outbreaks of H5N1 since 2003, H5N8 since 2013 and H5N6 since 2016. Although it was reported that long-distance migratory birds may play a major role in the global spread of avian influenza viruses (AIVs), transmission from such birds to poultry has not been confirmed. Intermediate hosts in the wild also may be a potential factor in viral transmission. Therefore, a total of 367 serum samples from wild animals were collected near major migratory bird habitats from 2011 to 2016 and tested by AIV-specific blocking ELISA and hemagglutination inhibition (HI) test. Two mammalian and eight avian species were seropositive according to the ELISA test. Among these, two mammalian (Hydropotes inermis and Prionailurus bengalensis) and three avian (Aegypius monachus, Cygnus cygnus, and Bubo bubo) species showed high HI titres (> 1,280) against one or two H5-subtype AIVs. As H. inermis (water deer), P. bengalensis (leopard cat), and B. bubo (Eurasian eagle owl) are indigenous animals in Korea, evidence of H5-subtype AIV in these animals implies that continuous monitoring of indigenous animals should be followed to understand interspecies transmission ecology of H5-subtype influenza viruses.

EVALUATION OF A COMMERCIAL COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF AVIAN INFLUENZA VIRUS SUBTYPE H5 ANTIBODIES IN ZOO BIRDS.

The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study compared a commercial ELISA for detection of AIV subtype H5 antibodies with HI test of 572 serum samples from zoo birds. There was no significant difference between the results of the two tests when statistically compared by a McNemar χ2 test (P = 0.86) and assessment of κ (κ = 0.87). With a specificity of 94.2% (95% confidence interval [CI], 0.92-0.97), a sensitivity of 93.9% (95% CI, 0.91-0.97), and an excellent correlation between the two tests, this ELISA can be recommended as an alternative to the HI test for preliminary screening of zoo bird sera for antibodies to AIV subtype H5.

Assessing evidence for avian-to-human transmission of influenza A/H9N2 virus in rural farming communities in northern Vietnam.

Rural farming communities in northern Vietnam do not routinely practice vaccination for influenza A viruses (IAV) for either humans or poultry, which enables us to study transmission intensity via seroepidemiology. Using samples from a longitudinal cohort of farming households, we determined the number of symptomatic and asymptomatic human infections for seasonal IAV and avian A/H9 over 2 years. As expected, we detected virologically confirmed acute cases of seasonal IAV in humans, as well as large numbers of subclinical seroconversions to A/H1pdm [55/265 (21 %)], A/H3 [95/265 (36 %)] and A/H9 [24/265 (9 %)]. Five of the A/H9 human seroconverters likely represented true infections rather than heterosubtypic immunity, because the individuals seroconverted solely to A/H9. Among co-located poultry, we found significantly higher seroprevalance for A/H5 compared to A/H9 in both chickens and ducks [for northern study sites overall, 337/1105 (30.5 %) seropositive for A/H5 and 123/1105 (11.1 %) seropositive for A/H9].

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission.

The 3D8 single chain variable fragment (scFv) is a mini-antibody that causes unusual sequence-independent nuclease activity against all types of nucleic acids. We used recombinant lentiviruses to generate transgenic chickens expressing the 3D8 scFv gene under the control of the chicken ß-actin promoter. From 420 injected embryos, 200 chicks (G0) hatched and were screened for the 3D8 scFv using PCR, and 15 chicks were identified as transgenic birds expressing the transgene in their semen. The G0 founder birds were mated with wild-type hens to produce seven transgenic chicks (G1). 3D8 scFv expression in the chicken embryonic fibroblasts (CEFs) was verified by RT-PCR and Western blot analysis. Immunofluorescence staining for 3D8 scFv in the CEFs revealed that the 3D8 scFv protein was primarily cytosolic. To identify 3D8 scFv anti-viral activity, wild-type and two transgenic CEF lines were infected with H9N2 avian influenza virus (AIV). We selected one line of transgenic chickens that exhibited the lowest number of plaque-forming units to be challenged with H9N2 virus. The challenge experiment revealed that contact exposed transgenic chickens expressing 3D8 scFv exhibited suppressed viral shedding. This results suggest that the transgenic chickens developed in this study could be useful for controlling potential within-flock AIV transmission.

Longitudinal Study of Avian Influenza and Newcastle Disease in Village Poultry, Mali, 2009-2011.

Newcastle disease (ND) is endemic in West Africa, which has also experienced outbreaks of highly pathogenic avian influenza (AI) H5N1 since 2006. We aimed to estimate the prevalence and incidence of AI and ND in village poultry in Mali and to identify associated risk factors. A longitudinal serologic study was conducted between November 2009 and February 2011 using ELISA commercial kits to detect antibodies. Sera (5963) were collected from 4890 different poultry. AI was rare, with a seroprevalence of 2.9% (95% confidence interval [CI] 2.3-3.5) and a seroincidence rate of 0.7 birds per 100 bird-months at risk (95% CI 0.4-1.0). AI antibodies were short lived, with a seroreversion rate of 25.4 birds per 100 bird-months at risk (95% CI 19.0-31.7). Risk factors for AI were limited: temporal variation occurred, but proximity to a water body was a risk factor only when large populations of wild waterbirds were present. ND was very common, with seroprevalence of 68.9% (95% CI 61.9-76.0) and a seroincidence rate of 15.9 birds per 100 bird-months at risk (95% CI 11.9-19.8). ND seroreversion rate was 6.2 birds per 100 bird-months at risk (95% CI 3.6-8.9). Regarding risk factors for ND, temporal variations occurred, and ND was more likely to be present in the Sudanian agro-ecological zone than in the Sahelian zone, in chickens than in other species, in flocks with higher numbers of Guinea fowl, and in flocks that had access to a waterbody. Control efforts would benefit from further increasing the ND vaccination coverage of village poultry, although this was already quite high (54.9%) for an African country. Seroconversion seemed satisfactory in vaccinated poultry, since 90.0% (95% CI 87.6-92.4) of these had ND antibodies. Further research should investigate the apparent lack of an epidemiologic role of domestic ducks for AI in Mali (unlike in Southeast Asia) and the potential role of Guinea fowl as a reservoir for ND.