Characterizing the domestic-wild bird interface through camera traps in an area at risk for avian influenza introduction in Northern Italy.

Direct or indirect interactions between sympatric wildlife and poultry can lead to interspecies disease transmission. Particularly, avian influenza (AI) is a viral epidemic disease for which the poultry-wild bird interface shapes the risks of new viral introductions into poultry holdings. Given this background, the study hereby presented aimed to identify wild bird species in poultry house surroundings and characterize the spatiotemporal patterns of these visits. Eight camera traps were deployed for a year (January to December 2021) in 3 commercial chicken layer farms, including free-range and barn-type setups, located in a densely populated poultry area in Northern Italy at high risk for AI introduction via wild birds. Camera traps' positions were chosen based on wildlife signs identified during preliminary visits to the establishments studied. Various methods, including time series analysis, correspondence analysis, and generalized linear models, were employed to analyze the daily wild bird visits. A total of 1,958 camera trap days yielded 5,978 videos of wild birds from 27 different species and 16 taxonomic families. The animals were predominantly engaged in foraging activities nearby poultry houses. Eurasian magpies (Pica pica), ring-necked pheasants (Phasianus colchicus), and Eurasian collared doves (Streptopelia decaocto) were the most frequent visitors. Mallards (Anas platyrhynchos), an AI reservoir species, were observed only in a farm located next to a fishing sport lake. Time series analysis indicated that wild bird visits increased during spring and winter. Farm and camera trap location also influenced visit frequencies. Overall, the results highlighted specific species that could be prioritized for future AI epidemiological surveys. However, further research is required to assess their susceptibility and infectivity to currently circulating AI viruses, essential for identifying novel bridge hosts.

Highly Pathogenic Avian Influenza (HPAI) H5 Clade 2.3.4.4b Virus Infection in Birds and Mammals.

Avian influenza viruses (AIVs) are highly contagious respiratory viruses of birds, leading to significant morbidity and mortality globally and causing substantial economic losses to the poultry industry and agriculture. Since their first isolation in 2013-2014, the Asian-origin H5 highly pathogenic avian influenza viruses (HPAI) of clade 2.3.4.4b have undergone unprecedented evolution and reassortment of internal gene segments. In just a few years, it supplanted other AIV clades, and now it is widespread in the wild migratory waterfowl, spreading to Asia, Europe, Africa, and the Americas. Wild waterfowl, the natural reservoir of LPAIVs and generally more resistant to the disease, also manifested high morbidity and mortality with HPAIV clade 2.3.4.4b. This clade also caused overt clinical signs and mass mortality in a variety of avian and mammalian species never reported before, such as raptors, seabirds, sealions, foxes, and others. Most notably, the recent outbreaks in dairy cattle were associated with the emergence of a few critical mutations related to mammalian adaptation, raising concerns about the possibility of jumping species and acquisition of sustained human-to-human transmission. The main clinical signs and anatomopathological findings associated with clade 2.3.4.4b virus infection in birds and non-human mammals are hereby summarized.

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.

Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Turkey adenovirus 3: ORF1 gene sequence comparison between vaccine-like and field strains.

Haemorrhagic enteritis is an economically significant disease reported in the majority of the countries where turkeys are raised intensively; it is caused by Turkey adenovirus 3 (TAdV-3). The aim of this study was to analyse and compare the ORF1 gene 3' region from turkey haemorrhagic enteritis virus (THEV) vaccine-like and field strains in order to develop a molecular diagnostic method to differentiate the strains from each other. Eighty samples were analysed by sequencing and phylogenetic analyses using a new set of polymerase chain reaction (PCR) primers targeting a genomic region spanning the partial ORF1, hyd and partial IVa2 gene sequences. A commercial live vaccine was also included in the analysis. The results showed that 56 of the 80 sequences obtained in this study showed ≥99.8% nucleotide identity with the homologous vaccine strain sequence. Three non-synonymous mutations - ntA1274G (aaI425V), ntA1420C (aaQ473H) and ntG1485A (aaR495Q) - were detected in the THEV field strains but not in the vaccine strain. Phylogenetic analysis confirmed the clustering of the field and vaccine-like strains in different phylogenetic branches. In conclusion, the method employed in this study could be a useful tool towards making a correct diagnosis. The data could contribute to the knowledge of field distribution of THEV strains and increase the limited existing information available on native isolates around the world.

Colistin Resistance Genes in Broiler Chickens in Tunisia.

Colistin is a polymyxin antibiotic that has been used in veterinary medicine for decades, as a treatment for enterobacterial digestive infections as well as a prophylactic treatment and growth promoter in livestock animals, leading to the emergence and spread of colistin-resistant Gram-negative bacteria and to a great public health concern, considering that colistin is one of the last-resort antibiotics against multidrug-resistant deadly infections in clinical practice. Previous studies performed on livestock animals in Tunisia using culture-dependent methods highlighted the presence of colistin-resistant Gram-negative bacteria. In the present survey, DNA extracted from cloacal swabs from 195 broiler chickens from six farms in Tunisia was tested via molecular methods for the ten mobilized colistin resistance (mcr) genes known so far. Of the 195 animals tested, 81 (41.5%) were mcr-1 positive. All the farms tested were positive, with a prevalence ranging from 13% to 93%. These results confirm the spread of colistin resistance in livestock animals in Tunisia and suggest that the investigation of antibiotic resistance genes by culture-independent methods could be a useful means of conducting epidemiological studies on the spread of antimicrobial resistance.

Migratory Wild Birds as Potential Long-Distance Transmitters of Toxoplasma gondii Infection.

Toxoplasma gondii is a worldwide distributed zoonotic protozoan capable of infecting a wide range of mammals (including humans) and birds as intermediate hosts. Migratory wild birds, through interconnecting countries along their flyways, can play a role in the spatial spread of T. gondii and could contribute to its sylvatic cycle. Additionally, hunted wild birds used for meat consumption could represent a further source of human infection. To determine the presence of T. gondii in wild birds, a total of 50 individuals belonging to the Anseriformes and Charadriiformes orders were sampled during the 2021-2022 hunting season in Northern Italy. Cardiac muscle samples of three Northern shovelers (Anas clypeata), two wild mallards (A. platyrhynchos), one Eurasian teal (A. crecca), and one Northern lapwing (Vanellus vanellus) were positive for the molecular detection of T. gondii based on a targeted amplification of the B1 gene. A 14% (7/50) overall positivity was observed in the sampled population. Results from this study suggest a moderate exposure of wild aquatic birds to T. gondii, highlighting the importance of a further characterization of T. gondii in its wildlife hosts.

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.

Marek's disease in genetically susceptible Cochin chickens in Italy: a case report.

The present study investigates an outbreak of classical Marek's disease (MD) in backyard Cochin chickens reared for hobby in Italy. Examined chickens showed spastic paralysis of the legs and at necropsy, enlargement and discoloration of the peripheral nerves and plexuses that matched microscopic A­ and B­ type MD lesions. Molecular analysis of the meq gene of the detected Gallid alphaherpesvirus 2 (GaHV­2) strain, showed typical markers of low virulence and the strain shared the entire meq gene sequence with strains circulating in Italian backyard chickens. Furthermore, the haplotype B19 of the major histocompatibility complex (MHC) was defined in the affected chickens, showing that the birds possessed a genetic profile of high susceptibility to MD, allowing the appearance of a classical nervous clinical form after infection with an apparently low pathogenicity GaHV­2 strain. Trade of live ornamental purebred chickens occurs frequently between hobby farmers and biosecurity practices, such as quarantine periods, should be applied to avoid the introduction of infected animals. Veterinarians should raise awareness of this issue and promote the use of vaccines against MD.

Effect of changes of vaccination strategies on IBV epidemiology, diagnosis and control: an Italian retrospective study.

Infectious bronchitis virus (IBV) is among the most impactful poultry pathogens, whose control, based on biosecurity and routine vaccination, is hampered by the existence of countless genetic variants sharing poor cross­protection. A retrospective study was conducted on IBV positive samples collected in Italian broiler farms from 2012 to 2019. In 2015, the adopted vaccination protocol shifted from a Mass and 793B­based vaccines to the administration of Mass and QX vaccines, allowing to study how changes in vaccination strategies may affect IBV epidemiology, control and diagnosis in the field. The most frequently detected lineages were QX (70.3%), 793B (15.8%) and Mass (11.9%). The relative frequencies of QX and 793B detections remained stable throughout the study, while Mass detections significantly increased after the vaccination change. Rather than to an actual growth of Mass population size, this finding may be attributable to different vaccine interactions, with Mass strains being more frequently concealed by 793B vaccines than by QX ones. Based on the obtained results, the two vaccination protocols appear to be similarly effective in fighting IB outbreaks, which in the last decade have been caused primarily by QX field strains in Italy. These results indicate that vaccination strategies may significantly affect IBV epidemiology and diagnosis, and should therefore be considered when choosing and interpreting diagnostic assays and planning control measures.

Disentangling the role of wild birds in avian metapneumovirus (aMPV) epidemiology: A systematic review and meta-analysis.

Given the avian metapneumovirus (aMPV) disease burden in poultry worldwide and the evidence of a possible role played by wild birds in the virus epidemiology, the present study summarizes aMPV serological and molecular data on free-ranging avifauna available in the literature by conducting a systematic review and meta-analysis. A computerized literature research was performed on PubMed, Scopus, CAB Direct and Web of Science to identify relevant publications across the period 1990-2021, along with the screening of reference lists. A random-effect model was applied to calculate pooled prevalence estimates with 95% confidence intervals. The inconsistency index statistic (I2 ) was applied to assess between-study heterogeneity. Subgroup analyses for molecular studies only were performed according to geographical area of samplings, taxonomic order, genus and migration patterns of the birds surveyed. A total of 11 publications on molecular surveys and 6 on serological ones were retained for analysis. The pooled molecular prevalence was 6% (95% CI: 1-13%) and a high between-study heterogeneity was detected (I2  = 96%, p < .01). Moderator analyses showed statistically significant differences according to geographical area studied, taxonomic order and genus. Concerning serological prevalence, a pooled estimate of 14% (95% CI: 1-39%), along with a high between-study heterogeneity, was obtained (I2  = 98%, p < .01). Moderator analysis was not performed due to the scarcity of eligible serological studies included. Overall, molecular and serological evidence suggests that some wild bird taxa could play a role in aMPV epidemiology. Particularly, wild ducks, geese, gulls and pheasants, according to scientific contributions hereby considered, proved to be susceptible to aMPV, and due to host ecology, may act as a viral carrier or reservoir. Further surveys of wild birds are encouraged for a better comprehension of the poultry/wild bird interface in aMPV epidemiology and for better characterizing the virus host breadth.

Rapid, Sensitive, and Species-Specific Detection of Conventional and Recombinant Herpesvirus of Turkeys Vaccines Using Loop-Mediated Isothermal Amplification Coupled With a Lateral Flow Device Readout.

Marek's disease, an economically important disease of chickens caused by virulent serotype 1 strains of the Mardivirus Marek's disease virus (MDV-1), is effectively controlled in the field by live attenuated vaccine viruses including herpesvirus of turkeys (HVT)-both conventional HVT (strain FC126) and, in recent years, recombinant HVT viruses carrying foreign genes from other avian viruses to protect against both Marek's disease and other avian viral diseases. Testing to monitor and confirm successful vaccination is important, but any such test must differentiate HVT from MDV-1 and MDV-2, as vaccination does not prevent infection with these serotypes. End-point and real-time PCR tests are widely used to detect and differentiate HVT, MDV-1 and MDV-2 but require expensive specialist laboratory equipment and trained operators. Here, we developed and validated two tube-based loop-mediated isothermal amplification tests coupled with detection by lateral flow device readout (LAMP-LFD): an HVT-specific test to detect both conventional and recombinant HVT strains, and a second test using novel LAMP primers to specifically detect the Vaxxitek® recombinant HVT. Specificity was confirmed using DNA extracted from virus-infected cultured cells, and limit of detection was determined using plasmid DNA carrying either the HVT or Vaxxitek® genome. The LAMP-LFD tests accurately detected all HVT vaccines, or Vaxxitek® only, in crude DNA as well as purified DNA extracted from field samples of organs, feathers, or poultry house dust that were confirmed positive for HVT by real-time PCR. These LAMP-LFD tests have potential for specific, rapid, simple, and inexpensive detection of HVT vaccines in the field.

Molecular Survey on A, B, C and New Avian Metapneumovirus (aMPV) Subtypes in Wild Birds of Northern-Central Italy.

Recent insights into the genetic and antigenic variability of avian metapneumovirus (aMPV), including the discovery of two new subtypes, have renewed interest in this virus. aMPV causes a well-known respiratory disease in poultry. Domestic species show different susceptibility to aMPV subtypes, whereas sporadic detections in wild birds have revealed links between epidemiology and migration routes. To explore the epidemiology of aMPV in wild species, a molecular survey was conducted on samples that were collected from wild birds during avian influenza surveillance activity in Italy. The samples were screened in pools by multiplex real time RT-PCR assays in order to detect and differentiate subtypes A, B, C, and those that have been newly identified. All the birds were negative, except for a mallard (Anas platyrhynchos) that was positive for aMPV subtype C (sampled in Padua, in the Veneto region, in 2018). The sequencing of partial M and full G genes placed the strain in an intermediate position between European and Chinese clusters. The absence of subtypes A and B supports the negligible role of wild birds, whereas subtype C detection follows previous serological and molecular identifications in Italy. Subtype C circulation in domestic and wild populations emphasizes the importance of molecular test development and adoption to allow the prompt detection of this likely emerging subtype.

First detection of avian metapneumovirus subtype C Eurasian lineage in a Eurasian wigeon (Mareca penelope) wintering in Northeastern Italy: an additional hint on the role of migrating birds in the viral epidemiology.

Avian metapneumovirus (aMPV) economically affects the global poultry industry causing respiratory and reproductive disorders. Considering the paucity of data on aMPV occurrence in European free-ranging avifauna, a molecular survey was conducted on wild birds of 23 species belonging to the orders Anseriformes, Charadriiformes or Passeriformes, captured alive and sampled in Northeast Italy as part of the national avian influenza virus (AIV) surveillance activities. A total of 492 oropharyngeal swabs, collected from 2007-2010, all AIV-negative, were screened from aMPV by subtype-specific qRT-PCR. An aMPV-C strain, named aMPV/C/IT/Wigeon/758/07, was found in a wintering young Eurasian wigeon (Mareca penelope) sampled in November 2007. The matrix, fusion, and attachment glycoprotein genes of the detected strain were subsequently amplified by specific independent RT-PCRs, then sequenced, and compared in a phylogenetic framework with known aMPV homologous sequences retrieved from GenBank. Close genetic relationships were found between the aMPV/C/IT/Wigeon/758/07 strain and subtype C Eurasian lineage strains isolated in the late 1990s in French domestic ducks, suggesting epidemiological links. Eurasian wigeons are medium/long-range migrant dabbling ducks that move along the Black Sea/Mediterranean flyway; our finding might, therefore, be related to migratory bridges between countries. To our knowledge, this is the first molecular evidence of the occurrence of aMPV subtype C in Italy and backdates the aMPV-C circulation to 2007. Moreover, the results suggest the susceptibility of Eurasian wigeons to aMPV. Broader investigations are needed to assess the role of wild ducks and the significance of the wildfowl/poultry interface in aMPV-C epidemiology.RESEARCH HIGHLIGHTSWild birds live-captured in Italy were tested for aMPV detection and characterization.aMPV-C Eurasian lineage was found for the first time in a wintering Eurasian wigeon.Migratory birds could be involved in the aMPV epidemiology.

Infectious bursal disease virus in free-living wild birds: A systematic review and meta-analysis of its sero-viroprevalence on a global scale.

Infectious bursal disease virus (IBDV) is an economically important pathogen for poultry, whereas knowledge of its occurrence in non-poultry hosts is limited. The objective of this systematic review and meta-analysis is to summarize the up-to-date knowledge about the sero-viroprevalence of IBDV in wild birds on a global scale. A computerized literature research was performed on PubMed, Scopus, CAB Direct and Web of Science to find relevant publications, along with the screening of reference lists. Journal articles, book chapters, scientific correspondences, conference proceedings and short communications on IBDV virological and/or serological surveys in free-living wild birds published between 1970 and 2021 were considered as eligible. Among 184 studies found, 36 original contributions met the pre-established criteria. A random-effect model was applied to calculate pooled seroprevalence estimates with 95% confidence intervals, whereas the paucity of virological studies (n = 6) only allowed a qualitative description of the data. The pooled seroprevalence was estimated to be 6% (95% CI: 3%-9%) and a high heterogeneity was detected (I2  = 96%). Sub-group analyses were not performed due to the scarcity of available information about hypothetical moderators. With respect to virological studies, IBDV was detected in Anseriformes, Columbiformes, Galliformes, Passeriformes and Pelecaniformes and different strains related to poultry infection were isolated. Our estimates of serological data showed a moderate exposure of wild birds to IBDV. The susceptibility of different species to IBDV infection underlines their potential role in its epidemiology at least as carriers or spreaders. Indeed, the isolation of IBDV in healthy wild birds with a migratory attitude might contribute to a long-distance spread of the virus and to strain diversity. While a wild reservoir host could not be clearly identified, we believe our work provides useful insights for conducting future surveys which are needed to broaden our knowledge of IBDV occurrence in wild birds.

Longitudinal Survey on aMPV Circulation in French Broiler Flocks following Different Vaccination Strategies.

In recent years, the impact of respiratory disease resulting from Avian Metapneumovirus (aMPV) infection has been generally rising in the broiler industry in Europe. In this context, in order to investigate aMPV contribution to the clinical picture and the potential benefits of diversified vaccination strategies compared to nonvaccination policies, a longitudinal monitoring was performed, also evaluating Infectious Bronchitis Virus (IBV) presence. Broiler flocks located in Western France, where aMPV has already proven to be a health and productivity issue, were screened by RT-PCR on rhino-pharyngeal swabs, and the viruses were genetically characterized by sequence analysis. For a more comprehensive picture of aMPV molecular epidemiology and evolution in France, aMPV subtype B strains detected from 1985 to 1998 were sequenced and included in the analysis. The survey confirmed the detection of aMPV subtype B in commercial broiler flocks in France, together with a certain heterogeneity demonstrated by the circulation of more recent and historical French field strains. No IBV field strains were detected. The implementation and evaluation of different management choices and vaccine strategies suggests once again that immunization does not prevent infection but contributes greatly to the containment of the clinical manifestations.

Genetic Heterogeneity among Chicken Infectious Anemia Viruses Detected in Italian Fowl.

Chicken infectious anemia virus (CIAV) is a pathogen of chickens associated with immunosuppression and with a disease named chicken infectious anemia. The present survey reports an epidemiological study on CIAV distribution in Italian broiler, broiler breeder and backyard chicken flocks. Twenty-five strains were detected by a specifically developed nested PCR protocol, and molecularly characterized by partial VP1 gene or complete genome sequencing. Viral DNA amplification was successfully obtained from non-invasive samples such as feathers and environmental dust. Sequence and phylogenetic analysis showed the circulation of field or potentially vaccine-derived strains with heterogeneous sequences clustered into genogroups II, IIIa, and IIIb. Marker genome positions, reported to be correlated with CIAV virulence, were evaluated in field strains. In conclusion, this is the first survey focused on the molecular characteristics of Italian CIAVs, which have proved to be highly heterogeneous, implementing at the same time a distribution map of field viruses worldwide.

High Frequency and Diversity of Tetracycline Resistance Genes in the Microbiota of Broiler Chickens in Tunisia.

Tetracycline resistance is still considered one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. The aim of this study was to evaluate the prevalence of tetracycline resistance (tet) genes in broiler chickens in Tunisia, and this was done by PCR. Individual cloacal swabs from 195 broiler chickens were collected at two slaughterhouses in the governorate of Ben Arous (Grand Tunis, Tunisia). Chickens were from 7 farms and belonged to 13 lots consisting of 15 animals randomly selected. DNA was extracted and tested for 14 tet genes. All the lots examined were positive for at least 9 tet genes, with an average number of 11 tet genes per lot. Of the 195 animals tested, 194 (99%) were positive for one or more tet genes. Tet(L), tet(M) and tet(O) genes were found in 98% of the samples, followed by tet(A) in 90.2%, tet(K) in 88.7% and tet(Q) in 80%. These results confirm the antimicrobial resistance impact in the Tunisian poultry sector and suggest the urgent need to establish a robust national antimicrobial resistance monitoring plan. Furthermore, the molecular detection of antibiotic resistance genes directly in biological samples seems to be a useful means for epidemiological investigations of the spread of resistance determinants.

Occurrence of Chicken Infectious Anemia Virus in Industrial and Backyard Tunisian Broilers: Preliminary Results.

Chicken infectious anemia virus (CIAV) is an economically important and widely distributed immunosuppressive agent in chickens. This study performed an epidemiological investigation on CIAV circulation in 195 Tunisian broilers, belonging to 13 lots from five industrial farms and in one rural farm. Fifteen animals were detected positive by a VP1 nested PCR. The amplicons were molecularly characterised by complete genome sequencing. All positive samples obtained in this study were from the rural farm, whereas the industrial farms sampled were negative. Nucleotide and amino acid sequence analyses showed a high degree of similarity among the sequences obtained, suggesting the circulation of a single CIAV strain in the positive lot. Phylogenetic analysis based on the CIAV VP1 nucleotide sequence and/or the complete genome showed that the sequences obtained in this study clustered with CIAV strains previously detected in Tunisia, Italy and Egypt, belonging to genogroup II. Our results highlight the need for constant CIAV surveillance in backyard chicken production.

What is new on molecular characteristics of Avian metapneumovirus strains circulating in Europe?

In the present study, one hundred and sixteen partial G gene sequences of Avian metapneumovirus (aMPV) subtype B, obtained during routine diagnostics in different European Countries in the last few years (2014-2019), were analysed by sequence and phylogenetic analyses in order to draw an updated picture of the molecular characteristics of circulating strains. Nucleotide sequences were compared with other sequences of European and non-European aMPV-Bs collected prior to that period or retrieved from GenBank. Phylogenetic relationships among the aMPV-B strains, reconstructed using the maximum likelihood method implemented in MEGA X, demonstrated that aMPV-B has evolved in Europe from its first appearance, frequently displaying a clear relation with the geographic area of detection. The 40% of aMPV-B viruses analysed were classified as vaccine-derived strains, being phylogenetically related, and showing high nucleotide identity with live commercial vaccine strains licensed in Europe. The remaining 60% were classified as field strains since they clustered separately and showed a low nucleotide identity with vaccines and vaccine-derived strains. The phylogenetic tree showed that the virus has continued to evolve from its first appearance in the '80s since more recently detected strains belonged to clades phylogenetically distant from the older strains. Unlike vaccine-derived strains, field strains tended to cluster according to their geographic origin and irrespective of the host species where the viruses had been detected. In conclusion, the molecular characterization of aMPV-B and the differentiation between vaccines and field strains through G gene sequence analysis can be a useful tool towards correct diagnosis and should be routinely applied in order to better address the control strategies.