Risk Factors for Exposure to Influenza A Viruses,Including Subtype H5 Viruses,in Thai Free‐Grazing Ducks

Free‐grazing ducks (FGD) have been associated with highly pathogenic avian influenza (HPAI) H5N1 outbreaks and may be a viral reservoir. In July–August 2010, we assessed influenza exposure of Thai FGD and risk factors thereof. Serum from 6254 ducks was analysed with enzyme‐linked immunosorbent assay (ELISA) to detect antibodies to influenza A nucleoprotein (NP), and haemagglutinin H5 protein. Eighty‐five per cent (5305 ducks) were seropositive for influenza A. Of the NP‐seropositive sera tested with H5 assays (n = 1423), 553 (39%) were H5 ELISA positive and 57 (4%) suspect. Twelve per cent (74 of 610) of H5 ELISA‐positive/suspect ducks had H5 titres ≥ 1 : 20 by haemagglutination inhibition. Risk factors for influenza A seropositivity include older age, poultry contact, flock visitors and older purchase age. Study flocks had H5 virus exposure as recently as March 2010, but no HPAI H5N1 outbreaks have been identified in Thailand since 2008, highlighting a need for rigorous FGD surveillance.     

Different environmental gradients associated to the spatiotemporal and genetic pattern of the H5N8 highly pathogenic avian influenza outbreaks in poultry in Italy

Comprehensive understanding of the patterns and drivers of avian influenza outbreaks is pivotal to inform surveillance systems and heighten nations’ ability to quickly detect and respond to the emergence of novel viruses. Starting in early 2017, the Italian poultry sector has been involved in the massive H5N8 highly pathogenic avian influenza epidemic that spread in the majority of the European countries in 2016/2017. Eighty‐three outbreaks were recorded in north‐eastern Italy, where a densely populated poultry area stretches along the Lombardy, Emilia‐Romagna and Veneto regions. The confirmed cases, affecting both the rural and industrial sectors, depicted two distinct epidemic waves. We adopted a combination of multivariate statistics techniques and multi‐model regression selection and inference, to investigate how environmental factors relate to the pattern of outbreaks diversity with respect to their spatiotemporal and genetic diversity. Results showed that a combination of eco‐climatic and host density predictors were associated with the outbreaks pattern, and variation along gradients was noticeable among genetically and geographically distinct groups of avian influenza cases. These regional contrasts may be indicative of a different mechanism driving the introduction and spreading routes of the influenza virus in the domestic poultry population. This methodological approach may be extended to different spatiotemporal scale to foster site‐specific, ecologically informed risk mitigating strategies.     

Spatial modelling for low pathogenicity avian influenza virus at the interface of wild birds and backyard poultry

Low pathogenicity avian influenza virus (LPAIV) is endemic in wild birds and poultry in Argentina, and active surveillance has been in place to prevent any eventual virus mutation into a highly pathogenic avian influenza virus (HPAIV), which is exotic in this country. Risk mapping can contribute effectively to disease surveillance and control systems, but it has proven a very challenging task in the absence of disease data. We used a combination of expert opinion elicitation, multicriteria decision analysis (MCDA) and ecological niche modelling (ENM) to identify the most suitable areas for the occurrence of LPAIV at the interface between backyard domestic poultry and wild birds in Argentina. This was achieved by calculating a spatially explicit risk index. As evidenced by the validation and sensitivity analyses, our model was successful in identifying high‐risk areas for LPAIV occurrence. Also, we show that the risk for virus occurrence is significantly higher in areas closer to commercial poultry farms. Although the active surveillance systems have been successful in detecting LPAIV‐positive backyard farms and wild birds in Argentina, our predictions suggest that surveillance efforts in those compartments could be improved by including high‐risk areas identified by our model. Our research provides a tool to guide surveillance activities in the future, and presents a mixed methodological approach which could be implemented in areas where the disease is exotic or rare and a knowledge‐driven modelling method is necessary.     

Factors Associated with the Emergence of Highly Pathogenic Avian Influenza A (H5N1) Poultry Outbreaks in China: Evidence from an Epidemiological Investigation in Ningxia, 2012

In April 2012, highly pathogenic avian influenza virus of the H5N1 subtype (HPAIV H5N1) emerged in poultry layers in Ningxia. A retrospective case–control study was conducted to identify possible risk factors associated with the emergence of H5N1 infection and describe and quantify the spatial variation in H5N1 infection. A multivariable logistic regression model was used to identify risk factors significantly associated with the presence of infection; residual spatial variation in H5N1 risk unaccounted by the factors included in the multivariable model was investigated using a semivariogram. Our results indicate that HPAIV H5N1‐infected farms were three times more likely to improperly dispose farm waste [adjusted OR = 0.37; 95% CI: 0.12–0.82] and five times more likely to have had visitors in their farm within the past month [adjusted OR = 5.47; 95% CI: 1.97–15.64] compared to H5N1‐non‐infected farms. The variables included in the final multivariable model accounted only 20% for the spatial clustering of H5N1 infection. The average size of a H5N1 cluster was 660 m. Bio‐exclusion practices should be strengthened on poultry farms to prevent further emergence of H5N1 infection. For future poultry depopulation, operations should consider H5N1 disease clusters to be as large as 700 m.     

Genetic characteristics,pathogenicity and transmission of H5N6 highly pathogenic avian influenza viruses in Southern China

Since 2014, H5 highly pathogenic avian influenza viruses (HPAIVs) from clade 2.3.4.4 have been persistently circulating in Southern China. This has caused huge losses in the poultry industry. In this study, we analysed the genetic characteristics of seven H5N6 HPAIVs of clade 2.3.4.4 that infected birds in Southern China in 2016. Phylogenetic analysis grouped the HA, PB2, PA, M and NS genes as MIX‐like, and the NA genes grouped into the Eurasian lineage. The PB1 genes of the GS24, GS25, CK46 and GS74 strains belonged to the VN 2014‐like group and the others were grouped as MIX‐like. The NP genes of GS24 and GS25 strains belonged to the ZJ‐like group, but the others were MIX‐like. Thus, these viruses came from different genotypes, and the GS24, GS25, CK46 and GS74 strains displayed genotype recombination. Additionally, our results showed that the mean death time of all chickens inoculated with 10⁵ EID₅₀ of CK46 or GS74 viruses was 3 and 3.38 days, respectively. The viruses replicated at high titers in all tested tissues of the inoculated chickens. They also replicated in all tested tissues of naive contact chickens, but their replication titers in some tissues were significantly different (p < 0.05). Thus, the viruses displayed high pathogenicity and variable transmission in chickens. Therefore, it is necessary to focus on the pathogenic variation and molecular evolution of H5N6 HPAIVs in order to prevent and control avian influenza in China.     

Re‐Emergence of a Novel H5N1 Avian Influenza Virus Variant Subclade 2.2.1.1 in Egypt During 2014

Large‐scale surveillance is crucial for understanding the evolution and the emergence of avian influenza viruses (AIVs) in endemic areas. Circulation of highly pathogenic avian influenza (HPAI) subtype H5N1 is continuously causing significant economic losses to the Egyptian poultry industry and is a threat to public health. In this report, a HPAI H5N1 strain (A/chicken/Egypt/Fadllah‐7/2014) was detected from a vaccinated flock showing clinical signs of infection. Genetic characterization of the isolate indicated a high level of nucleotide identity (95–98%) with variant and classical groups of H5N1. Moreover, multiple‐nucleotide and amino acid alignments revealed several prominent and characteristic substitutions in the surface glycoprotein, which may have biological relevance to the pathobiology of the virus. Phylogenetic analysis demonstrated that the reported isolate closely relates to H5N1 AIVs subclade 2.2.1.1 in spite of no reports of this subclade since 2011 from AI reported cases in Egyptian avian species. In conclusion, our results highlight the re‐emergence of a novel H5N1 AIV variant subclade 2.2.1.1 that could escape immunity induced by vaccines. This discovery illustrates the importance of continuous monitoring of poultry in this country for controlling AIV including identifying sources of vaccine seed viruses.     

Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)‐, 2.3.2.1c (H5N1)‐ and 2.3.4.4 (H5N6)‐infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a “mixing vessel” for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian–human influenza virus reassortment if they are also co‐infected with human influenza viruses.     

H7N6 low pathogenic avian influenza outbreak in commercial turkey farms in Chile caused by a native South American Lineage

In December 2016, low pathogenic avian influenza (LPAI) caused by an H7N6 subtype was confirmed in a grow‐out turkey farm located in Valparaiso Region, Chile. Depopulation of exposed animals, zoning, animal movement control and active surveillance were implemented to contain the outbreak. Two weeks later, a second grow‐out turkey farm located 70 km north of the first site was also infected by H7N6 LPAI, which subsequently spilled over to one backyard poultry flock. The virus involved in the outbreak shared a close genetic relationship with Chilean aquatic birds’ viruses collected in previous years. The A/turkey/Chile/2017(H7N6) LPAI virus belonged to a native South American lineage. Based on the H7 and most of the internal genes’ phylogenies, these viruses were also closely related to the ones that caused a highly pathogenic avian influenza outbreak in Chile in 2002. Results from this study help to understand the regional dynamics of influenza outbreaks, highlighting the importance of local native viruses circulating in the natural reservoir hosts.     

Assessment of Biosecurity Measures Against Highly Pathogenic Avian Influenza Risks in Small‐Scale Commercial Farms and Free‐Range Poultry Flocks in the Northcentral Nigeria

There is considerable global concern over the emergence of highly pathogenic avian influenza (HPAI) that has affected domestic poultry flocks in Nigeria and other parts of the world. There have been little investigations on the proposition that free‐range flocks are potentially at higher risk of HPAI than confined small‐scale commercial enterprises. The objective is to analyse the biosecurity measures instituted in the small‐scale commercial poultry farms and established free‐range bird flocks owned by households in the rural areas and qualitatively assess the risk status at the two levels of poultry management systems in northcentral Nigeria. We used data collected through questionnaire administration to farms and flock owners and subjected them to a traffic light system model to test for relative risks of HPAI infection based on the biosecurity measures put in place at the farm and flock levels. The results indicate that free‐range flocks are at lower risk compared to small‐scale commercial operations. These findings are plausible as birds from free‐range flocks have more opportunities to contact wild bird reservoirs of low‐pathogenic avian influenza (LPAI) strains than small‐scale commercial poultry, thus providing them with constant challenge and maintenance of flock immunity. The development of efficient and effective biosecurity measures against poultry diseases on small‐scale commercial farms requires adequate placement of barriers to provide segregation, cleaning and disinfection, while concerted community–led sanitary measures are required for free‐range poultry flocks in the developing topical and subtropical economies.     

Identification of Suitable Areas for African Horse Sickness Virus Infections in Spanish Equine Populations

African horse sickness (AHS) is one of the most important vector‐borne viral infectious diseases of equines, transmitted mainly by Culicoides spp. The re‐emergence of Culicoides‐borne diseases in Europe, such as the recent bluetongue (BT) or Schmallenberg outbreaks, has raised concern about the potential re‐introduction and further spread of AHS virus (AHSV) in Europe. Spain has one of the largest European equine populations. In addition, its geographical, environmental and entomological conditions favour AHSV infections, as shown by the historical outbreaks in the 1990s. The establishment of risk‐based surveillance strategies would allow the early detection and rapid control of any potential AHSV outbreak. This study aimed to identify the areas and time periods that are suitable or at high risk for AHS occurrence in Spain using a GIS‐based multicriteria decision framework. Specifically risk maps for AHS occurrence were produced using a weighted linear combination of the main risk factors of disease, namely extrinsic incubation period, equine density and distribution of competent Culicoides populations. Model results revealed that the south‐western and north‐central areas of Spain and the Balearic Islands are the areas at the highest risk for AHSV infections, particularly in late summer months. Conversely, Galicia, Castile and Leon and La Rioja can be considered as low‐risk regions. This result was validated with historical AHS and BT outbreaks in Spain, and with the Culicoides vector distribution area. The model results, together with current Spanish equine production features, should provide the foundations to design risk‐based and more cost‐effective surveillance strategies for the early detection and rapid control potential of AHS outbreaks in Spain.     

Association of wild bird densities around poultry farms with the risk of highly pathogenic avian influenza virus subtype H5N8 outbreaks in the Netherlands, 2016

Highly pathogenic (HP) avian influenza viruses (AIV) can spread globally through migratory birds and cause massive outbreaks in commercial poultry. AIV outbreaks have been associated with proximity to waterbodies, presence of waterfowl or wild bird cases near poultry farms. In this study, we compared densities of selected HPAI high‐risk wild bird species around 7 locations (H farms) infected with HPAIV H5N8 in the Netherlands in 2016–2017 to densities around 21 non‐infected reference farms. Nine reference farms were in low‐lying water‐rich areas (R‐W) and 12 in higher non‐water‐rich areas (R‐NW). Average monthly numbers/km² of Eurasian wigeons, tufted ducks, Anatidae (ducks, geese and swans) and Laridae (gulls) were calculated between September and April in rings of 0–1, 1–3, 3–6 and 6–10 km around the farms. Linear mixed model analyses showed generally higher bird densities for H and R‐W compared to R‐NW farms between October and March. This was most striking for Eurasian wigeons, with in peak month December 105 (95% CI:17–642) and 40 (7–214) times higher densities around H and R‐W farms, respectively, compared to R‐NW farms. Increased densities around H farms for Eurasian wigeons and Anatidae were more pronounced for distances up to 10 km compared to 0–1 km that mostly consists of the farm yard, which is an unattractive habitat for waterfowl. This distance effect was not observed in gulls, nor in tufted ducks that live on large open waterbodies which are unlikely to be within 0–1 km of farms. This study provides insights into spatio‐temporal density dynamics of HPAI high‐risk birds around farms and their associations with poultry outbreaks. The outcomes indicate that knowledge of environmental and ecological drivers for wild bird presence and abundance may facilitate identification of priority areas for surveillance and biosecurity measures and decisions on establishments of poultry farms to reduce risk of HPAI outbreaks.     

Genetic analysis identifies potential transmission of low pathogenic avian influenza viruses between poultry farms

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between‐farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10‐year surveillance period, we identified 35 potential cases of between‐farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between‐farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between‐farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p < 0.05). The results further suggest that between‐farm transmission was not restricted to holdings of the same poultry type and not related to the housing system. Although separate introductions from the wild bird reservoir cannot be excluded, our study indicates that between‐farm transmission occurred in seven of nine virologically analysed cases. Based on these findings, it is likely that between‐farm transmission contributes considerably to the incidence of LPAI virus infections in poultry.     

Circulation of bluetongue virus 8 in French cattle,before and after the re‐emergence in 2015

Bluetongue virus serotype 8 (BTV ‐8) re‐emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV ‐8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT ‐PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re‐emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDV et) and, when enough serum was available, ELISA ‐positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re‐emergence (N  = 14 of 511), significantly more on the following year (N  = 17 of 257), and eight animals (N  = 158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (N  = 12 of 150), and in winter 2015/2016 in three of them (N  = 21 of 555), where seven animals (N  = 154) seroconverted over 2015. These results suggest that BTV ‐8 may have spread at low levels before the re‐emergence, even in areas considered virus‐free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT ‐PCR .     

Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek^™ Avian Influenza Virus MultiS‐Screen^® Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP‐blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen‐based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample‐to‐negative (S/N) ratio of influenza‐inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated‐uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus‐infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.     

Genetic characterization of an H13N2 low pathogenic avian influenza virus isolated from gulls in China

Low pathogenic avian influenza viruses circulate in wild birds but are occasionally transmitted to other species, including poultry, mammals and humans. To date, infections with low pathogenic avian influenza viruses of HA subtype 6, HA subtype 7, HA subtype 9 and HA subtype 10 among humans have been reported. However, the epidemiology, genetics and ecology of low pathogenic avian influenza viruses have not been fully understood thus far. Therefore, persistent surveillance of low pathogenic avian influenza virus infections in wild birds and other species is needed. Here, we found a low pathogenic avian influenza virus of the subtype H13N2 (abbreviated as WH42) in black‐tailed gulls in China. All gene sequences of this H13N2 virus were determined and used for subsequent analysis. Phylogenetic analysis of the HA gene and NA gene indicated that WH42 was derived from the Eurasian lineage. We analysed the timing of the reassortment events and found that WH42 was a reassortant whose genes were transferred from avian influenza viruses circulating in Asia, Europe and North America. Additionally, WH42 possessed several molecular markers associated with mammalian virulence and mammalian transmissibility. Interestingly, we also found low but detectable haemagglutination inhibition antibodies against H13N2 low pathogenic avian influenza virus in serum samples collected from chickens. Taken together, our findings show that the H13 virus may have been introduced into poultry and that sustainable surveillance in gulls and poultry is required.     

Chasing Notifiable Avian Influenza in Domestic Poultry: A Case Report of Low‐Pathogenic Avian Influenza H5 Viruses in Two Belgian Holdings

In December 2008, bird species in two geographically distant holdings were found positive for H5 viruses following the annual Avian influenza serological screening in Belgium. The virological tests performed identified in one holding a low‐pathogenic avian influenza (LPAI) virus subtype H5N2, and a H5 LPAI virus was identified by real‐time PCR and direct sequencing at the second holding. The first farm was an outdoor mixed holding housing ornamental birds and poultry (n = 6000) and the second a free‐range geese breeding farm (n = 1500). No clinical signs or mortalities were reported. Control measures defined by Council Directive 2005/94/EC were followed, including notification to the European Commission via the Animal Disease Notification System and to the World Organization for Animal Health, and poultry were killed, while ornamental bird species were quarantined. Partial sequencing of the H5N2 virus haemagglutinin and neuraminidase N2 gene sequences revealed a close homology to some recent LPAI isolates identified from wild birds in Germany and Italy and from wild birds in Eurasia and Africa, respectively. It is noteworthy that, these two holdings were already H5 positive based on HI test results carried out during the previous serological screening; however, no virus was detected at that time. To have a better understanding of the potential ‘silent’ circulation of the H5N2 isolate in the field, experimental infections of chickens and turkeys were performed. The low excretion detected might in part explain viral persistence not associated with spread between gallinaceous birds in the same holding, indicating that the H5N2 LPAI isolate was not fully adapted to these two poultry species. Our results highlighted limitations to only using serological screening for the early detection of LPAI in an ‘at‐risk farm’, suggesting that virological and serological monitoring tests be applied simultaneously as a means of testing animals in ‘at‐risk farms’.     

Consecutive Natural Influenza A Virus Infections in Sentinel Mallards in the Evident Absence of Subtype‐Specific Hemagglutination Inhibiting Antibodies

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand‐raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT‐qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full‐length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian‐type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re‐introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein‐specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.