Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.     

Highly pathogenic virus recovered from chickens infected with mildly pathogenic 1986 isolates of H5N2 avian influenza virus

A combination of in vitro and in vivo selection procedures was used to examine the possibility that certain mildly pathogenic field isolates of avian influenza (AI) virus may contain minority subpopulations of highly pathogenic virus. Two mildly pathogenic H5N2 isolates, A/chicken/New Jersey/12508/86 (NJ12508) and A/chicken/Florida/27716/86 (FL27716), recovered from chickens epidemiologically associated with urban live-bird markets, were cloned in trypsin-free chicken embryo fibroblast cultures. Selected clones were inoculated intranasally and intratracheally (IN/IT) into specific-pathogen-free laying hens, and virus reisolated from the hens that died was serially passed in hens by IN/IT inoculation. Several highly pathogenic reisolates were recovered from hens infected with the cloned NJ12508 or FL27716 virus. A highly pathogenic NJ12508 reisolate killed 19 of 24 IN/IT-inoculated hens, and a FL27716 reisolate killed all 24 inoculated hens; signs and lesions were typical of fowl plague. In contrast, uncloned NJ12508 stock virus killed 1 of 24 hens and FL27716 stock virus killed 4 of 24 hens, and neither produced the complete spectrum of lesions associated with fowl plague. Recovery of highly pathogenic viruses from these isolates demonstrates the coexistence of pathogenically distinct subpopulations of virus. Competition for dominance among such subpopulations could explain the variable pathogenicity of some AI viruses.     

Intraocular vaccination with an inactivated highly pathogenic avian influenza virus induces protective antibody responses in chickens

Because it is expected to induce cross-reactive serum and mucosal antibody responses, mucosal vaccination against highly pathogenic avian influenza (HPAI) is potentially superior to conventional parenteral vaccination. Here, we tested whether intraocular vaccination with an inactivated AI virus induced protective antibody responses in chickens. Chickens were inoculated intraocularly twice with 10⁴ hemagglutination units of an inactivated H5N1 HPAI virus. Four weeks after the second vaccination, the chickens were challenged with a lethal dose of the homologous H5N1 HPAI virus. Results showed that most of the vaccinated chickens mounted positive antibody responses. The median serum hemagglutination inhibition titer was 1:80. Addition of CpG oligodeoxynucleotide 2006 or cholera toxin to the vaccine did not enhance serum antibody titers. Cross-reactive anti-hemagglutinin IgG, but not IgA, was detected in oropharyngeal secretions. In accordance with these antibody results, most vaccinated chickens survived a lethal challenge with the H5N1 HPAI virus and did not shed the challenge virus in respiratory or digestive tract secretions. Our results show that intraocular vaccination with an inactivated AI virus induces not only systemic but also mucosal antibody responses and confers protection against HPAI in chickens.     

Lesions in broiler and layer chickens in an outbreak of highly pathogenic avian influenza virus infection

Fifteen chickens, five broilers and ten layers, from the Pennsylvania 1983 outbreak of highly pathogenic avian influenza virus infection, were examined. Gross lesions in the broilers were limited to serosal petechiae and dehydration. In the layers there was comb edema, vesiculation, and necrosis. Microscopic lesions were mild to severe diffuse nonsuppurative encephalitis, very mild to severe diffuse necrotizing pancreatitis, and very mild to severe subacute necrotizing myositis involving numerous skeletal muscles and most severe in the external ocular muscles and limbs. While many of these lesions have been seen in experimental infections of chickens with influenza viruses, the pattern of organs involved in this group of chickens is distinctive.     

Highly pathogenic avian influenza virus subtype H5N1 in ducks in the Northern part of Cameroon

Highly pathogenic avian influenza (HPAI) virus was first detected in Cameroon in February 2006. Analysis of NA sequences of the virus demonstrated that it is closely related to the H5N1 isolates from Northern Nigeria, Sudan and Ivory Coast, suggesting a common virus ancestor.     

Highly pathogenic or low pathogenic avian influenza virus subtype H7N1 infection in chicken lungs: small differences in general acute responses

ABSTRACT: Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus (LPAI) based on their difference in virulence. To investigate if the difference in clinical outcome between LPAI and HPAI in chickens is due to immunological host responses in the lung within the first 24 hours post infection (hpi), chickens were infected with LPAI or HPAI of subtype H7N1. Virus was found in the caudal and cranial part of the lung. With LPAI, virus was localised around the intrapulmonary bronchus and secondary bronchi. In sharp contrast, HPAI was detected throughout the whole lung. However, based on viral RNA levels, no quantitative difference was observed between LPAI and HPAI infected birds. In infected areas of the lungs, an influx of CD8α+ cells as well as KUL01+ macrophages and dendritic cells (DC) occurred as fast as 8 hpi in both infected groups. No major difference between LPAI and HPAI infected birds in the induction of cytokines and interferons at mRNA level in lung tissue was found.In conclusion, the differences in lethality for chickens infected with LPAI or HPAI could be ascribed to difference in location of the virus. However similar amounts of viral RNA, similar cytokine mRNA levels, and similar influxes of CD8α+ and KUL01+ macrophages and DC were found between HPAI and LPAI in the lungs. A cytokine storm at mRNA level as described for mammals was not observed in the lungs of HPAI infected birds within 24 hpi.     

Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication,disease, and mortality in chickens

Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (10^6.9 EID₅₀) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (10^5.3–5.5 EID₅₀) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0237-5) contains supplementary material, which is available to authorized users.     

Highly pathogenic avian influenza H5N1 virus in cats and other carnivores

The Asian lineage highly pathogenic avian influenza (HPAI) H5N1 virus is a known pathogen of birds. Only recently, the virus has been reported to cause sporadic fatal disease in carnivores, and its zoonotic potential has been dominating the popular media. Attention to felids was drawn by two outbreaks with high mortality in tigers, leopards and other exotic felids in Thailand. Subsequently, domestic cats were found naturally infected and experimentally susceptible to H5N1 virus. A high susceptibility of the dog to H3N8 equine influenza A virus had been reported earlier, and recently also HPAI H5N1 virus has been identified as a canine pathogen. The ferret, hamster and mouse are suitable as experimental animals; importantly, these species are also kept as pets. Experimental intratracheal and oral infection of cats with an HPAI H5N1 virus isolate from a human case resulted in lethal disease; furthermore, cats have been infected by the feeding of infected chickens. Spread of the infection from experimentally infected to in-contact cats has been reported. The epidemiological role of the cat and other pet animal species in transmitting HPAI H5N1 virus to humans needs continuous consideration and attention.     

Myxosarcomas associated with avian leukosis virus subgroup A infection in fancy breed chickens

Formalin-fixed suspect tumors were submitted to the Poultry Diagnostic and Research Center at the University of Georgia (Athens, GA) for diagnosis. Samples were from fancy breed chickens with a history of increased tumor prevalence in both hens and roosters. Microscopically, in all the samples, there were neoplastic proliferations of spindle-shaped cells. The matrix surrounding tumor cells stained positively with Alcian blue at pH 2.5, but neoplastic cells did not stain with periodic acid-Schiff. Immunohistochemistry stains were positive for vimentin and neuron-specific enolase and negative for desmin, smooth muscle actin, and S-100 protein. Tumors were determined to be myxosarcomas. All samples were positive for PCR targeting the gp85 avian leukosis virus (ALV) envelope protein. However, analysis of the predicted amino acid sequences in the envelope gene from three separate samples showed high similarity between them and to ALV subgroup A.     

H9N2亚型禽流感病毒对樱桃谷肉鸭的致病性

为了研究H9N2亚型禽流感病毒（AIV）对樱桃谷肉鸭的致病性。本试验利用1株鸭源H9N2亚型AIV分别通过静脉注射和鼻内接种途径感染2周龄健康樱桃谷肉鸭,观察感染鸭的症状、排毒规律及组织学变化,流式细胞术分析外周血CD4^＋和CD8^＋T细胞变化规律,实时荧光定量（RT-qPCR）检测脾脏中细胞因子的表达水平,并测定血清HI抗体效价。结果显示,攻毒后,静脉注射组鸭出现体质量增长显著缓慢、呼吸道症状以及全身性感染,而鼻内接种组鸭无明显临床症状,仅发生局部感染;两攻毒组鸭泄殖腔较气管排毒量高且排毒时间长;外周血CD8^＋T细胞显著升高,并且脾脏IFNA、IFNB、IFNG和IL2表达水平上调,IL1B和IL6表达水平仅在静脉注射组上调;血清HI抗体水平低且维持时间短。结果表明,在实验室条件下,H9N2亚型AIV对樱桃谷肉鸭致病力较弱,仅静脉接种组引起鸭体质量增长缓慢及呼吸系统出血、淋巴细胞浸润。     

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.     

Different routes of inoculation impact infectivity and pathogenesis of H5N1 high pathogenicity avian influenza virus infection in chickens and domestic ducks

The H5N1 type A influenza viruses classified as Qinghai-like virus (clade 2.2) are a unique lineage of type A influenza viruses with the capacity to produce significant disease and mortality in gallinaceous and anseriform birds, including domestic and wild ducks. The objective of this study was to determine the susceptibility and pathogenesis of chickens and domestic ducks to A/Whooper Swan/Mongolia/224/05 (H5N1) high pathogenicity avian influenza (HPAI) virus when administered through respiratory or alimentary routes of exposure. The chickens and ducks were more susceptible to the H5N1 HPAI virus, as evidenced by low infectious and lethal viral doses, when exposed by intranasal as compared to alimentary routes of inoculation (intragastric or oral-fed infected chicken meat). In the alimentary exposure pathogenesis study, pathologic changes included hemorrhage, necrosis, and inflammation in association with virus detection. These changes were generally observed in most of the visceral organs of chickens, between 2 and 4 days postinoculation (DPI), and are similar to lesions and virus localization seen in birds in natural cases or in experimental studies using the intranasal route. Alimentary exposure to the virus caused systemic infection in the ducks, characterized by moderate lymphocytic encephalitis, necrotized hepatitis, and pancreatitis with a corresponding demonstration of virus within the lesions. In both chickens and ducks with alimentary exposure, lesions, virus, or both were first demonstrated in the upper alimentary tract on 1 DPI, suggesting that the alimentary tract was the initial site affected upon consumption of infected meat or on gavage of virus in liquid medium. However, as demonstrated in the infectivity study in chickens, alimentary infection required higher exposure doses to produce infection as compared to intranasal exposure in chickens. These data suggest that upper respiratory exposure to H5N1 HPAI virus in birds is more likely to result in virus infection and transmission than will consumption of infected meat, unless the latter contains high doses of virus, as found in cannibalized infected carcasses.     

重组禽流感病毒H5亚型二价灭活疫苗(H5N1,Re-5+Re-4)免疫鸡和鸭后抗体消长规律

禽流感(AI)是由A型流感病毒引起的一种禽类传染病.根据致病性的不同将禽流感分为高致病性禽流感(HPAI)和低致病性禽流感(IPAI),HPAI被世界动物卫生组织(OIE)列为A类传染病,我国将其列为一类动物疫病 [1].近几年来,禽流感在世界各地反复出现,2004年亚洲发生了高致病性禽流感,2006年底和2007年初东南亚又出现疫情,并发现了人感染禽流感.我国养禽业也一直受到禽流感的威胁.因此,它不仅给畜牧业造成了巨大的损失,而且给人们的公共卫生安全带来了严重的威胁.     

山西亚群H5N1亚型HPAIV对鸭及鸭胚的继代感染研究

2006年从山西省分离获得的H5N1亚型禽流感病毒(AIV)曾引起免疫鸡群的大量死亡,与我国之前分离的病毒抗原差异性较大,称为"山西鸡型"抗原变异株。本研究应用该亚群代表毒株CK/SX/2/06(H5N1),研究该病毒对SPF鸭和鸭胚的致病性,并通过病毒在鸭和鸭胚内的连续传代,探讨该亚群病毒在鸭和鸭胚中的进化规律。结果表明,病毒对3周龄鸭呈低致病性,在各脏器中的复制能力较弱;鸭感染病毒后无明显临床症状,排毒水平较低并且不能感染同居鸭。病毒在3周龄鸭和10日龄鸭胚中分别继代感染3代和5代后,对3周龄鸭仍呈低致病性且HA基因未发生氨基酸位点变化。病毒不能致死3周龄鸭和1日龄雏鸭,但能致死10日龄～23日龄鸭胚,而且对鸭胚的致病性随着鸭胚日龄的增长逐渐减弱,致死鸭胚时间从对10日龄鸭胚24h致死到对23日龄鸭胚72h致死直至对25日龄鸭胚只感染但不致死。病毒对鸭呈低致病性且在鸭群传播中保持着基因水平和致病性上的相对稳定,对不同日龄鸭胚、1日龄和3周龄鸭致病性的不同表现,与目前存在于我国的其他亚群H5N1HPAIV对鸭致病性和进化特点上呈现出明显的差异,并对家禽养殖业存在威胁。     

Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.     

Local and systemic immune responses following infection of broiler-type chickens with avian Metapneumovirus subtypes A and B

Infections with avian Metapneumovirus (aMPV) are often associated with swollen head syndrome in meat type chickens. Previous studies in turkeys have demonstrated that local humoral and cell-mediated immunity plays a role in aMPV-infection. Previous experimental and field observations indicated that the susceptibility of broilers and their immune reactions to aMPV may differ from turkeys. In the presented study local and systemic immune reactions of broilers were investigated after experimental infections with subtypes A and B aMPV of turkey origin. Both virus subtypes induced a mild respiratory disease. The recovery from respiratory signs correlated with the induction of local and systemic aMPV virus-neutralizing antibodies, which began to rise at 6 days post infection (dpi), when the peak of clinical signs was observed. In a different manner to the virus neutralizing (VN) and IgG-ELISA serum antibody titres, which showed high levels until the end of the experiments between 24 and 28 dpi, the specific IgA-ELISA and VN-antibody levels in tracheal washes decreased by 10 and 14 dpi, respectively, which may explain the recurring aMPV-infections in the field. Ex vivo cultured spleen cells from aMPV-infected broilers released at 3 and 6 dpi higher levels of IFN-γ after stimulation with Concanavalin A as compared to virus-free birds. In agreement with studies in turkeys, aMPV-infected broilers showed a clear CD4+ T cell accumulation in the Harderian gland (HG) at 6 dpi (P<0.05). In contrast to other investigations in turkeys aMPV-infected broilers showed an increase in the number of CD8alpha+ cells at 6 dpi compared to virus-free birds (P<0.05). The numbers of local B cells in the Harderian gland were not affected by the infection. Both aMPV A and B induced up-regulation of interferon (IFN)-γ mRNA-expression in the nasal turbinates, while in the Harderian gland only aMPV-A induced enhanced IFN-γ expression at 3 dpi. The differences in systemic and local T cell and possibly natural killer cell activity in the HG between turkeys and chickens may explain the differences in aMPV-pathogenesis between these two species.     

Highly pathogenic avian influenza virus subtype H5N1 in Mute swans in the Czech Republic

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in the Czech Republic extensive surveillance was carried out between January and April 2006. A total of 2101 samples representing 61 bird species were examined for the presence of influenza A by using PCR, sequencing and cultivation on chicken embryos. AIV subtype H5N1 was detected in 12 Mute swans (Cygnus olor). The viruses were determined as HPAI (highly pathogenic avian influenza) and the hemagglutinin sequence was closely similar to A/mallard/Italy/835/06 and A/turkey/Turkey/1194/05. Following the first H5N1 case, about 300 wild birds representing 33 species were collected from the outbreak region and tested for the presence of AIV without any positive result. This is the first report of highly pathogenic avian influenza subtype H5N1 in the Czech Republic. The potential role of swan as an effective vector of avian influenza virus is also discussed.