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Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

The water buffalo (Bubalus bubalis) is susceptible to bovine tuberculosis (TB), which receives increased attention in areas where buffalo breeding is prevalent, such as in Southern Italy, especially in the Campania region, where 70% of the buffalo stock is bred. Since 2012, TB testing in buffalo herds has been conducted using the Single Intradermal Test (SIT), with the Comparative Intradermal test (CIT) used in cases of inconclusive results. From 2012 to 2016, the interferon-gamma (IFN-γ) test was occasionally employed experimentally in herds with TB outbreaks to expedite eradication efforts. A local TB eradication program was implemented in officially TB-free buffalo herds between 2017 and 2019. This program involves initial screening with SIT, followed by confirmatory tests, including CIT and IFN-γ, for positive reactions. Since June 2019, the IFN-γ test has replaced the CIT in officially TB-free herds upon positive SIT reactions. Additionally, in suspected and confirmed TB-outbreak herds, the IFN-γ test was used at the discretion of the competent authority. Between 2017 and 2019, approximately 295,000 buffaloes in Campania were screened annually with in vivo tests provided by TB eradication programs. During this period, 32,040 animals from 855 herds were tested using the IFN-γ test and 4,895 tested positive. Since 2020, the use of IFN-γ testing has increased, and has become a prerequisite for the acquisition of TB-free status and is being systematically applied for TB outbreak-extinction procedures. The test was performed in all breeding buffaloes in cases of doubtful SIT results in TB-free herds and when TB lesions are detected at slaughter in animals from TB-free herds. This combined approach helped detect more TB outbreaks, and thereby led to a reduction in the TB prevalence and incidence rates. By 2022, the prevalence had decreased to 1.56%, and the incidence had decreased to 0.73%, after the increased use of the IFN-γ test. This study highlights the effectiveness of implemented strategies in reducing TB in this region. Overall, the data demonstrate the successful impact of TB eradication measures and surveillance activities in reducing bubaline TB prevalence and incidence in the Campania region.

Metabolic adaptations to negative energy balance, as well as lipomobilization, influence inflammatory responses, immune function, and oxidative stress in animals. This study aimed to evaluate the biochemical profile of Mediterranean buffaloes with different levels of lipomobilization from the prepartum to the postpartum period. A total of 76 Mediterranean buffaloes were enrolled, and a weekly blood sample was taken from 7 weeks before to 6 weeks after calving. The concentration of non-esterified fatty acids (NEFAs) was determined in serum and was used to categorize buffaloes into three lipomobilization groups 7 weeks before calving: mild (NEFA-I; NEFA ≤ 0.29 mEq/L; n = 18), medium (NEFA-II; 0.29 0.29 mEq/L; NEFA-II and NEFA-III) at 7 weeks before calving should be monitored more closely to reduce the risk of metabolic diseases. Furthermore, the medium (NEFA-II) and severe (NEFA-III) lipomobilization groups could be associated with differences in the animals’ ability to manage their metabolic status. Specifically, the severe mobilization group was most associated with a greater energy deficit during both the prepartum and postpartum periods without oxidative stress. On the contrary, the medium mobilization group was associated with a less severe energy deficit but was also associated with an inflammatory status and oxidative stress during the prepartum period. These distinctions highlight the need for tailored management strategies to address varying levels of metabolic stress in dairy buffaloes.

Antimicrobial resistance affects all environments, endangering the health of numerous species, including wildlife. Increasing anthropic pressure promotes the acquisition and dissemination of antibiotic resistance by wild animals. Sea turtles, being particularly exposed, are considered sentinels and carriers of potential zoonotic pathogens and resistant strains. Therefore, this study examined the antibiotic resistance profiles of bacteria isolated from loggerhead sea turtles hospitalised in a rescue centre of Southern Italy over a 9-year period. Resistance to ceftazidime, doxycycline, enrofloxacin, flumequine, gentamicin, oxytetracycline and sulfamethoxazole-trimethoprim was evaluated for 138 strains isolated from the clinical samples or organs of 60 animals. Gram-negative families were the most isolated: Vibrionaceae were predominant, followed by Shewanellaceae, Pseudomonadaceae, Enterobacteriaceae and Morganellaceae. These last three families exhibited the highest proportion of resistance and multidrug-resistant strains. Among the three Gram-positive families isolated, Enterococcaceae were the most represented and resistant. The opportunistic behaviour of all the isolated species is particularly concerning for diseased sea turtles, especially considering their resistance to commonly utilised antibiotics. Actually, the multiple antibiotic resistance was higher when the sea turtles were previously treated. Taken together, these findings highlight the need to improve antimicrobial stewardship and monitor antibiotic resistance in wildlife, to preserve the health of endangered species, along with public and environmental health.

We investigated the occurrence of eight potential zoonotic viruses in 91 exotic companion mammals from pet shops in southern Italy via real-time PCR and end-point PCR. The animals were screened for aichivirus, sapovirus, astrovirus, hepatitis A, noroviruses (GI and GII), rotavirus, circovirus, and SARS-CoV-2. Among the nine species of exotic pets studied, only one rat tested positive for aichivirus. The high sequence similarity to a murine kobuvirus-1 strain previously identified in China suggests that the virus may have been introduced into Italy through the importation of animals from Asia. Since exotic companion mammals live in close contact with humans, continuous sanitary monitoring is crucial to prevent the spread of new pathogens among domestic animals and humans. Further investigations on detecting and typing zoonotic viruses are needed to identify emerging and re-emerging viruses to safeguard public health.

Indirect transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been investigated but it is still not completely understood. The present study aimed to compare the persistence and viability of the lineage B.1 and omicron BA.1 subvariant in five daily-use materials to evaluate the role of fomites as a possible source of infection. Artificial contamination was performed in the first set of materials, ethylene vinyl acetate (EVA), cardboard, polystyrene, aluminium, and plastic. Further surfaces using BA.1 (glass, plexiglass, cotton, polyester, and tetrapak) were conducted. The persistence, viability of Vero E6 cell cultures and the residual infectivity of the two lineages were evaluated over 5 days. The results showed different stabilities between the tested matrices. In cotton and polyester, the RNA was undetectable in 24 and 48h post-contamination (p.c.), respectively, and the virus was not viable within 30 min, while in the other surfaces, both lineages, RNA was detectable until 120h p.c. A rapid decay of the viral load was revealed on cardboard, mostly for the omicron variant. Furthermore, on all the materials, longer stability of BA.1 was demonstrated, but showing a less intense CPE than the wild-type. EVA was the material that was able to better sustain virus stability as the virus developed CPE up to 72h p.c. In conclusion, the potential spread of SARS-CoV-2 through fomites is conceivable, albeit it is difficult to establish the real capacity to infect people. Nevertheless, thise information is fundamental to adopting the appropriate measures to mitigate the spread of SARS-CoV-2 and its variants.

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

As a result of the increase of game meat intended for human consumption through Europe, a plethora of food-borne diseases, including trichinellosis, may occur in consumers, posing a relevant public health threat.Thus, this study aims to a citizen science approach to monitor the occurrence of Trichinella spp. in wild boar meat intended for human consumption, evaluating the risk of infection for consumers.Following the European Regulation 2015/1375 (laying down specific rules on official controls for Trichinella in meat), from 2015 to 2021, hunters (n = 478) were involved to collect diaphragm pillar samples of wild boars from mainland southern Italy, which were tested for Trichinella spp. L1 larvae via HCl-pepsin digestion and Multiplex PCR.Overall, 139,160 animals were collected (average of 19,880 per year), being 14 (i.e., 0.01%) tested positive to Trichinella britovi by the combined biochemical and molecular approach. An average larval burden of 28.4 L1 per gram of meat was found (minimum 3.2 - maximum 132.6). A statistically significant difference was found in the prevalence according to hunting seasons (p

Caretta Caretta are endangered sea turtles leaving in the Mediterranean Sea. We carried out a study on 195 sea turtles stranded along the coast of Southern Italy, evaluating anthropogenic and opportunistic factors affecting animal health status and probably threatening the species. More than 60% of the animals showed body injuries, mainly caused by marine traffic and fishing. Ingestion of marine litter of various origins was observed in the digestive tract of 38.5% of the turtles investigated. Chelonid herpesvirus 5 was detected (for the first time in this basin) in 10.8% of the turtles, which were however free from fibropapillomatosis. Analyses on trace elements showed a time-dependent reduction in the mean concentrations of Cd and Hg, a decrease in the levels of Pb, and a time-dependent increase of As. Particularly, a site-dependent accumulation of As was detected in turtles from the coasts of Calabria. There is a significant correlation between the levels of As in the kidney of Campania turtles and their straight-line carapace length, indicating a size-dependent accumulation of this metal. Overall, besides dangerous debris items, high levels of trace elements may weaken the immune system of Caretta caretta, the most common turtle of the Mediterranean Sea, making it more likely vulnerable to viruses.

Brucella is a Gram-negative facultative intracellular pathogen that causes infection in sheep and goats (B. melitensis.); B. melitensis can also infect other animals. Sheep and goat brucellosis is still present in some regions of Italy, including Campania, and causes considerable economic losses and health threats. The aim of this study was to evaluate the possible risk factors influencing the spread of brucellosis among sheep and goat farms in the Campania region in order to provide the local veterinary services with practical support in evaluating and planning diagnostic, preventive and control interventions. The results of official controls for brucellosis carried out from 2015 to 2020 in the sheep and goat farms of the Campania Region were analyzed. Data were extracted from the National Veterinary Information Systems and the Laboratory Management System of the Experimental Zooprophylactic Institute of Southern Italy. Statistical analysis was carried out through the software R version 4.1.0; the dataset consisted of 37,442 observations, and 9 qualitative and quantitative variables were evaluated on 8487 farms, 248 of which were positive. The association between covariates and the outcome (presence/absence of the disease) was evaluated (Fisher and Wilcoxon tests). A logistic regression model with mixed effects was carried out. This study confirmed that brucellosis in sheep and goats in the Campania region mostly occurs through contact with infected animals imported from other farms (OR = 3.41—IC 95% [1.82–6.41]). Farms with a greater number of animals were seen to be at the greatest risk of infection (OR = 1.04—IC 95% [1.03–1.05]); previous suspension of healthy status also proved to be a risk factor (OR = 55.8—IC 95% [26.7–117]).

Abstract This study aimed to determine the lipidome of water buffalo milk with intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted lipidomic approach. Non-aureus Staphylococci are the most frequently isolated pathogens from dairy water buffalo milk during mastitis. A total of 17 milk samples from quarters affected by NAS-IMI were collected, and the lipidome was determined by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results were compared with the lipidome determined on samples collected from 16 healthy quarters. The study identified 1934 different lipids, which were classified into 15 classes. The abundance of 72 lipids changed in NAS-IMI milk compared to healthy quarters. Significant changes occurred primarily in the class of free fatty acids. The results of this study provided first-time insight into the lipidome of dairy water buffalo milk. Moreover, the present findings provide evidence that NAS-IMI induces changes in water buffalo milk's lipidome.

Abstract Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as “Infectious Bovine Rhinotracheitis” (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1.

Systematic wildlife surveillance is important to aid the prevention of zoonotic infections that jeopardize human health and undermine biodiversity. Toxoplasma gondii is an opportunistic zoonotic protozoan that can infect all endothermic vertebrates, causing severe disease in immunocompromised humans and cases of congenital transmission. Humans can be infected by ingestion of raw meat containing bradyzoites or water contaminated by oocysts. In our study, we assessed the potential circulation of Toxoplasma gondii in wild mammals by performing surveillance in the Campania region (southern Italy) and surveyed its presence from 2020 to 2022 within the framework of the Regional Plans for Wildlife Surveillance. In detail, 211 individuals belonging to five wild mammals (wolf, fox, wild boar, badger, and roe deer) underwent necropsy and the organs were analyzed by real-time PCR for the detection of the parasite. Toxoplasma gondii was found in 21.8% (46/211) of the subjects examined. No statistically significant differences were noticed between the prevalence and the host’s trophic level or age, rejecting the hypotheses that Toxoplasma gondii will have a higher prevalence in top predators and adult individuals, respectively. Our work emphasized the high circulation of Toxoplasma gondii in wildlife and remarked on the critical role of anthropized areas where domestic cats and wildlife may come into contact, urging a systematic surveillance.

The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular population, oocyte and embryo yields in summer and autumn in Podolic cattle. The effects of repeated OPU on cattle wellbeing was also documented. Twenty-six animals were used, and split into two groups, treatment (OPU; n = 18) and control (CG; n = 8). The OPU cattle were subsequently split into two subgroups (n = 9) and underwent repeated OPU, without and with eCG priming, for a total of 8 sessions (4 sessions/season). Follicular population, oocyte and embryo yields were recorded in those sub-groups. CG was handled in the same manner of OPU, except for epidural anaesthesia and follicular aspiration. Biochemical profile, serum protein electrophoresis and haptoglobin levels were analysed in OPU and CG. Hormonal priming increased the number of medium follicles (1.7 ± 0.2 vs 0.9 ± 0.2, p

Neutralizing monoclonal antibodies (mAbs) for pre- and post-exposure prophylaxis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are largely used to prevent the progression of the disease by blocking viral attachment, host cell entry, and infectivity. Sotrovimab, like other available mAbs, has been developed against the receptor binding Domain of the Spike (S) glycoprotein of the virus. Nevertheless, the latest Omicron variant has shown marked mutations within the S gene, thus opening the question of the efficacy of these neutralizing molecules towards this novel variant. In the present observational study, we describe the effects of Sotrovimab in the treatment of 15 fully vaccinated patients, infected by SARS-CoV-2 Omicron sub-variants, who were selected on the basis of factors widely considered to affect a worse prognosis: immune suppression (n = 12) and/or chronic kidney disease (n = 5) with evidence of interstitial pneumonia in nine patients. The effectiveness of Sotrovimab in the treatment of severe cases of COVID-19 was demonstrated by the regression of symptoms (mean 5.7 days), no need of hospitalisation, improvement of general health conditions and viral clearance within 30 days in all patients. In conclusion, although loss or reduction of mAbs neutralizing activity against the Omicron variant have been described, Sotrovimab has clinically proven to be a safe and useful treatment for patients with high risk of progression to severe COVID-19 infected by Omicron sub-variants.

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.

This study aims to investigate the presence of canine distemper virus (CDV) infection in 949 autochthonous or illegally imported dogs from Southern Italy, over a period of eight years (2014–2021). CDV RNA was detected in 6.8% (65/949) of the animals tested, with no detection of CDV in dogs sampled in 2020–2021. The frequency of CDV detection was higher in imported dogs (19/103, 18.3%) with respect to stray (27/365, 7.4%) and household dogs (19/481, 3.9%). On sequence and phylogenetic analyses of selected strains, the analyzed viruses belonged to the Arctic clade, which has already been reported in Italy and in Europe. The results of our study may suggest a reduction of CDV circulation in Southern Italy, while at the same time highlighting the need for strict controls on dog importation, in order to prevent the introduction of viruses from endemic countries.

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency.

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.

One serious concern associated with the SARS-CoV-2 pandemic is that the virus might spill back from humans to wildlife, which would render some animal species reservoirs of the human virus. We assessed the potential circulation of SARS-CoV-2 caused by reverse infection from humans to bats, by performing bat surveillance from different sites in Central–Southern Italy. We restricted our survey to sampling techniques that are minimally invasive and can therefore be broadly applied by non-medical operators such as bat workers. We collected 240 droppings or saliva from 129 bats and tested them using specific and general primers for SARS-CoV-2 and coronaviruses, respectively. All samples (127 nasal swabs and 113 faecal droppings) were negative for SARS-CoV-2, and these results were confirmed by testing the samples with the Droplet Digital PCR. Additionally, pancoronavirus end-point RT-PCR was performed, and no sample showed specific bands. This outcome is a first step towards a better understanding of the reverse transmission of this virus to bats. Although the occurrence of a reverse zoonotic pattern can only be fully established by serological testing, the latter might represent an in-depth follow-up to a broad-scale preliminary assessment performed with our approach. We encourage the systematic surveillance of bats to help prevent reverse zoonotic episodes that would jeopardize human health, as well as biodiversity conservation and management.

Bovine tuberculosis (bTB) is a worldwide zoonosis that affects many species of domestic and wild animals. Mycobaterium bovis is the main cause of infection in water buffalo (Bubalus bubalis) and bovines and is of great concern for human health and for buffalo producers in Italy. The bTB eradication programme is based on slaughterhouse surveillance and intradermal skin tests. Other in vivo diagnostic methods such as the interferon-gamma (IFN-γ) assay have been developed and are widely used in cattle to accelerate the elimination of bTB positive animals. The present study is the first to assess the use and performance of IFN-γ assays, which is used as an ancillary test for bTB diagnosis in water buffalo, and presents the results of a field-evaluation of the assay from 2012 to 2019 during the buffalo bTB eradication programme in Italy. The study involved 489 buffaloes with a positive result to the single intradermal tuberculin test (SITT). The IFN-γ assays and single intradermal comparative tuberculin test were used as confirmation tests. Then, a total of 458 buffaloes, reared on officially tuberculosis-free (OTF) herds, that were confirmed bTB-free for at least the last 6 years were subjected to IFN-γ testing. Furthermore, to evaluate the IFN-γ test in an OTF herd with Paratuberculosis (PTB) infection, 103 buffaloes were subjected to SITT and IFN-γ test simultaneously. Four interpretative criteria were used, and the IFN-γ test showed high levels of accuracy, with sensitivity levels between 75.3% (CI 95% 71.2–79.0%) and 98.4% (CI 95% 96.7–99.4%) and specificity levels between 94.3% (CI 95% 91.2–96.50%) and 98.5% (CI 95% 96.9–99.4%), depending on the criterion used. Finally, in the OTF herd with PTB infection, in buffalo, the IFN-γ test displayed high specificity values according to all 4 interpretative criteria, with specificity levels between 96.7% (CI 95% 88.4–99.5%) and 100% (CI 95% 96.2–100%), while SITT specificity proved unsatisfactory, with a level of 45.3% (CI 95% 35.0–55.7%). Our results showed that the IFN-γ test in the buffalo species could reach high Sensitivity and Specificity values, and that the level of Sensitivity and Specificity could be chosen based on the interpretative criterion and the antigens used depending on the health status of the herd and the epidemiological context of the territory. The IFN-γ test and the use of different interpretative criteria proved to be useful to implement bTB diagnostic strategies in buffalo herds, with the possibility of a flexible use of the assay.

The identification of cross-reactive monoclonal antibodies (mAbs) that recognize orthologous leukocyte differentiation molecules (LDM) in buffaloes has overcome a major impediment limiting research on the immune response to pathogens and development of vaccines. As reported, two pilot trials were conducted to accomplish two objectives: (1) demonstrate that multiparameter flow cytometry can be conducted equally well in buffalo with mAbs directly and indirectly labeled with fluorochromes in research and (2) flow cytometry can be used to compare and extend studies on diseases of economic importance to buffalo using bovine viral diarrhea virus (BVDV) as a model pathogen. Pregnant buffalo cows were infected with BVDV-1 at 81 (trial 1) and 203 (trial 2) days post artificial insemination and flow cytometric evaluations were performed at 0, 3, 4, and 14 days after infection (dpi). Fluorochrome conjugated mAbs were used in trial 1, and fluorochrome conjugated goat isotype specific anti-mouse antibodies were used to label mAbs in trial 2. Flow cytometric analysis revealed a transient lymphopenia occurs during the 1st days following infection similar to lymphopenia reported in cattle. In particular, significant differences were observed between pre- and post-infection absolute values of T lymphocytes (−56%, P < 0.01). CD21+ B lymphocytes (−65%, P = 0.04), and Natural Killer cells (−72%, P < 0.001). No significant differences were observed in monocytes and neutrophil absolute values, or the CD4:CD8 ratio. Animal health status was followed until 15 days after calving. No clinical signs of infection were observed during the evaluation period, however, animals in trial 1 developed complications later the infection. One cow aborted at 57 days post-infection, the second cow developed a prolapse a day after calving and died. These two animals also showed a more pronounced lymphopenia in comparison with animals infected at 203 days of pregnancy (e.g., −77 vs. −22% T lymphocytes at 3 dpi, respectively). The pilot studies have demonstrated that it is possible to use multicolour multiparameter flow cytometry to study the immune response to pathogens affecting the health of buffalo.

This case report describes for the first-time cases of severe gastroenteritis in water buffalo calves due to a new serovar of Salmonella enterica. The study was carried out on fecal matrix collected from live water buffalo calves that showed profuse diarrhea, severe dehydration and fever, exhibiting a systemic course. Culture and molecular investigations identified the pathogens isolated from intestinal contents as two Salmonella serovars, Salmonella enterica enterica O:35 and a new serovar of Salmonella enterica. The isolates showed multi-drug resistance. Timely diagnosis associated with a targeted antimicrobial treatment were found to be sufficient for the survival and recovery of the infected animals. Herd vaccines prepared from isolated pathogens were used to prevent further deaths of the calves.

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.

Listeria monocytogenes is a Gram-positive pathogen causing life-threatening infections both in humans and animals. In livestock farms, it can persist for a long time and primarily causes uterine infections and encephalitis in farmed animals. Whole genome sequencing (WGS) is currently becoming the best method for molecular typing of this pathogen due to its high discriminatory power and efficiency of characterization. This study describes the WGS-based characterization of an L. monocytogenes strain from an aborted water buffalo fetus in southern Italy. The strain under study was classified as molecular serogroup IVb, phylogenetic lineage I, MLST sequence type 6, Clonal Complex 6, and cgMLST type CT3331, sublineage 6. Molecular analysis indicated the presence of 61 virulence genes and 4 antibiotic resistance genes. Phylogenetic analysis, including all the publicly available European L. monocytogenes serogroup IVb isolates, indicated that our strain clusterized with all the other CC6 strains and that different CCs were variably distributed within countries and isolation sources. This study contributes to the current understanding of the genetic diversity of L. monocytogenes from animal sources and highlights how the WGS strategy can provide insights into the pathogenic potential of this microorganism, acting as an important tool for epidemiological studies.

This document describes the development of a tracing system for the buffalo supply chain, namely an online computer system in which farmers, dairies, and brokers must maintain records of the production of milk through to the production of derivatives. The system is jointly used throughout the Italian national territory by the Istituto Zooprofilattico Sperimentale del Mezzogiorno (IZSM) and the Sistema Informativo Agricolo Nazionale Italiano (SIAN), after being made mandatory and regulated with the publication of the Ministerial Decree of 9 September 2014. Farmers are obligated to communicate their daily production of bulk milk, the number of animals milked, the number of the delivery note of the sale, and the name of the purchaser; within the first week of the month, they must communicate the milk production of each animal milked. Dairies are required to communicate the milk and the processed product (mozzarella, yogurt, etc.) purchased on a daily basis. The intermediaries are required to communicate the daily milk purchased, both fresh and frozen, the semi-finished product, and the sale of the same. The tracing system linked to the project authorized by the Ministry of Health, called “Development, validation and verification of the applicability of an IT system to be used for the management of traceability in the buffalo industry”, provides operators with the monitoring of production and sales in real time through alerts and access logs. Currently, there are 1531 registered farmers, 601 non-PDO dairies, 102 PDO dairies, 68 non-PDO intermediaries, and 17 PDO intermediaries in Italy. The system provides support for the recovery of the buffalo sector; from the analysis of the data extrapolated from the tracing system of the buffalo supply chain for the years 2016 to 2019, this paper highlights that the application of the Ministerial Decree No. 9406 of 9 September 2014 and the tracing of the supply chain have increased the price of buffalo milk at barns from EUR 1.37/kg to EUR 1.55/kg from 2016 to 2019.

The microbiota of the gastrointestinal tract (GIT) are crucial for host health and production efficiency in ruminants. Its microbial composition can be influenced by several endogenous and exogenous factors. In the beef and dairy industry, the possibility to manipulate gut microbiota by diet and management can have important health and economic implications. The aims of this study were to characterize the different GIT site microbiota in water buffalo and evaluate the influence of diet on GIT microbiota in this animal species. We characterized and compared the microbiota of the rumen, large intestine and feces of water buffaloes fed two different diets with different non-structural carbohydrates/crude proteins (NSC/CP) ratios. Our results indicated that Bacteroidetes, Firmicutes and Proteobacteria were the most abundant phyla in all the GIT sites, with significant differences in microbiota composition between body sites both within and between groups. This result was particularly evident in the large intestine, where beta diversity analysis displayed clear clustering of samples depending on the diet. Moreover, we found a difference in diet digestibility linked to microbiota modification at the GIT level conditioned by NSC/CP levels. Diet strongly influences GIT microbiota and can therefore modulate specific GIT microorganisms able to affect the health status and performance efficiency of adult animals.

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.

Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

The aim of this study was to define the microbiota of water buffalo milk during sub-clinical and clinical mastitis, as compared to healthy status, by using high-throughput sequencing of the 16S rRNA gene. A total of 137 quarter samples were included in the experimental design: 27 samples derived from healthy, culture negative quarters, with a Somatic Cell Count (SCC) of less than 200,000 cells/ml; 27 samples from quarters with clinical mastitis; 83 samples were collected from quarters with subclinical mastitis, with a SCC number greater of 200,000 cells/ml and/or culture positive for udder pathogens, without clinical signs of mastitis. Bacterial DNA was purified and the 16S rRNA genes were individually amplified and sequenced. Significant differences were found in milk samples from healthy quarters and those with sub-clinical and clinical mastitis. The microbiota diversity of milk from healthy quarters was richer as compared to samples with sub-clinical mastitis, whose microbiota diversity was in turn richer as compared to those from clinical mastitis. The core microbiota of water buffalo milk, defined as the asset of microorganisms shared by all healthy milk samples, includes 15 genera, namely Micrococcus, Propionibacterium, 5-7N15, Solibacillus, Staphylococcus, Aerococcus, Facklamia, Trichococcus, Turicibacter, 02d06, SMB53, Clostridium, Acinetobacter, Psychrobacter and Pseudomonas. Only two genera (Acinetobacter and Pseudomonas) were present in all the samples from sub-clinical mastitis, and no genus was shared across all in clinical mastitis milk samples. The presence of mastitis was found to be related to the change in the relative abundance of genera, such as Psychrobacter, whose relative abundance decreased from 16.26% in the milk samples from healthy quarters to 3.2% in clinical mastitis. Other genera, such as SMB53 and Solibacillus, were decreased as well. Discriminant analysis presents the evidence that the microbial community of healthy and clinical mastitis could be discriminated on the background of their microbiota profiles.

Nel presente lavoro viene riportato il risultato del rilevamento degli spiaggiamenti e dei rinvenimenti in mare di Cetacei avvenuti negli anni 2006 - 2011 lungo le coste della Campania. I dati sono stati raccolti in base ad una collaborazione scientifica tra l’Università degli Studi di Napoli Federico II, l’Istituto Zooprofilattico Sperimentale del Mezzogiorno, il Centro Studi Cetacei e il Centro Studi Ecosistemi Marini, collaborazione, che nel 2008 è stata ratificata da un protocollo di intesa finalizzato agli interventi sui Cetacei spiaggiati lungo le coste della Campania. Nel 2009 la Giunta Regionale della Campania, su richiesta del Settore Veterinario dell’Assessorato alla Sanità, ha decretato la “Costituzione di un Comitato di Coordinamento per l’attuazione del protocollo di intesa per gli interventi in caso di Cetacei e tartarughe spiaggiate” formato dai rappresentanti dei citati enti, delle AA.SS.LL. costiere, della Direzione Marittima di Napoli e dell’A.R.P.A.C. al fine di approvare le linee guida relative alla procedura operativa da adottarsi in caso di intervento per il recupero di carcasse di Cetacei spiaggiati. In totale sono stati raccolti i dati riguardanti 73 esemplari di almeno 6 specie, così suddivisi: 1 Balaenoptera acutorostrata; 4 Balaenoptera physalus; 2 Grampus griseus; 1 Delphinus delphis; 9 Tursiops truncatus; 40 Stenella coeruleoalba; 10 Delfinidi indeterminati e 6 Cetacei indeterminati. Si riporta anche il primo caso di mal rossino diagnosticato in un esemplare di Stenella striata mai segnalato prima per le coste dell’Italia continentale.