Characterization of the semen microbiota of healthy stud dogs using 16S RNA sequencing.

The reproductive microbiota of male dogs has never been investigated using culture-independent sequencing techniques. The purpose of the present study was to get seminal knowledge on the microbiota of the ejaculate. Specifically, factors as the fraction of the ejaculate, the sperm quality (normospermia, teratozoospermia), and the living environment were evaluated. The sperm-rich and the prostatic fractions of the ejaculate were collected from healthy stud dogs. Following the sperm analysis, samples from twenty animals (normospermic n = 10 and teratozoospermic n = 10) were stored at - 80 °C until further processing including DNA extraction and 16S rRNA sequencing. Alpha- (Shannon index) and beta- (Bray-Curtis, Unweighted UniFrac) diversities were assessed and compared (PERMANOVA) based on the group of samples (biological samples from the ejaculate and controls), the fraction of the ejaculate (sperm-rich and prostatic fractions), the animal group (normospermia and teratozoospermia), and the living environment of the animal (kennel or pet living in-house). The most abundant bacterial phyla in canine semen samples were Proteobacteria, Firmicutes, and Actinobacteria. Overall, the dominant bacterial family was that of Pasteurellaceae The genus Mycoplasma was never detected. No differences in terms of bacterial composition were found based on the fraction of the ejaculate and based on the animal group (P > 0.05). On the other hand, differences in alpha and beta diversities were highlighted based on the living environment (P = 0.001). Overall, the results of the present study provide preliminary insights on dog semen microbiota, opening a new chapter in the field of canine andrology. Our results suggest that the environment may play a role in influencing the reproductive microbiota of male dogs and that the prostatic fraction of the ejaculate can be used for further research as a representative of the semen microbiota.

Complete Genome Sequence of Bovine Viral Diarrhea Virus Subgenotype 2a Strain CN10.2015.821, Isolated in Piedmont, Italy.

We sequenced the complete genome of noncytopathic bovine viral diarrhea virus (BVDV) type 2 strain CN10.2015.821. It belongs to the subgenotype 2a, and it was isolated from an immunotolerant and persistently infected calf identified during a serological investigation. The complete genome is composed of 12,273 nucleotides, organized as one open reading frame encoding 3,897 amino acids.

Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.

Expression and antigenic characterization of bubaline herpesvirus 1 (BuHV1) glycoprotein E and its potential application in the epidemiology and control of alphaherpesvirus infections in Mediterranean water buffalo.

Bubaline herpesvirus 1 (BuHV1) is a member of ruminant alphaherpesviruses antigenically related to bovine herpesvirus 1 (BoHV1). The impact of BuHV1 infection in infectious bovine rhinotracheitis control program is difficult to establish, due to the lack of specific diagnostic test. The ectodomain of glycoprotein E of BuHV1 was expressed as recombinant secreted protein and used in indirect ELISA as well as in a discriminatory test using the BoHV1 counterpart. A panel of monoclonal antibodies was produced against BuHV1; 6 out of 7 anti-gE monoclonal antibodies specifically recognized the BuHV1 gE. Results indicated BuHV1 gE as a sensitive marker of infection compared to seroneutralization (SN) test or blocking ELISA. When BoHV1 and BuHV1 gEs were immobilized in different wells of the same ELISA microplate, bovine and water buffalo sera were more reactive against the respective infecting virus. About one third of seropositive buffaloes with no history of contact with cattle and having higher SN titres, reacted in BoHV1 gE blocking ELISA, possibly because of steric hindrance. Since in two occasions BuHV1 was also isolated from water buffalo scoring gB+/gE+ BoHV1 blocking ELISA, we conclude that the combination of the two blocking ELISAs is not suitable to differentiate between BoHV1 and BuHV1.

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

A study to compare circulating flunixin, meloxicam and gabapentin concentrations with prostaglandin E2 levels in calves undergoing dehorning.

The purpose of this study was to investigate the pharmacokinetics of intravenous flunixin (2.2 mg/kg b.w.), oral meloxicam (1mg/kg b.w.), oral gabapentin (15 mg/kg b.w.) alone or co-administrated with meloxicam as well as the effects of these compounds on prostaglandin E2 (PGE2) synthesis in calves subjected to surgical dehorning. Plasma samples collected up to 24h after drug administration were analyzed by liquid chromatography/mass spectrometry, whereas blood PGE2 levels were measured by immunoenzymatic assay. In plasma, the terminal half-live of flunixin, meloxicam and gabapentin were 6.0 h (range, 3.4-11.0 h), 16.7h (range, 13.7-21.3h) and 15.3h (range, 11-32.9h), respectively. The co-administration of single doses of gabapentin and meloxicam did not seem to affect the pharmacokinetic profile of the two drugs except for gabapentin that reached significantly (P<0.05) higher maximum serum concentration (Cmax) when co-administered with meloxicam, than when administered alone. At 5, 360 and 720 min after dehorning, a significant (P<0.01) decrease in PGE2 concentration was observed in flunixin-treated animals compared with control calves. Moreover, circulating log PGE2 concentrations were inversely proportional to log flunixin concentrations (R(2)=0.75; P<0.0001). None of the other drugs significantly affected blood PGE2 levels. Further assessment of oral meloxicam and gabapentin in established pain models is required to formulate science based analgesic recommendations to enhance animal well-being after dehorning.

Characterization of new small ruminant lentivirus subtype B3 suggests animal trade within the Mediterranean Basin.

Small ruminant lentiviruses (SRLVs) represent a group of viruses infecting sheep and goats worldwide. Despite the high heterogeneity of genotype A strains, which cluster into as many as ten subtypes, genotype B was believed to be less complex and has, so far, been subdivided into only two subtypes. Here, we describe two novel full-length proviral sequences isolated from Sarda sheep in two Italian regions. Genome sequence as well as the main linear epitopes clearly placed this cluster into genotype B. However, owing to long-standing segregation of this sheep breed, the genetic distances that are clearly >15 % with respect to B1 and B2 subtypes suggest the designation of a novel subtype, B3. Moreover the close relationship with a gag sequence obtained from a Turkish sheep adds new evidence to historical data that suggest an anthropochorous dissemination of hosts (small ruminants) and their pathogens (SRLV) during the colonization of the Mediterranean from the Middle East.

In vitro properties of small ruminant lentivirus genotype E.

Small ruminant lentivirus genotype E lacks the dUTPase subunit and vpr-like gene. Two strains (Roccaverano and Seui) with identical genetic organization have been described, with the env HV1-HV2 domains being the most divergent. Although dUTPase and vpr-like deletions have been involved in the RT fidelity in non dividing cells, both strains were able to replicate efficiently in blood derived macrophages (BDM), while virus production of E1 subtype was reduced or abrogated in replicating fibroblastic-like cells. The transcriptional activity of genotype E was similar in these two cellular populations. When viral pseudotypes were generated with the env of both viruses, Roccaverano pseudotype displayed a paranuclear localization on BDM, suggesting a different mechanism of entry. Polymorphic GAS and TAS sites in the U3 region, further suggest that a population different from classically activated macrophages can be infected by these viruses, opening new insights into lentiviruses with low or null pathogenic potential.

Contemporary use of acepromazine in the anaesthetic management of male horses and ponies: a retrospective study and opinion poll.

REASON FOR PERFORMING STUDY: Current use of acepromazine in the anaesthetic management of male horses and ponies and associated risks are largely unknown. OBJECTIVES: To explore anaesthetic acepromazine use and related adverse effects in the male horse. METHODS: Of 8533 anaesthetised horses and ponies medical records of male animals treated perianaesthetically with acepromazine were reviewed. Demographic data, time and dose of acepromazine administration, co-administered drugs, quality of induction and recovery from anaesthesia, arterial blood pressures, and occurrence of penile dysfunction were recorded. Practising ACVA and ECVAA diplomates were polled on the use of acepromazine and its effects on blood pressure and penile dysfunction in the equine. RESULTS: Of all animals, 12% females and 11% males (n=575 including 42% stallions) received perianaesthetic acepromazine, predominantly for premedication. Anaesthetic induction was smooth in 566 animals. Lowest mean arterial pressures averaged 65±9 mmHg. Recovery was good or very good in 70% of all animals and 74% stood after 1-2 attempts. In 14 horses (2.4%; 7 stallions, 7 geldings), penile prolapse occurred for 0.5-4 h and in one stallion (0.2%) for >12 but <18 h post recovery. Most surveyed anaesthesiologists use acepromazine in stallions (occasionally 63%; frequently 17%) but more frequently in geldings (occasionally 34%; frequently 59%) and mares (occasionally 38%; frequently 59%), primarily for premedication with other sedatives and analgesics. Persistent intraoperative hypotension was not frequently reported. Only 5% of surveyed anaesthesiologists recall penile prolapse post acepromazine administration lasting for >12 h and only one recalls 3 cases of irreversible penile prolapse in 20 years of anaesthesia practice. CONCLUSIONS AND POTENTIAL RELEVANCE: The extremely low risk of permanent penile dysfunction (≤1 in 10,000 cases) does not justify more restricted use of acepromazine in the intact male vs. geldings and mares.

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses.

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.

Pharmacokinetics, metabolism and excretion of the glycine antagonist GV150526A in rat and dog.

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.

Barrett's ulcer and Campylobacter-like organisms infection in a child.

Barrett's esophagus is a gastrointestinal metaplasia of the esophageal epithelium occurring frequently in adults with long-standing peptic esophagitis. Recent reports of Barrett's esophagus in children with gastroesophageal reflux (GER) showed that also at the pediatric age intestinal metaplasia of the esophagus may occur in association with peptic esophagitis. Recently a close association between Campylobacter-like organisms (CLOs) and gastritis has been found in the stomach of both adults and children with a variety of peptic diseases, but evidence of such infection in specimens of Barrett's epithelium has never been described in children. We report here a child with Barrett's esophagus and GER, treated with H2 blockers, who showed a Barrett's ulcer in association with CLO infection. The addition of amoxicillin to antireflux treatment was accompanied by healing of the ulcer, suggesting that bacterial infection of Barrett's epithelium may have an important role in determining its inflammation and possibly ulceration.