Feline hypertrophic cardiomyopathy: Does the microRNA-mRNA regulatory network contribute to heart sarcomeric protein remodelling?

Feline primary hypertrophic cardiomyopathy (HCM) is an intrinsic myocardial disease characterized by concentric hypertrophy of the left ventricle. In the present study, we investigated the microRNA-mRNA regulatory network in feline myocardial tissue affected by primary (HCMI) and secondary HCM (HCMII). MRNA expression levels of sarcomeric genes, including, TNNT2, TNNI3, MYH7, MYBPC3, TPM1 and ACTC1 were assessed in the FFPE myocardial tissues. FFPE tissues from healthy cats were sequenced by the NGS, to explore, in the entire non-deposited miRNome, the expression level of microRNAs targeting the complementary sequences of selected sarcomeric mRNAs. The sarcomeric genes TNNT2, MYH7, MYBPC3 and TPM1 showed a statistically significant upregulation in HCMI compared to HCMII (p < .01), except ACTC1 which was downregulated (p < .01); TNNI3 showed no statistically significant difference. In HCMII miR-122-5p, miR-338-3p, miR-484, miR-370-3p, miR-92b-3p, miR-375 and miR-370-3p showed a significant upregulation (p < .01) compared to control. The exception was miR-30a-5p which showed downregulation. Worthy of note is the 4-fold higher expression of miR-370-3p, a key regulator of MYBPC3, in HMCI compared to HMCII. This research does not solve the aetiological mystery of HCM, but it may help to find a way to help diagnose and define the prognosis of HCM in cats.

Comparison between clinical evaluations and laboratory findings and the impact of biofilm on antimicrobial susceptibility in vitro in canine otitis externa.

BACKGROUND: In canine otitis externa (OE), biofilm-producing bacteria are frequently present but biofilm may be underdiagnosed clinically. HYPOTHESIS/OBJECTIVES: The study aimed to investigate an association between clinical and cytological findings with bacteriological data from dogs with OE, to establish, through Environmental Scanning Electron Microscope (ESEM) examination, whether the presence of biofilm in vivo can be predicted and to evaluate the impact of biofilm on antimicrobial susceptibility tests. MATERIALS AND METHODS: Fifty-six dogs showing clinical signs of OE were enrolled. One cotton swab each was collected for ESEM, bacterial culture and susceptibility testing and for cytology. Staphylococcus pseudintermedius (n = 42, 48.8%) and Pseudomonas aeruginosa (n = 26, 30.2%) were tested for their ability to form biofilm. Minimum Inhibitory Concentrations (MIC), Minimal Biofilm Inhibitory Concentrations (MBIC) and Minimal Biofilm Eradication Concentrations (MBEC) towards enrofloxacin, gentamicin, polymyxin B and rifampicin were determined. RESULTS: Pseudomonas aeruginosa was positively associated with the biofilm clinical evaluation (p < 0.01) and neutrophils (p < 0.05), nuclear streaks (p < 0.01) and rods bacteria (p < 0.01) on cytology. S. pseudintermedius was associated with a low presence of neutrophils. There was a statistical correlation between clinical and cytological biofilm presence (p ≤ 0.01), but none with the biofilm production assay nor ESEM biofilm detection. No differences were found comparing the results of MIC and MBIC. MBEC results showed higher values than MIC and MBIC for all antimicrobials tested (p ≤ 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Biofilm presence in OE was often underdiagnosed. Even if there is no specific clinical or cytological pattern related to biofilm, its presence should always be suspected.

Comparative Performances of Vitek-2, Disk Diffusion, and Broth Microdilution for Antimicrobial Susceptibility Testing of Canine Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is the primary cause of canine cutaneous infections and is sporadically isolated as a pathogen from humans. Rapidly emerging antibiotic-resistant strains are creating serious health concerns so that accurate and timely antimicrobial susceptibility testing (AST) is crucial for patient care. Here, the performances of the AST methods Vitek-2, disk diffusion (DD) and broth microdilution (BMD) were compared for the determination of susceptibility of 79 S. pseudintermedius isolates from canine cutaneous infections and one from human pyoderma to oxacillin (OXA), amoxicillin/clavulanate (AMC), cephalothin (CEF), gentamicin (GEN), enrofloxacin (ENR), doxycycline (DOX), clindamycin (CLI), inducible clindamycin resistance (ICR), mupirocin (MUP), and trimethoprim-sulfamethoxazole (SXT). Overall, the agreement of DD and Vitek-2 using the veterinary AST-GP80 card with reference BMD was ≥90%, suggesting reliable AST performances. While DD generated mainly minor errors and one major error for OXA, Vitek-2 produced one very major error for GEN, and it failed in identifying one ICR-positive isolate. Moreover, five bacteria were diagnosed as ICR-positive by Vitek-2, but they showed a noninduction resistance phenotype with manual methods. All S. pseudintermedius isolates were interpreted as susceptible or intermediately susceptible to DOX using CLSI breakpoints for human staphylococci that match the DOX concentration range included in AST-GP80. However, this could lead to inappropriate antimicrobial prescription for S. pseudintermedius infections in companion animals. Considering the clinical and epidemiological importance of S. pseudintermedius, we encourage updating action by the system manufacturer to address AST for this bacterium.

Detection of anti-HEV antibodies and RNA of HEV in pigs from a hyperendemic Italian region with high human seroprevalence.

BACKGROUND: Pigs are considered the main reservoir of genotypes 3 and 4 of hepatitis E virus (HEV), which is the major cause of acute hepatitis of viral origin in humans worldwide. An increasing number of autochthonous HEV infections have been observed in recent years in industrialized countries, most likely as a result of zoonotic transmission through the consumption of raw or undercooked meat products. METHODS: Two hundred and thirty-three blood and liver samples were collected at four different local slaughterhouses from domestic pigs bred in Abruzzo, a region of south-central Italy, where there is the highest human seroprevalence to HEV compared with the rest of Italy. An indirect enzyme-linked immunosorbent assay kit was used for detecting anti-HEV IgG in the sera, while the presence of HEV RNA was investigated by performing a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Between 87.3% and 100% of swine serum samples collected in different slaughterhouses of Abruzzo were positive for anti-HEV antibodies. Conversely, none of the liver samples collected from the same animals were positive for HEV by real-time RT-PCR. CONCLUSIONS: The hypothesis of foodborne zoonotic transmission from local pigs as responsible for the hyperendemic status of Abruzzo cannot be corroborated. However, the high seroprevalence observed in pigs indicates that HEV is highly circulating in these territories. We propose to further investigate the role of wild fauna and trade in carrier pigs, and the maintenance of HEV virulence in the environment and meat supply chain to shed light on the possible sources of human infection and the degree of occupational risk.

First molecular detection of ball python nidovirus in Italy - Short communication.

A retrospective study was conducted to investigate the presence of ferlavirus, ball python nidovirus and bacteria in 32 tracheobronchial lavages from ball pythons raised in captivity and affected by respiratory disease. A touchdown reverse transcription polymerase reaction (RT-PCR) was performed to detect ball python nidovirus RNA targeting a 260-bp portion of the ORF1a gene, while a nested RT-PCR was applied to identify RNA targeting the 518-bp ferlavirus partial L gene. RT-PCR positive products were submitted for Sanger's sequencing and phylogeny reconstruction. Bacteriological examinations were performed to diagnose a possible bacterial involvement. BLAST analysis revealed that the nucleotide sequences of the six (18.8%) RT-PCR positive amplicons were 90-97% identical to the partial sequence of the ORF1a gene of the recently described ball python nidovirus. All tested snakes were negative for ferlavirus. Thirteen out of 32 samples (40.6%) were bacteriologically positive. Respiratory tract diseases can be a substantial problem for snake breeders, considering the rapid transmission of respiratory pathogens. The results and published studies show that ball python nidovirus is circulating in python collections and could be linked to suboptimal management practices. Surveillance programs are desirable as part of the routine snake health assessment. Tracheobronchial lavage is a fast, practical, cost-effective procedure for sample collection.

Antimicrobial Strategies Proposed for the Treatment of S. pseudintermedius and Other Dermato-Pathogenic Staphylococcus spp. in Companion Animals: A Narrative Review.

The treatment of dermato-pathogenic Staphylococcus spp., particularly Staphylococcus pseudintermedius, in companion animals presents significant challenges due to rising antimicrobial resistance. This review explores innovative strategies to combat these infections. We examined novel antimicrobials and the repurposing of existing drugs to enhance their efficacy against resistant strains. Additionally, we evaluate the potential of natural products, nanomaterials, and skin antiseptics as alternative treatments. The review also investigates the use of antimicrobial peptides and bacteriophages, highlighting their targeted action against staphylococcal pathogens. Furthermore, the role of adjuvants in antibiotic treatments, such as antimicrobial resistance breakers, is discussed, emphasizing their ability to enhance therapeutic outcomes. Our analysis underscores the importance of a multifaceted approach in developing effective antimicrobial strategies for companion animals, aiming to mitigate resistance and improve clinical management of staphylococcal skin infections.

Antilisterial activity of olive-derived polyphenols: an experimental study on meat preparations.

Pork meat and processed pork products have been linked to multiple listeriosis outbreaks worldwide during the past few years. Specifically, it has been highlighted that minced pork meat is easily perishable and may increase the growth of Listeria monocytogenes, which could be harmful to the general public's health. This study aimed to investigate the potential application of olive oil mill wastewater polyphenolic and red beet extracts as natural antimicrobial agents for L. monocytogenes growth control in burgers. The minced pork meat was mixed with the extracts and experimentally inoculated with L. monocytogenes, then molded into vacuum-packaged and cold-stored (4±1°C) burgers kept under alternating exposure to fluorescent light. The L. monocytogenes enumeration was performed on burgers at 0, 2, 5, and 10 days of shelf life. In uninoculated burgers, physicochemical (pH, water activity, color) and sensory determination (descriptive sensory analysis) were also conducted. At the end of storage, the samples treated with olive-derived extract showed the lowest value of L. monocytogenes (approximately 1.3 Log CFU/g). The physicochemical and sensory traits of burgers have benefited from the addition of both olive-derived and red beet extracts. Results suggest that olive mill wastewater polyphenolic extracts could be added to minced pork meat products to act as a natural antimicrobial agent.

High Biofilm-Forming Multidrug-Resistant Salmonella Infantis Strains from the Poultry Production Chain.

The ability of Salmonella species to adhere to surfaces and form biofilms, leading to persistent environmental reservoirs, might represent a direct link between environmental contamination and food processing contamination. The purpose of this study was to investigate the biofilm-forming ability of 80 multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica serovar Infantis strains isolated from the broiler food chain production through whole genome sequencing (WGS), PCR, and morphotype association assays. Biofilm formation was quantified by testing the strains at two different temperatures, using 96-well polystyrene plates. The rough and dry colony (rdar) morphotype was assessed visually on Congo red agar (CRA) plates. Based on our results, all tested S. Infantis strains produced biofilm at 22 °C with an rdar morphotype, while at 37 °C, all the isolates tested negative, except one positive. Most isolates (58.75%) exhibited strong biofilm production, while 36.25% showed moderate production. Only 5 out of 80 (6.25%) were weak biofilm producers. WGS analysis showed the presence of the fim cluster (fimADF) and the csg cluster (csgBAC and csgDEFG), also described in S. Typhimurium, which are responsible for fimbriae production. PCR demonstrated the presence of csgD, csgB, and fimA in all 80 S. Infantis strains. To our knowledge, this is the first study comparing the effects of two different temperatures on the biofilm formation capacity of ESBL producing S. Infantis from the broiler production chain. This study highlights that the initial biofilm components, such as curli and cellulose, are specifically expressed at lower temperatures. It is important to emphasize that within the broiler farm, the environmental temperature ranges between 18-22 °C, which is the optimum temperature for in vitro biofilm formation by Salmonella spp. This temperature range facilitates the expression of biofilm-associated genes, contributing to the persistence of S. Infantis in the environment. This complicates biosecurity measures and makes disinfection protocols on the farm and in the production chain more difficult, posing serious public health concerns.

Upgrading hazelnut skins: Green extraction of polyphenols from lab to semi-industrial scale.

Hazelnut skins (HS) are usually managed as waste; however, this by-product is a source of bioactive compounds, with potential applications in feed and food sectors. Phenolic compounds can be extracted using green protocols combining enabling technologies and green solvents. This work investigates subcritical water extraction (SWE) of bioactive compounds from HS. A laboratory-scale study was performed on four different batches, with significant batch-to-batch heterogeneity. The evaluation of polyphenolic profiles and antioxidant activities afforded promising results compared to the benchmark of reflux maceration. To evaluate process effectiveness, the extraction protocol was replicated on a semi-industrial plant that processed 8 kg of matrix. Downstream processes have been optimized for scale-up, demonstrating the effectiveness of SWE in retaining product concentration and bioactivity avoiding excipients in spray-drying phase. Hazelnut extracts exhibited antibacterial properties against animal- and food-borne pathogens, supporting their potential use as sustainable feed ingredients for improved hazelnut production and animal farming practices.

Unusual Case of Biliary Peritonitis in a Dog Secondary to a Gastric Perforation.

Biliary peritonitis is a pathological condition representing a medical emergency with a high risk of mortality. This condition is reported in both human and veterinary medicine following biliary tract rupture, extrahepatic biliary obstructions, gallbladder rupture, trauma, or duodenal perforation. In this report, the first-ever case of biliary peritonitis due to gastric perforation in a Bobtail purebred dog is described, which was probably induced by the administration of nonsteroidal anti-inflammatory drugs (NSAIDs). After an elective splenectomy and castration, the dog was referred to our hospital for medical management for inappetence, mental depression, and multiple episodes of gastric vomits with traces of blood. Clinical diagnostic tests showed the presence of biliary peritonitis. Due to worsening clinical conditions, the patient was subjected to euthanasia. Macroscopic examination showed a free brownish abdominal effusion and the presence of perforating ulcer of the stomach pylorus region.

Inhibition of Staphylococcus pseudintermedius Efflux Pumps by Using Staphylococcus aureus NorA Efflux Pump Inhibitors.

One promising approach in treating antibiotic-resistant bacteria is to "break" resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated for their ability to inhibit norA-mediated efflux in Staphylococcus pseudintermedius and synergize with CIP, ethidium bromide (EtBr), gentamycin (GEN), and chlorhexidine digluconate (CHX). We focused efforts on S. pseudintermedius as a pathogenic bacterium of concern within veterinary and human medicine. By combining data from checkerboard assays and EtBr efflux inhibition experiments, the hits 2-arylquinoline 1, dihydropyridine 6, and 2-phenyl-4-carboxy-quinoline 8 were considered the best EPIs for S. pseudintermedius. Overall, most of the compounds, except for 2-arylquinoline compound 2, were able to fully restore the susceptibility of S. pseudintermedius to CIP and synergize with GEN as well, while the synergistic effect with CHX was less significant and often did not show a dose-dependent effect. These are valuable data for medicinal chemistry optimization of EPIs for S. pseudintermedius and lay the foundation for further studies on successful EPIs to treat staphylococcal infections.

Antimicrobial Susceptibility of Commensal E. coli Isolated from Wild Birds in Umbria (Central Italy).

The role of wildlife, including birds, in antimicrobial resistance is nowadays a speculative topic for the scientific community as they could be spreaders/sources of antimicrobial resistance genes. In this respect, we aimed to investigate the antimicrobial susceptibility of 100 commensal Escherichia coli strains, isolated from wild birds from an Umbrian rescue centre and admitted to the Veterinary Teaching Hospital of Perugia (Central Italy) mainly for traumatic injuries. The possible presence of Salmonella spp. and ESBL-producing E. coli was also estimated. The highest prevalence of resistance was observed for ampicillin (85%) and amoxicillin/clavulanic acid (47%), probably due to their extensive use in human and veterinary medicine. Seventeen out of the one hundred E. coli isolates (17%) displayed a multidrug-resistance profile, including the beta-lactam category, with the most common resistance patterns to three or four classes of antibiotics. Resistance to ciprofloxacin, cefotaxime and ceftazidime exhibited values of 18%, 17% and 15%, respectively. Eight out of the hundred E. coli isolates (8%) were ESBL and seven showed multidrug resistance profiles. Salmonella spp. was not isolated. Resistance to third-generation cephalosporins, also detected in long-distance migratory birds, suggests the need for monitoring studies to define the role of wild birds in antimicrobial resistance circuits.

Evaluation of ß-Lactamase Enzyme Activity in Outer Membrane Vesicles (OMVs) Isolated from Extended Spectrum ß-Lactamase (ESBL) Salmonella Infantis Strains.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including ß-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis' OMVs, the aim of the work was to collect OMVs from five S. Infantis ß-lactam resistant strains isolated from a broiler meat production chain and to investigate whether ß-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of ß-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of ß-lactamase enzymes within the OMVs. This suggests that ß-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

Effect of rearing systems on immune status, stress parameters, intestinal morphology, and mortality in conventional and local chicken breeds.

The majority of poultry meat used to be sourced from intensively housed birds. However, consumer preference has since demanded poultry producers develop more sustainable farming systems. Although free-range farming is considered beneficial for animal welfare, it is not as easy to standardize as an intensive system, which makes the choice of bird genotype appear crucial for alternative systems. In this study, we aimed to evaluate the effect of conventional and free-range rearing systems on the immune status, stress parameters, intestinal morphology and mortality in commercial hybrids (Ross 308) and local poultry strains, Bionda Piemontese (BP), Robusta Maculata (RM), BP x Sasso (BPxS), and RM x Sasso (RMxS). RNA was extracted from the jejunum and spleen to assess the mRNA expression of IL-2, IL-6, IL-10, IL-18, IL-1ß, inducible nitric oxide synthase (iNOS), toll-like receptor (TLR)-4, and interferon gamma (IFN-γ). The heterophil:lymphocyte (H/L) ratio and intestinal histomorphometric evaluation were also calculated. We found that compared to the conventional system, the rearing system significantly affected the jejunum expression of IL-10, iNOS, IL-2, and IL-6, where these genes were upregulated in free-range system. A significant interaction between the rearing system and the genotype was also shown. More specifically, local breeds showed a significantly higher expression (P < 0.001) of IL-6 in the free-range system compared to the same genotypes in the conventional system. Moreover, IL-6 is constantly upregulated in local breeds within the free-range system compared to Ross hybrids. We also found significantly increased H/L and mortality rates in the latter, compared to the local breeds in the free-range reared system. The jejunum morphology also demonstrated a significantly higher villus height in BP and BPxS compared to the Ross hybrids. Overall, the results of our study confirm that the intense selection for growth in broiler chickens may have reduced their ability to react to the environmental stimuli related to free-range systems, resulting in a lower adaptability to a free-range environment, thus making them inappropriate for any farming system other than the conventional one. On the contrary, local chicken breeds are able to adapt and survive in the free-range system of rearing, and represent a genetic resource especially when adaptability to free-range conditions is required.

Inclusion Body Disease and Columbid Alphaherpesvirus 1 Infection in a Eurasian Eagle-Owl (Bubo bubo) of Central Italy.

Hepatosplenitis or inclusion body disease is a fatal disease in owls caused by Columbid alphaherpesvirus 1 (CoHV-1). A few old case reports describe it worldwide. In Italy, knowledge regarding virus circulation and disease development is lacking. Four Eurasian eagle-owls (Bubo bubo), two adults and two juveniles, were submitted for postmortem examination showing aspecific clinical signs a few hours before death. Grossly disseminated petechial hemorrhages on serosal surfaces (n = 4), hepatic and splenic necrosis (n = 3), bilateral and symmetric necrosis of pharyngeal tonsils (n = 2), and diffuse and bilateral dark-red discoloration and firmness in lungs (n = 2) were seen. Tissues were sampled for histology, bacteriology, molecular testing, and transmission electron microscopy (TEM). On histology, disseminated petechial hemorrhages (n = 4) and necrosis of liver (n = 3) and spleen (n = 3) were seen, as well as lympho-histiocytic interstitial pneumonia and meningoencephalitis (n = 2). Intranuclear inclusion bodies (INIBs) were detected in one case. A panherpesviral PCR led to positive results in one case, identified in sequencing as CoHV-1. On TEM, intranuclear and intracytoplasmic virions with herpesviral morphology were seen in the same case. For the other three birds, the lack of PCR positivity, INIBs, and TEM detection could be linked to a possible reduction of the virus to undetectable levels. Death possibly occurred secondarily to bacterial infections, supposedly established during the acute phase of CoHV-1 infection. This paper reports the presence of CoHV-1in Italy and the development of a fatal form of the disease in a Eurasian eagle-owl.

Enfermedad con cuerpos de inclusión e infección por Alfaherpesvirus de las columbiformes 1 en un búho real euroasiático (Bubo bubo) del centro de Italia. La hepatoesplenitis o enfermedad con cuerpos de inclusión es una enfermedad mortal en los búhos causada por el Alfaherpesvirus de las columbiformes 1 (CoHV-1). Algunos informes de casos antiguos lo describen en todo el mundo. En Italia, falta conocimiento sobre la circulación del virus y el desarrollo de enfermedades. Cuatro búhos reales euroasiáticos (Bubo bubo), dos adultos y dos juveniles, fueron sometidos a examen post mortem mostrando signos clínicos específicos unas horas antes de la muerte. Se observaron hemorragias petequiales muy diseminadas en las superficies serosas (n = 4), necrosis hepática y esplénica (n = 2), necrosis bilateral y simétrica de las tonsilas faríngeas (n = 2) y decoloración difusa y bilateral de color rojo oscuro y firmeza en los pulmones (n = 2). Se recolectaron muestras de tejidos para histología, bacteriología, pruebas moleculares y microscopía electrónica de transmisión (TEM). En la histología se observaron hemorragias petequiales diseminadas (n = 4) y necrosis de hígado (n = 3) y bazo (n = 3), así como neumonía intersticial linfohistiocítica y meningoencefalitis (n = 2). En un caso se detectaron cuerpos de inclusión intranucleares (INIB). Un método de PCR panherpesviral arrojó resultados positivos en un caso, identificado en la secuenciación como CoHV-1. Mediante microscopía electrónica de transmisión, se observaron viriones intranucleares e intracitoplasmáticos con morfología herpesviral en el mismo caso. Para las otras tres aves, la falta de positividad de PCR, la ausencia de cuerpos de inclusión intranucleares y de detección por microscopía electrónica de transmisión podría estar relacionada con una posible reducción del virus a niveles no detectables. La muerte posiblemente ocurrió de forma secundaria a infecciones bacterianas, posiblemente establecidas durante la fase aguda de la infección por el CoHV-1. Este artículo reporta la presencia de CoHV-1 en Italia y el desarrollo de una forma mortal de la enfermedad en un búho real euroasiático.

mcr-1-Mediated Colistin Resistance and Genomic Characterization of Antimicrobial Resistance in ESBL-Producing Salmonella Infantis Strains from a Broiler Meat Production Chain in Italy.

This work aimed to evaluate phenotypically and genotypically the colistin susceptibility of 85 Salmonella Infantis strains isolated in Italy from the broiler production chain, and to apply a whole-genome approach for the determination of genes conferring antimicrobial resistance (AMR). All isolates were tested by the broth microdilution method to evaluate the colistin minimum inhibitory concentrations (MICs). A multiplex PCR was performed in all isolates for the screening of mcr-1, mcr-2, mcr-3 mcr-4, mcr-5 genes and whole-genome sequencing (WGS) of six S. Infantis was applied. Three out of 85 (3.5%) S. Infantis strains were colistin resistant (MIC values ranged from 4 to 8 mg/L) and mcr-1 positive. The mcr-1.1 and mcr-1.2 variants located on the IncX4 plasmid were detected in three different colistin-resistant isolates. The two allelic variants showed identical sequences. All six isolates harbored blaCTXM-1, aac(6')-Iaa and gyrA/parC genes, mediating, respectively, beta-lactam, aminoglycoside and quinolone resistance. The pESI-megaplasmid carrying tet(A) (tetracycline resistance), dfrA1, (trimethoprim resistance) sul1, (sulfonamide resistance) and qacE (quaternary ammonium resistance) genes was found in all isolates. To our knowledge, this is the first report of the mcr-1.2 variant described in S. Infantis isolated from broilers chickens. Our results also showed a low prevalence of colistin- resistance, probably due to a reduction in colistin use in poultry. This might suggest an optimization of biosecurity control both on farms and in slaughterhouses.

Detection of Testudinid alphaherpesvirus, Chlamydia spp., Mycoplasma spp., and Salmonella spp. in free­ranging and rescued Italian Testudo hermanni hermanni.

Testudo hermanni is included as near­threatened in the Red List of the International Union for Conservation of Nature, while T. hermanni hermanni is considered endangered in the Italian Red List. Appropriate management of smuggled or seized wild individuals is recommended before their reintroduction into the wild. Accordingly, a health monitoring study was carried out. During 2014­2016, 133 oral swabs and 121 cloacal swabs were collected from a total of approximately 180 free­ranging and rescued T. hermanni hermanni from eight different Italian regions to investigate the presence of DNA of Testudinid alphaherpesvirus (TeAHV), Chlamydia spp. and Mycoplasma spp. in the oral cavity, and Salmonella spp. isolates in the cloaca. Mycoplasma spp. was detected in 52 out of 87 (59.77%) of rescued and in 1 out of 46 free­ranging (2.17%) individuals; 33 out of 53 (62.26%) Mycoplasma spp. positive samples were typed as M. agassizii by PCR. Salmonella spp. was isolated from 45 out of 121 (37.19%) cloacal swabs, typed into 14 serovars, and characterized for complete antimicrobial susceptibility. A significantly different distribution of Salmonella spp. isolates was found in 2016 in comparison with 2014 and 2015, without any difference between free­ranging and rescued tortoises. All the tested tortoises were negative for TeAHV and Chlamydia spp. These results are considered a baseline information critical to monitor the dynamics of these microorganisms in free­ranging and rescued populations of T. h. hermanni, and to correctly approach the management of rescued animals and possible relocation programs.

Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test.

The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to ß-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the Staphylococcus genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.

Susceptibility of Commensal E. coli Isolated from Conventional, Antibiotic-Free, and Organic Meat Chickens on Farms and at Slaughter toward Antimicrobials with Public Health Relevance.

The spread of resistant bacteria from livestock to the food industry promoted an increase of alternative poultry production systems, such as organic and antibiotic-free ones, based on the lack of antimicrobial use, except in cases in which welfare is compromised. We aimed to investigate the antibiotic susceptibility of commensal Escherichia coli isolated from organic, antibiotic-free, and conventional broiler farms and slaughterhouses toward several antimicrobials critically important for human health. To assess antimicrobial susceptibility, all E. coli isolates and extended spectrum beta-lactamase (ESBL) E. coli were analysed by the microdilution method. The prevalence of tigecycline, azithromycin and gentamicin E. coli-resistant strains was highest in organic samplings. Conversely, the lowest prevalence of resistant E. coli strains was observed for cefotaxime, ceftazidime and ciprofloxacin in organic systems, representing a significant protective factor compared to conventional systems. All E. coli strains were colistin-susceptible. Contamination of the external environment by drug-resistant bacteria could play a role in the presence of resistant strains detected in organic systems. Of interest is the highest prevalence of cephalosporin resistance of E. coli in conventional samplings, since they are not permitted in poultry. Our results suggest that monitoring of antibiotic resistance of the production chain may be helpful to detect "risks" inherent to different rearing systems.

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas.

In horses, penile squamous cell carcinomas (epSCCs) are among the most common cutaneous neoplastic lesions. These tumors usually arise in benign lesions such as viral plaques and papillomas frequently induced by Equus caballus papillomavirus type 2 (EcPV2) infection. In the last decade, the introduction of immune checkpoint inhibitors (ICI) for the treatment of human cancers has demonstrated promising results. Among the most commonly targeted pathways, there is PD-1/PD-L1 and CTLA-4. The aim of this study is to investigate the expression of the PD-1/PD-L1 pathway and CTLA-4 in the tumor microenvironment of epSCCs to assess the feasibility of an immunotherapeutic approach. Twenty equine epithelial tumors were retrospectively selected and submitted to RT-qPCR for PD-1 and PD-L1 genes. After testing antibodies cross-reactivity by western blotting, immunohistochemistry for PD-L1 and CTLA-4 was performed. Results from RT-qPCR demonstrated that 3/20 cases expressed the PD-L1 gene, whereas the PD-1 gene was not detected. Immunohistochemical positivity for PD-L1 was found only in one case. CTLA-4-positive cells were observe in all cases but were few (Mdn = 4.8; IQR = 2.3-7.1 cells/HPF). In this study group, PD-1/PD-L1 and CTLA-4 do not appear to be highly expressed and therefore the use of ICI in epSCCs may not have promising rates of response.