Susceptibility of laughing gulls (Larus atricilla) to H5N1 and H5N3 highly pathogenic avian influenza viruses

This investigation detailed the clinical disease, gross and histologic lesions, and distribution of viral antigen in juvenile laughing gulls (Larus atricilla) intranasally inoculated with either the A/tern/South Africa/61 (H5N3) (tern/SA) influenza virus or the A/chicken/Hong Kong/220/97 (H5N1) (chicken/HK) influenza virus, which are both highly pathogenic for chickens. Neither morbidity nor mortality was observed in gulls inoculated with either virus within the 14-day investigative period. Gross lesions resultant from infection with either virus were only mild, with the tern/SA virus causing decreased lucency of the air sacs (2/6), splenomegaly (2/6), and pancreatic mottling (1/6) and the chicken/HK virus causing only decreased lucency of the air sacs (2/8) and conjunctival edema (2/8). Histologic lesions in the tern/SA-inoculated gulls included a mild to moderate heterophilic to lymphoplasmacytic airsacculitis (6/6), mild to moderate interstitial pneumonia (3/6), and moderate necrotizing pancreatitis and hepatitis at 14 days postinoculation (DPI) (2/6). Immunohistochemical demonstration of viral antigen occurred only in association with lesions in the liver and pancreas. In contrast, viral antigen was not demonstrated in any tissues from the chicken/HK-inoculated gulls, and inflammatory lesions were confined to the air sac (3/8) and lungs (3/8). Both viruses were isolated at low titers (<10(1.68) mean embryo lethal dose) from oropharyngeal and cloacal swabs up to 7 days postinoculation (DPI), from the lung and kidney of one of two tern/SA-inoculated gulls at 14 DPI, and from the lung of one of two chicken/HK-inoculated gulls at 7 DPI. Antibodies to influenza viruses as determined with the agar gel precipitin test at 14 DPI were detected only in the two tern/SA-inoculated gulls and not in the two chicken/HK-inoculated gulls.     

Evolution of highly pathogenic H7N3 avian influenza viruses in Mexico

Highly pathogenic H7N3 influenza A viruses have persisted in poultry in Mexico since 2012, diversifying into multiple lineages that have spread to three Mexican states, as of 2016. The H7N3 viruses segregate into three distinct clades that are geographically structured. All 2016 viruses are resistant to adamantane antiviral drugs and have an extended 24‐nucleotide insertion at the HA cleavage site that was acquired from host 28S ribosomal RNA.     

Limited susceptibility of pigeons experimentally inoculated with H5N1 highly pathogenic avian influenza viruses

An experimental infection study was performed using pigeons reared for racing or meat production in Japan and clade 2.2 and 2.3.2 isolates of H5N1 highly pathogenic avian influenza virus to evaluate the possible role of pigeons in virus transmission to poultry. In experiment 1, when 20 pigeons were intranasally inoculated with high or low viral doses, no inoculated pigeon exhibited clinical signs for 14 days. Drinking water and almost all swab samples were negative for virus isolation. Virus isolation was positive in 3 oral swab samples from 2 pigeons from day 2 through 4 postinoculation, but viral titers of positive samples were extremely low. Immunohistochemical analysis for virus detection was negative in all tissue samples. Along with seroconversion in a limited number of pigeons postinoculation, these results suggest that pigeons have limited susceptibility to the virus used for experimental infection. In experiment 2, when uninoculated chickens were housed with virus-inoculated pigeons, all pigeons and contact chickens survived for 14 days without exhibiting any clinical signs. According to serological analysis, the chickens did not exhibit seroconversion after close contact with inoculated pigeons. Our data suggest that the risk posed by pigeons with respect to the transmission of the H5N1 highly pathogenic avian influenza virus to poultry would be less than that for other susceptible avian species.     

Pathogenicity in quails and mice of H5N1 highly pathogenic avian influenza viruses isolated from ducks

In our study, the pathogenicity of H5N1 influenza A viruses circulating in waterfowls in Southern China was investigated. Three H5N1 highly pathogenic avian influenza (HPAI) viruses isolated from ducks, A/Duck/Guangdong/383/2008(DK383), A/Duck/Guangdong/378/2008(DK378) and A/Duck/Guangdong/212/2004(DK212) were inoculated at 10(6) fifty-percent egg infectious doses (EID(50)) into ducks, quails and mice and showed varying levels of pathogenicity. In ducks, the mortality rates ranged from 0 to 60% and the mean death time (MDT) was 0-6.7 days post-inoculation (DPI). While the viruses were highly pathogenic in quails, resulting in 83.3-100% mortality and the MDT of 2.3-3 DPI, they were completely lethal in mice (100% mortality). The viruses replicated in many organs of ducks and quails and were found in the brain, and kidney, lung and spleen of the mice. Phylogenetic analysis revealed that DK383 and DK378 viruses of clade 2.3.2 belonged to genotype 11, while DK212 virus of clade 9 was genotype 3. Our study illustrated H5N1 influenza viruses within Clade 2.3.2 and 9 from duck in Southern China had very highly pathogenicity to Japanese quails and BALB/c mice, but viruses within Clade 2.3.2 had more highly lethality than those of clade 9 to Muscovy ducks. Therefore, they had posed a continued challenge for disease control and public health.     

Origin and evolution of highly pathogenic H5N1 avian influenza in Asia

Outbreaks of highly pathogenic avian influenza caused by H5N1 viruses were reported almost simultaneously in eight neighbouring Asian countries between December 2003 and January 2004, with a ninth reporting in August 2004, suggesting that the viruses had spread recently and rapidly. However, they had been detected widely in the region in domestic waterfowl and terrestrial poultry for several years before this, and the absence of widespread disease in the region before 2003, apart from localised outbreaks in the Hong Kong Special Autonomous Region (SAR), is perplexing. Possible explanations include limited virus excretion by domestic waterfowl infected with H5N1, the confusion of avian influenza with other serious endemic diseases, the unsanctioned use of vaccines, and the under-reporting of disease as a result of limited surveillance. There is some evidence that the excretion of the viruses by domestic ducks had increased by early 2004, and there is circumstantial evidence that they can be transmitted by wild birds. The migratory birds from which viruses have been isolated were usually sick or dead, suggesting that they would have had limited potential for carrying the viruses over long distances unless subclinical infections were prevalent. However, there is strong circumstantial evidence that wild birds can become infected from domestic poultry and potentially can exchange viruses when they share the same environment. Nevertheless, there is little reason to believe that wild birds have played a more significant role in spreading disease than trade through live bird markets and movement of domestic waterfowl. Asian H5N1 viruses were first detected in domestic geese in southern China in 1996. By 2000, their host range had extended to domestic ducks, which played a key role in the genesis of the 2003/04 outbreaks. The epidemic was not due to the introduction and spread of a single virus but was caused by multiple viruses which were genotypically linked to the Goose/GD/96 lineage via the haemagglutinin gene. The H5N1 viruses isolated from China, including the Hong Kong SAR, between 1999 and 2004 had a range of genotypes and considerable variability within genotypes. The rising incidence and widespread reporting of disease in 2003/04 can probably be attributed to the increasing spread of the viruses from existing reservoirs of infection in domestic waterfowl and live bird markets leading to greater environmental contamination. When countries in the region started to report disease in December 2003, others were alerted to the risk and disease surveillance and reporting improved. The H5N1 viruses have reportedly been eliminated from three of the nine countries that reported disease in 2003/04, but they could be extremely difficult to eradicate from the remaining countries, owing to the existence of populations and, possibly, production and marketing sectors, in which apparently normal birds harbour the viruses.     

Pathogenicity and transmission of H5N1 avian influenza viruses in different birds

In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 10⁶ EID₅₀ of GSGD/1/96, 10³ EID₅₀ of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 10⁶ EID₅₀ of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses.     

Genetic variation of highly pathogenic H5N1 avian influenza viruses in Vietnam shows both species-specific and spatiotemporal associations

Domestic poultry act as a reservoir for persistent H5N1 endemicity in Vietnam, and the circulation of poultry flocks across farms and to market is thought to drive the spatial movement and evolution of avian influenza viruses. Using a dataset of complete or nearly full genomic sequences from highly pathogenic H5N1 avian influenza viruses collected in domestic poultry in Vietnam from 2003 to 2007, we explore potential differences in genetic characteristics according to species of isolation and the spatiotemporal characteristics of the viruses. Clustering algorithms and ANOVA indicate that H5N1 viruses in Vietnam show differences in the amount of genetic change that chicken viruses experience as compared to duck viruses, with duck viruses showing higher rates of molecular evolution on all eight of influenza's gene segments. There also exist distinct patterns of genetic differentiation according to the year in which they were isolated. These findings suggest that genetic evolution of avian influenza viruses is continuous through time but could also be mediated by the species in which the viruses occur, information that has implications for prevention efforts.     

Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses.

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.     

Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 10(6) EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2-4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 10(6) EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8-6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI.     

Experimental infection of highly pathogenic avian influenza virus H5N1 in black-headed gulls (Chroicocephalus ridibundus)

Historically, highly pathogenic avian influenza viruses (HPAIV) rarely resulted in infection or clinical disease in wild birds. However, since 2002, disease and mortality from natural HPAIV H5N1 infection have been observed in wild birds including gulls. We performed an experimental HPAIV H5N1 infection of black-headed gulls (Chroicocephalus ridibundus) to determine their susceptibility to infection and disease from this virus, pattern of viral shedding, clinical signs, pathological changes and viral tissue distribution. We inoculated sixteen black-headed gulls with 1 × 10⁴ median tissue culture infectious dose HPAIV H5N1 (A/turkey/Turkey/1/2005) intratracheally and intraesophageally. Birds were monitored daily until 12 days post inoculation (dpi). Oropharyngeal and cloacal swabs were collected daily to detect viral shedding. Necropsies from birds were performed at 2, 4, 5, 6, 7, and 12 dpi. Sampling from selected tissues was done for histopathology, immunohistochemical detection of viral antigen, PCR, and viral isolation. Our study shows that all inoculated birds were productively infected, developed systemic disease, and had a high morbidity and mortality rate. Virus was detected mainly in the respiratory tract on the first days after inoculation, and then concentrated more in pancreas and central nervous system from 4 dpi onwards. Birds shed infectious virus until 7 dpi from the pharynx and 6 dpi from the cloaca. We conclude that black-headed gulls are highly susceptible to disease with a high mortality rate and are thus more likely to act as sentinel species for the presence of the virus than as long-distance carriers of the virus to new geographical areas.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0084-9) contains supplementary material, which is available to authorized users.     

A single vaccination of commercial broilers does not reduce transmission of H5N1 highly pathogenic avian influenza

ABSTRACT: Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.     

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa)

ABSTRACT: An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.     

Assessing the risk of highly pathogenic avian influenza H5N1 transmission through poultry movements in Bali,Indonesia

Indonesia continues to report the highest number of human and poultry cases of highly pathogenic avian influenza H5N1. The disease is considered to be endemic on the island of Bali. Live bird markets are integral in the poultry supply chain on Bali and are important, nutritionally and culturally, for the rural and urban human populations. Due to the lack of biosecurity practiced along the supply chain from producer to live bird markets, there is a need to understand the risks associated with the spread of H5N1 through live bird movements for effective control. Resources to control H5N1 in Indonesia are very limited and cost effective strategies are needed. We assessed the probability a live bird market is infected through live poultry movements and assessed the effects of implementing two simple and low cost control measures on this risk. Results suggest there is a high risk a live bird market is infected (0.78), and risk mitigation strategies such as detecting and removing infected poultry from markets reduce this risk somewhat (range 0.67–0.76). The study demonstrates the key role live poultry movements play in transmitting H5N1 and the need to implement a variety of control measures to reduce disease spread.     

Impact of different husbandry conditions on contact and airborne transmission of H5N1 highly pathogenic avian influenza virus to chickens

Typically highly pathogenic avian influenza (HPAI) viruses spread very rapidly among chickens within sheds. However, the spread was slower than expected for the initial 10 days of the index farm in Japan during 2004. This slow spread, as well as the lack of gross lesions, clinical signs, or high mortality, hindered the field veterinarian from reporting a suspected HPAI outbreak to the veterinary office. To understand the field conditions for the slow virus spread, we examined contact and airborne transmission of the H5N1 virus to chickens in a negative-pressure isolator using various numbers of infected chickens and separate compartments. We found that the contact transmission did occur inefficiently when one or two chickens were infected, whereas the transmission was efficient when four chickens were infected. Airborne transmission of the HPAI virus was also dependent on the number of infected chickens and was less efficient than contact transmission. These data together with field observations suggested that number of infected chickens, chicken house types, and amount of environmental contamination might affect the virus transmission efficiency to chickens.     

The first specific detection of a highly pathogenic avian influenza virus (H5N1) in Ivory Coast

The Virology Laboratory of the Central Laboratory of Animal Diseases in Ivory Coast at Bingerville received samples of wild and domestic avian species between February and December 2006. An RT-PCR technique was used to test for avian influenza (AI) and highly pathogenic AI subtype viruses. Among 2125 samples, 16 were type A positive; of which, 12 were later confirmed to be H5N1. Fifteen of these 16 type A positive samples were inoculated into the chorioallantoic cavity of 11-day-old embryonated hens' eggs for virus isolation. Eight produced virus with hemagglutination titres from 1/64 to 1/512. The 4/16 M-RT-PCR positive samples, which were H5N1 negative, were shown to be H7 subtype negative. The diagnostic efficiency of the laboratory for the surveillance of H5N1 in Ivory Coast was demonstrated. The positive cases of H5N1 were from a sparrowhawk (Accipter nisus); live market poultry and in free-range poultry, where the mortality rate was approximately 20% (2/10) and 96.7% (29/30) respectively. Currently, investigations into intensive poultry farms have proved negative for H5N1. No human cases have been reported this time.