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.

Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus.

The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the "clades" described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.

Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal-maternal relationship. To this end, we used reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus-mother relationship can be hypothesized.

Characterization of genes encoding virulence determinants and toxins in Staphylococcus aureus from bovine milk in Central Italy.

The aim of this study was to evaluate the genotypic characteristics of Staphylococcus aureus isolates (n=170) from bovine milk collected from seven dairy farms in Italy. On the basis of cultural and biochemical properties and by amplification of the 23S rRNA specific to S. aureus, all isolates were identified as S. aureus. To genotypically characterize S. aureus isolates, genes encoding virulence determinants (nuc, clfA, spa-IgG-binding, spa-X-region, fnbA and fnbB, cap5 and cap8) and staphylococcal enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej) were investigated using a PCR technique. The results showed that the isolates of S. aureus in each farm had the same genotypic characteristics, while the isolates genotipically differed between the different farms. The present study might help to understand the distribution of prevalent S. aureus strains in dairy farms.

Phenotypic Characterization of Rhodococcus equi Biofilm Grown In Vitro and Inhibiting and Dissolving Activity of Azithromycin/Rifampicin Treatment.

Microbial biofilm has been implicated in a wide range of chronic infections. In spite of the fact that Rhodococcus equi is a recognized cause of chronic disease in animals and humans, few studies have focused on the sessile phenotype of R. equi. The aim of this research was to phenotypically characterize the biofilm development of R. equi and its answerability for hypo-responsiveness to macrolides and rifampicin. Biofilm formation is initiated by bacterial adhesion to the surface. In this work, the ability of R. equi to adhere to the surface of human lung epithelial cells was detected by a fluorometric adhesion test performed on 40 clinical isolates. Subsequently, the capability of R. equi to produce biofilm was investigated by colorimetric, fluorescence and scanning electron microscopy analysis, revealing a general slow growth of rhodococcal biofilm and different sessile phenotypes among field isolates, some also including filamented bacteria. Azithromycin treatment produced a higher long-term inhibition and dissolution of R. equi biofilms than rifampicin, while the two antibiotics combined boosted the anti-biofilm effect in a statistically significant manner, although this was not equally effective for all R. equi isolates. Increasing the MIC concentrations of drugs tenfold alone and in combination did not completely eradicate pre-formed R. equi biofilms, while a rifampicin-resistant isolate produced an exceptionally abundant extracellular matrix. These results have strengthened the hypothesis that biofilm production may occur as an antibiotic tolerance system in R. equi, potentially determining persistence and, eventually, chronic infection.

Temporal efficacy of antimicrobials against aerobic bacteria isolated from equine endometritis: an Italian retrospective analysis (2010-2017).

This study aimed to describe bacteria isolated from the reproductive tract of mares and to identify changes in antimicrobial susceptibility patterns to those antibiotics commonly used for the treatment of equine endometritis. A total of 4122 equine uterine swabs were collected from mares suffering from reproductive tract disorders in the period 2010-2017. Aerobic culture and antimicrobial susceptibility testing using agar disc diffusion were performed on each sample. Aerobic bacteria were isolated from 3171 of 4122 (76.9 per cent) samples. The most frequently isolated microorganisms were Escherichia coli (885/3171, 27.9 per cent) and Streptococcus equi subspecies zooepidemicus (791/3171, 24.9 per cent), confirming previous findings from the literature. Antimicrobial susceptibility patterns of E coli, S equi subspecies zooepidemicus and Klebsiella pneumoniae changed over time. A statistically significant decrease in antimicrobial efficacy of cefquinome against E coli was observed over the years, as well as of ampicillin, cefquinome and penicillin against S equi subspecies zooepidemicus The high frequency of resistant bacteria isolated in the present work proceeds in the same way as indicated by surveillance data on the huge antibiotic use in Italy. As a result, testing and monitoring programmes of antimicrobial efficacy are crucial to consciously using antibiotics and preserving their effectiveness both for veterinary and human medicine.

Equine Adipose-Derived Mesenchymal Stromal Cells Release Extracellular Vesicles Enclosing Different Subsets of Small RNAs.

BACKGROUND: Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role. METHODS: EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was isolated, enriched for small RNAs (<200 nt), and sequenced by Illumina technology. After bioinformatic analysis with state-of-the-art pipelines for short sequences, mapped reads were used to describe EV RNA cargo, reporting classes, and abundances. Enrichment analyses were performed to infer involved pathways and functional categories. RESULTS: Electron microscopy showed the presence of vesicles ranging in size from 30 to 300 nm and expressing typical markers. RNA analysis revealed that ribosomal RNA was the most abundant fraction, followed by small nucleolar RNAs (snoRNAs, 13.67%). Miscellaneous RNA (misc_RNA) reached 4.57% of the total where Y RNA, RNaseP, and vault RNA represented the main categories. miRNAs were sequenced at a lower level (3.51%) as well as protein-coding genes (1.33%). Pathway analyses on the protein-coding fraction revealed a significant enrichment for the "ribosome" pathway followed by "oxidative phosphorylation." Gene Ontology analysis showed enrichment for terms like "extracellular exosome," "organelle envelope," "RNA binding," and "small molecule metabolic process." The miRNA target pathway analysis revealed the presence of "signaling pathways regulating pluripotency of stem cells" coherent with the source of the samples. CONCLUSION: We herein demonstrated that e-AdMSC release EVs enclosing different subsets of small RNAs that potentially regulate a number of biological processes. These findings shed light on the role of EVs in the context of MSC biology.

Comparison of three blood transfusion guidelines applied to 31 feline donors to minimise the risk of transfusion-transmissible infections.

Objectives The increased demand for animal blood transfusions creates the need for an adequate number of donors. At the same time, a high level of blood safety must be guaranteed and different guidelines (GLs) deal with this topic. The aim of this study was to evaluate the appropriateness of different GLs in preventing transfusion-transmissible infections (TTI) in Italian feline blood donors. Methods Blood samples were collected from 31 cats enrolled as blood donors by the owners' voluntary choice over a period of approximately 1 year. Possible risk factors for TTI were recorded. Based on Italian, European and American GLs, specific TTI, including haemoplasmas, feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), Anaplasma phagocytophilum, Ehrlichia species, Bartonella species, Babesia species, Theileria species, Cytauxzoon species, Leishmania donovani sensu lato and feline coronavirus (FCoV), were screened. Rapid antigen and serological tests and biomolecular investigations (PCR) were used. Several PCR protocols for haemoplasma and FeLV DNA were compared. Results The presence of at least one recognised risk factor for TTI was reported in all cats. Results for FIV and FeLV infections were negative using rapid tests, whereas five (16.1%) cats were positive for FCoV antibodies. Four (12.9%) cats were PCR positive for haemoplasma DNA and one (3.2%) for FeLV provirus, the latter being positive only using the most sensitive PCR protocol applied. Other TTI were not detected using PCR. Conclusions and relevance Blood safety increases by combining the recommendations of different GLs. To reduce the risk of TTI, sensitive tests are needed and the choice of the best protocol is a critical step in improving blood safety. The cost and time of the screening procedures may be reduced if appropriate tests are selected. To this end, the GLs should include appropriate recruitment protocols and questionnaire-based risk profiles to identify suitable donors.

In vitro performances of novel co-spray-dried azithromycin/rifampicin microparticles for Rhodococcus equi disease treatment.

This work was aimed at providing clues on the in vitro performances of novel azithromycin/rifampicin combinations, in the form of co-spray-dried microparticles (AZM/RIF MP), against Rhodococcus equi, an animal and emerging human pathogen found responsible for worrying zoonosis. Various AZM/RIF combinations were spray-dried and characterized for their morphology and size. Susceptibility studies included determination of MIC, MBC, Fractional Inhibitory/Bactericidal Concentration Indexes and intracellular activity in R. equi-infected THP-1 cells. Cytotoxicity was tested on BEAS-2B cells through MTT assay and combination index assessment for drug interaction. Spray-dried MP were collapsed and 3-10 times smaller than commercial powders. Drug combinations showed an enhancement of in vitro antibacterial activity with a remarkable synergistic bactericidal effect. Azithromycin MP and AZM/RIF MP 2:1 led to a CFU reduction of >90% up to 4 days after treatment at all tested concentrations (p = 0.001) but AZM/RIF MP 2:1 were at least four-fold more potent than AZM MP alone. IC50 values of >100 mg/L supported low cytotoxicity of drug combinations and the combination index suggested an antagonistic toxic effect. Co-spray-drying enhanced powder dispersibility and solubility, which may improve bioavailability as well as provide administration alternatives. The novel AZM/RIF MP combinations could result a valid platform to develop new treatment strategies against R. equi infections in animals and humans.

Antimicrobial susceptibility survey on bacterial agents of canine and feline urinary tract infections: Weight of the empirical treatment.

OBJECTIVES: This work characterised the antimicrobial susceptibility of uropathogens isolated from empirically treated dogs and cats. Within-household transmission of uropathogens can involve humans and companion animals. Knowledge on the prevalence and susceptibility pattern of isolates from canine and feline urine samples and the impact of prior antimicrobial treatment is important to prevent the dissemination of antimicrobial resistance. METHODS: A retrospective study was conducted selecting antibiotic-treated companion animals. Urine samples were collected by cystocentesis and were submitted to an Italian diagnostic laboratory over a 2-year period (2013-2015). The antimicrobial susceptibility of the isolates was analysed both using Clinical and Laboratory Standards Institute (CLSI) guidelines and a formula to help select rational antimicrobial therapy. RESULTS: Gram-negative bacteria were clearly prevalent. Gentamicin had the highest impact factors. Amoxicillin/clavulanic acid and doxycycline appeared to be the most effective compounds against Gram-positive infections, whilst marbofloxacin may be a useful option against Gram-negative urinary tract infections (UTIs) as well as doxycycline and trimethoprim/sulfamethoxazole in cats and dogs, respectively. Consulting published studies, a comparable overall trend regarding bacterial species incriminated in canine and feline UTIs and their susceptibilities seems likely, despite different circumstances where the studies were conducted. CONCLUSIONS: Companion animals are potential reservoirs of drug-resistant uropathogens. Judicious use of antibiotics is necessary to maintain the efficacy of antimicrobials in human and veterinary medicine. Antimicrobial susceptibility monitoring programmes are therefore essential to facilitate the choice of antimicrobial agent that is most likely to be effective, particularly in cases of prior antimicrobial treatment.

Evaluation of immune responses in mice and sheep inoculated with a live attenuated Brucella melitensis REV1 vaccine produced in bioreactor.

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naïve cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naïve lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

DNA microarray assay and real-time PCR as useful tools for studying the respiratory tract Mycoplasma populations in young dairy calves.

PURPOSE: With more than 120 species, the genus Mycoplasma is one of the largest taxa in the class Mollicutes, a group of micro-organisms that are characterized by apparent simplicity and to which important animal pathogens belong. Mycoplasmabovis is the most frequently identified pathogenic Mycoplasma in cattle; however, the prevalence of other Mycoplasma species living in calves' airways is poorly understood. The aim of this work was to characterize the respiratory tract mycoplasma populations in calves on one of the largest dairy farms in Italy using a real-time PCR assay and a DNA microarray assay. METHODOLOGY: A total of 49 nasal swabs and 49 trans-tracheal aspirations from non-vaccinated veal calves were analysed. Genomic DNA was extracted from the samples and then tested using a real-time PCR targeting the oppD gene of M. bovis and a DNA microarray that was able to identify more than 70 Mycoplasma species. RESULTS: Forty-two out of 49 calves tested positive for Mycoplasma spp. (85.7 %). None of the samples tested positive for M. bovis. A majority (73.5 %) of the 98 samples tested positive for M. dispar, while 8 samples tested positive for M. bovirhinis (8.2 %). CONCLUSION: Our results expand our knowledge regarding the diversity of Mycoplasma populations in the respiratory airways of very young veal calves and add data regarding M. bovis prevalence in the Italian cattle population. However, the importance of these species in the respiratory diseases of calves still remains to be determined.

Investigation of the antibiotic resistance and biofilm formation of Staphylococcus pseudintermedius strains isolated from canine pyoderma.

The aim of the present study was to investigate the antibiotic resistance and bio lm formation among a collection of 51 clinical isolates of Staphylococcus pseudintermedius collected from canine pyoderma. All isolates were tested for the susceptibility to a panel of 14 antimicrobial agents by the disk di usion method in Müeller-Hinton agar. Oxacillin resistance was detected by subculture on oxacillin screening agar base. Bio lm formation was investigated by the Microtitre Plate test (MtP) and for some strains by transmission electron microscopy (TEM). Antibiotic resistance pro ling demonstrated that 45/51 S. pseudintermedius isolates had a multi drug resistant (MDR) phenotype, exhibiting simultaneous resistance to at least 3 antibiotics categories; whereas 6 isolates showed a non-MDR phenotype. Thirty strains (59%) were resistant in oxacillin resistant screening agar, the same strains were also positive for mecA by PCR assay. All S. pseudintermedius isolates showed bio lm production by MtP method. Seventeen out of 51 isolates were classi ed as weakly adherent, 26 as moderately adherent, and 8 as strongly adherent. Moreover, no di erence in bio lm formation between meticillin-resistant S. pseudintermedius (MRSP) and meticillin-suscebtible S. pseudintermedius (MSSP) (P value > 0.05) was noted. The antimicrobial resistance mechanisms and bio lm formation could explain the di culty in treating S. pseudintermedius canine infections, chemotherapeutic failure, and consequently persistent infections.

Detection and DNA quantification of Enterococcus casseliflavus in a foal with septic meningitis.

CASE DESCRIPTION A 3-month-old 180-kg (396-lb) Hanoverian colt was examined because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture. Initial treatment included empirical antimicrobial treatment and NSAIDs. CLINICAL FINDINGS Initial findings were consistent with CNS anomalies. Endoscopy revealed hyperemia, ecchymosis, and some mucopurulent exudate in the right guttural pouch. Hematologic findings were consistent with neutrophilic inflammation. On the third day of hospitalization, severe neurologic signs were observed. Computed tomography of the skull revealed a comminuted fracture of the axial aspect of the right mandibular condyle. Examination of CSF revealed turbidity, xanthochromia, and intracellular and extracellular cocci, consistent with septic meningitis. After DNA extraction from blood and CSF, sequenced products from a PCR assay for the bacterial 16S rRNA gene were 99% identical to Enterococcus casseliflavus. Microbial culture of CSF and blood samples yielded bacteria with Enterococcus spp morphology; antimicrobials were selected on the basis of susceptibility testing that identified the isolate as vancomycin resistant. A quantitative PCR assay was used to estimate Enterococcus DNA concentrations in CSF and blood. TREATMENT AND OUTCOME Treatment for E casseliflavus meningitis, including trimethoprim-sulfadiazine and ampicillin sodium administration, resulted in resolution of clinical signs. Culture of CSF and blood samples after 12 days of the targeted treatment yielded no growth. CLINICAL RELEVANCE To the authors' knowledge, this was the first report of E casseliflavus meningitis in a horse. Treatment was successful; vancomycin-resistant enterococci can be a clinical problem and may potentially be zoonotic.

High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has "captured" a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated.

Serological and biomolecular survey on canine herpesvirus-1 infection in a dog breeding kennel.

Canine herpesvirus-1 (CaHV-1) is a globally distributed pathogen causing reproductive, respiratory, ocular and neurological disorders in adult dogs and neonatal death in puppies. This pathogen is considered poorly immunogenic, and neutralizing antibodies are found for only a short time following exposure. Further, seroprevalence can be affected by several epidemiological factors. A virological survey was conducted in a high-density population breeding kennel in Central Italy. There were several factors predisposing animals to CaHV-1 infection, such as age, number of pregnancies, experience with mating and dog shows, cases of abortion, management and environmental hygiene. Serum neutralization (SN) and nested PCR assays were used to estimate prevalence of CaHV-1. None of the submitted samples tested positive for nested PCR, and none of the sera tested CaHV-1 positive. No association was observed between antibody titers and risk factors, and no sign of viral reactivation was detected in either males or females. These results suggest that CaHV-1 is not circulating within this kennel and that further studies are needed in order to better understand the distribution of the virus within Italy.

Detection of bacterial contamination and DNA quantification in stored blood units in 2 veterinary hospital blood banks.

Blood transfusions in veterinary medicine have become increasingly more common and are now an integral part of lifesaving and advanced treatment in small and large animals. Important risks associated with transfusion of blood products include the transmission of various infectious diseases. Several guidelines suggest what infectious agents to screen for in canine and feline transfusion medicine. However, while the risk of bacterial contamination of blood products during storage and administration has not been documented in veterinary medicine, it has emerged as a cause of morbidity and mortality in human transfusion medicine. Clinical experience shows that the majority of blood component bacterial contaminations are caused by only a few species. Unlike other types of bacteria, psychrotolerant species like Pseudomonas spp. and Serratia spp. can proliferate during the storage of blood units at 4°C from a very low titer at the time of blood collection to a clinically significant level (> 10(5) CFU/mL) causing clinical sepsis resulting from red blood cell concentrate transfusions in human medicine. The purpose of this report was to describe the detection and quantification procedures applied in 4 cases of bacterial contamination of canine and feline blood units, which suggest the need for further investigations to optimize patients' safety in veterinary transfusion medicine.

Microbiological and parasitological investigation on chelonians reared in Italian facilities.

The rapid rise in the number of pet chelonians and their illegal trade can modify the ecology, involving exotic pets, humans, and microbiological agents. Therefore, different epidemiological situations and the related risk to introduce and spread infectious diseases, especially zoonotic agents, have to be considered. The aim of this study was to investigate the microbiological and parasitological situation in 2 chelonian facilities (a private breeding of tortoises and a shelter for turtles) collecting oral/cloacal swabs and cloacal flushes to research viruses, bacteria, and parasites. No Chelonian Herperviruses, Cryptosporidium spp., and Giardia spp. infections were found. Salmonella spp. were detected in 8% of tortoises and in 37.5% of turtles and oxyurid eggs in 23.7% of tortoises and 15% of turtles; ascarid eggs were present only in tortoises. Moreover, 6 turtles showed cutaneous lesions, where Aeromonas sobria was isolated as main pathogen. Further studies should be performed to understand the zoonotic and infectious risk in each chelonian facility and to characterize the variables that could influence the microbiological patterns.

Phenotypic and genotypic characterization of canine pyoderma isolates of Staphylococcus pseudintermedius for biofilm formation.

Biofilm-forming ability is increasingly being recognized as an important virulence factor in several Staphylococcus species. This study evaluated the biofilm-forming ability of sixty canine derived clinical isolates of S. pseudintermedius, using three phenotypic methods, microtiter plate test (MtP), Congo red agar method (CRA) and tube adherence test, and the presence and impact of biofilm-associated genes (icaA and icaD). The results showed that icaA and icaD genes were detected concomitantly in 55 (91.7%) of 60 isolates. A majority (88.3%) of the strains screened had matching results by the tube adherence test, MtP and PCR analysis. Better agreement (95%) was found between the PCR-based analysis and the CRA. Results of the icaA and icaD gene PCRs showed good agreement with CRA results, with a kappa of 0.7. Comparing the phenotypic methods, the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good. Considering two classes (biofilm producer and biofilm non-producer), the percentage of matching results between the CRA method and the tube adherence test and between the CRA method and the MtP was 93.3%. A concordance of 100% was revealed between the MtP and the tube adherence test. The results indicate a high prevalence of the ica genes within S. pseudintermedius isolates, and their presence is associated with in vitro formation of a biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. pseudintermedius.