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Cowpox virus (CPXV) was isolated from skin lesions of a llama on a farm in Italy. Transmission electron microscopy showed brick-shaped particles consistent with orthopoxviruses. CPXV-antibodies were detected in llama and human serum samples; a CPXV isolate had a hemagglutinin sequence identical to CPXV-MonKre08/1-2-3 strains isolated from banded mongooses in Germany. [ABSTRACT FROM AUTHOR]

The mechanisms of the innate immunity control of equine infectious anemia virus in horses are not yet widely described. Equine monocytes isolated from the peripheral blood of three Equine infectious anemia (EIA) seronegative horses were differentiated in vitro into macrophages that gave rise to mixed cell populations morphologically referable to M1 and M2 phenotypes. The addition of two equine recombinant cytokines and two EIA virus reference strains, Miami and Wyoming, induced a more specific cell differentiation, and as for other species, IFNγ and IL4 stimulation polarized horse macrophages respectively towards the M1 and the M2 phenotypes. Infection with EIAV reference strains resulted in a morphological transformation of macrophages compatible with the M1 differentiation pattern. All samples were also analyzed by molecular analyses for equine herpesviruses that could have acted as an interference and were found to be negative. The mRNA expression level of the pro-inflammatory genes MMP13 and IL6 in treated equine monocyte-derived macrophages (eMDMs) was evaluated by a SYBR ® Green real-time PCR. In this study, MMP13 represented a reliable target gene to evaluate pro-inflammatory status of macrophages in horses because IFNγ and EIAV infection considerably increased its expression. A more in-depth study of the expression genes of both cytokine-induced and virus-induced markers of eMDM polarization may help us to understand whether these markers in horses are the same as those found in other animal species with similar pathways of innate immunity activation. The identified markers of each macrophage population would allow analysis of the differentiation profiles that could provide information on virus infectivity control in equid populations, envisioning their use in therapeutic strategies.

Introduction: This study examined the efficacy of a therapy based on a combination of Platelet Rich Plasma and hydroxyapatite nanoparticles in a severe clinical case involving a young Rottweiler with a complex spiral fracture of the tibia., Method: Following a worsening of the lesion after traditional surgical intervention, the subject was treated with the combined therapy. X-rays were taken at the following stages: immediately post-surgery, four weeks post-surgery, and 10 days post-treatment. Fracture gap and callus density measurements were obtained using ImageJ analysis, allowing for a detailed quantitative assessment of bone regeneration over time., Results: Post-operative radiographs indicated a clinical worsening of the fracture, revealing an increased fracture gap due to bone loss. However, significant improvements were observed ten days following the treatment, with a marked reduction in fracture gaps and increased callus density. These results demonstrated a notable acceleration in bone healing and callus formation compared to typical recovery times for similar lesions., Conclusion: The method showed potential for enhancing osteogenic regeneration, facilitating faster healing of serious orthopedic injuries compared to traditional methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Barbaro, Marconi, Innocenzi, Altigeri, Zepparoni, Monteleone, Alimonti, Marcoccia, Ghisellini, Rando, Ottoboni, Rau, Eggenhöffner and Scicluna.)

Background: Two outbreaks of swinepox were investigated in free-range domestic pig farms located in the northeastern side of Sicily, Italy. The disease is generally self-limiting with a low mortality rate, but morbidity can reach high rates in case of poor sanitary conditions, improper husbandry practices and ectoparasitic infestation. The presented cases are the first ever reported on the island and part of the few cases reported in domestic pigs., Case Presentation: Carcasses condemned at the slaughterhouse and deceased pigs from Farm A and Farm B respectively, were referred for post-mortem examination and further investigations, with a strong suspect of SwinePox virus (SWPV) infection. Twelve deceased pigs were examined in total, showing poor body condition and pustular lesions scattered all over the cutaneous surfaces. Moreover, pigs from Farm B showed ocular lesions classified from Grade I to IV (from mild conjunctivitis to severe keratoconjunctivitis with corneal oedema, opacity, and ulcers). Final diagnosis was pursued by the microscopic assessment of skin lesions in both farms, which revealed the typical SWPV-lesion appearance, such as severe and disseminated ulcerative dermatitis and suspected inclusion bodies multifocally observed in the epidermis. Moreover, negative staining Electron Microscopy (nsEM) was performed on skin lesions and ocular swabs from Farm B, revealing in two samples the presence of brick-shaped viral particles, 220 nm long and 160 nm wide, with irregularly arranged surface tubules, identified as SWPV. The gene encoding the 482-bp fragment of the virus late transcription factor-3 was detected by PCR and sequencing revealed 99.79% identity and 100% query-cover with a strain previously isolated in Germany. Field clinical assessment was then performed in Farm B, revealing high overcrowding, poor sanitary conditions and improper husbandry practices, which are relevant risk factors for SWPV transmission., Conclusions: The present is the first case report of SWPV in free-range pigs raised in Sicily, an island of the Southern coast of Italy, and wants to raise awareness on a neglected disease, and cause of animal health and welfare issues., (© 2024. The Author(s).)

The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy.

Equine hepacivirus (EqHV, Flaviviridae , hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Background: Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared between PCR-positive and -negative horses., Results: This cross-sectional study included 120 well-trained IS racehorses and was performed over a two-years period. The prevalence of T. equi was 36.3%, whereas all samples were negative for B. caballi. Red blood cells count, haemoglobin concentration, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities were significantly higher in PCR-positive horses, whereas blood urea nitrogen, globulin concentration and globulin-to-albumin ratio were significantly lower in PCR-positive horses compared to PCR-negative ones. Nonetheless, all values fell within the physiological range. The best racing time, which was selected as the most representative of the performance metrics at the principal component analysis, was not affected by PCR positivity, the muscle mass score or the training yard. The best racing time was significantly better in horses with a mild or no signs of muscular atrophy, within the PCR-positive group. The muscle mass score was associated with the training yard in PCR-negative horses., Conclusions: Prevalence of T. equi was high in IS racehorses in southern Italy. The absence of obvious changes in haematological and biochemical parameters, as well as performance metrics in positive horses, highlights the need for specific diagnostic tests to identify chronically infected horses., (© 2024. The Author(s).)

Rift Valley fever is a vector-borne zoonotic disease caused by the Rift Valley fever virus (Phlebovirus genus) listed among the eight pathogens included in the Bluepoint list by the WHO. The transmission is mainly vehicled by Aedes and Culex mosquito species. Symptoms of the disease are varied and non-specific, making clinical diagnosis often challenging, especially in the early stages. Due to the difficulty in distinguishing Rift Valley fever from other viral hemorrhagic fevers, as well as many other diseases that cause fever, an early diagnosis of the infection is important to limit its spread and to provide appropriate care to patients. To date, there is no validated point-of-care diagnostic tool. The virus can only be detected in the blood for a brief period, suggesting that molecular methods alone are not sufficient for case determination. For this, it is preferable to combine both molecular and serological tests. The wide distribution of competent vectors in non-endemic areas, together with global climate change, elicit the spread of RVFV to continents other than Africa, making surveillance activities vital to prevent or to limit the impact of human outbreaks and for a rapid identification of positive cases, making diagnosis a key factor for this achievement.

Background: Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance., Methods: Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories., Results: None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761)., Conclusions: This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy.

Subchondral bone cysts in horses represent one of the main causes of lameness that can occur in different anatomical locations. The study describes the treatment in regenerative therapy of the intracystic implantation of adipose tissue mesenchymal stromal cells (AMSCs) included in platelet-rich plasma (PRP). The ability of AMSCs to differentiate in osteogenic cells was tested in vitro and in vivo. Given the aim to investigate the application of AMSCs in bone defects and orthopedic pathologies in horses, a four-year-old male thoroughbred racing horse that had never raced before was treated for lameness of the left hind leg caused by a cyst of the medial femoral condyle. The horse underwent a new surgery performed with an arthroscopic approach in which the cystic cavity was filled with AMSCs contained in the PRP. Radiographs were taken 3, 5, and 10 months after the surgery to assess the development of newly regenerated bone tissue in the gap left by the cyst. Twelve months after the operation and after six months of regular daily training, the horse did not show any symptoms of lameness and started a racing career. According to the study, the use of AMSCs and PRP suggests promising benefits for treating subchondral bone cysts.

Between August and September 2023, three distinct autochthonous dengue virus transmission events occurred in Lazio, Italy, with the main event in Rome. The events involved three different dengue serotypes. No link with previous imported cases was identified. Here we describe the epidemiological and phylogenetic analysis of the first autochthonous cases and the implemented control actions. The multiple transmission events call for a strengthening of the vector control strategies and future research to better characterise the risk in countries like Italy.

European brown hare syndrome (EBHS) is a highly contagious and fatal viral disease, mainly affecting European brown hares ( Lepus europaeus ). The etiological agent, EBHS virus (EBHSV), belongs to the Lagovirus genus within the Caliciviridae family. The Italian hare ( Lepus corsicanus ) is endemic to Central-Southern Italy and Sicily and is classified as a vulnerable species. L. corsicanus is known to be susceptible to EBHS, but virological data available is scarce due to the few cases detected so far. In this study, we describe the occurrence of EBHS in two free-ranging L. corsicanus, found dead in a protected area of Central Italy. The two hares were identified as L. corsicanus using phenotypic criteria and confirmed through mitochondrial DNA analysis. Distinctive EBHS gross lesions were observed at necropsy and confirmed by subsequent histological examination. EBHSV was detected in the livers of the two animals initially using an antigen detection ELISA, followed by an EBHSV-specific reverse transcription-PCR, thus confirming the viral infection as the probable cause of death. The EBHS viruses detected in the two hares were identical, as based on blast analysis performed for the VP60 sequences and showed 98.86% nucleotide identity and 100% amino acid identity with strain EBHSV/GER-BY/EI97.L03477/2019, isolated in Germany in 2019. Phylogenetic analysis places our virus in group B, which includes strains that emerged after the mid-1980s. This study supports previous reports of EBHS in L. corsicanus and further expands the knowledge of the pathological and virological characteristics of the etiological agent. The ability of EBHSV to cause a fatal disease in the Italian hare represents a serious threat to the conservation of this vulnerable species, especially in populations kept in enclosed protected areas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Domanico, Cavadini, Nardini, Cecca, Mastrandrea, Eleni, Galietta, Attili, Pizzarelli, Onorati, Amoruso, Stilli, Pacchiarotti, Merzoni, Caprioli, Ricci, Battisti, Lavazza and Scicluna.)

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses., (© 2023. The Author(s).)

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022., Competing Interests: The authors declare no conflict of interest.

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse. Biopsies of the treated cartilage taken after 8 and 13 months of implantation for histological and immunohistochemical evaluation of the tissue demonstrate that the tissue was still immature 8 months after implantation, while at 13 months it was organized almost similarly to the original hyaline cartilage. Finally, a tissue perfectly comparable to native articular cartilage was detected 24 months after implantation. Histological investigations demonstrate the progressive maturation of the hyaline cartilage at the site of the lesion. The hyaline type of tracheal cartilage, used as a source of CCs, allows for the repair of joint cartilage injuries through the neosynthesis of hyaline cartilage that presents characteristics identical to the articular cartilage of the original tissue.

Lentiviruses, including equine infectious anemia virus (EIAV), are considered viral quasispecies because of their intrinsic genetic, structural and phenotypic variability. Immunoenzymatic tests (ELISA) for EIAV reported in the literature were obtained mainly by using the capsid protein p26, which is derived almost exclusively from a single strain (Wyoming), and do not reflect the great potential epitopic variability of the EIAV quasispecies. In this investigation, the GenBank database was exploited in a systematic approach to design a set of representative protein antigens useful for EIAV serodiagnosis. The main bioinformatic tools used were clustering, molecular modelling, epitope predictions and aggregative/ solubility predictions. This approach led to the design of two antigenic proteins, i.e. a full sequence p26 capsid protein and a doublestrain polypeptide derived from the gp45 transmembrane protein fused to Maltose Binding Protein (MBP) that were expressed by recombinant DNA technology starting from synthetic genes, and analyzed by circular dichroism (CD) spectroscopy. Both proteins were used in an indirect ELISA test that can address some of the high variability of EIAV. The novel addition of the gp45 double-strain antigen contributed to enhance the diagnostic sensitivity and could be also useful for immunoblotting application., 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.)

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

Background: To date, there is a scarcity of information and literature on Macaca maura health status relative to viral diseases. The objectives of the present study were to investigate on the potential spread of enteric and non-enteric viruses shed in the environment through a wild macaque feces and to understand the possible interrelation in the spread of zoonotic viruses in a poorly studied geographical area, the Sulawesi Island. This study will also contribute providing useful information on potential threats to the health of this endangered species., Methods: The sampling was conducted between 2014 and 2016 in the Bantimurung Bulusaraung National Park, in the south of the Sulawesi Island and non-invasive sampling methods were used to collect fresh stools of the M. maura, one of the seven macaque species endemic to the island of Sulawesi, Indonesia. The population under study consisted in two wild, neighboring social macaque groups with partially overlapping home ranges; twenty-four samples were collected and examined using negative staining electron microscopy and a panel of PCR protocols for the detection of ten RNA and two DNA viruses., Results: Viral particles resembling parvovirus (5 samples), picornavirus (13 samples) and calicivirus (13 samples) were detected by electron microscopy whereas the PCR panel was negative for the 12 viruses investigated, except for one sample positive for a mosquito flavivirus. The results did not correlate with animal sex; furthermore, because all of the animals were clinically healthy, it was not possible to correlate feces consistency with viral presence., Conclusions: As information on viral infections in wild moor macaques remains limited, further studies are yet required to identify the fecal-oral and blood transmitted potentially zoonotic viruses, which may infect the moor macaque and other macaque species endemic to the South Sulawesi Island., (© 2022. The Author(s).)

Equine piroplasmosis is a disease of equids, caused by tick-borne apicomplexan protozoan pathogens Babesia caballi and Theileria equi, which, according to the World Organisation for Animal Health (OIE), can be diagnosed by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR). The present study was conducted to evaluate and compare the assays available for the diagnosis of equine piroplasmosis. Data employed were obtained from 1300 blood samples collected between 2012-2014 from asymptomatic and symptomatic equines (horses and donkeys) of central-southern regions of Italy and analyzed by ELISA, IFAT, PCR (one commercial and one from literature) and blood smear microscopic examination. Statistical differences of the proportions of positivity for each parasite and group (asymptomatic and symptomatic) among the methods were verified by the z test to identify the most sensitive. The concordance between each pair of methods - for each parasite and within the groups - and trends in detection of suspect samples of four hypothetical diagnostic algorithms using serological and biomolecular assays were evaluated to identify the most suitable laboratory diagnostic workflow. The results of this study highlighted a lower capacity to detect suspect samples of commercial ELISA for B. caballi in all groups when compared to biomolecular methods and IFAT; and of the commercial PCRs in asymptomatic animals, identifying a PCR from literature and IFAT as the best choice for a combined diagnosis. For T. equi, IFAT detected more suspect samples than ELISA, even if the latter showed good performance and some samples were positive only by the ELISA and PCR, indicating that their simultaneous employment is still advantageous. Host-parasite interaction, amino-acid/genetic diversity and differences in detection limits among the assays could be among the reasons in explaining the present results. In view of further studies, ELISA should be used in combination with PCR, that should regularly be included in the laboratory diagnosis to maximise the detection of early infections and support the evaluation of pharmacological treatment., Competing Interests: Declaration of Competing Interest None, (Copyright © 2022. Published by Elsevier GmbH.)

Viral hepatitis has recently assumed relevance for equine veterinary medicine since a variety of new viruses have been discovered. Equine Hepacivirus (EqHV) is an RNA virus belonging to the Flaviviridae family that can cause subclinical hepatitis in horses, occasionally evolving into a chronic disease. EqHV, to date, is considered the closest known relative of human HCV. EqHV has been reported worldwide therefore assessing its features is relevant, considering both the wide use of blood products and transfusions in veterinary therapies and its similitude to HCV. The present review resumes the actual knowledge on EqHV epidemiology, risk factors and immunology, together with potential diagnostics and good practices for prevention. Moreover, adhering to PRISMA guidelines for systematic reviews a meta-analysis of serological and biomolecular prevalence and an updated phylogenetic description is presented as a benchmark for further studies.

Orthpoxvirus infection can spread more easily in a population with a waning immunity with the subsequent emergence/re-emergence of the viruses pertaining to this genus. In the last two decades, several cases of Orthopoxvirus, and in particular Cowpoxvirus infections in humans were reported in different parts of the world, possibly due to the suspension of smallpox vaccinations. To date, in Italy, few investigations were conducted on the presence of these infections, and because of this a serosurvey was carried out to evaluate Cowpoxvirus infection in feline colonies situated in the province of Rome, since these are also susceptible to other zoonotic viruses belonging to Orthopoxvirus, and from which humans may contract the infection. The sample design was set at an expected minimum seroprevalence of 7.5%, a 5% standard error and 95% confidence level. In parallel, a serological investigation was conducted using convenience sampling in domestic, exotic and wild susceptible animals of the Latium and Tuscany Regions, which are areas in the jurisdiction of the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, coordinating this study. The serological methods employed were indirect immunofluorescence for 36 sera of nonhuman primate and virus neutralization for 1198 sera of different species. All the 1234 sera examined were negative for the presence of antibodies against Cowpoxvirus, indicating its limited circulation in the areas of investigation. The methodology applied for the serosurveillance could be adopted in the case of outbreaks of this infection and for the evaluation of the spread of this infection in the area of interest, to obtain essential information crucial for animal and public health policies according to the One Health concept.

Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order to adopt this diagnostic method, the Italian National Reference Centre for Equine Diseases (NRC-ED) conducted a preliminary comparison between an end-point PCR, nested PCR, real-time PCR, and commercial real-time PCR, for the detection of T. equi and B. caballi, respectively. One hundred and three field samples, collected during spring-summer 2013 in Latium and Tuscany regions, were employed for the study, and results discordant between detection assays were confirmed by sequencing. The reference assay was defined as that showing the highest sensitivity, and the relative sensitivity (rSe) and specificity (rSp) of the other methods were estimated referring to this assay. Agreement between methods was estimated by calculating the concordance between each pair of methods. Although no statistical differences were detected among PCR-based methods, the non-commercial real-time PCR assays seemed to be the most suitable for detection of T. equi and B. caballi, respectively. An important advantage of direct PCR detection of the pathogen, in comparison to indirect detection using serological methods, is that it allows specific treatment against the causative pathogen species responsible of the infection as well as for the definition of the infectious status of an animal for international movement.

Background: West Nile (WNV) and Usutu (USUV) are emerging vector-borne zoonotic flaviviruses. They are antigenically very similar, sharing the same life cycle with birds as amplification host, Culicidae as vector, and man/horse as dead-end host. They can co-circulate in an overlapping geographic range. In Europe, surveillance plans annually detect several outbreaks., Methods: In Italy, a WNV/USUV surveillance plan is in place through passive and active surveillance. After a 2018 WNV outbreak, a reinforced integrated risk-based surveillance was performed in four municipalities through clinical and serological surveillance in horses, Culicidae catches, and testing on human blood-based products for transfusion., Results: Eight WNV cases in eight equine holdings were detected. Twenty-three mosquitoe catches were performed and 2367 specimens of Culex pipiens caught; 17 pools were USUV positive. A total of 8889 human blood donations were tested, and two asymptomatic donors were USUV positive., Conclusions: Different surveillance components simultaneously detected WNV only in horses and USUV only in humans and mosquitoes. While in endemic areas (i.e. northern Italy) entomological surveillance is successfully used as an early detection warning, this method in central Italy seems ineffective. To achieve a high level of sensitivity, the entomological trapping effort should probably exceed a reasonable balance between cost and performance. Besides, WNV/USUV early detection can be addressed by horses and birds. Further research is needed to adapt the surveillance components in different epidemiological contexts.

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the "Europe wildlife" lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies.

Babesia caballi and Theileria equi are widely recognized as causative agents of equine pirolasmosis (EP), an acute, sub-acute, and chronic disease of equines, with relevant economic impact on horse trade worldwide. Although several studies on EP prevalence from central Italy have been published, data on ticks responsible for its transmission are still lacking. In this study, we identified a potential competent vector, investigating main features of its ecology together with EP infection rates. A two-year sampling of questing ticks was carried out for the first time in Italy in an area known for high EP prevalence in horse sera, detecting the association between Rhipicephalus bursa and causative agents of EP. Most of the positive pools harbored a single infection (91.1%); mixed infections were also detected (8.9%). The infection rate for T. equi slightly decreased among years; B. caballi showed a lower, but increasing, infection rate. Tick phenology, climate variables, and peaks of EP prevalence indicated late May and second half of June as periods with the highest risk of new infections, especially during warm and dry days., (© 2020 The Society for Vector Ecology.)

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay. The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV. For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above. The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested. An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator. The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV. In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system., (Copyright © 2019. Published by Elsevier B.V.)

Bluetongue (BT) is a viral disease that affects ruminants and is transmitted by midges of the genus Culicoides spp. The seroprevalence, the clinical form and the occurrence rates significantly differ in relation to several factors such as bluetongue virus (BTV) serotype, host species, breed susceptibility, specific previous exposure, vector ecology, husbandry and health status. Following the 2001-2006 BTV2 and BTV16 epidemics in central Italy, a new epidemic caused by BTV1 occurred in 2013-2015 causing 398 outbreaks in a susceptible population of about 1 million ruminants. The present study assessed the BTV1 seroprevalence in the sheep population of central Italy by conducting two cross-sectional surveys, in the proximity of and within BT outbreak farms. A total of 2,984 sheep from 437 farms were sampled. The animal-level prevalence was 19% (95% CI: 17-21%), the between-herd prevalence was 46% (95% CI: 41-51%) and the within-herd prevalence was 21% (95% CI: 16-26%). Risk factors were investigated by logistic regression models. Living on a farm where an outbreak occurred and the number of outbreaks in proximity of the farm were identified as risk factors, while herd size was identified as a protective factor. This study represents the first BT survey in southern Europe and reports valuable findings on BTV epidemiology. Despite intensive virus circulation, the estimated seroprevalences were low. The assessment of the population immunity level is crucial for defining an efficient vaccination strategy and for predicting the impact of future virus circulation. In view of the low seroprevalence detected albeit an extensive BTV1 circulation, the population immunity was likely to be inadequate in preventing new BTV1 epidemics. Moreover, considering the recurrent introduction of new serotypes from North Africa and the Balkans, the control of multi-serotype BTV infections will continue to present a challenge in the near future., Competing Interests: The authors have declared that no competing interests exist.

The wide geographical distribution and genetic diversity of bat-associated lyssaviruses (LYSVs) across Europe suggest that similar viruses may also be harboured in Italian insectivorous bats. Indeed, bats were first included within the passive national surveillance programme for rabies in wildlife in the 1980s, while active surveillance has been performed since 2008. The active surveillance strategies implemented allowed us to detect neutralizing antibodies directed towards European bat 1 lyssavirus in six out of the nine maternity colonies object of the study across the whole country. Seropositive bats were Myotis myotis, M. blythii and Tadarida teniotis. On the contrary, the virus was neither detected through passive nor active surveillance, suggesting that fatal neurological infection is rare also in seropositive colonies. Although the number of tested samples has steadily increased in recent years, submission turned out to be rather sporadic and did not include carcasses from bat species that account for the majority of LYSVs cases in Europe, such as Eptesicus serotinus, M. daubentonii, M. dasycneme and M. nattereri. A closer collaboration with bat handlers is therefore mandatory to improve passive surveillance and decrypt the significance of serological data obtained up to now.

Crimean-Congo haemorrhagic fever (CCHF), endemic in Africa, Asia, Eastern Europe and the Middle East, is caused by a tibovirus (CCHFV) transmitted in particular by the Hyalomma genus of the Ixodidae family that can remain attached to the host for up to 26days, which in case of migratory birds allows long distance carriage. Although CCHF in domestic ruminants is usually subclinical, they may become reservoirs and act as sentinels for the introduction and/or circulation of CCHFV. In this study, possible CCHFV introduction and circulation in Italy were monitored by tick sampling on migratory birds and by a serosurvey conducted on sheep. While bird tick sampling was conducted in thirteen ringing sites of Central and Southern Italy, the serosurvey was performed on flocks grazing in coastal provinces of Central Italy that are stop over areas for birds flying from Africa, where Hyalomma ticks and CCHFV are endemic, to Central and Northern Europe. A total of 282 ticks (80.8% were Hyalomma spp.) were collected from 139 (0.28%) migratory birds of the 50,325 birds checked with 0.22% infested by Hyalomma spp., involving 22 avian species with a mean number of 1.6 Hyalomma spp. per infested bird. For the serosurvey, 540 sheep sera were randomly collected that resulted all negative when examined by an indirect IgG ELISA, employing a recombinant antigen coded by the CCHFV S gene. While the present study confirmed the introduction of CCHFV potential vectors in Central Italy, transported by migratory birds arriving from endemic areas, the serosurvey results did not put in evidence the concomitant arrival of the virus in the study area during the survey period. In general, in areas potentially at risk of CCHFV introduction and circulation, structured serological monitoring of susceptible domestic animals represents a rational system for an early detection of virus circulation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The National Reference Center for equine infectious anemia (EIA) validated a commercial ELISA (Eradikit ® EIAV Indirect ELISA, In3diagnostic ® , Turin, Italy) employing a chimeric recombinant gag and env peptide for the detection of EIA virus antibodies, following the guidelines of the World Organization for Animal Health. The validation parameters evaluated were: analytical sensitivity (Se) and specificity (Sp); diagnostic Se and Sp; precision, based on repeatability and reproducibility through the estimation of the standard deviation (SD) and the coefficient of variation (CV); accuracy, estimated from a multiple K and relative Sp and Se with respect to those of the agar gel immunodiffusion test (AGIDT). Positive and negative predictive values were also defined. The assay showed a high specificity and a limit of detection of 1.43 log 10 major than AGIDT. Diagnostic Se was 100% and Sp was 99.3%, while SD values ranged from 1.58 to 5.01 with a CV between 2.8% and 28.8%. Multiple K was 0.98 and relative Se and Sp were respectively 99.1% and 100%. The assay proved to be robust and to possess a high sensitivity in detecting first antibodies produced at onset of infection as well as high analytic and diagnostic Se and Sp values, confirming it as a serological assay fit for purpose within EIA surveillance programs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53 days post-infection (SA) while the other (DE) survived 5 months despite experiencing multiple febrile episodes. Nucleotide sequence identity between the isolates was 99.358% confirming infection with the same EIAV strain with most differences comprising single nucleotide polymorphisms in env and the second exon of rev. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity in vivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity., (© 2017 Blackwell Verlag GmbH.)

Background: ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and sensitivity (Se). Two serum panels, positive for non-equine retroviruses and the most frequent equine viruses, were employed to measure analytic Sp. ELISA precocity was also compared to that of one "in-house" and three commercial AGID kits, employing a panel of sera, collected weekly from horses infected with modified EIA viruses. Precision and accuracy were defined using results of a panel containing positive and negative sera examined in an inter-laboratory trial with the participation of the ten Official Laboratories. Furthermore, a questionnaire was used to assess the appropriateness of each kit for routine use., Results: Analytic Sp was 100%, while the 75th percentile of CVs for positive sera varied from 0.4% to 12.73% for repeatability and from 1.6% to 44.87% for reproducibility. Although CV of the negative serum was constantly high, its outcome was unaltered. Relative Se ranged from 98.2% to 100%, relative Sp was constantly 100% and multiple K ranged from 0.95 to 1. Precocity differed among the assays: three kits detected 4.8% and 42.9% positive samples on 21 days post infection (dpi), all assays detected positive samples on 28 dpi, between 47.6% and 95.2%. Precocity of ELISAs was superior to that of the AGIDs except for two assays. In view of the feedback obtained from the questionnaires, all kits were considered appropriate for routine use., Conclusion: All ELISAs having high Se and precocity are preferable as a screening test in EIA surveillance programmes to the AGID tests examined. These two tests can be incorporated in a serial diagnostic pathway to improve the efficacy of a surveillance plan.

In July 2011, in a zoological garden in Rome, Italy, malignant catarrhal fever (MCF), a fatal, systemic disease of Artiodactyla, was suspected on the basis of neurological signs and gross lesions observed in a banteng, the first animal to die of this infection. An MCF type-specific PCR with subsequent sequencing of the PCR amplicon confirmed the aetiological agent as ovine herpesvirus-2 (OvHV-2). Biological samples were collected from the dead animals for gross, histological, bacteriological, virological and serological examinations. An epidemiological investigation was conducted to identify the source of the outbreak, as further deaths due to OvHV-2 still occurred after the removal of the acknowledged reservoirs, domestic sheep and goats. For this purpose, samples from other susceptible species and reservoir hosts were collected for virological and serological analysis. In conjunction, a retrospective sero-investigation was conducted on sera collected between 1999 and 2010 from some of the species involved in the present episode. In total, 11 animals belonging to four different species (banteng, Himalayan tahr, Nile lechwe and sika deer) died between July 2011 and October 2012. The severe gross and histological lesions were consistent with the disease, namely haemorrhages and congestion of several organs as well as lymphoid cell infiltrates and vasculitis of varying severity. The virological tests confirmed that all animals had died of sheep-associated MCF. The investigation indicated that the OvHV-2 infection could have been due to the arrival of sheep in the petting zoo, with cases commencing after first lambing and subsequent shedding of virus. This was also supported by the serological retrospective study that indicated limited previous MCF virus circulation. Further MCF cases that occurred even after the removal of the domestic sheep and goats were attributed to the mouflon. This episode confirms the importance of biosecurity measures in zoos, which house MCF susceptible species, especially those endangered., (© 2015 Blackwell Verlag GmbH.)

Information on equine infectious anaemia (EIA) in mules, including those with an equivocal reaction in agar gel immunodiffusion test (AGIDT), is scarce. For this, a study was conducted to evaluate the clinical, viral loads and pathological findings of two groups of naturally infected asymptomatic mules, respectively with a negative/equivocal and positive AGIDT reactivity, which were subjected to pharmacological immune suppression (IS). A non-infected control was included in the study that remained negative during the observation period. Throughout the whole study, even repeated episodes of recrudescence of EIA were observed in 9 infected mules, independently from their AGIDT reactivity. These events were generally characterised by mild, transient alterations, typical of the EIA acute form represented by hyperthermia and thrombocytopenia, in concomitance with viral RNA (vRNA) peaks that were higher in the Post-IS period, reaching values similar to those of horses during the clinical acute phase of EIA. Total tissue viral nucleic acid loads were greatest in animals with the major vRNA activity and in particular in those with negative/equivocal AGIDT reactivity. vRNA replication levels were around 10-1000 times lower than those reported in horses, with the animals still presenting typical alterations of EIA reactivation. Macroscopic lesions were absent in all the infected animals while histological alterations were characterised by lymphomonocyte infiltrates and moderate hemosiderosis in the cytoplasm of macrophages. On the basis of the above results, even mules with an equivocal/negative AGIDT reaction may act as EIAV reservoirs. Moreover, such animals could escape detection due to the low AGIDT sensitivity and therefore contribute to the maintenance and spread of the infection., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The Italian National Reference Center for equine infectious anemia (CRAIE; Rome, Italy) developed and validated a monoclonal, recombinant p26-based competitive enzyme-linked immunosorbent assay (cELISA) for the detection of EIA virus antibodies employing the 2010 criteria of the World Organization for Animal Health (OIE). The following parameters were evaluated: cutoff values, repeatability, reproducibility, concordance, analytical sensitivity (Se), absolute analytical specificity (Sp), and diagnostic Se and Sp. Positive and negative predictive values were also defined in relation to the estimated prevalence. When the cELISA was used as a screening test for 96,468 samples in the Italian EIA surveillance program, 17% more EIA cases were detected than by the agar gel immunodiffusion test, and the apparent diagnostic Sp estimated from these samples was 99.8%, which was more than the diagnostic Sp (80.2%) estimated from validation. The high Se and Sp of the cELISA confirm its fit for purpose as a screening test., (© 2015 The Author(s).)

To improve the efficiency of the National equine infectious anaemia (EIA) surveillance program in Italy, a three-tiered diagnostic system has been adopted. This procedure involves initial screening by ELISA (Tier 1) with test-positive samples confirmed by the agar gel immunodiffusion test (AGIDT) (Tier 2) and, in the case of ELISA positive/AGIDT negative results, final determination by immunoblot (IB) (Tier 3). During this evaluation, 74,880 samples, principally collected from two Regions of Central Italy (Latium and Abruzzo) were examined, with 44 identified as negative in AGIDT but positive in both ELISA and IB. As the majority of these reactions occurred in mules, an observational study was conducted in this hybrid equid species to investigate if there is a correlation between plasma-associated viral loads and serological reactivity, to test the hypothesis that false-negative or very weak positive AGIDT results are associated with elite control of EIA virus (EIAV) replication accompanied by reduced transmission risks. The study animals consisted of 5 mules with positive AGIDT readings, along with another 5 giving negative or very weak positive results in this test. All mules were seropositive in Elisa and IB. Samples were collected routinely during an initial 56-day observation period, prior to dexamethasone treatment lasting 10 days, to determine the effect of immune suppression (IS) on clinical, humoral and virological responses. All mules were monitored for a further 28 days from day 0 of IS. None of the animals experienced relevant clinical responses before IS and there were no significant changes in antibody levels in ELISA, IB or AGIDT. However, plasma-associated viral-RNA (vRNA) loads, as determined using TaqMan(®) based RT-PCR, showed unexpectedly high sample to sample variation in all mules, demonstrating host-mediated control of viral replication is not constant over time. Furthermore, there was no apparent correlation between vRNA loads and antibody reactivity in serological tests. Analysis of PCR products established all mules were infected with viruses possessing nucleotide sequence similarity, varying from 77 to 96%, to previously identified European EIAV strains. Following IS, all mules showed increases in plasma-associated vRNA loads, suggesting control of EIAV replication is mediated by immune responses in this hybrid species. However, only three mules showed anamnestic humoral responses to rises in viral loads, as defined by at least a four-fold increase in ELISA titre, while two remained AGIDT-negative. This study demonstrates that viral loads in equids with consistent ELISA/IB positive-AGIDT negative to very weak positive test results (Group N) can be equivalent to those that produce clearly positive results in all three serologic tests (Group P). Therefore, such animals do not pose inherently lower risks for the transmission of EIAV. Consequently, the exclusive use of the AGIDT, as prescribed by the World Organization of Animal Health (OIE) for diagnosis of EIA prior to the international movement of horses, can report as negative some EIAV-infected equids. These results dramatically underscore the necessity of combining the specificity of AGIDT with tests with higher sensitivity, such as the ELISA and the power of the IB to enhance the accuracy of EIA diagnosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Serological diagnosis of equine infectious anaemia virus (EIAV) infections has depended mainly on the agar gel immunodiffusion test (AGIDT). This study documents the presence of EIAV genetic sequences in a number of persistently infected horses and mules whose serums were interpreted as negative/equivocal on AGIDT, but positive on more than one ELISA test and in immunoblot tests. Strategies designed to take advantage of the combined strengths of the ELISA and AGIDT are shown effective in a national surveillance program for EIA in Italy where 17 per cent (25/149) of the equids considered to be infected with EIAV on combined/comparative serological data had reactions in the AGIDT that were interpreted as negative or equivocal. These data document the benefits of using a three-tiered laboratory system for the diagnosis of EIA. Although the ELISA-first strategy introduces some confusing results, the discovery of up to 20 per cent more cases of EIA makes it compelling. In our opinion, it is better and more defensible to find two samples in 1000 with resolvable but falsely positive ELISA tests for EIA than to release two to three horses in 10,000 with falsely negative test results for EIA (the rates seen in the Italian surveillance presented here).

Background: This preliminary study was aimed at evaluating the association between single nucleotide polymorphisms (SNPs) on Toll like receptor 9 (TLR9) gene and some immunological parameters in a population of Italian Holstein calves., Methods: The study was carried out in a commercial farm on 68 Holstein calves aging about 6 months. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) and genotyped for nine SNPs on TLR9. Immunological parameters considered were the immunoglobulin (Ig) G titers against bovine herpesvirus 1, and the proliferative response of peripheral blood mononuclear cells to mitogens. For the association study, only results relative to the SNP located in the promoter region have been discussed., Results: Among the nine SNPs expected, only eight were detected. Considering the SNP located in the promoter region, all three possible genotypes were observed, and their distribution was as follows: genotype a (n=34), b (n=19), and c (n=8). On the basis of their response to vaccine, calves were categorized as low (L, n=8), medium (M, n=45) and high responders (H, n=8). Although no significant association was found between genotypes and L, M or H categories, the genotype estimated as the less represented within the population (c) had no calves categorized as H, the highest frequency of L (25%), and mean values of IgG lower (P < 0.005) compared to genotype b. Furthermore, IgG titers were positively correlated with responses of PBMC to mitogens., Conclusions: Genotype c appeared to be "non advantageous" in terms of immune response. It was characterized by the presence of the mutation in homozygosity and, not surprisingly, it was the most rare genotype in the population. Larger studies are necessary in order to confirm these observations.

Only limited information is available on the epidemiology and pathogenesis of Bovine Herpesvirus 1 (BoHV-1) in domestic buffalos. In this study, a virulent BoHV-1 field strain isolated from cattle was inoculated into buffaloes to evaluate their susceptibility to the virus and to investigate the establishment of viral latency through clinical, virological and serological investigations. Latency was also studied by attempting viral reactivation using pharmacological induction. Six of seven male, 5 months old buffaloes were intranasally inoculated with BoHV-1; the other animal was kept as negative control. The animals were clinically monitored during the post-infection (P.I.) and the post-pharmacological induction (P.P.) periods. During these periods, nasal and rectal swabs, and blood samples, with and without anticoagulant, were collected at 2-3 day intervals. On culling the animals, 206 days P.I., their trigeminal ganglia and tonsils were collected. No clinical signs referable to BoHV-1 were observed throughout the experimental period. However, seropositivity was detected in all infected animals within day 20 P.I., using BoHV-1 glycoprotein E and glycoprotein B competitive ELISAs (IDEXX) and virus neutralisation test. In real-time PCR (RT-PCR), five of these animals were positive, at least once, for nasal or rectal swabs, during the P.I. period. The sixth infected animal was found positive only in the trigeminal ganglia after culling. Ganglia were also positive for two other animals. Virus isolation in permissive cell-lines was successful for a part of the RT-PCR positive samples. The detected viruses were confirmed by genetic analysis as identical to the inoculated strain. No evidence of infection was observed in the negative control. This study represents the first experimental transmission of BoHV-1 in buffaloes, confirming their susceptibility to infection and their possible role as host/reservoirs of BoHV-1., (Copyright 2010 Elsevier B.V. All rights reserved.)

West Nile virus (WNV) is a flavivirus that is maintained in a bird-mosquito transmission cycle. Humans, horses and other non-avian vertebrates are usually incidental hosts. However, WNV is a neurotropic virus, which requires an efficient humoral response for the control of a neuroinvasive infection. The WNV genome encodes three structural (capsid, premembrane/membrane and envelope) and seven non-structural proteins. Bioinformatic analysis performed on the WNV genomes detected a conserved alternative open reading frame restricted to the lineage I virus. To quickly verify the existence of this putative protein, entitled West Nile Alternative Reading Frame 4 (WARF4), we produced a prokaryotic recombinant source of WARF4 and verified its immunogenicity in vivo by analyzing 43 horse serum samples, of which 15 were positive for antibodies to WNV premembrane and envelope (prM-E) proteins. Specific antibodies to WARF4 were significantly detected in 5 out of the 15 serum samples testing positive for antibodies to prM-E WNV proteins. Our findings provide evidence of a significant antibody response to the WARF4 protein in the serum of the horse testing positive for antibodies to prM-E proteins, thus indicating that this antigen might be a potential tool for further characterization of the immune response of WNV infections in humans as well.

During local respiratory disease outbreaks, occurring in 2003 and 2004 in horse training stables within race-tracks in Rome, and on a stud horse farm in Bari in 2005, four strains of equine influenza (EI) virus were isolated. All outbreaks occurred in flu-vaccinated horses. Here, we are reporting the results of the genetic characterization of these isolates, together with that of another EI virus strain isolated in 1999 from a dead foal presenting pulmonary lesions. Alignment and phylogenetic analyses were carried out using the haemagglutinin amino acid sequences. The Rome and Bari isolates were identified as members of the American lineage, closely related to other recent strains isolated in America as well as in Europe, including the latest recommended American lineage vaccine prototype A/eq/SouthAfrica/4/2003. In contrast, the Italian 1999 isolate was clustered within the European lineage. In Italy, the most recent outbreaks of EI have been caused by the currently circulating American-like strains, even in vaccinated populations, confirming that vaccines should contain an updated representative strain of this lineage. Presently, companies are still in the process of registering updated vaccines but no product is yet available on the market.

Several seroconversions occurring in 2002 among sentinel cattle during the bluetongue-vaccination campaign in Lazio and Tuscany (central Italy) led to the suspicion of vaccine-virus circulation. Therefore in 2003, 17 seroconverting sentinel herds were investigated for the characteristics of the virus involved. From these farms, 91 unvaccinated animals and 57 Culicoides pools were tested for the presence of the bluetongue vaccine virus (serotype-2) or other strains. The presence of vaccine virus serotype-2 was confirmed by PCR followed by restriction analysis in the whole blood of 17 unvaccinated sentinel cattle and 12 pools of Culicoides imicola or C. obsoletus. Of the 17 herds, five were positive only for vaccine virus serotype-2, four were positive for other strains and two for both the vaccine and other strains; the remaining premises were virologicaly negative. The vaccine virus serotype-2 also was detected in areas not included in the vaccination campaign.

During the epidemic of bluetongue (BT) in Lazio and Tuscany between 2001 and 2003, the distribution pattern of Culicoides imicola did not always correspond either geographically or seasonally, with virus circulation. Culicoides obsoletus was observed to be abundant, ubiquitous and active throughout the year. The geographical and seasonal distribution of BT virus (BTV), C. imicola and C. obsoletus was compared. The territory of the two regions was divided into 30 cells each measuring 1 600 km(2). The presence of C. obsoletus was recorded in every cell, while C. imicola was detected in 18 of the 30 cells, but was absent in 6 of the 21 cells that indicated the presence of BTV. The occurrence of seroconversions appeared to be positively correlated with maximum C. obsoletus catches. Seroconversions were recorded throughout the year, even when C. imicola was not active, whereas C. obsoletus was detected during the entire period. The occurrence of BTV circulation in areas and periods where C. imicola was absent, and the abundant and constant presence of adult C. obsoletus in all the cells, suggest the active role of the latter species in BTV circulation in central Italy.

During the late summer of 1998, veterinary authorities in Tuscany, Italy, received reports of cases of neurologic disease among horses residing in a large wetland area located in the provinces of Florence and Pistoia. West Nile virus was isolated from two of the six horses that died or were euthanized. A retrospective epidemiologic study identified 14 clinical neurologic cases that occurred from August 20 to October 6 (attack rate of 2.8%). A serologic survey conducted over a 700-km2 area in stables with and without apparent clinical cases confirmed a wider spread of the infection, with an overall seroprevalence rate of 38% in the affected area. No significant differences in age-specific prevalence were observed, suggesting that the horses residing in the area had not been exposed previously to West Nile virus and supporting the hypothesis of its introduction in the wetland area during the first half of 1998.

Seventy-eight bovine viral diarrhoea viruses (BVDV) recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5'-untranslated (5'UTR) and autoprotease (Npro) regions of the pestivirus genome. Seventy-six of the isolates were BVDV-1 and two French isolates were of the BVDV-2 genotype. Phylogenetic analysis of the 5'UTR (245 nt), including additional BVDV-1 sequences from USA, Canada, Germany, New Zealand, Mozambique and Sweden, taken from GenBank and from our previous works, indicated that these viruses were clustered not only into the two generally accepted groups (BVDV-1a-"NADL like" and BVDV-1b-"Osloss like"), but altogether into 11 phylogenetic groups. Similar clustering was observed with Npro region sequences (385 nt) and the highest bootstrap values (over 95%) were obtained by phylogeny combining 5'UTR and Npro sequences. Some associations between the genetic grouping and the origin of the isolates were apparent, probably reflecting historical trade contacts. Considering the variability of isolates it is recommended that diagnostic PCR primers should be re-examined to ensure coverage of all BVDV-1 groups. The genogroups were less clearly differentiated by monoclonal antibody typing, suggesting significant antigenic similarities within the BVDV-1 genotype.

Equine arteritis viruses (EAV) from Europe and America were compared by phylogenetic analysis of 43 isolates obtained over four decades. An additional 22 virus sequences were retrieved from GenBank. Fragments of the glycoprotein G(L) and the replicase genes were amplified by RT-PCR, prior to sequencing and construction of phylogenetic trees. The trees revealed many distinctive lineages, consistent with prolonged diversification within geographically separated host populations. Two large groups and five subgroups were distinguished. Group I consisted mainly of viruses from North America, whilst group II consisted mainly of European isolates. In most instances, where the geographic origin of the viruses appeared to be at variance with the phylogenetically predicted relationships, the horses from which the viruses were recovered had been transported between Europe and America or vice versa. Analysis of the replicase gene revealed similar phylogenetic relationships although not all of the groups were as clearly defined. Virus strains CH1 (Switzerland, 1964) and S1 (Sweden, 1989) represented separate 'outgroups' based on analysis of both genomic regions. The results of this study confirm the value of the G(L) gene of EAV for estimating virus genetic diversity and as a useful tool for tracing routes by which EAV is spread. In addition, computer-assisted predictions of antigenic sites on the G(L) protein revealed considerable variability among the isolates, especially with respect to regions associated with neutralization domains.

A BVD control programme based on the identification and removal of persistently infected (PI) animals is being undertaken in an area in the Rome province, where BVD outbreaks had been previously detected. It involves 174 mainly dairy herds, from which blood samples of all bovines older than 1 year are obtained through the national brucellosis and leukosis eradication programme. Samples sufficient to detect the presence of seropositive animals at a prevalence of 5% or more are initially screened for antibodies against BVD virus (BVDV) using an immunoenzymatic assay. Upon identification of seroreagents additional blood samples are tested from the 6-12-month age category not included in the initial samples. Animals are considered immunotolerant if BVDV is demonstrated twice at a minimum 30-day interval. When no seropositive animals are detected during the first serological screening the herd is declared BVD-free if a second testing, preferably carried on the same animals previously tested, confirms the seronegative status of the herd. At present 147 farms have been tested, of which 63 (42.9%) are negative with respect to antibodies against BVDV. Of the 84 remaining herds in which one or more seropositives are detected, 13 are classified as recently infected. In eight of these recently infected herds, 22 PI animals have been identified.