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Aleutian mink disease virus (AMDV) is one the most relevant pathogens of domestic mink, where it can cause significant economic losses, and wild species, which are considered a threat to mink farms. Despite their relevance, many aspects of the origin, evolution, and geographic and host spreading patterns of AMDV have never been investigated on a global scale using a comprehensive biostatistical approach. The present study, benefitting from a large dataset of sequences collected worldwide and several phylodynamic-based approaches, demonstrates the ancient origin of AMDV and its broad, unconstrained circulation from the initial intercontinental spread to the massive among-country circulation, especially within Europe, combined with local persistence and evolution. Clear expansion of the viral population size occurred over time until more effective control measures started to be applied. The role of frequent changes in epidemiological niches, including different hosts, in driving the high nucleotide and amino acid evolutionary rates was also explored by comparing the strengths of selective pressures acting on different populations. The obtained results suggest that the viral passage among locations and between wild and domesticated animals poses a double threat to farm profitability and animal welfare and health, which is particularly relevant for endangered species. Therefore, further efforts must be made to limit viral circulation and to refine our knowledge of factors enhancing AMDV spread, particularly at the wild-domestic interface.

Vaccination is currently one of the most relevant control strategies in poultry production to reduce infectious disease–induced economic losses and decrease antimicrobial use. Besides intrinsic vaccine efficacy, a proper administration is fundamental to achieve an adequate coverage and protection. Hatchery vaccination is becoming the standard approach for routine vaccination because of administration easiness, the possibility to standardize and optimize the overall process, and the lower impact on animal welfare compared with different types of on-farm vaccination. However, a continuous maintenance, refinement, and training of the personnel are the key to success. In the present work, the effect of longitudinal hatchery audits, performed using a standardized, expert-developed questionnaire was evaluated in 169 hatcheries, located in 11 European countries, over a period of more than 4 yr. A dedicated tablet-based application was implemented for data collection, storage, and analysis, and the obtained scores were used in the evaluation, reporting to the hatchery management and improvement of critical points. A positive significant association was demonstrated between the variation in global and process-specific hatchery scores and the number of performed audits. Similarly, when the longitudinal nature of the data (i.e., multiple visits) was accounted for using linear mixed models, including the hatchery and country as random factors, a significant trend in performance improvement was observed visit after visit, although with certain differences based on the specific score and country. The present study demonstrates the benefits of an objective evaluation of hatchery performances through a standardized questionnaire, followed by the discussion on the major required actions. The widespread application of this approach should lead to a significant improvement in vaccine administration performances, with direct consequences on infectious disease occurrence and animal production performances, and indirectly on therapeutic and control-related costs.

Canine circovirus (CanineCV) is a recently identified virus affecting both domestic and wild carnivores, including foxes, sometimes in presence of severe clinical signs. Its circulation in wild animals can thus represent a potential threat for endangered species conservation and an infection source for dogs. Nevertheless, no data were available on its circulation in the Alps region of Northern Italy. In the present study, samples collected from 186 foxes in the period 2009–2020 from Valle d’Aosta and Veneto regions were tested using a real-time PCR assay, demonstrating a viral circulation of approximatively 2–5%, depending on the considered regions. Two complete or almost complete genome sequences were obtained, highlighting that the detected strains were part of a so defined “fox only” clade, which suggests that, despite common contact opportunities, Alps foxes are not involved in frequent transmission events to domestic dogs. Such genetic isolation could be at least partially attributed to some sort of independent evolution occurred in the foxes, leading to species barrier. Additionally, CanineCV strains in foxes from Italy were unexpectedly related to those previously identified in foxes from the United Kingdom and Scandinavian area. Combining the history of fox distribution in Europe since the last glacial maximum (LGM) with the viral history allowed us to speculate a long-standing coexistence between European canine circovirus and this host, justifying the peculiar geographic distribution and evolutionary paths of the fox infecting clade.

The control of infectious bronchitis (IB) is essential in intensive broiler production and is pursued through strict biosecurity and mass vaccination. Despite effective and routinely adopted, hatchery spray vaccination has been hypothesized to affect chicks’ body temperature and wellbeing. Recently, gel administration has been proposed as an alternative and proved feasible in experimental settings. In this study, IBV spray and gel vaccination methods were compared in field conditions. One hundred birds from the same hatch were enrolled in the study and vaccinated, half by spray and half by gel, with 793B and Mass vaccines. After vaccination, rectal temperature was measured and vaccine intake assessed. The two groups were housed for 35 days in separate pens and swabs and blood samples were collected at multiple time points for genotype-specific molecular analyses and serology, respectively. The temperature was significantly lower in spray-vaccinated chicks 10 min and an hour after administration. A similar trend in 793B titres was observed in both groups, while the Mass vaccine was detected later but persisted longer in gel-vaccinated chicks. No differences were observed in mean antibody titres. Compared to spray, gel administration appears equally effective and less impactful on body temperature, thus supporting its application for IBV vaccination.

Altres ajuts: Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria RTA2017-00007-00-00 Porcine circovirus 3 (PCV-3) was discovered in 2015 using next-generation sequencing (NGS) methods. Since then, the virus has been detected worldwide in pigs displaying several clinical-pathological outcomes as well as in healthy animals. The objective of this review is to critically discuss the evidence existing so far regarding PCV-3 as a swine pathogen. In fact, a significant number of publications claim PCV-3 as a disease causal infectious agent, but very few of them have shown strong evidence of such potential causality. The most convincing proofs of disease association are those that demonstrate a clinical picture linked to multisystemic lymphoplasmacytic to lymphohistiocytic perivascular inflammation and presence of viral nucleic acid within these lesions. Based on these evidence, individual case definitions for PCV-3-reproductive disease and PCV-3-systemic disease are proposed to standardize diagnostic criteria for PCV-3-associated diseases. However, the real frequency of these clinical-pathological conditions linked to the novel virus is unknown, and the most frequent outcome of PCV-3 infection is likely subclinical based on its worlwide distribution.

West Nile Virus (WNV) is a mosquito-borne virus enzootically maintained in birds. However, it can incidentally infect other species, leading to sometimes severe clinical consequences like in horses and especially human beings. Despite the topic relevance, the presence and distribution of WNV are currently unknown in Namibia. Several countries implement surveillance systems based on virus detection in birds, mosquitoes, and vertebrate species including horses. The present study aimed to fill this knowledge gap by serologically evaluating WNV exposure in Namibian donkeys, whose population is remarkably bigger than the horse one. Forty-seven out of 260 sampled animals showed neutralizing antibodies against WNV (18.07% [95% CI = 13.59–23.30%]), demonstrating its circulation in all country territory, although, with apparent regional differences. On the contrary, no association with animal age or sex could be identified. The present study demonstrates the widespread presence of WNV in Namibia as well as the practical utility and effectiveness of donkeys as sentinels for infection surveillance. Due to clinical relevance, vaccination campaigns should be considered for horses of high economic or genetic value. Additionally, the burden of WNV infection on human health should be carefully evaluated.

Porcine circovirus type 3 (PCV-3) infection is widely distributed in domestic pig populations in America, Europe, and Asia. However, no data is currently available about its presence and distribution in Africa. This study investigated the presence of PCV-3 in pigs (n = 122) in Namibia, by means of biomolecular methods. The pig samples collected (n = 122) were representative of the swine industry in Namibia, covering the major pig production facilities in the country. All of the samples tested were negative for PCV-3, and this indicated that the virus was either not present in the country or was circulating at low levels. Further studies are needed to better understand the distribution, if any, of PCV-3 in Namibia.

Archival swine DNA samples from Indonesia and Mongolia, some of which were previously shown to be positive for African swine fever virus, were screened for the presence of porcine circovirus 2 (PCV-2) and porcine circovirus 3 (PCV-3) by PCR. Samples from both countries were positive for PCV-2 (three from Mongolia and two from Indonesia), while none were positive for PCV-3. The PCV-2 amplicons were sequenced, and phylogenetic analysis revealed that the PCV-2 strains belonged to four different genotypes: PCV-2a (Mongolia), PCV-2b (Mongolia and Indonesia), PCV-2d (Indonesia), and PCV-2g (Mongolia). This is the first report of ASFV/PCV-2 coinfection in pigs and the first report of the presence of PCV-2 in Mongolia.

Vaccination against porcine circovirus 2 (PCV-2) is a common practice all over the world. Vaccines can prevent PCV-2-systemic disease (PCV-2-SD) outbreaks but not PCV-2 infection, which can be detectable in a percentage of vaccinated animals. Occasionally, PCV-2-SD is diagnosed in vaccinated farms. The objective of this study was to genotype the PCV-2 strains detected in vaccinated animals diagnosed with PCV-2-SD. Additionally, the evolution of the frequency of PCV-2 genotype detection at Spanish, European, and world levels was assessed. Fifty cases diagnosed as PCV-2-SD between 2009 and 2020 were included in this study. PCV-2 genotype was determined by sequencing the Cap gene region. Among them, only PCV-2b (23/50, 46%) and PCV-2d (27/50, 54%) genotypes were detected. Although the frequency of detection of these two genotypes was similar, their temporal distribution was different. Whereas most PCV-2b sequences (17/23, 74%) were detected between 2009 and 2012, PCV-2d sequences were obtained from 2013 to 2020. Indeed, a predominance of the PCV-2d genotype was observed from 2013 onwards, a trend also noticed at European and world levels. The results suggest that detection of particular genotypes in vaccinated animals probably reflects the general prevalence of the genotypes over time rather than genotype-specific vaccine-immunity escaping. info:eu-repo/semantics/publishedVersion

Porcine circovirus 2 (PCV-2) is the causative agent of porcine circovirus diseases (PCVD). A study was undertaken to determine whether PCV-2 was present in samples collected from commercial pigs (n = 46) and warthogs (n = 42) in Namibia between 2019 and 2020. Twenty-three of the collected samples were positive by PCR (13 from pigs and 10 from warthogs), and a phylogenetic analysis of ORF2 identified three genotypes (PCV-2b and PCV-2d in pigs and PCV-2c in warthogs). This is the first time that PCV-2 has been identified in warthogs and in Namibia. It is also the first report of PCV-2c in Africa.

Poultry production plays a relevant role in the Ethiopian economy and represents a source of poverty alleviation for several social classes. Infectious diseases can therefore significantly impact the economy and welfare. Despite infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) being present, the knowledge of their epidemiology and impact is extremely limited. In the present work, a cross-sectional study based on 500 tracheal swabs collected from 50 intensive and backyard unvaccinated flocks of the Jimma Zone was performed to investigate the circulation of these viruses and molecularly characterize them. IBV and aMPV presence was tested by molecular assays, and genotyping was carried out on positive samples. Accordingly, 6% (95% CI 2.06% to 16.22%) and 8% (95% CI 3.15% to 18.84%) of flocks tested IBV and aMPV positive, respectively. Particularly, IBV 793B (GI-13) strains were detected in backyard flocks only, and identical or closely related sequences (p-distance &lt, 2%) were detected in distantly spaced flocks, suggesting relevant viral circulation. On the contrary, both backyard and intensive flocks were affected by aMPV subtype B. Potential epidemiological links associated to the importation of parental birds from foreign countries could be established. These results highlight non-negligible circulation of these viruses, warranting further epidemiological studies and the evaluation of control measure implementation.

The prevention of avian colibacillosis has historically been investigated through vaccination, with variable outcomes. Commercial live (attenuated) and inactivated vaccines are reported to have limited efficacy in the context of heterologous challenge. Autogenous vaccination, using field isolates, is widely used, but scarcely documented. Different vaccination programs, including a live commercial vaccine and/or an inactivated autogenous vaccine, were compared for three different avian pathogenic Escherichia coli (APEC) strain (serotypes O78, O18 and O111) challenges. On the pullet farm, four groups of conventional pullets received different vaccination protocols. Group A was kept unvaccinated (control group). Group B was vaccinated three times with a live commercial O78 E. coli vaccine (at one day old, 59 and 110 days of age). Group C was immunized twice (at 79 and 110 days) with a three-valence autogenous vaccine (O78, O18 and O111). Group D was vaccinated first with the commercial vaccine (at one day old and 59 days), then with the autogenous vaccine (110 days). Birds were transferred to the experimental facility at 121 days of age and were challenged 10 days later. In each group, 20 birds were challenged with one of the three APEC strains (O78, O18, O111), in total, 80 birds were challenged by the same strains (20 per group). The recorded outcomes were: mortality rate, macroscopic lesion score in target organs and the bacterial recovery of the challenge strain from bone marrow and pooled organs. When challenged with O78 or O111 strains, birds from groups C and D proved to be significantly better protected, in terms of lesion scoring and bacteriological isolation, than those of groups A and B. With the O18 challenge, only birds of group D presented a statistically significant reduction of their lesion score. To the authors&rsquo, knowledge, this is the first report on the efficacy of an immunization program in poultry that combines commercial and autogenous vaccines.

RNA viruses are characterized by high mutation and recombination rates, which allow a rapid adaptation to new environments. Most of the emerging diseases and host jumps are therefore sustained by these viruses. Rapid evolution may also hinder the understanding of molecular epidemiology, affect the sensitivity of diagnostic assays, limit the vaccine efficacy and favor episodes of immune escape, thus significantly complicating the control of even well-known pathogens. The history of infectious bronchitis virus (IBV) fits well with the above-mentioned scenario. Despite being known since the 1930s, it still represents one of the main causes of disease and economic losses for the poultry industry. A plethora of strategies have been developed and applied over time, with variable success, to limit its impact. However, they have rarely been evaluated objectively and on an adequate scale. Therefore, the actual advantages and disadvantages of IBV detection and control strategies, as well as their implementation, still largely depend on individual sensibility. The present manuscript aims to review the main features of IBV biology and evolution, focusing on their relevance and potential applications in terms of diagnosis and control.

Outbreaks of inclusion body hepatitis (IBH) and adenoviral gizzard erosion have been anecdotally reported in Greece since approximately 2011. However, a relevant increase in clinical outbreaks compatible with IBH has been described since 2014. Unfortunately, with limited exceptions, only serological assays were performed, and involved strains were not properly characterized. In the present study, 35 outbreaks were investigated in the period between July 2017 and February 2018 in Greece. In addition to clinical and histopathological diagnosis, fowl adenovirus (FAdV) presence was investigated by PCR and sequencing. Thirty-four out of 35 samples tested FAdV positive. Twenty-nine (85.29%) and 5 (14.71%) strains were classified as FAdV-E and FAdV-D, respectively. Fowl adenovirus-E strains were genetically homogeneous and formed an independent cluster of Greek-only sequences, including the sole previously available sequence, suggesting the prolonged circulation of this species in Greece. On the contrary, FAdV-D strains were more heterogeneous and closely related to strains sampled in other European countries, testifying the occurrence of multiple introduction events. The evaluation of phylogenetic relationships, geographic clustering, age of infection, and origin of the broiler breeder flocks suggests that both vertical and horizontal transmission are important in FAdV epidemiology in Greece and highlights the limited efficacy of currently implemented control measures. Of note, a significantly higher mortality was observed in precociously infected flocks, likely because of the higher susceptibility of younger animals. This evidence stresses the need of preventing vertical and/or early infection to limit the economic impact of adenovirus-induced diseases.

Porcine circovirus 2 (PCV2) is a single stranded DNA virus with one of the highest mutation rates among DNA viruses. This ability allows it to generate a cloud of mutants constantly providing new opportunities to adapt and evade the immune system. This pig pathogen is associated to many diseases, globally called porcine circovirus diseases (PCVD) and has been a threat to pig industry since its discovery in the early 90’s. Although 11 ORFs have been predicted from its genome, only two main proteins have been deeply characterized, i.e. Rep and Cap. The structural Cap protein possesses the majority of the epitopic determinants of this non-enveloped virus. The evolution of PCV2 is affected by both natural and vaccine-induced immune responses, which enhances the genetic variability, especially in the most immunogenic Cap region. Intra-host variability has been also demonstrated in infected animals where long-lasting infections can take place. However, the association between this intra-host variability and pathogenesis has never been studied for this virus. Here, the within-host PCV2 variability was monitored over time by next generation sequencing during an experimental infection, demonstrating the presence of large heterogeneity. Remarkably, the level of quasispecies diversity, affecting particularly the Cap coding region, was statistically different depending on viremia levels and clinical signs detected after infection. Moreover, we proved the existence of hyper mutant subjects harboring a remarkably higher number of genetic variants. Altogether, these results suggest an interaction between genetic diversity, host immune system and disease severity.

Infectious bronchitis (IB) control has a strong impact on poultry farming, because of the necessary epidemiological knowledge for planning the best strategy, the optimal strain association, the priming and boosting interventions. Broiler farming is even more problematic given the short and intense productive cycle, which requires an early onset of protection against most of the infectious threats, possibly with limited respiratory post-vaccination reactions that would have a direct impact on the bird health and productivity. For this purpose, gel vaccination has been proposed as a new approach for infectious bronchitis virus (IBV) control and vaccine intake, kinetics and compatibility of combined strains administered by gel have been analyzed in this study. After gel vaccination with single and combined 1/96 and B-48 strains on 4 groups of commercial broilers, a 21-d-long experimental trial has been conducted to monitor the vaccine safety by clinical assessment and vaccine kinetics by strain-specific real-time RT-PCR on choanal cleft swabs. The vaccine strains administered by gel were safe and negligible respiratory signs were detected, even when combined. Vaccine titers were compared among groups and within the same group among a 10-bird pooled sample and 10 swabs from individually sampled birds. 1/96 strain early reached high titers in all animals, while B-48 presence was less constant even though it was detected in almost all birds before the trial end. The individual and pooled sample comparison revealed a partial overestimation of vaccine titers in the pooled samples and the loss of the prevalence data, although the trend portrayed by the pooled swabs closely followed the individual ones.

Infectious Bronchitis virus (IBV) genotype Q1 was detected for the first time in China in 1996, and then spread worldwide. The first report of Q1 genotype in Italy occurred in 2011 and a deep molecular investigation of a Q1 isolated in Italy in 2013 has led to speculation regarding the origin of this genotype. Phylogenetic analysis of the S1 sequence of a Q1 Italian strain revealed a close relationship with sequences of the 624I strains circulating in Italy in the early 1990s and this led to the idea that 624I was an ancestor of the Q1 genotype. Despite the fact that most heterogeneity of IBVs occurs in the S1 gene, the sequence analysis of this gene alone was not sufficient to confirm or deny this hypothesis. In the present study, an Italian 624I (gammaCoV/AvCov/Ck/Italy/IP14425/96) was fully sequenced for the first time and compared to all available complete Q1 genome sequences. This analysis confirmed the genetic correlation between GammaCoV/AvCov/Ck/Italy/IP14425/96 and Q1 strains, suggesting a common origin between 624I and Q1 genotypes.

Background Diarrhoea is a major cause of death in neonate pigs and most of the viruses that cause it are RNA viruses. Next Generation Sequencing (NGS) deeply characterize the genetic diversity among rapidly mutating virus populations at the interspecific as well as the intraspecific level. The diversity of RNA viruses present in faeces of neonatal piglets suffering from diarrhoea in 47 farms, plus 4 samples from non-diarrhoeic piglets has been evaluated by NGS. Samples were selected among the cases submitted to the Veterinary Diagnostic Laboratories of Infectious Diseases of the Universitat Autònoma de Barcelona (Barcelona, Spain) and Universidad de León (León, Spain). Results The analyses identified the presence of 12 virus species corresponding to 8 genera of RNA viruses. Most samples were co-infected by several viruses. Kobuvirus and Rotavirus were more commonly reported, with Sapovirus, Astrovirus 3, 4 and 5, Enterovirus G, Porcine epidemic diarrhoea virus, Pasivirus and Posavirus being less frequently detected. Most sequences showed a low identity with the sequences deposited in GenBank, allowing us to propose several new VP4 and VP7 genotypes for Rotavirus B and Rotavirus C. Conclusions Among the cases analysed, Rotaviruses were the main aetiological agents of diarrhoea in neonate pigs. Besides, in a small number of cases Kobuvirus and Sapovirus may also have an aetiological role. Even most animals were co-infected in early life, the association with enteric disease among the other examined viruses was unclear. The NGS method applied successfully characterized the RNA virome present in faeces and detected a high level of unreported intraspecific diversity.

Frailty is defined as a decline in an organism’s physiological reserves resulting in increased vulnerability to stressors. In humans, a single continuous variable, the so-called Frailty Index (FI), can be obtained by multidimensionally assessing the biological complexity of an ageing organism. Here, we evaluate this variability in dogs and compare it to the data available for humans. In dogs, there was a moderate correlation between age and the FI, and the distribution of the FI increased with age. Deficit accumulation was strongly related to mortality. The effect of age, when combined with the FI, was negligible. No sex-related differences were evident. The FI could be considered in epidemiological studies and/or experimental trials to account for the potential confounding effects of the health status of individual dogs. The age-related deficit accumulation reported in dogs is similar to that demonstrated in humans. Therefore, dogs might represent an excellent model for human aging studies.

In a recent study, an emerging infectious bursal disease virus (IBDV) genotype (ITA) was detected in IBDV-live vaccinated broilers without clinical signs of infectious bursal disease (IBD). VP2 sequence analysis showed that strains of the ITA genotype clustered separately from vaccine strains and from other IBDV reference strains, either classic or very virulent. In order to obtain a more exhaustive molecular characterization of the IBDV ITA genotype and speculate on its origin, genome sequencing of the field isolate IBDV/Italy/1829/2011, previously assigned to the ITA genotype, was performed, and the sequences obtained were compared to the currently available corresponding sequences. In addition, phylogenetic and recombination analyses were performed. Interestingly, multiple amino acid (AA) sequence alignments revealed that the IBDV/Italy/1829/2011 strain shared several AA residues with very virulent IBDV strains as well as some virulence markers, especially in the VP1 protein. Nevertheless, sequence analysis demonstrated the presence of several residues typical of IBDV strains at a low degree of virulence in the IBDV/Italy/1829/2011 strain. Although homologous recombination and reassortant phenomena may occur naturally among different IBDV strains, no evidence of those events was found in the genome of the IBDV/Italy/1829/2011 strain, which was confirmed to be a genetically distinctive IBDV genotype.

Infectious Bursal Disease Virus (IBDV) of the ITA genotype (G6) was shown to have peculiar molecular characteristics and, despite a subclinical course, aggressiveness towards lymphoid tissues after experimental infection of specific-pathogen-free (SPF) chickens. The aim of the present study was to evaluate and compare with a Classical IBDV strain, ITA IBDV distribution and persistence in various tissues (bursa of Fabricious, spleen, thymus, bone marrow, caecal tonsils, Harderian gland, kidney, liver and proventriculus), its cloacal shedding and the involvement of gut TLR-3 in duodenum tissues. The 35-day-old SPF chickens were experimentally infected and sampled up to 28 days post infection (dpi) for IBDV detection and TLR-3 quantification by qRT-PCR. The ITA IBDV strain was detected in lymphoid and most non-lymphoid tissues up to the end of the trial, with higher loads compared to the Classical IBDV. Most of those differences were found during the first 2 weeks post-infection. Notably, bone marrow and caecal tonsils presented higher viral loads until 28 dpi, allowing to speculate that these organs may serve as non-bursal lymphoid tissues supporting virus replication. Differences in relative TLR-3 gene expression between ITA IBDV-infected birds and Classical-IBDV infected ones were observed at 4, 14 and 21 dpi, being initially higher in Classical group and later in ITA group. Our results provide new insights into IBDV pathogenesis showing that IBDV of ITA genotype leads to a high and persistent viral load in lymphoid tissues and to a delayed antiviral response.

Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), account for severe economic losses in the poultry industry. The continuous emergence of a multitude of IBV variants poses many challenges for its diagnosis and control, and live attenuated vaccines, despite their routine use, still plays a significant role in driving IBV evolution, further complicating the epidemiological scenario. Unfortunately, the impact of different vaccination strategies on IB control, epidemiology, and diagnosis has rarely been investigated. This work presents the results of a large-scale diagnostic survey performed in Poland to study IBV molecular epidemiology and how vaccination may affect the viral circulation in the field. To this purpose, 589 samples were collected between May 2017 and January 2019, tested by reverse transcription-PCR for IBV and sequenced. Vaccine and field strains were discriminated based on genetic and anamnestic information. The most commonly detected lineages were 793B (79%) and variant 2 (17.4%), with sporadic detections of QX, Mass, and D274-like strains. Most of the detected strains had a vaccine origin: 46.3% matched one of the applied vaccines, while 36.5% were genetically related to vaccines not implemented in the respective protocol. Besides their practical value for the proper planning of vaccination protocols in Poland, these results suggest that only a fraction (17.2%) of the circulating strains are field ones, imposing a careful assessment of the actual IBV field menaces. Moreover, phenomena like vaccine spreading and persistence seem to occur commonly, stressing the need to further study the epidemiological consequences of the extensive use of live vaccines.

Respiratory diseases like infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) have been held accountable for major losses for poultry production. Nevertheless, scarce information was present dealing with the prevalence and molecular epidemiology of these infections in Greece and the efficacy of currently applied control strategies. To fill this gap, a specific epidemiological study was designed. A total of 106 broiler and layer farms, including 10 backyard and 96 commercial flocks, were sampled between March 2016 and May 2017, and the obtained tracheal swabs were tested for IBV and aMPV using RT-PCR based techniques followed by sequencing. For each farm, data regarding production type, flock features, clinical signs, and vaccination program were also recorded. Different associations between vaccination protocol, production type, animal category, birds density, age, presence of clinical signs, and IBV and/or aMPV infection were tested. Both IBV and aMPV field strain prevalence were proven high, approximately 20 and 30%, respectively, being the GI-19 lineage (14 out of 19; 73.6%) and B subtype (30 out of 30; 100%), the most commonly detected IBV and aMPV genetic types. Infection with IBV field strains was significantly associated with clinical sign presence (odds ratio = 8.55 [95CI = 2.17–42.90]). Remarkably, only the vaccination protocol involving a double vaccination at 1 D of age was proven protective against IBV-induced symptomatology, with the odds of developing disease being 4.14 [95CI = 1.34–14.51] times lower. No association was demonstrated between aMPV infection and clinical outbreaks or between aMPV and IBV detection, suggesting the marginal role of the former pathogen in poultry farming. Globally, the present study provides the first detailed investigation of the epidemiological scenario of 2 viruses traditionally considered of pivotal relevance in poultry farming and demonstrates that remarkable benefits could be obtained with just minor adjustments in vaccination protocols.

Background Canine parvovirus (CPV) represents one of the major infections in dogs. While supportive therapy significantly reduces mortality, other approaches have been reported to provide significant benefits. Unfortunately, the high cost of these treatments is typically a limiting factor. Consequently, a reliable prognostic tool allowing for an informed therapeutic approach would be of great interest. However, current methods are essentially based on ‘a priori’ selection of predictive variables, which could limit their predictive potential. Methods In the present study, the predictive performances in terms of CPV enteritis survival likelihood of an operator-validated logistic regression were compared with those of more flexible methods featured by automatic variable selection. Several anamnestic, clinical, haematological and biochemical parameters were collected from 134 dogs at admission in a veterinary practice. Animal status was monitored until dismissal or death (mortality=21.6%). Results The best automatic variable selection method (random forest) showed excellent discriminatory capabilities (AUC=0.997, sensitivity=0.941 and specificity=1) compared with the logistic regression model (AUC=0.831, sensitivity=0.882 and specificity=0.652), when evaluated on a fully independent test data set. The implemented approaches allowed to identify antithrombin, serum aspartate aminotransferase, serum lipase, monocyte and lymphocyte count as the clinical parameter combination with the highest predictive capability, thus limiting the panel of required tests. Conclusion The model validated in the present study allows prompt prediction of disease severity at admission and provides objective and reliable criteria to support the clinician in selection of the therapeutic approach.

Anaplasma phagocytophilum (A. phagocytophilum) is a tick-borne pathogen causing disease in both humans and animals. Human granulocytic anaplasmosis (HGA) is an emerging disease, but despite the remarkable prevalence in European ticks and wild animals, human infection appears underdiagnosed. Several genetic variants are circulating in Europe, including the zoonotic ecotype I. This study investigated A. phagocytophilum occurrence in wild ungulates and their ectoparasites in an area where HGA has been reported. Blood samples from wild ungulates and ectoparasites were screened by biomolecular methods targeting the mps2 gene. The groEL gene was amplified and sequenced to perform genetic characterization and phylogenetic analysis. A total of 188 blood samples were collected from different wild ungulates species showing an overall prevalence of 63.8% (88.7% in wild ruminants and 3.6% in wild boars). The prevalence of A. phagocytophilum DNA in ticks (manly Ixodes ricinus), and keds collected from wild ruminants was high, reflecting the high infection rates obtained in their hosts. Among ticks collected from wild boars (Hyalomma marginatum and Dermacentor marginatus) no DNA was detected. Phylogenetic analysis demonstrated the presence of ecotype I and II. To date, this is the first Italian report of ecotype I in alpine chamois, mouflon, and wild boar species. These findings suggest their role in HGA epidemiology, and the high prevalence detected in this study highlights that this human tick-borne disease deserves further attention.

Differently from alpha- and betaherpesviruses affecting swine, interest in the recently discovered Suid gammaherpesvirus 3, Suid gammaherpesvirus 4, and Suid gammaherpesvirus 5, also known as porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, and PLHV-3), has largely focused on their role as potential zoonotic agents in cases of xenotransplantation. However, their role as primary pathogens of swine or as co-factors for other lymphotropic infections has essentially been neglected. The present study aims at filling this gap, evaluating the association between PLHVs infection and different clinical conditions and/or porcine circovirus (PCV) co-infection. One hundred seventy-six samples were obtained from different animals located in a high-density pig area of Northern Italy in the period 2017&ndash, 2020. The presence of PLHVs and PCVs was tested and quantified by specific real-time PCR: PLHVs were widespread among pigs (PLHV-1, PLHV-2, and PLHV-3 prevalence was 28.97%, 10.79%, and 4.54%, respectively) and detected in all considered tissues and clinical conditions. Frequent co-infections were also observed among PLHVs and with PCVs, although a significant association was not detected with the exception of a positive interaction between PLHV-1 and PLHV-3, and a negative one between PLHV-2 and PCV-2. Significantly, no association between PLHVs, alone or in co-infection, emerged with any of the considered clinical signs, their frequency being comparable between healthy and diseased animals. Based on these pieces of evidence and despite their high prevalence, PLHVs&rsquo, relevance for the swine industry appears negligible, either as primary pathogens or as predisposing factors for circovirus-induced diseases.

Since early 2017, in Spain there was an apparent increase in reports on rotavirus involvement in neonatal diarrhoea outbreaks, affecting also adult sows. In this study, 16 unrelated outbreaks of diarrhoea in suckling pigs and sows, where rotavirus A was the only pathogen detected, were investigated. Deep-sequencing was performed on total RNA from twenty-four positive faecal samples. Genotyping, phylogenetic and bayesian analyses showed that all isolates had a common ancestor of porcine, or human porcine-like, origin. The new strain was introduced in the population shortly before the onset of the outbreaks. Besides, a high diversification of the VP7 and VP4 genes occurred in a short time. Isolates presented a high number of amino acid changes in the neutralizing epitopes compared to vaccine sequences. The present report illustrates how a new rotavirus A strain may disseminate rapidly and the extremely high diversification that this pathogen may undergo in a short period.

The genus Circovirus includes one of the most relevant infectious agents affecting domestic pigs, Porcine circovirus type 2 (PCV-2). The wild boar susceptibility to this pathogen has also been demonstrated although the actual epidemiological role of wild populations is still debated. In recent times, a new circovirus, Porcine circovirus type 3 (PCV-3), has been discovered and reported in the presence of several clinical conditions. However, no information is currently available about PCV-3 circulation and prevalence in wild boar. To fill this gap, 187 wild boar serum samples were collected in the Colli Euganei Regional Park (Northern Italy) and screened for PCV-3, demonstrating a high viral prevalence (approximately 30%). No gender differences were demonstrated while a lower infection prevalence was observed in animals younger than 12 months compared to older ones, differently from what described in commercial pigs. Almost all sampled animals were in good health conditions and no association was proven between PCV-3 status and clinical syndromes in wild animals. The genetic characterization of selected strains enlightened a relevant variability and the absence of closely related strains originating from domestic pigs. Therefore, the observed scenario is suggestive of multiple introductions from other wild or domestic swine populations followed by prolonged circulation and independent evolution. Worldwide, this study reports for the first time the high susceptibility of the wild boar to PCV-3 infection. The high prevalence and the absence of association with clinical signs support the marginal role of this virus in the wild boar population ecology. However, its epidemiological role as reservoir endangering commercial swine cannot be excluded and will require further investigations.

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.

The recent discovery of porcine circovirus 3 (PCV3) has caused some concern among field veterinarians and brought the attention of the scientific world, likely because of some similarities with porcine circovirus 2 (PCV2). Several manuscripts reporting the detection of PCV3 and the presence of different clinical conditions have been published to date.1–5 A priori emphasis has been attributed to the association between PCV3 infections and clinical pathological conditions, even if little evidence is currently available to sustain this association. We conducted a brief exploratory study in northern Italy: 116 samples (from 17 gilts, …

In the present study the subtype B aMPV, strain aMPV/B/IT/GuineaFowl/1818/12, was detected in Guinea fowls affected by respiratory signs, sequenced and molecularly characterized. Comparisons among several F and G gene full sequences of aMPVs subtype B, showed that no consistent pattern related to host-tropism could be identified. Moreover, analysis of partial G gene revealed the perfect identity of the Guinea fowl strain with four Italian aMPVs isolated from turkeys or chickens in a time frame of three years, in the same geographic area. Phylogenetic analysis of both genes showed an evolutionary trend of subtype B circulating in Northern Italy from its first appearance in 1987, to date. The co-presence in the same geographic area of farms housing different avian species sensitive to aMPV, vaccinated with different vaccination programs or not vaccinated (like Guinea fowls), and often belonging to the same integrate poultry company, could be a crucial factor for the establishment of an endemic infection.