Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability

Field vaccinated chickens with low antibody titres show equally insufficient protection against matching and non-matching genotypesof virulent Newcastle disease virus

Newcastle disease (ND) is a severe threat to the poultry industry and is caused by virulent strains of Newcastle disease virus (NDV). Many countries maintain a vaccination policy, but NDV is rapidly evolving as shown by the discovery of several new genotypes in the last decades. We tested the efficacy of the currently used classical commercial ND vaccine based on the genotype II strain VG/GA, applied under standard field conditions, against outbreak strains. Field vaccinated broilers were challenged with four different viruses belonging to genotype II, V or VII. A large proportion of field vaccinated broilers showed suboptimal immunity and the protection level against early and recent NDV isolates was dramatically low. Furthermore, there were no significant differences in protection afforded by a genotype II vaccine against a genotype II virus challenge compared to a challenge with viruses belonging to the other genotypes. This study suggests that the susceptibility of vaccinated poultry to NDV infection is not the result of vaccine mismatch, but rather of poor vaccination practice

Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy

After avian influenza (AI) vaccination, hens will produce progeny chickens with maternally derived AI-specific antibodies. In the present study we examined the effect of maternal immunity in young chickens on the protection against highly pathogenic AI H5N1 virus infection and on the effectiveness of AI vaccination. The mean haemagglutination inhibition antibody titre in sera of 14-day-old progeny chickens was approximately eight-fold lower than the mean titre in sera of vaccinated hens. After H5N1 infection at the age of 14 days, chickens with maternal antibody titres lived a few days longer than control chickens. However, only a low proportion of chickens with maternal immunity survived challenge with H5N1. In most progeny chickens with maternal immunity, high virus titres (104 median embryo infective dose) were present in the trachea during the first 4 days after H5N1 infection. In the cloaca, only low virus titres were present in most chickens. In 14-day-old progeny chickens with maternal immunity, the induction of antibody titres by vaccination was severely inhibited, with only a few chickens showing responses similar to the control chickens. It is concluded that high maternal antibody titres are required for clinical protection and reduction of virus titres after infection of chickens, whereas low antibody titres already interfere with vaccine efficacy

Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy

After avian influenza (AI) vaccination, hens will produce progeny chickens with maternally derived AI-specific antibodies. In the present study we examined the effect of maternal immunity in young chickens on the protection against highly pathogenic AI H5N1 virus infection and on the effectiveness of AI vaccination. The mean haemagglutination inhibition antibody titre in sera of 14-day-old progeny chickens was approximately eight-fold lower than the mean titre in sera of vaccinated hens. After H5N1 infection at the age of 14 days, chickens with maternal antibody titres lived a few days longer than control chickens. However, only a low proportion of chickens with maternal immunity survived challenge with H5N1. In most progeny chickens with maternal immunity, high virus titres (104 median embryo infective dose) were present in the trachea during the first 4 days after H5N1 infection. In the cloaca, only low virus titres were present in most chickens. In 14-day-old progeny chickens with maternal immunity, the induction of antibody titres by vaccination was severely inhibited, with only a few chickens showing responses similar to the control chickens. It is concluded that high maternal antibody titres are required for clinical protection and reduction of virus titres after infection of chickens, whereas low antibody titres already interfere with vaccine efficacy

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 virusus

Highly pathogenic avian influenza (HPAI) viruses of H5 clade 2.3.4.4 have spread to many countries in Asia, Europe and North America by migratory wild birds, causing outbreaks on hundreds of poultry farms. Strategies to control spread by wild birds appear limited, hence timely characterization of novel viruses is important to limit the risk for the poultry sector and human health. In this study we characterize three recent viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the H5N8 group A and B viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent virus, the H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with the group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds, and between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, showing that chickens are efficiently infected by (fecal)contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021–2022 outbreak in the Netherlands included red fox (Vulpes vulpes),polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals weresubmitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal.Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness