Comparative molecular characterization,pathogenicity and seroprevalence of avian influenza virus H9N2 in commercial and backyard poultry flocks

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections.     

The risk factors for avian influenza on poultry farms: A meta-analysis

Background

Avian influenza is a severe threat both to humans and poultry, but so far, no systematic review on the identification and evaluation of the risk factors of avian influenza infection has been published. The objective of this meta-analysis is to provide evidence for decision-making and further research on AI prevention through identifying the risk factors associated with AI infection on poultry farms.

Methods

The results from 15 selected studies on risk factors for AI infections on poultry farms were analyzed quantitatively by meta-analysis.

Results

Open water source (OR = 2.89), infections on nearby farms (OR = 4.54), other livestock (OR = 1.90) and disinfection of farm (OR = 0.54) have significant association with AI infection on poultry farms. The subgroup analysis results indicate that there exist different risk factors for AI infections in different types of farms.

Conclusions

The main risk factors for AI infection in poultry farms are environmental conditions (open water source, infections on nearby farms), keeping other livestock on the same farm and no disinfection of the farm.     

Comparative susceptibility of chickens and turkeys to avian influenza A H7N2 virus infection and protective efficacy of a commercial avian influenza H7N2 virus vaccine

During the spring of 2002, a low pathogenic avian influenza (LPAI) A (H7N2) virus caused a major outbreak among commercial poultry in Virginia and adjacent states. The virus primarily affected turkey flocks, causing respiratory distress and decreased egg production. Experimentally, turkeys were more susceptible than chickens to H7N2 virus infection, with 50% bird infectious dose titers equal to 10(0.8) and 10(2.8-3.2), respectively. Comparison of virus shedding from the cloaca and oropharynx demonstrated that recent H7N2 virus isolates were readily isolated from the upper respiratory tract but rarely from the gastrointestinal tract. The outbreak of H7N2 virus raised concerns regarding the availability of vaccines that could be used for the prevention and control of this virus in poultry. We sought to determine if an existing commercial avian influenza (AI) vaccine prepared from a 1997 seed stock virus could provide protection against a 2002 LPAI H7N2 virus isolated from a turkey (A/turkey/Virginia/158512/02 [TV/02]) in Virginia that was from the same lineage as the vaccine virus. The inactivated AI vaccine, prepared from A/chicken/ Pennsylvania/21342/97 (CP/97) virus, significantly reduced viral shedding from vaccinated turkeys in comparison with sham controls but did not prevent infection. The protective effect of vaccination correlated with the level of virus-specific antibody because a second dose of vaccine increased antiviral serum immunoglobulin G and hemagglutination inhibition (HI) reactivity titers in two different turkey age groups. Serum from CP/97-vaccinated turkeys reacted equally well to CP/97 and TV/02 antigens by HI and enzyme-linked immunosorbent assay. These results demonstrate the potential benefit of using an antigenically related 1997 H7N2 virus as a vaccine candidate for protection in poultry against a H7N2 virus isolate from 2002.     

不同H9N2亚型禽流感病毒株致病力的比较试验

参照NDV评价毒力的方法对1998—2008年间收集的25株H9N2AIV的致病性进行了比较研究。结果显示,25株H9N2AIV不同毒株间致病性有较大差异,大致分为3类：致病性偏强、致病性中等和致病性较弱。从中选出致病力有差异的8个毒株进行了1日龄雏鸡脑内接种指数（ICPI）、6周龄鸡静脉接种指数（IVPI）以及8周龄SPF鸡人工感染排毒试验,结果证明大部分毒株ICPI和IVPI基本上为0,排毒散毒的高峰期在攻毒后第5天到第6天。在25个毒株中,3#和12#表现出了比较高的致病性,不仅其EID50值最高（分别为10-8.8/0.2mL和10-8.8/0.2mL）、ELD50值最高（分别为10-7.9/0.2mL和10-8.7/0.2mL）,而且鸡胚平均死亡时间也最短（分别为66.5,69h）。3#、12#还出现了1日龄雏鸡脑内接种指数（分别为0.238,0.437）和6周鸡静脉内接种指数（分别为0.34,0.51）。在8周龄SPF鸡人工感染排毒试验中3#和12#毒株的排毒量大,排毒时间也明显长于其他毒株（第2～9天）。以上试验结果表明我国H9N2AIV不同毒株致病性有明显差异,呈多态性,致病性偏强的毒株造成鸡群较高的死亡率。     

Molecular characterization of low pathogenicity H7N3 avian influenza viruses isolated in Italy

The complete coding regions of the surface glycoproteins, nucleoprotein (NP), polymerase 2 (PB2), and matrix (M) of A/turkey/214845/02 and A/turkey/220158/99 (H7N3) low pathogenicity avian influenza (LPAI) viruses isolated in October 2002 in Italy were amplified and sequenced to determine the epidemiologic relationships with an A/turkey/Italy/4603/99 (H7N1/4603/99) LPAI virus isolated during the 1999-2001 epizootic in Italy. The hemagglutinin (HA) of H7N3 viruses showed 97.8% nucleotide similarity with A/turkey/Italy/4603/99 (H7N1), and NP, M, and PB2 gene similarities were 93.6%, 98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed that H7N3 and H7N1 viruses were closely related. Sequence analysis revealed a 23 amino acid deletion in the stalk of the neuraminidase of H7N3 viruses and a unique deletion of amino acid glycine in position 17 in the NP gene of H7N1 virus.     

H9N2亚型禽流感病毒对鸡输卵管致病性的研究

为了研究H9N2亚型禽流感病毒(H9N2 AIV)对鸡输卵管的致病性,本研究首先采用凝集素亲和组化染色法检验了流感病毒受体SAα-2,3Gal在鸡输卵管上的分布;然后用H9N2 AIV人工感染产蛋母鸡,制作输卵管的病理组织切片观察其病理变化;最后,采用胶原酶Ⅰ灌注消化法培养鸡输卵管膨大部的原代上皮细胞,接毒H9N2 AIV后观察细胞病变.试验结果表明,鸡输卵管除漏斗部没发现SAα-2,3Gal受体,其余部分均呈强阳性分布;攻毒后病理组织切片观察,输卵管膨大部上皮细胞绒毛脱落,部分细胞坏死、脱落,组织间隙变大,子宫内部组织内部充血出血,其他部分变化不明显;原代细胞接毒72 h后,细胞病变不明显,但是上清中HA滴度可以达到3log2.从以上三方面可以看出,鸡输卵管上皮细胞膜上存在禽流感病毒受体,H9N2 AIV可以引起侵害鸡输卵管,并引起部分输卵管病变,而且H9N2 AIV可以感染鸡输卵管膨大部原代上皮细胞,H9N2 AIV有可能通过输卵管感染母鸡.     

Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus

In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.     

两株H4N3禽流感病毒全基因组序列分析及对小鼠的致病性研究

2009年在我国南方活禽交易市场进行流行病学调查时,从鸭体内分离到2株H4N3亚型禽流感病毒(AIV),DK/FJ/S1419/09(H4N3)(FJ/419/2009)和DK/HUN/S1010/09(H4N3)(HuN/010/2009)。为了解这2株H4N3亚型AIV的生物学特性,本研究对其进行全基因组分析及对小鼠致病性研究。结果显示:其HA基因来源于近两年流行的H4亚型病毒株,NA基因来源于其它亚型病毒株。内部基因来源较复杂,与FJ/419/2009内部基因同源性最高的病毒株均来自国内H5、H6等亚型分离株,但与HuN/010/2009同源的内部基因则差异较大,与PA、M和NS同源性最高的病毒株分别为A/wild/duck/Korea/UP122/2007(H1N1),A/muscovy/duck/Thailand/CU-LM1983/2009(H4N6)and A/avian/Japan 8KI0068/2008(H3N6)。用106EID50病毒剂量感染6周龄BALB/c小鼠,结果显示试验组小鼠感染后第3 d采脏器样品,仅在鼻甲和肺部能检测到病毒存在。以上数据表明,尽管这2株H4N3特殊亚型组合病毒来源复杂,但对小鼠的致病性较低。     

Comparative pathobiology of low and high pathogenicity H7N3 Chilean avian influenza viruses in chickens

Chickens were intranasally inoculated with Chilean H7N3 avian influenza (AI) viruses of low pathogenicity (LP) (H7N3/LP), high pathogenicity (HP) (H7N3/HP), and a laboratory derivative (02-AI-15-#9) (H7N3/14D) from the LPAI virus to determine pathobiologic effects. All chickens inoculated with H7N3/HP AI virus became infected and abruptly died 2 or 3 days postinoculation, but a few showed moderate depression before death. The H7N3/HP AI virus produced focal hemorrhages of the comb, petechial hemorrhage at the esophageal-proventricular junction and proventricular mucosa, edema and congestion of the lung, petechiation of the spleen, and generalized decrease in body fat. Histologically, severe necrosis, hemorrhage, and inflammation were primarily identified in lungs and the lymphoid tissues. All tissues sampled from the H7N3/HP AI group were positive for the AI viral antigen, predominantly in endothelium of blood vessels throughout most tissues and less frequently in histiocytes and cellular debris of lymphoid tissues. Even less consistently, cardiac myocytes, hepatocytes, Kupffer cells, glandular epithelial cells, microglial cells, and neurons became infected. These studies suggest the Chilean H7N3/LP AI virus was poorly infectious for chickens and may have been recently introduced from a nongalliform host. By contrast, the H7N3/HP AI virus was highly infectious and lethal for chickens. The H7N3/HP AI virus had a strong tropism for the cardiovascular system, principally vascular endothelium, which is similar to the viral tropism demonstrated previously with other H5 and H7 HPAI viruses. Interestingly, the H7N3/LP AI virus on intravenous inoculation replicated in cardiac myocytes, a feature of HPAI and not LPAI viruses, which further supports the theory that the H7N3/LP AI virus was in transition from LP to HP.     

Transmission characteristics of low pathogenic avian influenza virus of H7N7 and H5N7 subtypes in layer chickens

Low pathogenic avian influenza virus (LPAIv) infections of H5 and H7 subtypes in poultry are notifiable to the OIE, hence surveillance programmes are implemented. The rate at which LPAIv strains spread within a flock determines the prevalence of infected birds and the time it takes to reach that prevalence and, consequently, optimal sample size and sampling frequency. The aim of this study was to investigate the transmission characteristics of an H7N7 and an H5N7 LPAIv in layer chickens. Two transmission experiments were performed, which consisted of 30 (first experiment) and 20 (second experiment) pairs of conventional layers, respectively. At the start of the experiments, one chicken per pair was inoculated with LPAIv and the other chicken was contact-exposed. Occurrence of infection was monitored by regularly collecting tracheal and cloacal swab samples, which were examined for the presence of virus RNA by RT-PCR. The results of the test were used to estimate the transmission rate parameter (β), the infectious period (T) and the basic reproduction ratio (R(0)). In addition, egg production and virus shedding patterns were quantified. For the H7N7 virus, the β, T and R(0) estimates were 0.10 (95% confidence interval (CI): 0.04-0.18) day(-1), 7.1 (95% CI: 6.5-7.8) days and 0.7 (95% CI: 0.0-1.7), respectively. With the H5N7 virus, only a few inoculated chickens (5 out of 20) became infected and no transmission was observed. This study shows that transmission characteristics of LPAIv strains may vary considerably, which has to be taken into account when designing surveillance programmes.     

Risk factors for the introduction of avian influenza virus into commercial layer chicken farms during the outbreaks caused by a low-pathogenic H5N2 virus in Japan in 2005

Avian influenza outbreaks caused by a low-pathogenic H5N2 virus occurred in Japan from June to December 2005. All 41 affected farms housed layer chickens. Therefore, we conducted a case-control study targeting all commercial layer chicken farms within the movement restriction areas in Ibaraki prefecture, where most outbreaks were detected, to investigate the risk factors for the introduction of avian influenza virus (AIV). Four variables were identified as risk factors associated with the introduction of AIV by multivariate logistic regression: 'introduction of end-of-lay chickens ' (odds ratio (OR) = 36.6), 'sharing of farm equipment among farms' (OR = 29.4), 'incomplete hygiene measures of farm visitors on shoes, clothes and hands' (OR = 7.0), and 'direct distance to the nearest case farm' (0-500 m, OR = 8.6; 500-1000 m, OR = 0.8; 1000-1500 m, OR = 20.1; referenced more than 1500 m). We fully believe that strict biosecurity measures should be applied against any incursion points so as not to introduce AIV into more farms.     

Risk factors for highly pathogenic H7N1 avian influenza virus infection in poultry during the 1999-2000 epidemic in Italy

In 1999-2000, Italian poultry production was disrupted by an H7N1 virus subtype epidemic of highly pathogenic avian influenza (HPAI). The objectives of the present study were to identify risk factors for infection on poultry farms located in regions that had the highest number of outbreaks (Veneto and Lombardia) and the impact of pre-emptive culling as a complementary measure for eradicating infection. A Cox regression model that included spatial factors, such as the G index, was used. The results confirmed the relationship between risk of infection and poultry species, production type and size of farms. The effectiveness of pre-emptive culling was confirmed. An increased risk of infection was observed for poultry farms located near an infected farm and those at altitudes less than 150m above sea level. The measures for the control and eradication of AI virus infection need to consider species differences in susceptibility, the types of production and the density of poultry farms in the affected areas.     

Putative human and avian risk factors for avian influenza virus infections in backyard poultry in Egypt

Highly pathogenic influenza A virus subtype H5N1 causes significant poultry mortality in the six countries where it is endemic and can also infect humans. Egypt has reported the third highest number of poultry outbreaks (n = 1084) globally. The objective of this cross-sectional study was to identify putative risk factors for H5N1 infections in backyard poultry in 16 villages in Damietta, El Gharbia, Fayoum, and Menofia governorates from 2010–2012. Cloacal and tracheal swabs and serum samples from domestic (n = 1242) and wild birds (n = 807) were tested for H5N1 via RT-PCR and hemagglutination inhibition, respectively. We measured poultry rearing practices with questionnaires (n = 306 households) and contact rates among domestic and wild bird species with scan sampling. Domestic birds (chickens, ducks, and geese, n = 51) in three governorates tested positive for H5N1 by PCR or serology. A regression model identified a significant correlation between H5N1 in poultry and the practice of disposing of dead poultry and poultry feces in the garbage (F = 15.7, p < 0.0001). In addition, contact between domestic and wild birds was more frequent in villages where we detected H5N1 in backyard flocks (F = 29.5, p < 0.0001).     

Transmission parameters of highly pathogenic avian influenza (H7N1) among industrial poultry farms in northern Italy in 1999-2000

We estimated between-farm transmission parameters of the highly pathogenic avian-influenza (HPAI) epidemic that struck the poultry industry of northern Italy (including turkeys, layer hens, broilers, gamebirds, and waterfowl) from December 1999 through April 2000. We estimated the average number of susceptible farms that were infected with HPAI virus by each infectious farm during a day (β) with a generalised linear model (GLM). The HPAI's reproductive ratios (R_h; the average number of new infected farms (IFs) that were caused by an infectious farm) were calculated separately for the regions of Lombardy and Veneto, where 382 out of 413 (92.5%) of IFs were located. In both regions, R_h decreased to 1 during the second month of the epidemic (showing that its containment had been initiated). Subsequently, during the last two months of the epidemic, β and R_h were reduced to 0.04/day and 0.6, respectively, in Veneto and to 0.07/day and 0.8 in Lombardy. The reduction of the susceptible population through strict control measures, including pre-emptive slaughter of at-risk poultry flocks, was implemented to a greatest extent in Veneto and this might have been associated with a more rapid control of the epidemic in this region than in Lombardy.     

Identification of risk factors associated with highly pathogenic avian influenza H5N1 virus infection in poultry farms, in Nigeria during the epidemic of 2006-2007

We conducted a matched case-control study to evaluate risk factors for infection with highly pathogenic avian influenza (HPAI) H5N1 virus in poultry farms during the epidemic of 2006-2007 in Nigeria. Epidemiologic data were collected through the use of a questionnaire from 32 case farms and 83 control farms. The frequency of investigated exposure factors was compared between case and control farms by using conditional logistic regression analysis. In the multivariable analysis, the variables for (i) receiving visitors on farm premises (odds ratio [OR]=8.32; 95% confidence interval [CI]=1.87, 36.97; P<0.01), (ii) purchased live poultry/products (OR=11.91; 95% CI=3.11-45.59; P<0.01), and (iii) farm workers live outside the premises (OR=8.98; 95% CI=1.97, 40.77; P<0.01) were identified as risk factors for HPAI in poultry farms. Improving farm hygiene and biosecurity should help reduce the risk for influenza (H5N1) infection in poultry farms in Nigeria.     

Evaluation of disinfectants with the addition of antifreezing compounds against nonpathogenic H7N2 avian influenza virus.

In the winter of 1997 and 1998, in the midst of the H7N2 avian influenza outbreak in Pennsylvania, producers added antifreeze or windshield washer fluid to disinfectant solutions in wash stations to prevent freezing. The purpose of this study was to determine if the addition of these products to the disinfectant solutions would have deleterious effects. Four disinfectants (two phenols, one quarternary ammonium, and one combination product: quarternary ammonium and formaldehyde) and one sodium hypochlorite detergent product currently used in the poultry industry were studied. Each product was diluted according to the manufacturer's recommendation in sterile distilled water and compared with dilutions of the disinfectants with the addition of antifreeze products (ethylene glycol or propylene glycol) or windshield washer fluid for their effectiveness in killing nonpathogenic H7N2 avian influenza virus. All products diluted according to the manufacturer's recommendation killed the nonpathogenic H7N2 avian influenza virus in this test system. The phenol products and the quaternary ammonium product were still efficacious with the addition of the antifreeze containing ethylene glycol. Both the combination product and the sodium hypochlorite detergent had decreased efficacy when the ethylene glycol product was added. When the propylene glycol product was added, the efficacy of all disinfectants remained unaffected, whereas the efficacy of the sodium hypochlorite detergent decreased. With the addition of the windshield washer fluid (methyl alcohol), all products remained efficacious except for the combination product.     

对麻鸭不同致病力H5N1亚型禽法流感病毒的鉴定

禽流感病毒(avain influenza virus,AIV)根据其HA和NA蛋白分为不同亚型,目前16种HA亚型和9种NA亚型均已从水禽中分离到[1].     

两株鸭源H6N2亚型禽流感病毒的序列分析及对鸡的致病性研究

为了解鸭源H6N2亚型禽流感病毒(AIV)的生物学特性,本研究对两株鸭源H6N2亚型AIV [A/duck/Hube/Sd061/2008(HB/061/08)和A/duck/Fujian/S2080/2009(FJ/080/09)]进行序列分析和致病性试验.序列分析显示:两株病毒的HA和NA基因均来源于我国近年流行的H6亚型病毒株,但是HB/061/08株的内部基因可能来源于H9、H5等其他亚型.与病毒株HB/061/08 NP、PA、PB2基因的核苷酸同源性最高的病毒株为A/environment/Hunan/2-84/2007(H9N2);与M、PB1和NS基因的核苷酸同源性最高的病毒株分别为A/duck/Zhejiang/11/2000(H5N1)、A/chicken/Hebei/7/2008(H9N2)和A/chicken/Henan/L1/2008(H9N2).两株病毒的抗原差异性较显著,相关系数为0.49;用106 EID50病毒剂量感染4周龄SPF鸡,结果显示FJ/080/09株不能感染鸡,而HB/061/08株在鸡体内能够高效复制,并通过咽喉和泄殖腔持续排毒.鸡群感染后第3d采取脏器样品,在气管和肺部能够检测到病毒存在,部分脏器和器官的组织学观察显示存在一定程度的病理变化.以上数据表明,H6亚型AIV在跨越不同宿主感染的传播过程中,对新宿主适应能力的差异导致对其致病性的差异.