Evidence and antibiotic resistance profiles of clinical Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) and non-ACB complex members in companion animals: A 2020-2022 retrospective study.

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020-2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles.

Fungal metabolite 6-pentyl-α-pyrone reduces canine coronavirus infection.

Canine coronavirus (CCoV) can produce a self-limited enteric disease in dogs but, because of notable biological plasticity of coronaviruses (CoVs), numerous mutations as well as recombination events happen leading to the emergence of variants often more dangerous for both animals and humans. Indeed, the emergence of new canine-feline recombinant alphacoronaviruses, recently isolated from humans, highlight the cross-species transmission potential of CoVs. Consequently, new effective antiviral agents are required to treat CoV infections. Among the candidates for the development of drugs against CoVs infection, fungal secondary metabolites (SMs) represent an important source to investigate. Herein, antiviral ability of 6-pentyl-α-pyrone (6 PP), a SM obtained by Trichoderma atroviride, was assessed against CCoV. During in vitro infection, nontoxic concentration of 6 PP significantly increased cell viability, reduced morphological signs of cell death, and inhibited viral replication of CCoV. In addition, we found a noticeable lessening in the expression of aryl hydrocarbon receptor (AhR), a strategic modulator of CoVs infection. Overall, due to the variety of their chemical and biological properties, fungal SMs can decrease the replication of CoVs, thus identifying a suitable in vitro model to screen for potential drugs against CoVs, using a reference strain of CCoV (S/378), non-pathogenic for humans.

Antiviral activity of Taurisolo® during bovine alphaherpesvirus 1 infection.

Bovine alphaherpesvirus 1 (BoAHV-1), the pathogen causing Infectious Bovine Rhinotracheitis (IBR) and predisposing to polymicrobial infections in cattle, provokes farm economic losses and trading restrictions in the world. However, nontoxic antiviral agents for BoAHV-1 infection are still unavailable, but plant extracts, such as flavonoid derivatives possess activity against BoAHV-1. Taurisolo®, a nutraceutical produced by Aglianico grape pomace, has recently shown promising antiviral activity. Herein, the potential activity of Taurisolo® during BoAHV-1 infection in Madin Darby bovine kidney (MDBK) cells was tested. Taurisolo® enhanced cell viability and reduced morphological death signs in BoAHV-1-infected cells. Moreover, Taurisolo® influenced the expression of bICP0, the key regulatory protein of BoAHV-1, and it strongly diminished virus yield. These effects were associated with an up-regulation of aryl hydrocarbon receptor (AhR), a transcription factor involved in microbial metabolism and immune response. In conclusion, our findings indicate that Taurisolo® may represent a potential antiviral agent against BoAHV-1 infection. Noteworthy, AhR could be involved in the observed effects and become a new target in antiviral therapy.

In Vitro Evaluation of Antiviral Activities of Funicone-like Compounds Vermistatin and Penisimplicissin against Canine Coronavirus Infection.

Recent studies have demonstrated that 3-O-methylfunicone (OMF), a fungal secondary metabolite from Talaromyces pinophilus belonging to the class of funicone-like compounds, has antiviral activity against canine coronaviruses (CCoV), which causes enteritis in dogs. Herein, we selected two additional funicone-like compounds named vermistatin (VER) and penisimplicissin (PS) and investigated their inhibitory activity towards CCoV infection. Thus, both compounds have been tested for their cytotoxicity and for antiviral activity against CCoV in A72 cells, a fibrosarcoma cell line suitable for investigating CCoV. Our findings showed an increase in cell viability, with an improvement of morphological features in CCoV-infected cells at the non-toxic doses of 1 µM for VER and 0.5 µM for PS. In addition, we observed that these compounds caused a strong inhibition in the expression of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which is activated during CCoV infection. Our results also showed the alkalinization of lysosomes in the presence of VER or PS, which may be involved in the observed antiviral activities.

First serological evidence of SARS-CoV-2 natural infection in small ruminants : Brief report.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late December 2019 and spread worldwide, quickly becoming a pandemic. This zoonotic coronavirus shows a broad host range, including wildlife and domestic animals. Small ruminants are shown to be susceptible to SARS-CoV-2 but, to date, no natural infection has been reported. Herein, we performed a survey for SARS-CoV-2 among sheep and goats in the Campania region of Italy using an indirect multispecies ELISA. Next, positive sera were submitted to virus serum neutralization for the quantification of specific neutralizing antibodies. Out of 612 sheep and goats, 23 were found ELISA positive (3.75%) and 1 of them showed 1:20 neutralizing antibodies titer. No significant difference was found between the two species, as well as between male and female, geographical location and age. Our findings demonstrate that natural infection can occur in flocks in a field situation. Moreover, low susceptibility to SARS-CoV-2 is reported for sheep and goats, nevertheless, the continuous mutations of this virus open new scenarios on viral host range and tropism, highlighting the importance of investigating animal species that could represent ongoing or future possible hosts.

Effectiveness of the Fungal Metabolite 3-O-Methylfunicone towards Canine Coronavirus in a Canine Fibrosarcoma Cell Line (A72).

Canine coronavirus (CCoV), an alphacoronavirus, may cause self-limiting enteric disease in dogs, especially in puppies. The noteworthy plasticity of coronaviruses (CoVs) occurs through mutation and recombination processes, which sometimes generate new dangerous variants. The ongoing SARS-CoV-2 pandemic and the isolation of a novel canine-feline recombinant alphacoronavirus from humans emphasizes the cross-species transmission ability of CoVs. In this context, exploring antiviral compounds is essential to find new tools for fighting against CoVs infections. Fungi produce secondary metabolites, which are often developed as antibiotics, fungicides, hormones, and plant growth regulators. Previous examinations of benzo-γ-pyrone 3-O-methylfunicone (OMF), obtained from Talaromyces pinophilus, showed that it reduces the infectivity of hepatitis C virus and bovine herpesvirus 1. Based on this evidence, this study evaluated the antiviral ability of OMF against CCoV infection in a canine fibrosarcoma (A72) cell line. During CCoV infection, a non-toxic dose of OMF markedly increased features of cell viability. Moreover, OMF induced a significant reduction in virus yield in the presence of an intense downregulation of the viral nucleocapsid protein (NP). These findings occurred in the presence of a marked reduction in the aryl hydrocarbon receptor (AhR) expression. Taken together, preliminary findings suggest that OMF inhibiting AhR shows promising activity against CCoV infection.

Canine Coronavirus Activates Aryl Hydrocarbon Receptor during In Vitro Infection.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that interacts with substrates, including microbial metabolites. Recent advances reveal that AhR is involved in the host response to coronaviruses (CoVs) infection. Particularly, AhR antagonists decrease the expression of angiotensin-converting enzyme 2 (ACE2) via AhR up-regulation, resulting in suppression of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in mammalian cells. Herein, we report that AhR is expressed in canine fibrosarcoma (A72) cells, where it is considerably activated by infection with genotype II of canine coronavirus (CCoV-II). The pharmacological inhibition of AhR, by CH223191, suppressed cell death signs and increased cell viability. Furthermore, the AhR antagonist induced a meaningful decline in virus yield, accompanied by the inhibition of the expression of viral nuclear protein (NP). Fascinatingly, during CCoV infection, a novel co-expression of NP and AhR expression was found. Taken together, our preliminary findings show that infection with CCoV activates AhR, and pharmacologic AhR inhibition reduces CCoV replication, identifying AhR as a possible candidate target for CCoV antiviral therapy.

Bioaccumulation of PCDD/Fs and PCBs in free-range hens: Congener fingerprints and biotransfer factors.

The bioaccumulation of lipophilic environmental contaminants in farm animals is an important issue to control and prevent human exposure to toxic pollutants. Free-range hens were used as a model to assess the transfer and the bioaccumulation of PCDD/Fs, DL-PCBs and NDL-PCBs from the soil to eggs in an area in the Campania region particularly affected by pollution, the "Land of Fires". We determined the following ranges of concentrations in eggs: 0.90-5.51 pg WHO-TEQ g-1 fat for PCDD/Fs; 1.63-4.24 pg WHO-TEQ g-1 fat for DL-PCBs; 2.77-9.75 pg WHO-TEQ g-1 fat for the sum of PCDD/Fs and DL-PCBs and 12.53-30.94 ng g-1 fat for NDL-PCBs. Higher contamination levels were observed in the eggs from free-range hens, compared to those of the control group, that is hens raised indoors; this indicates that soil remains a major source of contamination. Livers showed low levels of contamination for both outdoor and indoor reared hens. Biotransfer factors (BTFs) were calculated for each of the 35 PCDD/F and PCB congeners analysed. The BTFs highlighted that the transfer and bioaccumulation depend on chlorination degree and substitution pattern, in particular they increase with increasing chlorination degree up to the hexa-chlorinated congeners and then decrease. An exception to these finding was observed for 2,3,7,8 TCDD and 2,3,7,8 TCDF, which showed very high BTFs. These results demonstrate the importance of promoting studies on the environmental contaminants bioaccumulation as they provide an effective support for the risk assessment and management of exposure to toxic chemicals.

First Description of Serological Evidence for SARS-CoV-2 in Lactating Cows.

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.

Antiviral Property of the Fungal Metabolite 3-O-Methylfunicone in Bovine Herpesvirus 1 Infection.

Bovine herpesvirus type-1 (BoHV-1) is a widespread pathogen that provokes infectious rhinotracheitis and polymicrobial infections in cattle, resulting in serious economic losses to the farm animal industry and trade restrictions. To date, non-toxic active drugs against BoHV-1 are not available. The exploitation of bioactive properties of microbial products is of great pharmaceutical interest. In fact, fungi are a promising source of novel drugs with a broad spectrum of activities and functions, including antiviral properties. Hence, the potential antiviral properties of 3-O-methylfunicone (OMF), a secondary metabolite produced by Talaromyces pinophilus, were evaluated on BoHV-1. In this study, during BoHV-1 infection in bovine cells (MDBK), the non-toxic concentration of 5 µM OMF considerably reduced signs of cell death and increased cell proliferation. Furthermore, OMF significantly decreased the virus titer as well as the cytopathic effect and strongly inhibited the expression of bICP0, the major regulatory protein in the BoHV-1 lytic cycle. These findings were accompanied by a considerable up-regulation in the expression of the aryl hydrocarbon receptor (AhR), a multifunctional transcription factor also linked to the host's response to a herpesvirus infection. Overall, our results suggest that by involving AhR, OMF shows potential against a BoHV-1 infection.

Involvement of herpesviruses in cases of abortion among water buffaloes in southern Italy.

A six-year study on water buffaloes from the Campania Region (Southern Italy) was conducted to evaluate the presence of bovine/bubaline herpesviruses in cases of abortion. A total of 244 buffalo foetuses were analysed by real-time PCR to detect the presence of: bovine alphaherpesvirus 1(BoHV-1), bubaline alphaherpesvirus 1 (BuHV-1), bovine alphaherpesvirus 2 (BuHV-2), and bovine gammaherpesvirus 4 (BoHV-4). The foetuses of 14 water buffaloes that showed abortions were positive for BuHV-1 (4 animals) and/or BoHV-4 (11 animals), with one of these cases showing co-infection with BuHV-1 and BoHV-4. This study reports the first identification of BoHV-4 in water buffaloes. Cases of abortion were analysed using both molecular and cultural assays for the presence of other pathogens. In nearly all the abortion cases positive for BoHV-4, the virus was identified as a co-infecting agent together with other microorganisms, whereas in two abortion cases, it was the only pathogen found.

Oyster Crassostrea gigas, a good model for correlating viral and chemical contamination in the marine environment.

To establish a relationship between viruses and chemicals, they were analysed in oyster Crassostrea gigas from an Italian experimental station. The chemicals concentrations were: Σ6 NDL-PCBs 0.82-7.12 ng g-1; BaP LOQ (<0.2 µg kg-1) to 1.2 µg kg-1; PAH4 LOQ (<0.2 µg kg-1) to 9.8 µg kg-1; Cd 0.073-0.365 mg kg-1; Pb 0.010-0.487 mg kg-1; and Hg < LOQ (0.089 mg kg-1). The viruses identified included: noroviruses (NoVGI/GII), astrovirus (AsV), rotavirus (RV), adenovirus (AdV), and sapovirus (SaV), while hepatitis A, hepatitis E, and Aichi viruses were not detected. Significant correlations were observed for NDL-PCBs with NoVGI, NoVGII, and AdV; BaP and PAH4 with NoVGI and AsV; Cd with RV; Pb with NoVGI and AsV; PAHs with Pb; AsV with NoVGI; and AdV with NoVGII. The study indicated as C. gigas is a model for correlating pollutants and foodborne viruses, whose co-presence may represent an additional food safety risk.

On Gram-Positive- and Gram-Negative-Bacteria-Associated Canine and Feline Skin Infections: A 4-Year Retrospective Study of the University Veterinary Microbiology Diagnostic Laboratory of Naples, Italy.

A 4-year retrospective study (2016-2019) of selected routine bacteriological examinations of the veterinary microbiology laboratory of the University Veterinary Teaching Hospital of Naples (Italy) was carried out. A total of 189 bacteriological samples were collected from 171 dogs and 18 cats suffering from skin infections. In dogs, the most common cutaneous infection was otitis externa, while pyoderma was found to be prevalent in cats. The number of recorded Gram-positive strains over the study period did not vary considerably from year to year and was always significantly higher (p-value = 0.0007) in comparison with Gram-negative bacterial isolations. In dogs, Staphylococcus pseudintermedius was the most common identified Gram-positive bacterium (65%), while Pseudomonas aeruginosa (36%) was the one among the isolated Gram-negative bacteria. In cats, coagulase-negative staphylococci were the most predominant isolated bacteria (47%). The phenotypic profiles of antibiotic resistance showed that most of the strains were resistant to amoxicillin-clavulanate, penicillin, clindamycin, and trimethoprim-sulfamethoxazole. Several multi-drug-resistant strains (35%) were detected in canine isolates. An updating of antibiotic resistance profiles of the main Gram-positive and Gram-negative bacteria principally associated with skin infections of pet animals is necessary to improve stewardship programs of veterinary hospitals and clinics.

MG-132 interferes with iron cellular homeostasis and alters virulence of bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) requires an iron-replete cell host to replicate efficiently. BoHV-1 infection provokes an increase in ferritin levels and a decrease of transferrin receptor 1 (TfR-1) expression, ultimately lowering iron pool extent. Thus, cells try to limit iron availability for virus spread. It has been demonstrated that MG-132, a proteasome inhibitor, reduces BoHV-1 release. Since ferritin, the major iron storage protein in mammalian cells, undergoes proteasome-mediated degradation, herein, the influence of MG-132 on iron metabolism during BoHV-1 infection was examined. Following infection in bovine cells (MDBK), MG-132 reduced cell death and viral yield. Western blot analysis showed a significant ferritin accumulation, likely due to the inhibition of its proteasome-mediated degradation pathway. In addition, the concomitant down-regulation of TfR-1 expression, observed during infection, was counteracted by proteasome inhibitor. This trend may be explained by enhanced acidic vesicular organelles, detected by acridine orange staining, determining a reduction of intracellular pH, that promotes new synthesis of TfR-1 degraded in a recycling pathway. In addition, MG-132 influences cellular iron distribution during BoHV-1 infection, as revealed by Perls' Prussian blue staining. However, cellular iron content, evaluated by Atomic Absorption Spectrophotometry, resulted essentially unaltered. These findings reveal that MG-132 may contribute to limit cellular iron availability for virus replication thereby enhancing cell survival.

Importance of broth-enrichment culture in equine endometritis diagnosis.

The objective of this study was to investigate the diagnostic accuracy of the standard microbiological protocol to assure the evaluation of bacterial endometritis in the equine clinical practice. Four hundred fifty-two equine uterine swabs were seeded on different types of agar plates and then in a broth-enrichment (Brain Heart Infusion Broth) before plating by using the same media the day after. The prevalence of positivity was 33.7% following direct plating and 66.3% following use of added enrichment-broth phase before seeding on solid media. Furthermore, the prevalence of isolated bacteria included E. coli (29.7%) and Streptococcus equi subsp. zooepidemicus (15.2%), both frequently associated with equine endometritis. Our results indicate that the addition enrichment-broth culture significantly increases the rate of positivity for the detection of bacteria in equine uterine swabs compared to the direct plating of samples alone. Thus, this diagnostic technique may be recommended to increase the sensitivity of bacteriological analysis in mares with endometritis.

Occurrence and Antimicrobial Susceptibility Profiles of Streptococcus equi subsp. zooepidemicus Strains Isolated from Mares with Fertility Problems.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus), is a ß-hemolytic Streptococcus belonging to the Lancefield group C; it is a rare human pathogen, but in horses, it is frequently associated with endometritis. This study aimed to isolate S. zooepidemicus strains, associated with bacterial endometritis in mares, and to define their antimicrobial resistance profile. Twenty-three isolates were recovered from one hundred ninety-six equine uterine swabs (11.7%). Bacterial identification was carried out by Api 20 Strep and confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), while antimicrobial susceptibility testing was performed by disk diffusion method on Muller Hinton agar plates. The antibiotic resistance profiles of the isolates revealed a high percentage of resistance to amikacin (95.6%), ampicillin (73.9%) and tetracycline (69.6%), while ceftiofur and ceftriaxone were highly effective with 82.6% and 78.3% of the isolates inhibited, respectively. An intriguing value of resistance to penicillin (34.8%), which represents the first-choice antibiotic in equine S. zooepidemicus infections, was observed. Furthermore, a high prevalence of multidrug-resistant strains (82.6%) was recorded. Continuous surveillance of this potential zoonotic pathogen and an appropriate antimicrobial stewardship program with the promotion of correct use of antimicrobials, after a proper diagnosis, are needed to allow an effective therapy.

A Preliminary Study on Antimicrobial Susceptibility of Staphylococcus spp. and Enterococcus spp. Grown on Mannitol Salt Agar in European Wild Boar (Sus scrofa) Hunted in Campania Region-Italy.

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-ß-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.

Polychlorinated organic pollutants (PCDD/Fs and DL-PCBs) in loggerhead (Caretta caretta) and green (Chelonia mydas) turtles from Central-Southern Tyrrhenian Sea.

This study assesses for the first time the levels of PCDD/Fs and DL-PCBs in sea turtles coming from Tyrrhenian Sea. The concentrations measured in liver of the 24 specimens analysed were 6.90 vs 5.65 pg g-1 wet weight (ww) for PCDD/Fs and 10.95 vs 0.79 ng g-1 ww for DL-PCBs in Caretta caretta and Chelonia mydas, respectively. The DL-PCB levels resulted very higher in Caretta caretta than Chelonia mydas probably due to the different eating habits between the two species investigated. Furthermore, the highest levels of DL-PCBs were determined in livers of the adult Caretta caretta turtles of male sex. Positive correlations were found out between PCB-81 and the body mass (BM) of turtles (r2 = 0.6561; p = 0.001) and between PCB-81 and the curved carapace length (CCL) (r2 = 0.6250; p = 0.006) suggesting that the body burden of contaminants is related to the body size. The mean TEQ values, as a matter of risk assessment for turtles, were 3.64 vs 1.62 pg TEQ g-1 ww for PCDD/Fs and 8.72 vs 2.16 pg TEQ g-1 ww for DL-PCBs in Caretta caretta and Chelonia mydas, respectively. The results reported in this study increase the data available about the consequences of the Mediterranean Sea contamination by organochlorine pollutants and highlight an evident PCDD/F and PCB bioaccumulation in sea turtle tissues that threatens the survival of these marine organisms.

Antimicrobial Activity of Some Essential Oils against Methicillin-Susceptible and Methicillin-Resistant Staphylococcus pseudintermedius-Associated Pyoderma in Dogs.

This study aimed to test in vitro the antimicrobial activity of 11 essential oils (EOs) against four methicillin-resistant Staphylococcus pseudintermedius (MRSP) and four methicillin-susceptible S. pseudintermedius (MSSP) clinical isolates. The obtained findings demonstrated a clear in vitro efficacy of some tested EOs against both MRSP and MSSP strains. Particularly, modal minimum inhibitory concentration (MIC) values ranging from 1:2048 v/v for Melissa officinalis against an MSSP strain to 1:256 v/v for Cymbopogoncitratus against all MRSP strains were observed. The best results, highlighting a modal MIC value of 1:1024 v/v for all tested isolates, was provided by Cinnamomum zeylanicum. Intriguingly, Cinnamomum zeylanicum showed, in many cases, a correspondence between minimum bactericidal concentration (MBC) and MIC values, indicating that the inhibiting dose is also often bactericidal. Moreover, a mild antibacterial and bactericidal activity against both MRSP and MSSP isolates was detected for the other tested EOs. Considering the zoonotic potential of S. pseudintermedius and the increased dissemination of multidrug-resistant strains, the employment of EOs could be useful for the treatment of canine pyoderma. Since antibiotic resistance has become the most urgent issue, from the perspective of the One Health initiative, alternative therapeutic approaches are desirable to limit the use of antibiotics or to improve the efficacy of conventional therapies.

Bovine herpesvirus-1 infection in mouse neuroblastoma (Neuro-2A) cells.

Bovine herpesvirus 1 (BoHV-1) is an important cattle pathogen, that may cause rhinotracheitis, abortions and shipping fever. Virus establishes latency in sensory neurons, but periodically could reactivate. Recent studies identified mouse neuroblastoma (Neuro-2A) cells as a novel cell culture model to study factors that regulate BoHV-1 productive infection in neuronal cells. Herein, following BoHV-1 infection in Neuro-2A, a reduced cell viability occurred. Membrane damage and death morphological alterations, features of apoptosis and necrosis, were distinguished in infected cells. In addition, biochemical signs of apoptosis (caspase 3 activation and PARP cleavage) were observed. These results were accompanied by incomplete autophagy due to enhanced amounts of autophagic markers (LC3-II, ATG5 and Beclin 1), in the presence of increased levels of p62. Interestingly, protein expression of viral infected cell protein 0 (bICP0) was detected in Neuro-2A cells, although BoHV-1 inefficiently replicates in these cells, because just low levels of viral yield were found. Taken together, our results suggest that BoHV-1 may exert its potential neurotoxicity through a combined mechanism of necrosis and apoptosis. Moreover, incomplete autophagy occurred during BoHV-1 replication in Neuro-2A cells, which were favourable for viral persistence.