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Abstract Background Feline Herpesvirus type-1 (FHV-1) is a worldwide spread pathogen responsible for viral rhinotracheitis and conjunctivitis in cats that, in the most severe cases, can lead to death. Despite the availability of a variety of antiviral medications to treat this illness, mainly characterized by virostatic drugs that alter DNA replication, their use is often debated. Phytotherapeutic treatments are a little-explored field for FHV-1 infections and reactivations. In this scenario, natural compounds could provide several advantages, such as reduced side effects, less resistance and low toxicity. The purpose of this study was to explore the potential inhibitory effects of the green tea extract (GTE), consisting of 50% of polyphenols, on FHV-1 infection and reactive oxygen species (ROS) production. Results Crandell-Reese feline kidney (CRFK) cells were treated with different doses of GTE (10–400 µg/mL) during the viral adsorption and throughout the following 24 h. The MTT and TCID50 assays were performed to determine the cytotoxicity and the EC50 of the extract, determining the amounts of GTE used for the subsequent investigations. The western blot assay showed a drastic reduction in the expression of viral glycoproteins (i.e., gB and gI) after GTE treatment. GTE induced not only a suppression in viral proliferation but also in the phosphorylation of Akt protein, generally involved in viral entry. Moreover, the increase in cell proliferation observed in infected cells upon GTE addition was supported by enhanced expression of Bcl-2 and Bcl-xL anti-apoptotic proteins. Finally, GTE antioxidant activity was evaluated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) and total antioxidant capacity (TAC) assays. The ROS burst observed during FHV-1 infection was mitigated after GTE treatment, leading to a reduction in the oxidative imbalance. Conclusions Although further clinical trials are necessary, this study demonstrated that the GTE could potentially serve as natural inhibitor of FHV-1 proliferation, by reducing viral entry. Moreover, it is plausible that the extract could inhibit apoptosis by modulating the intrinsic pathway, thus affecting ROS production.

Abstract Background Pseudorabies is an infection of domestic and wild pigs that has occasionally been reported in dogs with fatal encephalitis. Hunting dogs are predisposed to pseudorabies exposure due to incorrect practices (administration of raw infected meat) or close contact with infected wild boars. This study described an outbreak of pseudorabies in two hunting dogs in the Campania region, southern Italy. Case presentation Two hunting dogs were hospitalized after a hunting trip, with fever, itching, and self-inflicted lesions. Laboratory tests showed mild anemia and marked leukocytosis. Despite conservative therapy, both animals died 48 h after the presentation of symptoms. One of the carcasses was sent to the Department of Veterinary Medicine and Animal Production in Naples to confirm the suspicion of pseudorabies. DNA was extracted from different matrices and used as a template for real-time PCR to detect PRV. Several samples (brain, cerebellum, brainstem, lung, and liver) tested positive. Subsequent sequence analyses of glycoprotein E from DNA extracted from the brain stem revealed a sequence similarity to those described in previous cases of pseudorabies in dogs in Italy, France and Belgium. One month after the outbreak, blood samples were collected from 42 dogs belonging to the same hunting team and from 245 dogs (cohort population) living in the Campania region. All samples were tested with two commercial ELISAs to detect seroconversion against glycoproteins B and E. A seroprevalence of 19% was observed in the hunting team affected by the outbreak, while only 0.8% was observed in the regional dog population. Conclusions The data reported in this study demonstrate potential exposure to PRV by dead-end hosts, particularly hunting dogs. The sequencing results indicated the homogeneity of PRV strains circulating in the different Italian regions.

Q fever is a zoonosis whose main reservoirs are domestic ruminants. Surveillance in these species is carried out mainly with serological tests, which, however, have limited diagnostic performance, and their manufacturing requires laboratories equipped with high biosafety requirements for antigen production. Recombinant ELISAs do not depend on these requirements and, being based on a single antigen, can reduce potential false positivity by identifying antibodies specific to a phase of infection. The aim of this study was to apply a new technology (dual antigen test) to a recombinant protein (Ybgf), an antigen produced in recombinant form and already used in previous studies for the design of an indirect ELISA. The successfully produced recombinant antigen was used to coat 96-well plates and, at the same time, another antigen aliquot was conjugated with HRP to obtain an HRP-conjugated Ybgf. After setting the test conditions, the results obtained with the recombinant double antigen test were compared with those obtained with a commercial assay (considered as reference assay) testing a total of 514 ruminant samples (280 goats and 234 cattle). A concordance of 86.2 and a Cohen's Kappa value of 0.72 were obtained, with no significant difference between the two species tested. Notably, the test proved to be highly specific, having correctly identified 250 out of 253 animals. This research represents an additional effort to use recombinant antigens to enhance serological methods in veterinary medicine. In a “one-health scenario”, improving the performance of serological tests used in veterinary practice also means improving the surveillance of this infection.

Abstract Background Pigs are susceptible to several ruminant pathogens, including Coxiella burnetti, Schmallenberg virus (SBV) and bovine viral diarrhea virus (BVDV). These pathogens have already been described in the pig population, although the dynamics of the infection and the impact on pig farms are currently unclear. The aim of this work was to evaluate the presence of these infections in the pig population of the Campania region, southern Italy, and to evaluate the risk factors associated with a greater risk of exposure. Results A total of 414 serum samples belonging to 32 herds were tested for the presence of antibodies against SBV, Coxiella, and BVD using commercial multispecies ELISA kits. SBV (5.3%) was the most prevalent pathogen, followed by Coxiella (4.1%) and BVD (3%). The risk factors included in the study (age, sex, province, farming system, ruminant density and major ruminant species) had no influence on the probability of being exposed to BVD and Coxiella, except for the location, in fact more pigs seropositive to Coxiella were found in the province of Caserta. However, the univariate analysis highlighted the influence of age, location, and sex on exposure to SBV. The subsequent multivariate analysis statistically confirmed the importance of these factors. The presence of neutralizing antibodies for SBV and BVDV, or antibodies directed towards a specific phase of infection for Coxiella was further confirmed with virus-neutralization assays and phase-specific ELISAs in a large proportion of positive samples. The presence of high neutralizing antibody titers (especially for SBV) could indicate recent exposures. Twelve of the 17 positive samples tested positive for antibodies against Coxiella phase I or II antigens, indicating the presence of both acute and chronic infections (one animal tested positive for both phases antibodies). Conclusions Our study indicates a non-negligible exposure of pigs from southern Italy to the above pathogens. Further studies are necessary to fully understand the dynamics of these infections in pigs, the impact on productivity, and the public health consequences in the case of Coxiella.

Abstract Monitoring disease among wildlife is critical to preserving health in both domestic animals and wildlife, and it becomes much more critical when the diseases cause significant economic damage to the livestock industry or threaten public health. Given the continuous increase in populations and its role as a reservoir for several infections, wild boar (Sus scrofa) requires special attention regarding disease surveillance and monitoring. In this study, we investigated the molecular prevalence of selected pathogens in the wild boar population of Campania, southern Italy. The prevalence of pathogens causing reproductive problems in pigs (Sus domesticus), including porcine parvovirus (PPV), porcine circovirus types 2 and 3 (PCV-2 and PCV-3), pseudorabies virus (PRV), Coxiella burnetii, and Brucella suis, was evaluated by testing the reproductive organs collected from 63 wild boars with polymerase chain reaction. The most common pathogens were PPV (44.4%) and two porcine circoviruses (14.3%). PRV and C. burnetii, on the other hand, showed a significantly lower prevalence (1.6%). No reproductive organs tested were positive for B. suis. Risk factor analysis revealed a correlation between age and PCV-2 positivity, with animals less than 12 months old having significantly higher prevalence rates. Our findings suggest that wild boars hunted in the Campania region harbour several infections potentially transmissible to other mammals’ reproductive tracts. Furthermore, our results emphasized the importance of strict adherence to biosecurity protocols on domestic swine farms, especially on free-range farms, to avoid interactions between domestic and wild animals.

Abstract Background Although FeHV-1 is a primary feline pathogen, little is known about its interactions with host cells. Its relationship with several cellular pathways has recently been described, whereas its interplay with the apoptotic process, unlike other herpesviruses, has not yet been clarified. The aim of this work was to evaluate whether FeHV-1 induces apoptosis in its permissive cells, as well as the pathway involved and the effects of induction and inhibition of apoptosis on viral replication. Methods Monolayers of CRFK cells were infected at different times with different viral doses. A cytofluorimetric approach allowed the quantification of cells in early and late apoptosis. All infections and related controls were also subjected to Western blot analysis to assess the expression of apoptotic markers (caspase 3-8-9, Bcl-2, Bcl-xL, NF-κB). An inhibitor (Z-VAD-FMK) and an inducer (ionomycin) were used to evaluate the role of apoptosis in viral replication. Finally, the expression of autophagy markers during the apoptosis inhibition/induction and the expression of apoptosis markers during autophagy inhibition/induction were evaluated to highlight any crosstalk between the two pathways. Results FeHV-1 triggered apoptosis in a time- and dose-dependent manner. Caspase 3 cleavage was evident 48 h after infection, indicating the completeness of the process at this stage. While caspase 8 was not involved, caspase 9 cleavage started 24 h post-infection. The expression of other mitochondrial damage markers also changed, suggesting that apoptosis was induced via the intrinsic pathway. NF- κB was up-regulated at 12 h, followed by a gradual decrease in levels up to 72 h. The effects of apoptosis inhibitors and inducers on viral replication and autophagy were also investigated. Inhibition of caspases resulted in an increase in viral glycoprotein expression, higher titers, and enhanced autophagy, whereas induction of apoptosis resulted in a decrease in viral protein expression, lower viral titer, and attenuated autophagy. On the other hand, the induction of autophagy reduced the cleavage of caspase 3. Conclusions In this study, we established how FeHV-1 induces the apoptotic process, contributing to the understanding of the relationship between FeHV-1 and this pathway.

Infectious laryngotracheitis virus (ILTV) is responsible for avian infectious laryngotracheitis (ILT), a highly contagious acute respiratory disease affecting chickens. However, there is limited information on ILTV and its distribution in Ethiopia, particularly in the southeastern region. The aim of this study was to establish the serological prevalence and molecular evidence in commercial and backyard chickens from Robe town, Southeastern Ethiopia. A cross-sectional study was conducted between December 2021 and June 2022, collecting 240 serum samples from randomly selected chickens belonging to eight kebeles (farms) using systematic random sampling. ILTV-specific antibodies were detected using a commercial indirect enzyme-linked immunosorbent assay (ELISA). From 240 serum samples, 26.7% were positive for ILTV antibodies. Logistic regression analysis identified the type of poultry farm (backyard) and the introduction of chickens from other farms as potential risk factors associated with ILTV exposure. Tracheal tissue and oropharyngeal and tracheal swabs were collected from suspected chickens for isolation and molecular detection. A total of six samples were successfully isolated in embryonated eggs (40%), with four of them verified with a specific PCR. These findings documented the presence of ILTV in the study area, which needs further insight to fully understand the actual spread of ILTV and quantify the damage caused to the poultry sector.

Abstract The expansion of urbanization in natural environments increases interactions between wildlife, domestic animals, and humans. In Italy, the red fox (Vulpes vulpes) is one of the most common wild carnivores. This species can serve as a reservoir and sentinel host for several infectious diseases. We aimed to improve knowledge about the exposure of red foxes to selected zoonotic (Anaplasma spp, Ehrlichia spp., Borrelia spp., and hepatitis E virus) and carnivore-specific pathogens (canine parvovirus, canine distemper virus, pseudorabies virus, and Dirofilaria spp.) through a retrospective survey performed in the Tuscany region during the spring season of 2013. Using specific ELISAs and serum samples (n = 38) collected during a culling campaign, a prevalence of 2.6% for canine distemper virus, 18.4% for canine parvovirus, 5.2% for Anaplasma spp., 2.6% for Ehrlichia spp., 7.9% for Dirofilaria spp., 21.05% for hepatitis E virus, and 10.5% for pseudorabies virus was observed. Conversely, antibodies against Borrelia spp. were not identified in any of the animals. Our results revealed no significant sex-related differences in seroprevalence and confirmed hepatitis E virus as the most common pathogen in the analyzed samples. All of the animals that tested positive for tick-borne zoonotic agents presented ticks at the time of sampling. Our study confirms the exposure of red foxes in the Tuscany region to viral and bacterial infections raising medical and veterinary concern and indicating the need for large-scale surveillance to fully assess the epidemiological significance of these findings.

Abstract Background Schmallenberg virus (SBV) is a vector-borne pathogen that mainly affects ruminants. Schmallenberg disease has never been described in southern Italy, although this geographic area displays climatic features suitable for Culicoides biting midges, which transmit the pathogen. An observational study was carried out in the Campania region in 2020 to evaluate the seroprevalence in cattle and water buffalo as well as to identify the risk factors involved in the distribution of SBV. Results Relatively high seroprevalences of 38.2% (cattle) and 43% (water buffalo) were found by using a commercial SBV ELISA, which is comparable to the prevalence obtained in other countries under post-epidemic conditions. A virus neutralization assay performed on positive samples showed high titers in a large percentage of animals which is assumed to indicate recent exposure. Bivariate analysis of several variables revealed some environmental factors associated with higher seroprevalence, such as mean annual temperature, distance from the coast, and altitude. Multivariate logistic regression confirmed the statistical association only for mean annual temperature, that was found to be the main factor responsible for the distribution of the virus in southern Italy. In addition, molecular diagnosis attempts were performed on serum samples and resulted in the detection of SBV RNA in two herds and six animals. Conclusions In this work we have demonstrated the circulation of SBV in southern Italy using both molecular and serological assays. This study emphasized the essential role of monitoring in preventing the re-emergence of vector-borne diseases in ruminants.

The lumpy skin disease (LSD) vaccination status and epidemiological distribution remain unknown in some parts of Ethiopia, including the Sidama regional state. In this study, a serological survey of LSD was performed using a specific virus neutralization assay in selected districts of the Sidama regional state representing three agroecological zones from September 2021 to June 2022. Moreover, an assessment of community awareness and LSD vaccine-related problems was conducted using a questionnaire. Our results showed an overall animal and herd level seroprevalence of 40.8% (95%CI = 35.8, 45.8) and 81% (95%CI = 77, 85), respectively. High and low seroprevalence were observed in lowland (48%) and highland (28%) areas, although they were not statistically significant. However, risk factors such as management systems and breeds showed substantial differences in their LSD prevalence. The results obtained through the questionnaire showed that a small portion of respondents (29.2%) know about LSD and vaccinate their cattle (23.3%) against this disease. Only 20.8% of the respondents stated that there was LSD occurrence in their vaccinated cattle. In conclusion, both qualitative and quantitative study results showed the need for intervention in terms of community-based awareness creation about LSD clinical signs and vaccination advantages together with the frequently updated information on LSD prevalence.

Feline herpesvirus type 1 (FeHV-1) is a primary pathogen in cats responsible for respiratory and ocular signs. There are presently no antiviral drugs that are officially licensed for veterinary use in several countries. Consequently, veterinarians must depend on off-label antivirals designed for human use. Recent advances in virus–host cell interaction have resulted in new insights into FeHV-1 replication, establishing the importance of the PI3K/Akt axis. The aim of this study was to employ this new information to assess the efficacy of two compounds whose activities involve this pathway. The antiviral properties of miltefosine and nitazoxanide were examined using seven different concentrations, evaluating cell viability and viral titers after 24 h of infection. Furthermore, selected concentrations were supplied at different time points to investigate the influence of the timing of the addition. The best results were obtained when the drugs were added both before and after viral adsorption (in particular for nitazoxanide). Each compound was further investigated by real-time PCR, western blot, and immunofluorescence. Nitazoxanide was the most effective treatment, reducing the expression of viral glycoproteins as measured by western blot and immunofluorescence, as well as reducing the release of virions in the supernatant (measured by real-time PCR). Moreover, treatment with nitazoxanide and miltefosine was associated with a decrease in Akt phosphorylation. This work emphasized the significance of comprehending the pathways necessary for viral replication and their use in the assessment of novel and effective antivirals.

IntroductionFeline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are well-known retroviruses causing important infections in domestic cats worldwide. The goal of this study was to determine the prevalence of FeLV and FIV infections in cat living indoor and outdoor in southern Italy.MethodsThe survey was conducted on 1322 stray and owned cats from the regions of Campania, Basilicata, and Calabria. It was carried out over a 10-year period to obtain a more realistic picture of the prevalence of these retroviral diseases in the country. FIV and FeLV status was determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (SNAP Combo Plus FeLV/FIV, IDEXX). Risk factors were analysed by logistic regression.Results and DiscussionThe results showed that 101/1322 (7.64%) cats were positive for FeLV antigen and 110/1322 (8.32%) cats were positive for FIV antibody. Twenty-six of the 1322 cats (1.97%) were positive for both FIV and FeLV infection. Our results are similar to those published in recent studies in Europe. A statistically significant association (p

FeHV-1 is a member of the Herpesviridae family that is distributed worldwide and causes feline viral rhinotracheitis (FVR). Since its relationship with the autophagic process has not yet been elucidated, the aim of this work was to evaluate the autophagy mediated by FeHV-1 and to determine its proviral or antiviral role. Our data showed that autophagy is induced by FeHV-1 in a viral dose and time-dependent manner. Phenotypic changes in LC3/p62 axis (increase of LC3-II and degradation of p62) were detected from 12 h post infection using western blot and immuno-fluorescence assays. In a second step, by using late autophagy inhibitors and inducers, the possible proviral role of autophagy during FeHV-1 infection was investigating by assessing the effects of each chemical in terms of viral yield, cytotoxic effects, and expression of viral glycoproteins. Our findings suggest that late-stage autophagy inhibitors (bafilomycin and chloroquine) have a negative impact on viral replication. Interestingly, we observed an accumulation of gB, a viral protein, when cells were pretreated with bafilomycin, whereas the opposite effect was observed when an autophagy inducer was used. The importance of autophagy during FeHV-1 infection was further supported by the results obtained with ATG5 siRNA. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, and the negative impact of late autophagy inhibitors on viral replication.

FeHV-1 is the causative agent of infectious rhinotracheitis in cats. The relationship between viral infection and the PI3K/Akt/mTOR pathway, as well as its function in crucial physiological processes like as autophagy, apoptosis or the IFN induction cascade is known for other varicelloviruses. However, there is no information on whether autophagy is activated during FeHV-1 infection nor on how this infection modifies PI3K/Akt/mTOR pathway. In this work, we aim to elucidate the involvement of this pathway during cytolytic infection by FeHV-1 in permissive cell lines. Using a phenotypic approach, the expression of proteins involved in the PI3K/Akt/mTOR pathway was examined by Western blot analysis. The findings demonstrated the lack of modifications in relation to viral dose (except for phospho-mTOR), whereas there were changes in the expression of several markers in relation to time as well as a mismatch in the time of activation of this axis. These results suggest that FeHV-1 may interact independently with different autophagic signaling pathways. In addition, we found an early phosphorylation of Akt, approximately 3 h after infection, without a concomitant decrease in constitutive Akt. This result suggests a possible role for this axis in viral entry. In a second phase, the use of early autophagy inhibitors was examined for viral yield, cytotoxic effects, viral glycoprotein expression, and autophagy markers and resulted in inefficient inhibition of viral replication (12 h post-infection for LY294002 and 48 h post-infection for 3-methyladenine). The same markers were examined during Akt knockdown, and we observed no differences in viral replication. This result could be explained by the presence of a protein kinase in the FeHV-1 genome (encoded by the Us3 gene) that can phosphorylate various Akt substrates as an Akt surrogate, as has already been demonstrated in genetically related viruses (HSV-1, PRV, etc.). For the same reasons, the use of LY294002 at the beginning of infection did not affect FeHV-1-mediated Akt phosphorylation. Our findings highlight changes in the PI3K/Akt/mTOR pathway during FeHV-1 infection, although further research is needed to understand the importance of these changes and how they affect cellular processes and viral propagation.

Zearalenone (ZEN) is a mycotoxin produced by fungi belonging to the genera Fusarium spp. and commonly found in feed and food. It is frequently related to reproductive disorders in farm animals and, occasionally, to hyperestrogenic syndromes in humans. Nowadays, knowledge about ZEN effects on wild boars (Sus scrofa) is extremely scarce, despite the fact that they represent one of the most hunted game species in Italy. The aim of this study was to investigate how ZEN affects the liver, kidney, and muscle oxidative status and morphology of wild boars hunted in various locations throughout the province of Avellino, Campania Region, Southern Italy, during the 2021–2022 hunting season. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, as well as the malondialdehyde (MDA) levels, were assessed by colorimetric assays; tissue morphology was evaluated by hematoxylin–eosin and Masson’s stains. Our data showed that ZEN contamination might result in oxidative stress (OS) and some histopathological alterations in wild boars’ livers and kidneys rather than in muscles, emphasizing the importance of developing a wildlife monitoring and management strategy for dealing not only with the problem of ZEN but the surveillance of mycotoxins in general.

We conducted a serologic survey among dogs and cats in Italy to detect antibodies against severe acute respiratory syndrome virus 2 (SARS-CoV-2). We found that SARS-CoV-2 seroprevalence was higher among cats (16.2%) than dogs (2.3%). In addition, seroprevalence was higher among animals living in close contact with SARS-CoV-2–positive owners.

Leukocyte-Platelet-Rich Fibrin (L-PRF) is a second generation of platelet concentrates; it was widely used, as an autologous platelet-based wound sealant and hemostatic agent in surgical wound healing. L-PRF clot or membrane is a solid fibrin-based biomaterial, with a specific 3D distribution of the leukocytes and platelet aggregates. This biological scaffold releases growth factors (i.e., TGF- β1, PDGF-AB, VEGF) and matrix proteins (fibronectin, vitronectin and thrombospondin-1) during the healing process after the application. To the Authors' knowledge both in human and veterinary medicine a single standardised protocol was not reported. This prospective study aimed to apply Crisci's L-PRF protocol (which is characterised by 30” of acceleration, 2' at 2,700 rpm, 4' at 2,400 rpm, 3' at 3,000 rpm, and 36” of deceleration and arrest) sin canine species, evaluate macroscopically and histologically the L-PRF membranes obtained by using Wound Box to standardise the L-PRF protocol in dogs and to evaluate the clinical feasibility of using L-PRF membranes by quantitative in vitro analysis of growth factors over 7 days. One hundred twenty-eight dogs in good general condition with no history of recent NSAIDs intake (15 days of washout) and/or any medication or disease related to coagulation process met inclusion criteria and therefore were enrolled. We obtained 172 membrane L-PRF membranes by 86 dogs: half of them underwent macroscopic and histological analysis, the other 86 underwent ELISA analysis. The Wound Box gave a membrane of mean (±SD) length (cm), width (cm) and weight (g) of 1.97 (±0.89), 0.95 (±0.36), 0.46 (±0.20) respectively. Histology analysis confirmed a well-defined histoarchitecture with five layers reproducing density and distribution of blood cells in this biomaterial. Finally, the ELISA assay performed with 22 L-PRF membranes showed a peak in growth factors at 6 h after membrane production, followed by a decrease in release at 24 and 72 h and a second peak in release at 168 h after production. Statistical analysis of demographic variables (age, sex, and body condition score BCS) and the average of growth factors determined by the ELISA assay did not reveal statistical significance, except for the BCS factor compared with the production of VEGF. Our data confirm the effectiveness of this protocol and of Wound Box to produce L-PRF membranes in dogs.

Cattle and water buffalo are the main livestock species that are raised in the Campania region, southern Italy, and they contribute significantly to the regional rural economy. Currently there are limited data on the prevalence of relevant impact infections, such as bovine coronavirus (BCov), an RNA virus that causes acute enteric and respiratory disease. Although these diseases are described primarily in cattle, there have been reports of spillovers to other ruminants, including water buffalo. Here, we determined the seroprevalence of BCoV in cattle and water buffalo in the Campania region of southern Italy. An overall seroprevalence of 30.8% was determined after testing 720 sampled animals with a commercial enzyme-linked immunosorbent assay. A risk factor analysis revealed that the seropositivity rates in cattle (49.2%) were higher than in water buffalo (5.3%). In addition, higher seroprevalence rates were observed in older and purchased animals. In cattle, housing type and location were not associated with higher seroprevalence. The presence of BCoV antibodies in water buffalo was associated with the practice of co-inhabiting with cattle, demonstrating that this practice is incorrect and promotes the transmission of pathogens between different species. Our study found a considerable seroprevalence, which is consistent with previous research from other countries. Our results provide information on the widespread distribution of this pathogen as well as the risk factors that are involved in its transmission. This information could be useful in the control and surveillance of this infection.

Porcine coronaviruses and reproductive and respiratory syndrome (PRRS) are responsible for severe outbreaks that cause huge economic losses worldwide. In Italy, three coronaviruses have been reported historically: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Although repeated outbreaks have been described, especially in northern Italy, where intensive pig farming is common, there is a worrying lack of information on the spread of these pathogens in Europe. In this work, we determined the seroprevalence of three porcine coronaviruses and PRRSV in the Campania region, southern Italy. A total of 443 samples were tested for the presence of antibodies against porcine coronaviruses and PRRSV using four different commercial ELISAs. Our results indicated that PEDV is the most prevalent among porcine coronaviruses, followed by TGEV, and finally PRCV. PRRSV appeared to be the most prevalent virus (16.7%). For coronaviruses, seroprevalence was higher in pigs raised in intensive farming systems. In terms of distribution, TGEV is more widespread in the province of Avellino, while PEDV and PRRSV are more prevalent in the province of Naples, emphasizing the epidemic nature of both infections. Interestingly, TGEV-positive animals are more common among growers, while seropositivity for PEDV and PRRSV was higher in adults. Our research provides new insights into the spread of swine coronaviruses and PRRSV in southern Italy, as well as a warning about the need for viral surveillance.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of the Fusarium genera, and is a contaminant of cereals and plant products. ZEN and its metabolites are considered endocrine disruptors, and could have various toxic effects on animals and humans. In recent years, there has been a significant demographic increase in wild boar (Sus scrofa) in many mountainous and hilly areas of Italy, including the Campania region, mainly due to global climate change. The wild boar can be defined as a generalist and omnivorous species capable of varying its diet; therefore, it can play a role as an environmental bioindicator towards contaminants such as mycotoxins. This study was conducted to evaluate, for the first time, the concentrations of ZEN and its metabolites in the liver, kidney, and muscle of 82 wild boars shot in their habitat by hunters with hunting permits in different localities of Avellino province (Campania region, Southern Italy) from 2021 to 2022. The samples were collected and analyzed with an SPE clean-up and high-pressure liquid chromatography method with fluorescence detection. The results indicated that ZEN and α-Zearalenol were present in most of the samples, suggesting that a plan to monitor these mycoestrogens is essential to achieve the goals of “One Health”.

From 2019 to 2021, a retrospective molecular study was conducted in the Campania region (southern Italy) to determine the prevalence of viral diseases in domestic cats. A total of 328 dead animals were analyzed by Real-Time PCR for the presence of feline panleukopenia virus (FPV), feline leukemia virus (FeLV), feline enteric coronavirus (FCoV), rotavirus (RVA), feline herpesvirus type 1 (FHV-1), and feline calicivirus (FCV). The possible presence of SARS-CoV-2 was also investigated by Real-Time PCR. The cats included in this study were specifically sourced and referred by local veterinarians and local authorities to the Zooprofilactic Experimental Institute of Southern Italy (IZSM) for pathological evaluation. The samples consisted of owners, catteries, and stray cats. Results revealed: 73.5% positive cats for FPV (189/257), 23.6% for FeLV (21/89), 21.5% for FCoV (56/266), 11.4% for RVA (16/140), 9.05% for FeHV-1 (21/232), and 7.04 for FCV (15/213). In contrast, SARS-CoV-2 was never detected. FPV was more prevalent in winter (p = 0.0027). FCoV FHV-1, FCV, and RVA predominated in autumn, whereas FeLV predominated in summer. As expected, viral infections were found more frequently in outdoor and shelter cats than in indoor ones, although no statistical association was found between animal lifestyle and viral presence. The study showed a high prevalence of FPV, FeLV, and FCoV and a moderate prevalence of RVA, FHV-1, and FCV. Moreover, the prevalence of these pathogens varied among the cat populations investigated.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that activates multiple signaling pathways, causing cells to produce higher levels of reactive oxygen species (ROS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major generator of ROS in leukemia, and marine natural products have shown promising activities for the treatment of hematopoietic malignancies. In the present study, we investigated the effect of the marine microalga Skeletonema marinoi (S.M.), a ubiquitous diatom that forms massive blooms in the oceans, on the human leukemia cell line K562. The effects of S.M. extract on cell viability, production of ROS, nitric oxide (NO), and apoptosis were examined. In this preliminary work, S.M. was able to decrease cell viability (p < 0.05) and increase apoptosis levels (p < 0.05) in K562 cells after 48 h of treatment. In addition, the levels of NOX, NO, and malondialdehyde (MDA) were reduced in K562-treated cells (p < 0.05), whereas the levels of SOD, CAT, and GPx increased during treatment (p < 0.05). Finally, analyzing Bax and Bcl-2 expression, we found a significant increase in the proapoptotic protein Bax and a sustained decrease in the antiapoptotic protein Bcl-2 (p < 0.05) in the K562-treated cells.

Due to its economic impact on livestock and its zoonotic effect, Q fever is a public and animal health problem. Information on this infection in Italy is presently supported by reports of reproductive problems in livestock farms and is, therefore, insufficient to properly understand the impact of the disease. This study aimed to describe for the first time the seroprevalence of Q fever in dairy cows and water buffalos in the Campania region (Southern Italy). A total of 424 dairy cattle and 214 water buffalo were tested using a commercial indirect ELISA kit. An overall seroprevalence of 11.7% confirmed the wide distribution of C. burnetii in this region. Several factors were positively associated with higher seroprevalence, such as species (higher in cattle than in water buffalo), age, and coexistence with other ruminant species. The final model of logistic regression included only age (older) and species (cattle), which were positively associated with the presence of Q fever antibodies. Our findings support the widespread presence of Coxiella burnettii in Campania and show a seroprevalence similar to that observed in previous studies in other Italian regions and European countries. Since human cases are typically linked to contact with infected ruminants, there is a need to improve surveillance for this infection.

Following the COVID-19 epidemic outbreak in Ariano Irpino, Campania region (Italy), we tested lactating cows for the presence of SARS-CoV-2 on a cattle farm at which, prior to the investigation, 13 of the 20 farmworkers showed COVID-19-like symptoms, and one of them died. Twenty-four lactating cows were sampled to detect SARS-CoV-2. All nasal and rectal swabs and milk samples were negative for SARS-CoV-2 RNA. Of the 24 collected serum samples, 11 showed antibodies against SARS-CoV-2 nucleocapsid protein, 14 showed antibodies against SARS-CoV-2 spike protein, and 13 developed neutralising antibodies for SARS-COV-2; all samples were negative for Bovine Coronavirus (BCoV), another betacoronavirus. To our knowledge, this is the first report of natural serological evidence of SARS-CoV-2 infection in lactating cows. We hypothesise that this may be a case of reverse zoonosis. However, the role of cattle in SARS-CoV-2 infection and transmission seems to be negligible.

The problem of residues of toxic contaminants in food products has assumed considerable importance in terms of food safety. Naturally occurring contaminants, such as mycotoxins, are monitored routinely in the agricultural and food industries. Unfortunately, the consequences of the presence of mycotoxins in foodstuffs are evident in livestock farms, where both subacute and chronic effects on animal health are observed and could have non-negligible effects on human health. Ochratoxin A (OTA) is a common mycotoxin that contaminates food and feeds. Due to its thermal stability, the eradication of OTA from the food chain is very difficult. Consequently, humans and animals are frequently exposed to OTA in daily life. In this review article, we will devote time to highlighting the redox-based nephrotoxicity that occurs during OTA intoxication. In the past few decades, the literature has improved on the main molecules and enzymes involved in the redox signaling pathway as well as on some new antioxidant compounds as therapeutic strategies to counteract oxidative stress. The knowledge shown in this work will address the use of nutraceutical substances as dietary supplements, which would in turn improve the prophylactic and pharmacological treatment of redox-associated kidney diseases during OTA exposure, and will attempt to promote animal feed supplementation.

The etiology of spontaneous cranial cruciate ligament rupture in dogs is unknown despite being one of the most impacting orthopedic diseases in dogs. Numerous studies have contributed to the understanding of a multifactorial pathogenesis, this, however, without identifying a pivotal link to explain progressive collagen degeneration and osteoarthritic changes. In human medicine, recent reports have identified relaxin as a triggering factor in ligament ruptures in knee and metacarpal joints. We thus hypothesized that relaxin might also play a role in canine cruciate ligament rupture. Relaxin’s primarily known property is connective tissue remodeling through collagenolysis. We therefore investigated relaxin and its cognate receptors LGR7/LGR8 in 18 dogs with cranial cruciate ligament disease (CCLD) and compared them to a group of dogs with normal stifle joints. Applying immunohistochemistry (IHC), double immunofluorescence (dIF), and western blot analysis (WB), we found strong and significantly increased expression of both relaxin and its receptors in ruptured cruciate ligaments, and in synovial membranes. Pattern of immuno-staining on dIF strongly suggests relaxin binding to primed receptors and activation of signaling properties, which in turn may have affected collagen matrix metabolism. Thus, in canine cranial cruciate ligament disease, relaxin/receptor signaling may be a primary trigger for collagen fiber degradation and collagen lysis, eventually followed by ligament rupture.

Malignant mesothelioma (MM) is an aggressive asbestos-related cancer, against which no curative modalities exist. Oncolytic virotherapy is a promising therapeutic approach, for which MM is an ideal candidate; indeed, the pleural location provides direct access for the intra-tumoral injection of oncolytic viruses (OVs). Some non-human OVs offer advantages over human OVs, including the non-pathogenicity in humans and the absence of pre-existing immunity. We previously showed that caprine herpesvirus 1 (CpHV-1), a non-pathogenic virus for humans, can kill different human cancer cell lines. Here, we assessed CpHV-1 effects on MM (NCI-H28, MSTO, NCI-H2052) and non-tumor mesothelial (MET-5A) cells. We found that CpHV-1 reduced cell viability and clonogenic potential in all MM cell lines without affecting non-tumor cells, in which, indeed, we did not detect intracellular viral DNA after treatment. In particular, CpHV-1 induced MM cell apoptosis and accumulation in G0/G1 or S cell cycle phases. Moreover, CpHV-1 strongly synergized with cisplatin, the drug currently used in MM chemotherapy, and this agent combination did not affect normal mesothelial cells. Although further studies are required to elucidate the mechanisms underlying the selective CpHV-1 action on MM cells, our data suggest that the CpHV-1-cisplatin combination could be a feasible strategy against MM.

The importance of wild boar lies in its role as a bioindicator for the control of numerous zoonotic and non-zoonotic diseases, including antibiotic resistance. Mannitol Salt Agar (MSA) is a selective medium used for isolation, enumeration, and differentiation of pathogenic staphylococci. Other genera such as Enterococcus spp. are also salt tolerant and able to grow on MSA. The present study focused on the identification, by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), of bacteria grown on MSA isolated from the nasal cavities of 50 healthy wild boars hunted in Campania Region (southern Italy) in the year 2019. In addition, the antimicrobial resistance phenotype of the isolated strains was determined by disk diffusion method. Among genus Staphylococcus, coagulase-negative Staphylococcus (CoNS) were the most common isolated species, with Staphylococcus xylosus as the most prevalent species (33.3%). Furthermore, Enterococcus spp. strains were isolated, and Enterococcus faecalis was the species showing the highest frequency of isolation (93.8%). For staphylococci, high levels of resistance to oxacillin (93.3%) were recorded. Differently, they exhibited low frequencies of resistance to tested non-β-lactams antibiotics. Among enterococci, the highest resistances were observed for penicillin (93.7%), followed by ampicillin (75%), and ciprofloxacin (68.7%). Interestingly, 43.7% of the isolated strains were vancomycin-resistant. In conclusion, this study reports the phenotypic antibiotic resistance profiles of Staphylococcus spp. and Enterococcus spp. strains isolated from nasal cavities of wild boars hunted in Campania Region, highlighting that these wild animals are carriers of antibiotic resistant bacteria.

In recent years, porcine circovirus (PCV) infection has been documented as an important and emerging health concern for livestock and wildlife worldwide. The purpose of the present study was to assess the molecular prevalence of PCV-2 and PCV-3 and to clarify the epidemiological role of wild boars in the circulation of this virus in Campania, Southern Italy. For this purpose, samples from several organs were collected during the hunting season 2017–2018 from 148 wild boars in the Campania region. Quantitative real-time PCR was used for the detection and quantification of PCV-2 and PCV-3 genomes. The combined prevalence of PCV-2 and PCV-3 was 74.32% in the wild boars tested. The proportions of wild boars positive for PCV-2 or PCV-3, or coinfected, were 47.30%, 49.32%, and 22.30%, respectively. No link was detected between PCV positivity and location, but gender was a risk factor for the disease (female; p < 0.0001; OR 0.29). Furthermore, our study provides a snapshot of PCV-2 and PCV-3 circulation in wild boars in the Campania region: our findings can help us to better understand the role of wildlife in PCV circulation.

Aujeszky’s disease (AD, pseudorabies) is a viral disease of suids caused by Suid Herpesvirus 1 (SHV-1) also referred as Aujeszky’s disease virus (ADV) or Pseudorabies virus (ADV). Domestic pig and Wild boar (Sus scrofa) are the natural host, but many species can be infected with ADV. The aim of our study was to evaluate seroprevalence of AD in wild boar hunted in the Campania Region, during the 2016–2017 hunting season. A total of 503 serum samples from wild boars hunted in the provinces of Campania Region (Southern Italy) were collected and were tested for antibody against ADV using an AD, blocking ELISA assay. A Seroprevalence of 23.85% (120/503, 95% Confidence Interval (CI): 20.15–27.55) was found. Gender was not significantly associated with of ADV seropositivity (p > 0.05), while the presence of ADV antibodies was statistically associated with age (>36-month, p < 0.0001) and location (Avellino, p = 0.0161). Our prevalence values are like those obtained in 2010 in our laboratory (30.7%), demonstrating a constant circulation of ADV in the area.

Canine vector-borne diseases (CVBD) are an important and emerging health concern for humans and animals worldwide. The purpose of the presented study was to assess, from 2016 to 2019, the seroprevalence of CVBD agents and clarify the epidemiology of tick-borne disease in stray dogs living in the Campania Region, Southern Italy. For this purpose, blood samples were collected from January 2016 to December 2019 from 1023 dogs in authorized kennels located in the five municipalities of the Campania Region. SNAP® 4DX® from IDEXX® Laboratories was used for detection of Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi sensu lato (s.l.), and Dirofilaria immitis antibodies. The overall seroprevalence of CVBD in stray dogs was 19.6% (95% Confidence Intervals (CI): 17.2–22.8%; 201/1023). The most common pathogen was Ehrlichia spp., with a percentage of positivity of 16.03%, followed by Anaplasma spp. with 7.8%. B. burgdorferi s.l. and D. immitis were detected in only 0.2% of dogs; co-infection was detected in 4.5% of stray dogs tested. No link was detected between the gender, age, location, and CVBD seropositivity, except for Ehrlichia spp. for which location (Avellino Province; p = 0.007) and gender (male, p = 0.002) were risk factors for seropositivity. Our results demonstrated that animals are exposed to at least one of the four etiological agents (Ehrlichia spp., Anaplasma spp. Borrelia burgdorferi s.l., and Dirofilaria immitis) transmitted by vectors. Finally, this study highlighted the utility of serological monitoring in stray dogs, housed in kennels, given the threat posed by CVBD to animals and the zoonotic implications of these etiological agents and their vectors on human health.

Background: A retrospective sero-survey for evidence of West Nile virus (WNV) infection in European wild boar (Sus scorfa) was conducted in the Latium region, Italy, on stored serum samples of the period November 2011 to January 2012. Methods: Sera were collected from 168 European wild boars and screened for antibodies to WNV and other Flaviviruses by competitive enzyme linked immunosorbent assay (cELISA). All sera positive for Flavivirus antibodies by cELISA were further examined by virus neutralization test (VNT). To test the presence of Flavivirus RNA in samples, an RT-PCR was performed using a pan-Flavivirus primers pair. Results: Thirteen wild boars (7.73%) were seropositive for Flaviviruses. The hemolysis of serum samples limited the interpretation of the VNT for 7 samples, confirming the presence of specific antibody against WNV in a single European wild boar serum sample. The presence of ELISA positive/VNT negative samples suggests the occurrence of non-neutralizing antibodies against WNV or other antigen-related Flaviviruses. No samples resulted positive for Flavivirus by RT-PCR assay. Conclusion: Although a moderately high percentage of animals with specific antibody for WNV has been detected in wild boar in other surveillance studies in Europe, this has not been reported previously in Italy. Together, these data indicate that European wild boar are exposed to WNV and/or other related-Flavivirus in central Italy and confirm the usefulness of wild ungulates, as suitable Flavivirus sentinels.