Genetic Diversity of Avian Influenza Viruses Detected in Waterbirds in Northeast Italy Using Two Different Sampling Strategies.

Avian influenza viruses (AIVs), which circulate endemically in wild aquatic birds, pose a significant threat to poultry and raise concerns for their zoonotic potential. From August 2021 to April 2022, a multi-site cross-sectional study involving active AIV epidemiological monitoring was conducted in wetlands of the Emilia-Romagna region, northern Italy, adjacent to densely populated poultry areas. A total of 129 cloacal swab samples (CSs) and 407 avian faecal droppings samples (FDs) were collected, with 7 CSs (5.4%) and 4 FDs (1%) testing positive for the AIV matrix gene through rRT-PCR. A COI-barcoding protocol was applied to recognize the species of origin of AIV-positive FDs. Multiple low-pathogenic AIV subtypes were identified, and five of these were isolated, including an H5N3, an H1N1, and three H9N2 in wild ducks. Following whole-genome sequencing, phylogenetic analyses of the hereby obtained strains showed close genetic relationships with AIVs detected in countries along the Black Sea/Mediterranean migratory flyway. Notably, none of the analyzed gene segments were genetically related to HPAI H5N1 viruses of clade 2.3.4.4b isolated from Italian poultry during the concurrent 2021-2022 epidemic. Overall, the detected AIV genetic diversity emphasizes the necessity for ongoing monitoring in wild hosts using diverse sampling strategies and whole-genome sequencing.

Vaccination of poultry against highly pathogenic avian influenza - Part 2. Surveillance and mitigation measures.

Selecting appropriate diagnostic methods that take account of the type of vaccine used is important when implementing a vaccination programme against highly pathogenic avian influenza (HPAI). If vaccination is effective, a decreased viral load is expected in the samples used for diagnosis, making molecular methods with high sensitivity the best choice. Although serological methods can be reasonably sensitive, they may produce results that are difficult to interpret. In addition to routine molecular monitoring, it is recommended to conduct viral isolation, genetic sequencing and phenotypic characterisation of any HPAI virus detected in vaccinated flocks to detect escape mutants early. Following emergency vaccination, various surveillance options based on virological testing of dead birds ('bucket sampling') at defined intervals were assessed to be effective for early detection of HPAIV and prove disease freedom in vaccinated populations. For ducks, virological or serological testing of live birds was assessed as an effective strategy. This surveillance could be also applied in the peri-vaccination zone on vaccinated establishments, while maintaining passive surveillance in unvaccinated chicken layers and turkeys, and weekly bucket sampling in unvaccinated ducks. To demonstrate disease freedom with > 99% confidence and to detect HPAI virus sufficiently early following preventive vaccination, monthly virological testing of all dead birds up to 15 per flock, coupled with passive surveillance in both vaccinated and unvaccinated flocks, is recommended. Reducing the sampling intervals increases the sensitivity of early detection up to 100%. To enable the safe movement of vaccinated poultry during emergency vaccination, laboratory examinations in the 72 h prior to the movement can be considered as a risk mitigation measure, in addition to clinical inspection; sampling results from existing surveillance activities carried out in these 72 h could be used. In this Opinion, several schemes are recommended to enable the safe movement of vaccinated poultry following preventive vaccination.

Avian influenza overview December 2023-March 2024.

Between 2 December 2023 and 15 March 2024, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (227) and wild (414) birds across 26 countries in Europe. Compared to previous years, although still widespread, the overall number of HPAI virus detections in birds was significantly lower, among other reasons, possibly due to some level of flock immunity in previously affected wild bird species, resulting in reduced contamination of the environment, and a different composition of circulating A(H5N1) genotypes. Most HPAI outbreaks reported in poultry were primary outbreaks following the introduction of the virus by wild birds. Outside Europe, the majority of outbreaks in poultry were still clustered in North America, while the spread of A(H5) to more naïve wild bird populations on mainland Antarctica is of particular concern. For mammals, A(H5N5) was reported for the first time in Europe, while goat kids in the United States of America represented the first natural A(H5N1) infection in ruminants. Since the last report and as of 12 March 2024, five human avian influenza A(H5N1) infections, including one death, three of which were clade 2.3.2.1c viruses, have been reported by Cambodia. China has reported two human infections, including one fatal case, with avian influenza A(H5N6), four human infections with avian influenza A(H9N2) and one fatal case with co-infection of seasonal influenza A(H3N2) and avian influenza A(H10N5). The latter case was the first documented human infection with avian influenza A(H10N5). Human infections with avian influenza remain rare and no sustained human-to-human infection has been observed. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for those occupationally or otherwise exposed to infected animals.

Avian influenza overview September-December 2023.

Between 2 September and 1 December 2023, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (88) and wild (175) birds across 23 countries in Europe. Compared to previous years, the increase in the number of HPAI virus detections in waterfowl has been delayed, possibly due to a later start of the autumn migration of several wild bird species. Common cranes were the most frequently affected species during this reporting period with mortality events being described in several European countries. Most HPAI outbreaks reported in poultry were primary outbreaks following the introduction of the virus by wild birds, with the exception of Hungary, where two clusters involving secondary spread occurred. HPAI viruses identified in Europe belonged to eleven different genotypes, seven of which were new. With regard to mammals, the serological survey conducted in all fur farms in Finland revealed 29 additional serologically positive farms during this reporting period. Wild mammals continued to be affected mostly in the Americas, from where further spread into wild birds and mammals in the Antarctic region was described for the first time. Since the last report and as of 1 December 2023, three fatal and one severe human A(H5N1) infection with clade 2.3.2.1c viruses have been reported by Cambodia, and one A(H9N2) infection was reported from China. No human infections related to the avian influenza detections in animals in fur farms in Finland have been reported, and human infections with avian influenza remain a rare event. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated); this assessment covers different situations that depend on the level of exposure.

Tracking the Selective Pressure Profile and Gene Flow of SARS-CoV-2 Delta Variant in Italy from April to October 2021 and Frequencies of Key Mutations from Three Representative Italian Regions.

The SARS-CoV-2 Delta variant of concern (VOC) was often associated with serious clinical course of the COVID-19 disease. Herein, we investigated the selective pressure, gene flow and evaluation on the frequencies of mutations causing amino acid substitutions in the Delta variant in three Italian regions. A total of 1500 SARS-CoV-2 Delta genomes, collected in Italy from April to October 2021 were investigated, including a subset of 596 from three Italian regions. The selective pressure and the frequency of amino acid substitutions and the prediction of their possible impact on the stability of the proteins were investigated. Delta variant dataset, in this study, identified 68 sites under positive selection: 16 in the spike (23.5%), 11 in nsp2 (16.2%) and 10 in nsp12 (14.7%) genes. Three of the positive sites in the spike were located in the receptor-binding domain (RBD). In Delta genomes from the three regions, 6 changes were identified as very common (>83.7%), 4 as common (>64.0%), 21 at low frequency (2.1%-25.0%) and 29 rare (≤2.0%). The detection of positive selection on key mutations may represent a model to identify recurrent signature mutations of the virus.

Avian influenza overview June-September 2023.

Between 24 June and 1 September 2023, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (25) and wild (482) birds across 21 countries in Europe. Most of these outbreaks appeared to be clustered along coastlines with only few HPAI virus detections inland. In poultry, all HPAI outbreaks were primary and sporadic with most of them occurring in the United Kingdom. In wild birds, colony-breeding seabirds continued to be most heavily affected, but an increasing number of HPAI virus detections in waterfowl is expected in the coming weeks. The current epidemic in wild birds has already surpassed the one of the previous epidemiological year in terms of total number of HPAI virus detections. As regards mammals, A(H5N1) virus was identified in 26 fur animal farms in Finland. Affected species included American mink, red and Arctic fox, and common raccoon dog. The most likely source of introduction was contact with gulls. Wild mammals continued to be affected worldwide, mostly red foxes and different seal species. Since the last report and as of 28 September 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans have been reported by the United Kingdom, and three human infections with A(H5N6) and two with A(H9N2) were reported from China, respectively. No human infection related to the avian influenza detections in animals on fur farms in Finland or in cats in Poland have been reported, and human infections with avian influenza remain a rare event. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated); this assessment covers different situations that depend on the level of exposure.

Vaccination of poultry against highly pathogenic avian influenza - part 1. Available vaccines and vaccination strategies.

Several vaccines have been developed against highly pathogenic avian influenza (HPAI), mostly inactivated whole-virus vaccines for chickens. In the EU, one vaccine is authorised in chickens but is not fully efficacious to stop transmission, highlighting the need for vaccines tailored to diverse poultry species and production types. Off-label use of vaccines is possible, but effectiveness varies. Vaccines are usually injectable, a time-consuming process. Mass-application vaccines outside hatcheries remain rare. First vaccination varies from in-ovo to 6 weeks of age. Data about immunity onset and duration in the target species are often unavailable, despite being key for effective planning. Minimising antigenic distance between vaccines and field strains is essential, requiring rapid updates of vaccines to match circulating strains. Generating harmonised vaccine efficacy data showing vaccine ability to reduce transmission is crucial and this ability should be also assessed in field trials. Planning vaccination requires selecting the most adequate vaccine type and vaccination scheme. Emergency protective vaccination is limited to vaccines that are not restricted by species, age or pre-existing vector-immunity, while preventive vaccination should prioritise achieving the highest protection, especially for the most susceptible species in high-risk transmission areas. Model simulations in France, Italy and The Netherlands revealed that (i) duck and turkey farms are more infectious than chickens, (ii) depopulating infected farms only showed limitations in controlling disease spread, while 1-km ring-culling performed better than or similar to emergency preventive ring-vaccination scenarios, although with the highest number of depopulated farms, (iii) preventive vaccination of the most susceptible species in high-risk transmission areas was the best option to minimise the outbreaks' number and duration, (iv) during outbreaks in such areas, emergency protective vaccination in a 3-km radius was more effective than 1- and 10-km radius. Vaccine efficacy should be monitored and complement other surveillance and preventive efforts.

Emergence and Persistent Circulation of Highly Pathogenic Avian Influenza Virus A (H5N8) in Kosovo, May 2021-May 2022.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on 19 May 2021. The outbreak consisted of three phases: May-June 2021, September-November 2021, and January-May 2022. In total, 32 backyards and 10 commercial holdings tested positive for the virus. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that had been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of three H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.

A novel array of real-time RT-PCR assays for the rapid pathotyping of type I avian paramyxovirus (APMV-1).

Newcastle disease (ND) caused by virulent avian paramyxovirus type I (APMV-1) is a WOAH and EU listed disease affecting poultry worldwide. ND exhibits different clinical manifestations that may either be neurological, respiratory and/or gastrointestinal, accompanied by high mortality. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid discrimination of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1. The appropriateness of molecular methods for APMV-1 pathotyping is often hampered by the high genetic variability of these viruses that affects sensitivity and inclusivity. This work presents a new array of real-time RT-PCR (RT-qPCR) assays that enable the identification of virulent and avirulent viruses in dual mode, i.e., through pathotype-specific probes and subsequent Sanger sequencing of the amplification product. Validation was performed according to the WOAH recommendations. Performance indicators on sensitivity, specificity, repeatability and reproducibility yielded favourable results. Reproducibility highlighted the need for assays optimization whenever major changes are made to the procedure. Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples.

Outbreak of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cats, Poland, June to July 2023.

BackgroundOver a 3-week period in late June/early July 2023, Poland experienced an outbreak caused by highly pathogenic avian influenza (HPAI) A(H5N1) virus in cats.AimThis study aimed to characterise the identified virus and investigate possible sources of infection.MethodsWe performed next generation sequencing and phylogenetic analysis of detected viruses in cats.ResultsWe sampled 46 cats, and 25 tested positive for avian influenza virus. The identified viruses belong to clade 2.3.4.4b, genotype CH (H5N1 A/Eurasian wigeon/Netherlands/3/2022-like). In Poland, this genotype was responsible for several poultry outbreaks between December 2022 and January 2023 and has been identified only sporadically since February 2023. Viruses from cats were very similar to each other, indicating one common source of infection. In addition, the most closely related virus was detected in a dead white stork in early June. Influenza A(H5N1) viruses from cats possessed two amino acid substitutions in the PB2 protein (526R and 627K) which are two molecular markers of virus adaptation in mammals. The virus detected in the white stork presented one of those mutations (627K), which suggests that the virus that had spilled over to cats was already partially adapted to mammalian species.ConclusionThe scale of HPAI H5N1 virus infection in cats in Poland is worrying. One of the possible sources seems to be poultry meat, but to date no such meat has been identified with certainty. Surveillance should be stepped up on poultry, but also on certain species of farmed mammals kept close to infected poultry farms.

Asymptomatic infection with clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) in carnivore pets, Italy, April 2023.

In April 2023, an outbreak of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses carrying the T271A mammalian adaptive mutation in the PB2 protein was detected in a backyard poultry farm in Italy. Five domestic dogs and one cat living on the premises had seroconverted in the absence of clinical signs. Virological and serological monitoring of individuals exposed to the virus proved the absence of human transmission, however, asymptomatic influenza A(H5N1) infections in mammalian pets may have important public health implications.

Avian influenza overview April - June 2023.

Between 29 April and 23 June 2023, highly pathogenic avian influenza (HPAI) A(H5N1) virus (clade 2.3.4.4b) outbreaks were reported in domestic (98) and wild (634) birds across 25 countries in Europe. A cluster of outbreaks in mulard ducks for foie gras production was concentrated in Southwest France, whereas the overall A(H5N1) situation in poultry in Europe and worldwide has eased. In wild birds, black-headed gulls and several new seabird species, mostly gulls and terns (e.g. sandwich terns), were heavily affected, with increased mortality being observed in both adults and juveniles after hatching. Compared to the same period last year, dead seabirds have been increasingly found inland and not only along European coastlines. As regards mammals, A(H5N1) virus was identified in 24 domestic cats and one caracal in Poland between 10 and 30 June 2023. Affected animals showed neurological and respiratory signs, sometimes mortality, and were widely scattered across nine voivodeships in the country. All cases are genetically closely related and identified viruses cluster with viruses detected in poultry (since October 2022, but now only sporadic) and wild birds (December 2022-January 2023) in the past. Uncertainties still exist around their possible source of infection, with no feline-to-feline or feline-to-human transmission reported so far. Since 10 May 2023 and as of 4 July 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans were reported from the United Kingdom, and two A(H9N2) and one A(H5N6) human infections in China. In addition, one person infected with A(H3N8) in China has died. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA, low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated).

Isolation and genome characterization of Lloviu virus from Italian Schreibers's bats.

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses.

Avian influenza overview March - April 2023.

Between 2 March and 28 April 2023, highly pathogenic avian influenza (HPAI) A(H5Nx) virus, clade 2.3.4.4b, outbreaks were reported in domestic (106) and wild (610) birds across 24 countries in Europe. Poultry outbreaks occurred less frequently compared to the previous reporting period and compared to spring 2022. Most of these outbreaks were classified as primary outbreaks without secondary spread and some of them associated with atypical disease presentation, in particular low mortality. In wild birds, black-headed gulls continued to be heavily affected, while also other threatened wild bird species, such as the peregrine falcon, showed increased mortality. The ongoing epidemic in black-headed gulls, many of which breed inland, may increase the risk for poultry, especially in July-August, when first-year birds disperse from the breeding colonies. HPAI A(H5N1) virus also continued to expand in the Americas, including in mammalian species, and is expected to reach the Antarctic in the near future. HPAI virus infections were detected in six mammal species, particularly in marine mammals and mustelids, for the first time, while the viruses currently circulating in Europe retain a preferential binding for avian-like receptors. Since 13 March 2022 and as of 10 May 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans were reported from China (1), and Chile (1), as well as three A(H9N2) and one A(H3N8) human infections in China. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA, and low to moderate for occupationally or otherwise exposed people.

One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

The Evolution of Highly Pathogenic Avian Influenza A (H5) in Poultry in Nigeria, 2021-2022.

In 2021, amidst the COVID-19 pandemic and global food insecurity, the Nigerian poultry sector was exposed to the highly pathogenic avian influenza (HPAI) virus and its economic challenges. Between 2021 and 2022, HPAI caused 467 outbreaks reported in 31 of the 37 administrative regions in Nigeria. In this study, we characterized the genomes of 97 influenza A viruses of the subtypes H5N1, H5N2, and H5N8, which were identified in different agro-ecological zones and farms during the 2021-2022 epidemic. The phylogenetic analysis of the HA genes showed a widespread distribution of the H5Nx clade 2.3.4.4b and similarity with the HPAI H5Nx viruses that have been detected in Europe since late 2020. The topology of the phylogenetic trees indicated the occurrence of several independent introductions of the virus into the country, followed by a regional evolution of the virus that was most probably linked to its persistent circulation in West African territories. Additional evidence of the evolutionary potential of the HPAI viruses circulating in this region is the identification in this study of a putative H5N1/H9N2 reassortant virus in a mixed-species commercial poultry farm. Our data confirm Nigeria as a crucial hotspot for HPAI virus introduction from the Eurasian territories and reveal a dynamic pattern of avian influenza virus evolution within the Nigerian poultry population.

Highly pathogenic avian influenza A (H5N1) virus infections in wild carnivores connected to mass mortalities of pheasants in Finland.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe during 2020-2022. Virus types H5N8 and H5N1 have dominated the epidemic. Isolated spill-over infections in mammals started to emerge as the epidemic continued. In autumn 2021, HPAI H5N1 caused a series of mass mortality events in farmed and released pheasants (Phasianus colchicus) in a restricted area in southern Finland. Later, in the same area, an otter (Lutra lutra), two red foxes (Vulpes vulpes) and a lynx (Lynx lynx) were found moribund or dead and infected with H5N1 HPAI virus. Phylogenetically, H5N1 strains from pheasants and mammals clustered together. Molecular analyses of the four mammalian virus strains revealed mutations in the PB2 gene segment (PB2-E627K and PB2-D701N) that are known to facilitate viral replication in mammals. This study revealed that avian influenza cases in mammals were spatially and temporally connected with avian mass mortalities suggesting increased infection pressure from birds to mammals.

Vaccine Interaction and Protection against Virulent Avian Metapneumovirus (aMPV) Challenge after Combined Administration of Newcastle Disease and aMPV Live Vaccines to Day-Old Turkeys.

Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) are among the most impactful pathogens affecting the turkey industry. Since turkeys are routinely immunized against both diseases, the hatchery administration of the combined respective live vaccines would offer remarkable practical advantages. However, the compatibility of NDV and aMPV vaccines has not yet been experimentally demonstrated in this species. To address this issue, an aMPV subtype B live vaccine was administered to day-old poults either alone or in combination with one of two different ND vaccines. The birds were then challenged with a virulent aMPV subtype B strain, clinical signs were recorded and aMPV and NDV vaccine replication and humoral immune response were assessed. All results supported the absence of any interference hampering protection against aMPV, with no significant differences in terms of clinical scoring. In addition, the mean aMPV vaccine viral titers and antibody titers measured in the dual vaccinated groups were comparable or even higher than in the group vaccinated solely against aMPV. Lastly, based on the NDV viral and antibody titers, the combined aMPV and NDV vaccination does not seem to interfere with protection against NDV, although further studies involving an actual ND challenge will be necessary to fully demonstrate this hypothesis.