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The clinical relevance of avian metapneumovirus (aMPV) is growing in the poultry sector, especially in broiler farming, where no vaccination is administered in Italy. Given the naïve status of the birds, a serological survey was conducted in a densely populated area of Northern Italy, to evaluate aMPV circulation. Seven farms were selected and sampled in summer/fall, then sampling was repeated in the following season (winter/spring) to assess a possible seasonal effect. In each farm, fifteen birds were blood sampled towards the end of the cycle and sera were analyzed with an ELISA test. Clinical signs were reported in 5 out of 7 farms, although all farms were positive at both sampling points, except for one, which was negative at the first sampling. The seroprevalence within farm ranged from 26.6% to 100%, and antibody titres appear to increase with age. No seasonality effect was evidenced, whereas a farm effect was more distinct. aMPV circulation appears wide in Northern Italian farms, with different clinical outcomes that could be modulated by intrinsic characteristics of the farms. In absence of vaccination, serological monitoring can be a useful tool for viral entrance monitoring, although sampling timing should be evaluated in order to spot seroconversion after late infections., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Porcine circovirus type 2 (PCV-2) is among the most burdensome viruses of the swine industry globally. Several genotypes have been periodically emerging, but just three of them (PCV-2a, PCV-2b, and PCV-2d) seem to circulate worldwide and be associated with the disease. Conversely, the spatial-temporal distribution of minor genotypes appears limited and their clinical relevance is still unclear. Recently PCV-2e was incidentally detected for the first time in Europe in a breeding farm in Northeastern Italy, while no connection could be established with countries where this genotype had been previously detected. To investigate circulating genotypes in the neglected rural context and provide a comparison with the most explored industrial context, a molecular survey was performed on samples collected in rural (n = 72) and industrial farms (n = 110) located in the same geographic area. Phylogenetic analysis surprisingly evidenced PCV-2e circulation only in pigs reared in backyard farms (n = 5), while major genotypes (PCV-2a, -2b, -2d) circulate in both rearing contexts. However, the close genetic similarity between the herein detected PCV-2e strains and the previously reported one testify that, although unusual, such rural-to-industrial strains exchange affected also PCV-2e. The greater genetic and phenotypic diversity of PCV-2e genotype compared to other ones might threaten the protection granted by current vaccines. The present study suggests the rural context as an ecological niche for the circulation of PCV-2e, and even of other minor genotypes. PCV-2e detection in pigs with outdoor access further stresses the epidemiological role of backyard farms as interfaces for pathogen introduction, potentially ascribable to the different rearing approaches, lower managerial and biosecurity capabilities, and easier contacts with wildlife., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 cross‑protection. 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 793B‑based 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.

Porcine reproductive and respiratory syndrome (PRRS) is probably the most relevant viral disease affecting pig farming. Despite the remarkable efforts paid in terms of vaccination administration and biosecurity, eradication and long-term control have often been frustrated. Unfortunately, few studies are currently available that objectively link, using a formal statistical approach, viral molecular epidemiology to the risk factors determining the observed scenario. The purpose of the present study is to contribute to filling this knowledge gap taking advantage of the advancements in the field of phylodynamics. Approximately one-thousand ORF7 sequences were obtained from strains collected between 2004 and 2021 from the largest Italian pig company, which implements strict compartmentalization among independent three-sites (i.e., sow herds, nurseries and finishing units) pig flows. The history and dynamics of the viral population and its evolution over time were reconstructed and linked to managerial choices. The viral fluxes within and among independent pig flows were evaluated, and the contribution of other integrated pig companies and rurally risen pigs in mediating such spreading was investigated. Moreover, viral circulation in Northern Italy was reconstructed using a continuous phylogeographic approach, and the impact of several environmental features on PRRSV strain persistence and spreading velocity was assessed. The results demonstrate that PRRSV epidemiology is shaped by a multitude of factors, including pig herd management (e.g., immunization strategy), implementation of strict-independent pig flows, and environmental features (e.g., climate, altitude, pig density, road density, etc.) among the others. Small farms and rurally raised animals also emerged as a potential threat for larger, integrated companies. These pieces of evidence suggest that none of the implemented measures can be considered effective alone, and a multidimensional approach, ranging from individual herd management to collaboration and information sharing among different companies, is mandatory for effective infection control.

Avian metapneumovirus (aMPV) is a viral pathogen that causes respiratory signs and drops in egg production most notably in turkeys but also affects chickens, ducks and other species. Four subgroups (aMPV-A, -B, -C and -D) have been characterized to date, which differ genetically, antigenically and in terms of geographic distribution and host range. Despite the rising awareness towards aMPV, many aspects of its epidemiology are still poorly understood. In this pilot study, a serological survey was carried out to assess the possible circulation of subgroups A, B, and C in intensively raised ducks in Northern Italy. A total of 220 sera from 10 Pekin duck flocks and one mallard flock were collected at slaughter and tested by subgroup-specific aMPV-A, B, and C indirect ELISA assays. None of the tested birds had been vaccinated against aMPV and no symptoms had been reported. No aMPV-A, B or C antibodies were detected in any bird of the Pekin duck flocks, whereas the entire mallard flock tested positive for aMPV-C antibodies. This is the first report of aMPV-C antibodies in ducks in Italy, where only aMPV-B has been reported to circulate in recent years. Further research efforts will be targeted towards the screening of a larger panel of samples, in the hope of obtaining positive samples from which full length genome sequences and infectious viruses can be isolated for virus characterization. Finally, the demonstration of aMPV-C specific antibodies in the serum of all sampled mallards indirectly suggests that this species is readily infected by aMPV-C and supports a possible role of wild anatids as a transmission vector of the virus., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Feline panleukopenia is a severe disease of cats caused by feline parvovirus (FPV), and marginally canine parvovirus (CPV). Despite being less rapid than CPV, FPV evolution deserves attention, especially since outbreaks of particular severity are currently reported. This apparently different virulence needs monitoring from genetic and clinical points of view. This manuscript explored FPV molecular epidemiology at both Italian and international levels and the possible association between viral phylogeny and disease severity. Sequences from clinical cases of feline panleukopenia in Italy were obtained from 2011 to 2019, and the etiological agent was characterized, distinguishing FPV from CPV. Phylogenetic and phylodynamic analyses were conducted on Italian and international sequences. Moreover, the association between the viral sequence and clinical variables was evaluated on a group of highly characterized patients. After its origin in the 1920s, FPV showed a constant population size until a more recent expansion since 2000. Few long-distance introduction events characterized FPV spreading, however, most of its evolution occurred locally. Although without a strong statistical association, several clinical variables appeared influenced by viral phylogeny, suggesting a differential virulence potentially characterizing FPV strains. These results stress the importance of the continuous study of viral evolution and its repercussions on the disease clinical aspects.

Infectious bronchitis virus (IBV) control is mainly based on wide vaccine administration. Although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. Despite this, the determinants of IBV epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. In the present study, 361 IBV QX (the most relevant field genotype in Italy) sequences were obtained between 2012 and 2016 from the two main Italian integrated poultry companies. Several biostatistical and bioinformatics approaches were used to reconstruct the history of the QX genotype in Italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. Moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third "ghost" deme, representative of minor industrial poultry companies and the rural sector. The obtained results suggest that the integration of the poultry companies is an effective barrier against IBV spreading, since the strains sampled from the two companies formed two essentially-independent clades. Remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of Northern Italy. The inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in Northern Italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of Northern Italy. Accordingly, when the effect of different environmental and urban parameters on IBV geographic spreading was investigated, no factor seems to contribute to IBV dispersal velocity, being poultry population density the only exception. Finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on IBV epidemiology. Overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale.

Molecular-based tools sometimes are the only laboratory techniques available to detect a recently discovered agent, and their validation without the existence of previously described 'gold standard' methods poses a challenge for the diagnosticians. A good example within this scenario is the recently described porcine circovirus 3 (PCV-3) in the swine population worldwide, from which only few PCR methods have been described. Therefore, the primary objective of this study was to estimate the diagnostic accuracy of a direct PCR (dPCR) and a real-time qPCR (qPCR) for detection of PCV-3 in Italian swine population. Bayesian latent class analysis approach was used to rigorously assess their features and applicability in routine diagnostic activity. Data on dPCR and qPCR were available from 116 domestic pigs, which were randomly selected from 55 farms located at different regions in Northern Italy. The sensitivity (Se) estimates of dPCR (94%; posterior credible interval (PCI%) 84-100) and qPCR (96%; PCI% 90-100) were high and similar. The estimated specificity (Sp) of both dPCR and qPCR assays was around 97%. dPCR and qPCR assays showed a high and comparable Se and Sp estimates for the detection of PCV-3 in Italian swine population. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous discovery of new pathogens poses a challenge in the development and evaluation of adequate diagnostic tools. In fact, since molecular-based tools sometimes are the only available laboratory techniques, it is typically difficult to evaluate their diagnostic performances in the absence of a gold standard. The present study assesses this issue, demonstrating the excellent performances of two PCR-based assays for porcine circovirus 3 (PCV-3) detection using a Bayesian latent class analysis approach. Therefore, the molecular tests evaluated under this study constitute reliable tools for the routine diagnosis and surveillance programs of PCV-3 circulating in swine populations., (© 2019 The Society for Applied Microbiology.)

Despite the fact that vaccine resistance has been typically considered a rare phenomenon, some episodes of vaccine failure have been reported with increasing frequency in intensively-raised livestock. Infectious bronchitis virus (IBV) is a widespread avian coronavirus, whose control relies mainly on extensive vaccine administration. Unfortunately, the continuous emergence of new vaccine-immunity escaping variants prompts the development of new vaccines. In the present work, a molecular epidemiology study was performed to evaluate the potential role of homologous vaccination in driving IBV evolution. This was undertaken by assessing IBV viral RNA sequences from the ORF encoding the S1 portion of viral surface glycoprotein (S) before and after the introduction of a new live vaccine on broiler farms in northern-Italy. The results of several biostatistics analyses consistently demonstrate the presence of a higher pressure in the post-vaccination period. Natural selection was detected essentially on sites located on the protein surface, within or nearby domains involved in viral attachment or related functions. This evidence strongly supports the action of vaccine-induced immunity in conditioning viral evolution, potentially leading to the emergence of new vaccine-escape variants. The great plasticity of rapidly-evolving RNA-viruses in response to human intervention, which extends beyond the poultry industry, is demonstrated, claiming further attention due to their relevance for animal and especially human health.

Canine parvovirus is one of the most frequent pathogens of young dogs, causing severe clinical manifestations. Its phenotypic variability led to an antigenic-based classification into different variants, which have been reported worldwide. To update the Italian distribution of CPV, a molecular survey was performed on 100 geographically-annotated samples collected from 2008 to 2015 by full VP2 sequencing. All three antigenic variants (CPV-2a, 2b, 2c) were detected during the studied period, being CPV-2a the most prevalent (60%). Locally, CPV appeared widely distributed, without any regional or temporal clustering, demonstrating an outstanding and uncontrolled within-country viral spreading. The Italian sequences were also contextualized in the International scenario. The analysis of CPV worldwide molecular epidemiology highlighted the remarkable genetic heterogeneity of the circulating strains and their broad distribution. In fact, a frequent viral exchange among Countries, was proven both over short and long distances, involving haplotypes persisting through time. The reported information on viral dissemination patterns appears crucial for understanding the introduction routes of new variants or strains, which could complicate the epidemiological scenario, affect the disease patterns, show possible differential virulence and clinical relevance of emerging strains especially, and impair the vaccine efficacy., (Copyright © 2018 Elsevier B.V. All rights reserved.)