Evaluating surveillance strategies for the early detection of low pathogenicity avian influenza infections

In recent years, the early detection of low pathogenicity avian influenza (LPAI) viruses in poultry has become increasingly important, given their potential to mutate into highly pathogenic viruses. However, evaluations of LPAI surveillance have mainly focused on prevalence and not on the ability to act as an early warning system. We used a simulation model based on data from Italian LPAI epidemics in turkeys to evaluate different surveillance strategies in terms of their performance as early warning systems. The strategies differed in terms of sample size, sampling frequency, diagnostic tests, and whether or not active surveillance (i.e., routine laboratory testing of farms) was performed, and were also tested under different epidemiological scenarios. We compared surveillance strategies by simulating within-farm outbreaks. The output measures were the proportion of infected farms that are detected and the farm reproduction number (R(h)). The first one provides an indication of the sensitivity of the surveillance system to detect within-farm infections, whereas R(h) reflects the effectiveness of outbreak detection (i.e., if detection occurs soon enough to bring an epidemic under control). Increasing the sampling frequency was the most effective means of improving the timeliness of detection (i.e., it occurs earlier), whereas increasing the sample size increased the likelihood of detection. Surveillance was only effective in preventing an epidemic if actions were taken within two days of sampling. The strategies were not affected by the quality of the diagnostic test, although performing both serological and virological assays increased the sensitivity of active surveillance. Early detection of LPAI outbreaks in turkeys can be achieved by increasing the sampling frequency for active surveillance, though very frequent sampling may not be sustainable in the long term. We suggest that, when no LPAI virus is circulating yet and there is a low risk of virus introduction, a less frequent sampling approach might be admitted, provided that the surveillance is intensified as soon as the first outbreak is detected.     

Highly pathogenic avian influenza outbreak mitigated by seasonal low pathogenic strains: Insights from dynamic modeling

The spread of highly pathogenic avian influenza (HPAI) H5N1 remains a threat for both wild and domestic bird populations, while low pathogenic avian influenza (LPAI) strains have been reported to induce partial immunity to HPAI in poultry and some wild birds inoculated with both HPAI and LPAI strains. Here, based on the reported data and experiments, we develop a two-strain avian influenza model to examine the extent to which this partial immunity observed at the individual level can affect the outcome of the outbreaks among migratory birds in the wild at the population level during different seasons. We find a distinct mitigating effect of LPAI on the death toll induced by HPAI strain, and this effect is particularly important for populations previously exposed to and recovered from LPAI. We further investigate the effect of the dominant mode of transmission of an HPAI strain on the outcome of the epidemic. Four combinations of contact based direct transmission and indirect fecal-to-oral (or environmental) routes are examined. For a given infection peak of HPAI, indirect fecal-to-oral transmission of HPAI can lead to a higher death toll than that associated with direct transmission. The mitigating effect of LPAI can, in turn, be dependent on the route of infection of HPAI.     

Homo- and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.     

Replication and adaptive mutations of low pathogenic avian influenza viruses in tracheal organ cultures of different avian species

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.     

Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses

Genetic reassortment of H5N1 highly pathogenic avian influenza viruses (HPAI) with currently circulating human influenza A strains is one possibility that could lead to efficient human-to-human transmissibility. Domestic pigs which are susceptible to infection with both human and avian influenza A viruses are one of the natural hosts where such reassortment events could occur. Virological, histological and serological features of H5N1 virus infection in pigs were characterized in this study. Two- to three-week-old domestic piglets were intranasally inoculated with 10(6) EID(50) of A/Vietnam/1203/04 (VN/04), A/chicken/Indonesia/7/03 (Ck/Indo/03), A/Whooper swan/Mongolia/244/05 (WS/Mong/05), and A/Muscovy duck/Vietnam/ 209/05 (MDk/VN/05) viruses. Swine H3N2 and H1N1 viruses were studied as a positive control for swine influenza virus infection. The pathogenicity of the H5N1 HPAI viruses was also characterized in mouse and ferret animal models. Intranasal inoculation of pigs with H5N1 viruses or consumption of infected chicken meat did not result in severe disease. Mild weight loss was seen in pigs inoculated with WS/Mong/05, Ck/Indo/03 H5N1 and H1N1 swine influenza viruses. WS/Mong/05, Ck/Indo/03 and VN/04 viruses were detected in nasal swabs of inoculated pigs mainly on days 1 and 3. Titers of H5N1 viruses in nasal swabs were remarkably lower compared with those of swine influenza viruses. Replication of all four H5N1 viruses in pigs was restricted to the respiratory tract, mainly to the lungs. Titers of H5N1 viruses in the lungs were lower than those of swine viruses. WS/Mong/05 virus was isolated from trachea and tonsils, and MDk/VN/05 virus was isolated from nasal turbinate of infected pigs. Histological examination revealed mild to moderate bronchiolitis and multifocal alveolitis in the lungs of pigs infected with H5N1 viruses, while infection with swine influenza viruses resulted in severe tracheobronchitis and bronchointerstitial pneumonia. Pigs had low susceptibility to infection with H5N1 HPAI viruses. Inoculation of pigs with H5N1 viruses resulted in asymptomatic to mild symptomatic infection restricted to the respiratory tract and tonsils in contrast to mouse and ferrets animal models, where some of the viruses studied were highly pathogenic and replicated systemically.     

Detection of low pathogenic avian influenza viruses in wild ducks from Canada: comparison of two sampling methods

Surveillance for avian influenza viruses in wild birds was initiated in Canada in 2005. In 2006, in order to maximize detection of highly pathogenic avian influenza viruses, the sampling protocol used in Canada's Inter-agency Wild Bird Influenza Survey was changed. Instead of collecting a single cloacal swab, as previously done in 2005, cloacal and oropharyngeal swabs were combined in a single vial at collection. In order to compare the two sampling methods, duplicate samples were collected from 798 wild dabbling ducks (tribe Anatini) in Canada between 24 July and 7 September 2006. Low pathogenic avian influenza viruses were detected significantly more often (P<0.0001) in combined oropharyngeal and cloacal samples (261/798, 33%) than in cloacal swabs alone (205/798, 26%). Compared to traditional single cloacal samples, combined samples improved virus detection at minimal additional cost.     

高致病性禽流感防控难点的分析

禽流感(avian influenza,AI)是由禽流感病毒引起的一种严重危害畜牧业的急性传染病,特别是高致病性禽流感引起禽类的呼吸系统感染以及全身性败血症,死亡率极高。多年来,许多国家和地区都爆发过此病,造成巨大经济损失,而2004年亚洲爆发的H5N1亚型禽流感造成经济损失的同时还出现了众多的禽流感病毒直接感染人类、造成人员死亡病例,再一次把人类的目光转移向此病。AI抗原类型众多,变异频繁,不同的类型抗原之间无交叉反应,同时,病毒具有复杂的感染和复制机制以及复杂的传播网络等多种因素单独和,协同作用,导致高致病性禽流感防控困难。     

Modelling the wind-borne spread of highly pathogenic avian influenza virus between farms

A quantitative understanding of the spread of contaminated farm dust between locations is a prerequisite for obtaining much-needed insight into one of the possible mechanisms of disease spread between farms. Here, we develop a model to calculate the quantity of contaminated farm-dust particles deposited at various locations downwind of a source farm and apply the model to assess the possible contribution of the wind-borne route to the transmission of Highly Pathogenic Avian Influenza virus (HPAI) during the 2003 epidemic in the Netherlands. The model is obtained from a Gaussian Plume Model by incorporating the dust deposition process, pathogen decay, and a model for the infection process on exposed farms. Using poultry- and avian influenza-specific parameter values we calculate the distance-dependent probability of between-farm transmission by this route. A comparison between the transmission risk pattern predicted by the model and the pattern observed during the 2003 epidemic reveals that the wind-borne route alone is insufficient to explain the observations although it could contribute substantially to the spread over short distance ranges, for example, explaining 24% of the transmission over distances up to 25 km.     

Comparative analysis of avian influenza virus diversity in poultry and humans during a highly pathogenic avian influenza A (H7N7) virus outbreak

Although increasing data have become available that link human adaptation with specific molecular changes in nonhuman influenza viruses, the molecular changes of these viruses during a large highly pathogenic avian influenza virus (HPAI) outbreak in poultry along with avian-to-human transmission have never been documented. By comprehensive virologic analysis of combined veterinary and human samples obtained during a large HPAI A (H7N7) outbreak in the Netherlands in 2003, we mapped the acquisition of human adaptation markers to identify the public health risk associated with an HPAI outbreak in poultry. Full-length hemagglutinin (HA), neuraminidase (NA), and PB2 sequencing of A (H7N7) viruses obtained from 45 human cases showed amino acid variations at different codons in HA (n=20), NA (n=23), and PB2 (n=23). Identification of the avian sources of human virus infections based on 232 farm sequences demonstrated that for each gene about 50% of the variation was already present in poultry. Polygenic accumulation and farm-to-farm spread of known virulence and human adaptation markers in A (H7N7) virus-infected poultry occurred prior to farm-to-human transmission. These include the independent emergence of HA A143T mutants, accumulation of four NA mutations, and farm-to-farm spread of virus variants harboring mammalian host determinants D701N and S714I in PB2. This implies that HPAI viruses with pandemic potential can emerge directly from poultry. Since the public health risk of an avian influenza virus outbreak in poultry can rapidly change, we recommend virologic monitoring for human adaptation markers among poultry as well as among humans during the course of an outbreak in poultry.     

Transient expression of hemagglutinin antigen from low pathogenic avian influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.     

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.     

Transmission dynamics of low pathogenicity avian influenza infections in Turkey flocks

Low pathogenicity avian influenza (LPAI) viruses of H5 and H7 subtypes have the potential to mutate into highly pathogenic strains (HPAI), which can threaten human health and cause huge economic losses. The current knowledge on the mechanisms of mutation from LPAI to HPAI is insufficient for predicting which H5 or H7 strains will mutate into an HPAI strain, and since the molecular changes necessary for the change in virulence seemingly occur at random, the probability of mutation depends on the number of virus replicates, which is associated with the number of birds that acquire infection. We estimated the transmission dynamics of LPAI viruses in turkeys using serosurveillance data from past epidemics in Italy. We fitted the proportions of birds infected in 36 flocks into a hierarchical model to estimate the basic reproduction number (R(0)) and possible variations in R(0) among flocks caused by differences among farms. We also estimated the distributions of the latent and infectious periods, using experimental infection data with outbreak strains. These were then combined with the R(0) to simulate LPAI outbreaks and characterise the resulting dynamics. The estimated mean within-flock R(0) in the population of infected flocks was 5.5, indicating that an infectious bird would infect an average of more than five susceptible birds. The results also indicate that the presence of seropositive birds does not necessarily mean that the virus has already been cleared and the flock is no longer infective, so that seropositive flocks may still constitute a risk of infection for other flocks. In light of these results, the enforcement of appropriate restrictions, the culling of seropositive flocks, or pre-emptive slaughtering may be useful. The model and parameter estimates presented in this paper provide the first complete picture of LPAI dynamics in turkey flocks and could be used for designing a suitable surveillance program.     

First case of highly pathogenic H5N1 avian influenza virus in Spain

Background  

In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.     

Highly pathogenic avian influenza in magpies (Pica pica sericea) in South Korea

Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease of birds that produces high mortality and morbidity. HPAI was diagnosed in the three dead magpies (Pica pica sericea) submitted to the National Veterinary Research and Quarantine Service. At necropsy, the prominent lesions were multifocal or coalescing necrosis of the pancreas with enlargement of the livers and spleens. Microscopically, there were severely necrotizing pancreatitis and lymphocytic meningoencephalitis. Influenza viral antigen was also detected in areas closely associated with histologic lesions. Avian influenza virus was isolated from cecal tonsils and feces of the magpies. The isolated virus was identified as a highly pathogenic H5N1, with hemagglutinin proteolytic cleavage site deduced amino acid sequence of QREKRKKR/GLFGAIAG. To determine the pathogenicity of the isolate, eight 6-wk-old specific-pathogen-free chickens were inoculated intravenously with the virus, and all birds died within 24 hr after inoculation. This is the first report of HPAI in magpies.     

Control of a highly pathogenic H5N1 avian influenza outbreak in the GB poultry flock

The identification of H5N1 in domestic poultry in Europe has increased the risk of infection reaching most industrialized poultry populations. Here, using detailed data on the poultry population in Great Britain (GB), we show that currently planned interventions based on movement restrictions can be expected to control the majority of outbreaks. The probability that controls fail to keep an outbreak small only rises to significant levels if most transmission occurs via mechanisms which are both untraceable and largely independent of the local density of premises. We show that a predictor of the need to intensify control efforts in GB is whether an outbreak exceeds 20 infected premises. In such a scenario neither localized reactive vaccination nor localized culling are likely to have a substantial impact. The most effective of these contingent interventions are large radius (10 km) localized culling and national vaccination. However, the modest impact of these approaches must be balanced against their substantial inconvenience and cost.     

Detection of avian influenza viruses from shorebirds: evaluation of surveillance and testing approaches

Although influenza A viruses have been isolated from numerous shorebird species (Family: Scolopacidae) worldwide, our understanding of natural history of these viruses in this diverse group is incomplete. Gaining this information can be complicated by sampling difficulties related to live capture, the need for large sample sizes related to a potentially low prevalence of infection, and the need to maintain flexibility in diagnostic approaches related to varied capabilities and resources. To provide information relevant to improving sampling and testing of shorebirds for influenza A viruses, we retrospectively evaluated a combined data set from Delaware Bay, USA, collected from 2000 to 2009. Our results indicate that prevalence trends and subtype diversity can be effectively determined by either direct sampling of birds or indirect sampling of feces; however, the extent of detected subtype diversity is a function of the number of viruses recovered during that year. Even in cases where a large number of viruses are identified, an underestimate of true subtype diversity is likely. Influenza A virus isolation from Ruddy Turnstones can be enhanced by testing both cloacal and tracheal samples, and matrix real-time PCR can be used as an effective screening tool. Serologic testing to target species of interest also has application to shorebird surveillance. Overall, all of the sampling and diagnostic approaches have utility as applied to shorebird surveillance, but all are associated with inherent biases that need to be considered when comparing results from independent studies.