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Bovine herpesvirus 1 (BoHV-1) can provoke conjunctivitis, abortions and shipping fever. BoHV-1 infection can also cause immunosuppression and increased susceptibility to secondary bacterial infections, leading to pneumonia and occasionally to death. Herein, we investigated the influence of MG-132, a proteasome inhibitor, on BoHV-1 infection in bovine kidney (MDBK) cells. Infection of MDBK cells with BoHV-1 induces apoptotic cell death that enhances virus release. Whereas, MG-132 inhibited virus-induced apoptosis and stimulated autophagy. Protein expression of viral infected cell protein 0 (bICP0), which is constitutively expressed during infection and is able to stimulate Nuclear factor kappa B (NF-κB), was completely inhibited by MG-132. These results were accompanied by a significant delay in the NF-κB activation. Interestingly, the efficient virus release provoked by BoHV-1-induced apoptosis was significantly reduced by MG-132. Overall, this study suggests that MG-132, through the activation of autophagy, may limit BoHV-1 replication during productive infection, by providing an antiviral defense mechanism.

Introduction Bovine herpesvirus 1 (BoHV1) andbubaline herpesvirus 1 (BuHV1) are ruminant alphaherpesviruses.BoHV-1 can provoke infectious bovine rhinotracheitis (IBR), genital disorders, conjunctivitis, abortions, encephalitis or immune suppression which may lead to secondary bacterial infections and cause pneumonia. Hence, BoHV-1 infection mightcause substantial economic loss and trade restrictions in cattle industry.IBR control and eradication programshave been implemented in many Europeancountries.Recent serological surveys described a prevalence of antibodies against BuHV-1 in water buffalo farms in Campania Region. Objectives The aim of this study was to compare the results of two different serological surveys, performed respectively in 2009 and in 2016, to evaluate the prevalence of BoHV-1 and BuHV-1 infections in water buffalo farms in the Province of Caserta, Campania Region (Italy), whereIBR control programs wererecently implemented. Animals and methods In both surveys, serum samples were collected from buffalo farms (precisely,862 animals/28 farms in 2009 and 309/16 farms in 2016).To detect antibodies for IBR,two commercial ELISA kitswere used, and,precisely, in 2009 IBR-gB and IBR-gE (IDEXX), in 2016 Eradikit Discrimination BoHV-1/BuHV-1 (IN3 diagnostic). Both tests provide the most correct diagnosis on serum samples (Nogarol et al., 2014;Tignon et al., 2017). Results and Discussion In 2009, we detected a seroprevalence of BuHV-1 (49,55%) vs. BoHV-1 (23,06%).In 2016, 57,60% of buffaloes were positive to BuHV-1 vs. BoHV-1 (1,62%), according with another survey carried out in the same Region (Caruso et al., 2016). Overall, we detected a time-dependent increase in BuHV-1 prevalence and a significantdecrease in BoHV-1 infection rates between the two surveys.To explain the difference in the decrease of BoHV-1, we suppose thata possible explanation could be in the existence of antigenic cross-reactionsbetween these viruses and in their abilityto crossthe speciesbarrier. Indeed,both BoHV-1 and BuHV-1 have been reported to cross the species barrier (Thiry et al., 2006). Conclusion Currently, the high percentage of sera reactive to BuHV-1 (57,60%) indicates that BuHV-1may be the main circulating alphaherpesvirus infection in Mediterranean water buffalo.

The distribution pattern of calbindin (CB)-D28k-expressing neurons results to be altered in several brain regions of chronic morphine exposed adult mice. In this study, the influence of chronic maternal exposure to morphine on the distribution pattern of CB-D28k-expressing neurons in the brain of mouse offspring was investigated. Females of CD-1 mice were daily administered with saline or morphine for 7 days before mating, during the whole gestation period, and until 21 day post-partum. Their offspring were sacrificed on postnatal day 18, and the brains were examined by histology using cresyl violet and by immunohistochemistry using a rabbit polyclonal anti-CB-D28k antibody. Histology revealed no significant differences in the distribution pattern and the number of neurons between the offspring forebrain of the control group of mice and the two groups of mice treated with different doses of morphine. However, immunohistochemical analysis revealed that the number of CB-D28k-immunoreactive neurons remarkably decreased in the cingulate cortex, in the layers II-IV of the parietal cortex and in all regions of the hippocampus, while it increased in the layers V-VI of the parietal cortex and in the subicular region of the offspring brain of morphine treated mice. Overall, our findings demonstrate that maternal exposure to morphine alters the pattern of CB-D28k-expressing neuron pattern in specific regions of murine developing brain, in a layer- and dose-dependent way, thus suggesting that these alterations might represent a mechanism by which morphine modifies the functional aspects of developing brain.

Introduction: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly known as dioxin, is a toxic and persistent environmental contaminant which induces immune suppression and increased susceptibility to infectious agents. Bovine herpesvirus 1 (BHV-1), a cattle pathogen, can provoke infectious bovine rhinotracheitis (IBR), genital infections, conjunctivitis, abortions, encephalitis or other fatal diseases in newborns. BHV-1 induced immune suppression which may lead to secondary bacterial infections that can cause pneumonia. Hence, BHV- 1 infection might cause substantial economic loss in the cattle industry. Following BHV-1 infection in bovine cells (MDBK), we previously showed that TCDD influences the infection by increasing virus replication. Moreover, TCDD anticipates BHV-1- induced apoptosis, by accelerating the down-regulation of telomerase activity when virus-induced apoptosis occurred, and by anticipating in the cytoplasm the presence of bICP0, the main transcriptional regulatory protein of BHV-1. Furthermore, TCDD enhances the free intracellular iron availability, which might promote the onset of BHV-1 infection and render bovine cells more vulnerable to the virus. Objectives: Herein, we performed an epidemiological analysis on prevalence of IBR in some areas where high levels of TCDD have been detected in dairy products. Materials and Methods: We collected serum and plasma samples to detect antibodies for IBR from cattle raised on farms in Campania Region (Italy), by using IBR-gB and IBR-gE E.L.I.S.A. kit, which represents the test procedure of choice in many European IBR programs. Results: We revealed a significant prevalence of IBR on samples collected from farms in contaminated areas compared to uncontaminated areas. Discussion and Conclusions: Very low doses of TCDD cause the damage previously showed in bovine cells. In addition, herein we provided evidence that TCDD influences BHV-1 infection, promoting the prevalence of IBR in cattle. Thus, TCDD may act as an additional risk factor for progression of BHV-1 infection in cattle. We suggest that this risk should be given adequate consideration in the care of farm animal health.

Introduction: Staphylococcus intermedius (S. intermedius) is a coagulase positive zoonotic staphylococcus found in several domestic animals. It is a common commensal of oral, nasal and skin flora in healthy dogs, where it can also cause invasive diseases. Studies on genotypic characterization of antibiotic resistance have shown that the distribution of the antibiotic resistance genes seems to be vary among staphylococci of different animal origin. Materials and Methods: The specimens were collected from diseased dogs which attended the Microbiology Laboratory of the Department of Veterinary Medicine and Animal Production of the University of Naples Federico II. The swabs from different origins were inoculated onto Mannitol Salt Agar (MSA) and identified by automated VITEK 2 (bioMérieux®). The isolated strains were tested for their susceptibility to 14 antibiotics by using Kirby-Bauer disk diffusion test. Multiplex PCR were performed to determine genetic profiles of antibiotic resistance. Results: We isolated 25 strains of S. intermedius. Phenotypically penicillin resistance was present in 18 bacterial strains; however, only 4 of these isolated strains carried the mecA gene. The resistance rates for tetracycline and erythromycin were 60 and 56 per cent, respectively. Tetracycline resistance genes tet(K) and tet(M) were found positive in 15 isolates which were phenotypically tetracycline-resistant. Erythromycin resistance gene erm(B) was found positive in 14 isolates which were phenotypically erythromycin-resistant. In particular, nine of the tetracycline-resistant strains carried only tet(K) gene, while six carried both tet(K) and tet(M) genes. All the erythromycin- resistant isolates had erm(B) gene. Discussion and Conclusions: We noticed 56 per cent multidrug resistant S. intermedius strains. Moreover, our results show that the phenotypic and genotypic characterization of methicillin resistance are not correlated and so further studies are needed, whereas the phenotypic and genotypic characterization of tetracycline and erythromycin resistance are well correlated. The distribution and the prevalence of the antibiotic resistance genes among Staphylococcus spp. suggest that effective measures should be taken to control antibiotic use in pet animals.

RIASSUNTO Durante il periodo dicembre 2014 e giugno 2015 è stato condotto uno studio di prevalenza, nella specie bufalina, per conoscere le caratteristiche epidemiologiche di herpesvirus 1 in Campania (distretto di Caserta). In modo particolare è stata valutata la prevalenza di Bovine Herpesvirus type 1 (BoHV-1) e Bubaline Herpesvirus type 1 (BuHV-1). Sono state campionate aziende (ad indirizzo produttivo latte) dove non era stato vietato l’accesso per ragioni sanitarie (brucellosi, tubercolosi, etc.) e dove i proprietari hanno dato il consenso informato a poter eseguire prelievi di sangue, tamponi nasali e vaginali sui capi. Sono stati processati 217 sieri appartenenti a 16 diverse aziende casertane. I risultati ai test sierologici effettuati mediante IBR gB Ab test e IBR gE Ab test ELISA (IDEXX), comunemente in uso, hanno sottolineato una netta diffusione di BoHV-1 (85,25%) rispetto a BuHV-1 (11,52%). I valori ottenuti, pur affetti da un certo grado di bias dovuto ai metodi di campionamento adottati, sono stati comunque indicativi della circolazione di questi due virus erpetici nella specie presa in esame. INTRODUZIONE Il bufalo mediterraneo (Bubalus bubalis), chiamato dagli anglosassoni water buffalo, è l’ospite primario e reservoir di Bubaline Herpesvirus type 1 (BuHV-1), patogeno originariamente isolato in Australia dal prepuzio di un maschio bufalino (15) e più recentemente nel sud Italia dopo riattivazione farmacologica in una bufala (2). Nella specie bufalina è noto che circola anche Bovine Herpesvirus type 1 (BoHV-1) (9,2,13,14). Il BuHV-1 è un virus antigeneticamente e geneticamente correlato al BoHV-1. L’analisi filogenetica dei due li classifica come virus diversi, ma strettamente omologhi (7,3); sequenze di virus specifiche sono state messe in evidenza anche da feti abortiti (1). BuHV-1 e BoHV-1 sono responsabili di patologie altamente contagiose come la Rinotracheite Infettiva Bovina (IBR), la Vulvovaginite Pustolosa (IPV), la Balanopostite (IPB), trasmissibili rispettivamente per via aerogena e venerea, benchè il contagio avvenga anche in modo indiretto per via diaplacentare e per contatto con secrezioni infette. Entrambi i virus sono capaci di stabilire latenza nei gangli craniali (in particolare nei gangli del trigemino) e/o sacrali, questo consente loro di persistere nel soggetto infetto per tempi indefiniti (5,12,16,17,8,4). Ciò assume enorme rilevanza sul piano della pato¬logia e dell'epidemiologia in quanto, a seguito di stress endogeni o esogeni, si verifica riattivazione dell'infe¬zione latente con conseguente disseminazio¬ne dei virus nell'ambiente, anche in forma silente. I due patogeni inducono sia danni sanitari diretti (malattia clinica o subclinica) che indiretti (economici: ipofertilità/infertilità, calo della produzione lattea, aborto, encefaliti, spese veterinarie, etc.). In Italia, all’Anagrafe Nazionale Zootecnica (dati 31/05/15) si contano 376.705 capi bufalini, di cui oltre il 90% è concentrato nelle regioni di centro-sud, prevalentemente in Campania (278.721 capi). La domanda di latte bufalino per la produzione di formaggi (mozzarella, ricotta, etc.) è in costante ascesa e il latte di bufala non è soggetto a limitazioni o quote di produzione previste invece dalla CE per il latte bovino. Considerando che la presenza di IBR nel nostro territorio può avere implicazioni negative nell’ambito della commercializzazione e movimentazione degli animali sieropositivi tra una zona e l’altra, e ancor di più in ambito internazionale (11,3) (diversi Paesi hanno attuato piani di eradicazione ed esigono un certificato rilasciato da Enti autorizzati - IIZZSS - che attesti la sieronegatività a IBR), si rende necessaria la conoscenza della presenza di herpesvirus negli allevamenti bufalini. Inoltre la sieronegatività a IBR è richiesta soprattutto per i tori destinati ai centri di riproduzione (si ha una riduzione del numero di stalle dalle quali è possibile prelevare soggetti, con un’evidente rischio di forte riduzione della variabilità e del miglioramento genetico) e quindi bisogna adottare misure di routine per il controllo dell’infezione. In molti Paesi europei (eccezione fatta per Svizzera, Austria, Svezia, Norvegia, Finlandia, Danimarca, alcune aree della Baviera considerate indenni IBR) e in alcune regioni italiane (Trentino, Lombardia, Piemonte, Veneto, Friuli, Lazio, Campania; eccetto Bolzano e Trento reputate indenni IBR dall’UE) esistono già specifici piani di risanamento/eradicazione della malattia su base obbligatoria o volontaria (11) che sono indirizzati in modo particolare al comparto bovino, e non sempre estesi a quello bufalino. Indagini epidemiologiche, attualmente disponibili, condotte su alcuni territori italiani, confermano la diffusione di questi virus nei bufali (13,3), ma si tratta ancora di dati frammentari. Ciò rende importante quantificare il reale impatto economico che la malattia determina in una popolazione infetta, chiarire il ruolo patogeno che gli herpesvirus rivestono, valutare la funzione di serbatoio e di diffusore che il bufalo rappresenta. Obiettivo del presente studio di prevalenza è stato quello di effettuare una valutazione preliminare sulla diffusione di BoHV-1 e BuHV-1 nel comparto bufalino casertano al fine di poter disporre, in un futuro prossimo, di un quadro epidemiologico più completo anche per i bufali che permetterà la messa a punto di misure di eradicazione più efficaci, e attiverà un programma di sorveglianza nella regione Campania che comprenda anche questa specie. MATERIALI E METODI L'indagine è stata condotta in un periodo di 6 mesi (compreso tra dicembre 2014 e giugno 2015) in una specifica area del territorio: la provincia di Caserta. Tale scelta è stata operata considerando che la realtà zootecnica casertana (188.779 capi bufalini; dati BDN al 31/05/15) è la più rappresentativa della regione per l’alta densità di bufale da latte, per la produzione di un latte di altissima qualità da indirizzare alla caseificazione, per la presenza di allevamenti costituiti da un numero cospicuo di capi. Nelle aziende oggetto di studio è stato effettuato un campionamento di convenienza dopo il consenso dei proprietari. I campioni di sangue e i tamponi nasali e vaginali sono stati effettuati nel rispetto delle normative vigenti che regolano il benessere animale. I sieri collezionati sono stati complessivamente n.217 provenienti da n.16 allevamenti. I capi campionati avevano un‘età compresa tra i 6 mesi e i 18 anni. I 217 sieri sono stati processati utilizzando in serie due test competitivi, IBR gB Ab e IBR gE Ab ELISA (IDEXX). Lo status dell’infezione veniva definito come segue: gB-pos/gE-pos animale positivo a BoHV-1, gB-pos/gE-neg animale positivo a BuHV-1, gB-neg/gE-neg animale negativo a IBR (13). Le analisi sierologiche per la ricerca degli anticorpi anti-IBR sono state effettuate con un kit del commercio dotato di una specificità del 99% e una sensibilità del 99% (IDEXX kit). I tamponi saranno esaminati con prove di isolamento per la ricerca di herpesvirus 1 in uno studio seguente. A conferma dell’isolamento virale saranno effettuate prove di biologia molecolare. RISULTATI E DISCUSSIONE Le analisi sierologiche hanno fornito i seguenti risultati preliminari (TABELLA 1), da cui si evince che la prevalenza degli herpesvirus 1 tra le diverse aziende casertane esaminate è del 96,77%, e più interessante sottolineare come il patogeno BoHV-1 sia largamente più diffuso (85,25%) di quello bufalino, BuHV-1 (11,52%). Tali percentuali contraddicono studi effettuati precedentemente (13,10,3) dove invece il BuHV-1 era maggiormente rappresentativo (rispettivamente 42% nel centro Italia, 50% nelle Marche, 53% in Campania). Questi risultati contrastanti possono dipendere dalla modalità di campionamento eseguita: nel nostro caso non probabilistica e negli altri tre studi randomizzata, ma anche dai kit sierologici impiegati: nella nostra ricerca due ELISA test attualmente in commercio messi a punto per il BoHV-1, e per contro un ELISA test ideato specificamente per differenziare il BuHV-1 e il BoHV-1 nei bufali (6). Altra motivazione potrebbe essere che in 4 dei 16 allevamenti esaminati, a pochi metri di distanza dai bufali erano stabulati anche vacche e tori da latte positivi a IBR.

Introduction: In recent years, oxidative stress has been postulated to be an important factor in pathogenesis and development of diseases. Reactive Oxygen Species (ROS) are well known for being both beneficial and deleterious and have long been known to be a component of the killing response of immune cells to microbial invasion. Measuring the free radical activity in serum samples is the best way to investigate the association between oxidative stress and infectious diseases. The aim of this study is to determine the association between alphaherpesvirus infection and serum reactive oxygen metabolites (ROM) in Italian Mediterranean buffaloes (Bubalus bubalis). Materials and Methods: The presence of antibodies against bovine herpesvirus type 1 (BoHV-1) or bubaline herpesvirus type 1 (BuHV-1) was investigated by enzyme-linked immunosorbent assay (anti- gB/gE blocking ELISA) (Idexx). We carried out the derivatives of ROM (d-ROM) and the antioxidant activity (anti-ROM) tests by spectrophotometry in sera of BoHV-1 (A) or BuHV-1 (B) infected animals compared to seronegative animals (C). Results: The concentrations of d-ROM in the three groups, expressed in Carratelli Units (U-CARR) were, respectively, 64.6 ± 11.6, 85.0 ± 10.7 and 56.2 ± 10.5. Although the serum d-ROM levels in group A and B were higher than that in group C, only the group B compared with the control group C was significant at p < 0.05. At anti-ROM test, the three groups of animals showed values respectively of: 287.5 ± 66.1, 318 ± 64.3 and 276 ± 53.0. The results of group A and B compared to that of control group C were not significant. The oxidative stress index (OSI), calculated as d-ROM/anti-ROM x 100, showed significant differences between the groups B (26) and C (20) whereas no significant difference was detected between the groups A (22) and C (20). Discussion and Conclusions: Our data indicate that in alphaherpesvirus infected buffaloes, in particular those in BuHV-1 infected, the d-ROM and the OSI values were significantly increased. These results suggest that the presence of BuHV-1, the virus species-specific, seems to induce a worsening balance in ROM levels, whereas that of BoHV-1 has no substantial effects. Future studies are needed to assess the role of oxidative stress in Bubalus bubalis infected by alphaherpesvirus.

Introduction: 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) influences infection of bovine cells (MDBK) with Bovine Herpesvirus 1 (BHV- 1) by increasing virus replication (Fiorito et al., 2008a) and anticipating BHV-1-induced apoptosis (Fiorito et al., 2008b). Moreover, TCDD accelerates the down-regulation of telomerase activity when virus-induced apoptosis occurred (Fiorito et al., 2014), anticipates the presence of bICP0 in the cytoplasm (Fiorito et al., 2010), and enhances the free intracellular iron availability (Fiorito et al., 2013). Several viruses control key cellular signaling pathways to facilitate the infection. For example, BHV-1 increases p53 levels during infection (Devireddy and Jones, 1999), and activates both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signalregulated kinase 1/2 (MAPK/Erk1/2) signaling pathways (Zhu et al., 2011). Here to elucidate the influence of TCDD on BHV-1-infection we examined Akt/mTOR, ERK pathways and their downstream effectors including FOXO3, a transcription factor involved in promoting virus-induced apoptosis, as in canine coronavirus type II infection (Marfè et al., 2011).

Bovine herpesvirus 1 (BHV-1) is a cattle pathogen that can provoke infectoius bovine rhinotracheitis (IBR), conjunctivitis, abortions and shipping fever. 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contamination, which suppresses immune responses and decreases host resistance to infectious disease. Following BHV-1 infection in bovine kidney (MDBK) cells we showed that TCDD increases BHV-1 replication, accelerates BHV-1-induced apoptosis as well as, significantly decreased telomerase activity when virus-induced apoptosis occurred. It is known that TCDD acts on the cells through aryl hydrocarbon receptor (AhR). In our recent study in MDBK clls we demonstrated a dose-dependent overxpression of AhR in the presence of TCDD. Moreover, in the presence of TCDD, we detected an increase in the levels of both gene and protein expression of the immediate-early BHV-1 infected cell protein 0 (bICP0), which regulates all the three phases of viral replication by acting as a strong activator or as a repressor of specific viral promoters. Since, BHV-1 is a DNA virus which requires an iron-replete host for efficient replication, we investigated the role of TCDD in virus infection and demonstrated that TCDD increases the free intracellular iron availability. This might promote the onset of BHV-1 infection and render bovine cells more vulnerable to the virus. These results suggest that TCDD may act as an additional risk factor for progression of BHV-1 infection in cattle.

In nature, herpesviruses are mostly associated with a single host species. Italian Mediterranean buffaloes (Bubalus bubalis) is associated with Bubaline herpesvirus 1 (BuHV-1) but also with Bovine herpesvirus 1 (BoHV-1). Like different alphaherpesvirinae subfamily members, the primary site for BuHV-1 or BoHV-1 latency is sensory neurons within trigeminal ganglia (TG). Recently, it has been shown that cattle infected by BoHV-1 seemed to have more oxidative stress and low antioxidant defense (Durgut et al., 2013). Thus, to better understand the physiopathology of BuHV-1 or BoHV-1 infection in Bubalus bubalis, we collected blood through iugulation and TG after decapitation. Samples from each TG were placed in a single 50-ml conical tube, and the tube were placed in a dry ice ethanol bath. TG samples were then stored at -80°C. Animals submitted for sampling were chosen within the > 5 year age category because considered at higher risk of infection. The presence of antibodies against BuHV-1 or BoHV-1 was investigated in 15 blood samples by enzyme-linked immunosorbent assay (anti-gB/gE blocking ELISA) (Idexx). Through the combined use of gB-gE ELISA tests, we assigned a specific infection status, for the BuHV1 infection status (gB-pos/gE-neg), and for the BoHV1 status (gBpos/gE-pos) as reported by Scicluna et al. (2007). The ELISA results showed that 40% of tested samples was positive at BoHV-1 whereas 33.3% resulted to be BuHV-1 positive. Moreover 26.7% were BoHV-1 and BuHV-1 negative (gB-neg/gE-neg). Furthermore, we examined trigeminal ganglia tissues of the 15 animals. Oxidative status was assessed using the Reactive Oxygen Metabolites-derived compounds (d-ROMs) test and the antioxidant activity (anti-ROMs) test through spectrophotometric procedures. TG of seropositive or seronegative buffaloes were also used to measure the concentration of lipid peroxides using the Lipotiss test. We detected in TG of seropositive BuHV-1 animals a significantly reduction of d-ROMs values (P < 0.001) as well as anti-ROMs values (P < 0.001) compared with those in TG of seronegative animals. Whereas TG of seropositive BoHV-1 animals had significantly higher d-ROMs values (P < 0.001) and lower anti-ROMs values (P < 0.001) compared with those in TG of seronegative animals. Furthermore, the results of lipotiss test showed that samples of both seropositive BuHV-1 and BoHV-1 animals were significantly lower (P