Are Italian-Polish veterinarians and breeders prepared to control an outbreak of Brucella canis infection in dogs?

Brucella canis infection is one of the most important causes of infertility in dogs and is a zoonosis for which no effective treatment or vaccines exist. It is not a mandatory notifiable disease. Following an increase of cases in Europe and worldwide, an investigation was performed to evaluate how much Italian and Polish veterinarians and breeders know about canine brucellosis and understand their perceptions of this infection. For this reason, two questionnaires were prepared, in Italian and Polish. Eighteen Italian and Polish veterinarians, specialists in canine reproduction, responded to the first survey and 44.4% of them affirmed having diagnosed canine brucellosis at least once in their clinical practice, and different perceptions emerged regarding the infection in the two countries. The second survey was completed by 145 Italian and Polish breeders; the disease was completely unknown to 22.8% of them, whereas 2.1% had diagnosed infection by B. canis in their kennels. In conclusion, knowledge of B. canis infection differs between these countries, with extremes ranging from diagnosed cases to complete underestimation of the presence of the problem. However, based on international data and reporting of a recent large outbreak in Italy, awareness of this contagious infectious disease and its management must be increased.

Detection of environmental contamination with feline and canine parvoviruses: new perspectives and challenges.

AIMS: To develop a protocol for environmental sampling to detect parvoviruses of dogs and cats in the environment. METHODS AND RESULTS: Environmental contamination was carried out using different dilutions of parvovirus-contaminated materials; further field samplings were performed in areas in which clinical cases of parvovirus infections were present. Sterile cotton swabs and sponges for microbial surface sampling were used. Viruses were detected in these samples with different methods: conventional PCR, nested PCR and real-time PCR, detecting viral DNA; virus isolation, detecting infectious virus; and a commercial rapid enzyme immunoassay, detecting viral antigen. No substantial differences were observed in the two sampling methods, although the sponge was more convenient for sampling rough surfaces. Molecular assays were the most sensitive methods, identifying even very low amounts of viral DNA (up to 10 copies of viral DNA/10 µl of sample). Virus isolation and the rapid test detected the viruses only at the highest viral concentrations, both in the experimental setting and field conditions. CONCLUSIONS: Environmental sampling and molecular protocols were effective in detecting environmental contamination with parvoviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol will be useful to identify possible sources of infection and to assess the efficacy of disinfection protocols in the environment.

Porencephaly and Periventricular Encephalitis in a 4-month-old Puppy: Detection of Canine Parvovirus Type 2 and Potential Role in Brain Lesions.

A 4-month-old puppy died after showing intracranial signs a few days after a suspected viral enteritis. Grossly, the right cerebral hemisphere had a large irregular cavity external to the internal capsule. Histopathological examination revealed a cystic lesion in the right hemisphere and non-suppurative inflammation of the diencephalon and periaqueductal nervous tissue. Porencephaly associated with periventricular non-suppurative encephalitis was diagnosed. A nested polymerase chain reaction (PCR) identified the presence of parvovirus DNA in the brain and real-time PCR typed this as canine parvovirus (CPV) type 2a. Immunohistochemistry revealed the presence of CPV antigen in the cytoplasm of scattered cells in the subependymal layers and choroid plexus epithelium. The porencephaly was not associated with inflammatory lesions or CPV antigen and was considered to have preceded the neurological signs. In contrast, the detection of CPV antigen in the subependymal layers and choroid plexus epithelium supported the association of this virus with the periventricular encephalitis.

Molecular characterization and virulence of an alphaherpesvirus isolated from a BoHV1 gB-seropositive and gE-seronegative Italian buffalo.

During a serological survey, 157 out of 681 unvaccinated buffaloes resulted seropositive for bovine alphaherpesvirus 1 (BoHV1) glycoprotein B (gB) and seronegative for BoHV1 glycoprotein E (gE). These serological results were generally expected in animals vaccinated with a BoHV1 gE-deleted vaccine but not in unvaccinated animals. Seroneutralization tests on 36 selected sera detected neutralizing antibody titers more than three times higher for BuHV1 than for BoHV1. In order to investigate the virus, one of these buffaloes was injected with dexamethasone, and from nasal and vaginal swabs collected at different time points, a ruminant herpesvirus was isolated, characterized and also detected by PCR. Restriction enzyme analysis, sequencing and phylogenic analysis of gB and gD genes showed that the virus was genetically similar but not identical to BuHV1 strain b6. Intranasal inoculation of the virus in a healthy seronegative buffalo resulted in a mild and transient upper respiratory disease; the virus was isolated from clinical specimens and DNA was detected by PCR in nasal and vaginal swabs up to 9 days after infection. Further investigations should be aimed at sequencing the whole viral genome and at evaluating the host-range of this virus. Specific tests are needed to discriminate infections by different ruminant herpesviruses and to improve eradication programs of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis in cattle.

Microbiological and parasitological survey of zoonotic agents in apparently healthy feral pigeons.

Microbiological and parasitological investigation was carried out on a colony of feral pigeons, located in a green area near the main hospital of a Central Italy city. One hundred pigeons were submitted to clinical examination. Cloacal swabs, grouped in pool of 4 samples, were analyzed to detect the presence of Coxiella burnetii, Chlamydia psittaci, Chlamydophila spp. using a biomolecular procedure, while individual cloacal samples were examined for Salmonella spp., Campylobacter spp., and yeasts by means of a specific culture media. An ELISA test was used to determine the presence of Giardia spp., and Cryptosporidium spp. coproantigens. Individual serological samples were also tested with the modified agglutination test (MAT) in order to detect antibodies against Toxoplasma gondii. The pigeons did not show any clinical signs. The cloacal pools proved to be negative for C. burnetii DNA while three pools were positive for C. psittaci or Chlamydophila spp. DNAs. Salmonella spp. was not detected. C. jejuni and C. coli were found in 13% and 4% of the samples, respectively. No Giardia spp. and Cryptosporidium spp. were detected. Thirty-three out of 100 samples (33%) were positive for yeast colonies. The seroprevalence for T. gondii was 8%. Although with moderate incidence, potentially zoonotic agents were present thus highlighting the need for sanitary surveillance on feral pigeon colonies.

Intra-articular administration of lidocaine plus adrenaline in dogs: Pharmacokinetic profile and evaluation of toxicity in vivo and in vitro.

The aim of this study was to evaluate the safety of intra-articular (IA) lidocaine plus adrenaline for improving peri-operative analgesia in anaesthetized dogs undergoing arthroscopy of the elbow. A solution of lidocaine (L) 1.98% plus adrenaline 1:100.000 was administered via the IA route and its safety evaluated in terms of cardio-, neuro-, and chondro-toxicity. No bradycardia or hypotension was recorded from induction to the last observational time point. Signs of toxicity of the nervous system could have been masked by the general anaesthesia but lidocaine concentrations detected in the blood were lower than those thought to be capable of producing toxicity. The assessment of in vitro chondrotoxicity showed a dose- and time-dependent effect of lidocaine on the viability of articular cells. Adrenaline appeared to reduce the chondrotoxicity of 1% lidocaine, following an exposure of up to 30 min.

Rhodococcus equi pneumonia in foals: an assessment of the early diagnostic value of serum amyloid A and plasma fibrinogen concentrations in equine clinical practice.

Early diagnosis and prevention of Rhodococcus equi pneumonia in foals represent important goals for equine clinicians. Recent protocols for diagnosis and treatment of Rhodococcosis in foals typically rely on a multimodal approach based on sonographic evidence suggestive of pyogranulomas, sonographic abscess scores and laboratory findings including plasma fibrinogen concentrations, blood biochemistry testing and platelet and leukocyte counts. The aim of this study was to assess the utility of weekly testing of serum amyloid A (SAA) and plasma fibrinogen concentrations in foals to achieve early diagnosis of R. equi pneumonia prior to the onset of clinical signs. This testing was used to simulate a clinically practical screening procedure and compared with thoracic ultrasonography performed in parallel. The present study suggests that SAA does not represent a reliable early marker of Rhodococcosis when plasma concentrations are tested weekly. However, when clinical signs of R. equi pneumonia are present, SAA concentrations may allow clinicians to obtain 'real-time' indications concerning both the progress of infection and the effectiveness of therapy. This study raises the possibility that plasma fibrinogen monitoring starting at 1 week of age and repeated on a weekly basis, could serve as a screening test allowing clinicians to identify foals as suspected of R. equi infection. Future investigations regarding both physiological plasma fibrinogen concentrations in foals as well as fibrinogen kinetics in foals affected with R. equi pneumonia, including the establishment of appropriate reference intervals for the test method employed in this study, will be necessary in order to clarify this possibility.

Intra-articular administration of lidocaine in anaesthetized dogs: pharmacokinetic profile and safety on cardiovascular and nervous systems.

The intra-articular administration of lidocaine is a frequent practice in human orthopaedic surgical procedures, but an eventual absorption of the drug into the bloodstream can lead to toxicity, mainly concerning the central nervous system and the cardiovascular systems. The purpose of this study was to determine the pharmacokinetic profile and the safety, in terms of cardiovascular and CNS toxicity, of lidocaine after intra-articular administration to anesthetized dogs undergoing arthroscopy. Lidocaine 2% was administered to eight dogs before surgery in differing amounts, depending on the volume of the joints involved, and blood samples were taken at predetermined time points. The maximum serum concentration of lidocaine ranged from 0.50 to 3.01 µg/mL (mean ± SD: 2.18 ± 0.91 µg/mL), and the time to reach it was 28.75 ± 15.74 min. No signs of cardiac toxicity were detected during the entire procedure, and possible signs of CNS toxicity were masked by the anaesthesia. However, concentrations reported in literature as responsible for neurotoxicity in dog were achieved in three of eight investigated subjects. Pending further studies, veterinarians should consider the possibility of side effects occurring following the intra-articular administration of local anaesthetics.

Extensive myenteric ganglionitis in a case of equine chronic intestinal pseudo-obstruction associated with EHV-1 infection.

A 7-year-old male trotter horse with a history of recurrent colic displayed clinical findings consistent with chronic intestinal pseudo-obstruction (CIP). At laparotomy, an impaction of the descending colon associated with marked atrophy of the right dorsal colon was found. The horse was humanely destroyed and tissues collected at necropsy examination revealed diffuse enteric ganglionitis comprising an infiltrate of CD3(+) T lymphocytes and plasma cells. At all levels of the intestinal tract the number of myenteric ganglia and of normal ganglion cells was decreased significantly. There were chromatolytic or necrotic neurons and the amount of connective tissue surrounding ganglia was increased. Immunohistochemical studies demonstrated slightly reduced expression of neuron-specific enolase and a moderate increase in expression of S100 and glial fibrillary acidic protein in a sample of right dorsal colon taken during the necropsy examination compared with a biopsy sample taken from the same location. Immunolabelling and semi-nested polymerase chain reaction for equine herpesvirus (EHV)-1 performed on the gut were positive, supporting an aetiological relationship between EHV-1 infection and the enteric ganglionitis.

Insertion sequence IS256 in canine pyoderma isolates of Staphylococcus pseudintermedius associated with antibiotic resistance.

Staphylococcus pseudintermedius is the most frequent staphylococcal species isolated from canine pyoderma. The control of S. pseudintermedius infection is often difficult due to the expanded antimicrobial resistance phenotypes. Antibiotic resistance in staphylococcal pathogens is often associated to mobile genetic elements such as the insertion sequence IS256 that was first described as a part of the transposon Tn4001, which confers aminoglycoside resistance in Staphylococcus aureus and in Staphylococcus epidermidis. In this study a collection of 70 S. pseudintermedius isolates from canine pyoderma was used to investigate antimicrobial susceptibility to 15 antibiotics and the presence of IS256, not revealed in S. pseudintermedius yet. Antibiotic resistance profiling demonstrated that all S. pseudintermedius isolates had a multi-drug resistance phenotype, exhibiting simultaneous resistance to at least five antibiotics; indeed methicillin resistant S. pseudintermedius isolates were simultaneously resistant to at least nine antibiotics and all were also gentamicin resistant. PCR analyses revealed the presence of IS256 in 43/70 S. pseudintemedius isolates. The association between the presence of IS256 and the resistance was particularly significant for certain antibiotics: cefovecin, amikacin, gentamicin and oxacillin (χ(2)p-value<0.05). However, there was a striking result in frequency of strains resistant to gentamicin and oxacillin, suggesting a specific association between the presence of the IS256 element and the determinants for the resistance to these antibiotics. To the best of our knowledge, this is the first report showing the detection of IS256 in S. pseudintermedius isolates and its association with antibiotic resistance. Our findings suggest that S. pseudintermedius may acquire antibiotic resistance genes through mobile genetic elements which may play a predominant role in the dissemination of multi-drug resistance.

Isolation and characterization of ß-haemolytic-Streptococci from endometritis in mares.

The objective of this manuscript was to validate published PCR-based methods for detection of ß-haemolytic Streptococci by comparison with established bacteriological techniques using 85 clinical isolates recovered from uterine swabs of mares with clinical signs of endometritis and to determine the distribution of SeeL/SeeM and SzeL/SzeM superantigens in isolates of Streptococcus equi subsp. equi (S. equi) and S. equi subsp. zooepidemicus (S. zooepidemicus). The conventional bacteriological techniques showed the vast majority of these isolates (78) were S. zooepidemicus with just 5 Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis) and 2 S. equi strains detected. The PCR analyses confirmed the bacteriological results demonstrating the reliability of the 16S rRNA PCR assay for detecting Streptococci, the multiplex PCR for differentiating between S. zooepidemicus, and S. equi, and PCR assays based on streptokinase genes for identification of S. equisimilis. PCRs for genes encoding superantigens revealed seeL and seeM specific amplicons with size of approximately 800 and 810 bp respectively for the S. equi strains and for 2 S. zooepidemicus strains. To our knowledge, this is the first report of szeL and szeM possession by S. zooepidemicus isolates derived from endometritis in mares.

Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals.

Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by PCR and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the pcr for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to EHV-1 and/or EHV-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.

Seroepidemiological survey of Rhodoccocus equi infection in asymptomatic horses from Bursa, Izmir and Istanbul provinces, Turkey.

In order to assess the Rhodococcus equi infection in three provinces of Turkey (Bursa, Izmir and Istanbul), 696 sera from healthy foals and adult horses were tested by indirect ELISA using a R. equi reference strain (ATCC 6939) as antigen. 103 sera (14.80%) with titres >0.646 resulted positive. Seroprevalence was significantly higher (P=0.0053) in male than in female horses of Istanbul province, although higher antibody titres (mean value) were observed in the female group of Bursa and Izmir provinces with differences estimated between provinces (P=0.0002). Seroprevalence was correlated with age: foals aged less than 1 year (P<10(-4)) and horses from 5 to 10 years old (P=0.018) resulted more infected in Bursa and Izmir provinces. Our findings indicate that R. equi infection actually occurs in all investigated provinces, suggesting the importance of serological survey to diagnose the infection and to prevent the zoonotic risk.

Staphylococcus aureus: study of genomic similarity of strains isolated in veterinary pathology using amplified fragment length polymorphism (AFLP).

Staphylococcus aureus is a widespread pathogen causing infections in different animal species. The extensive use of antibiotics, particularly methicillin, causes the rise of antibiotic-resistant strains (MRSA). In order to verify the epidemiology and genetic relatedness among MRSA and sensible strains (MSSA), an accurate fingerprinting technique, the amplified fragment length polymorphism (AFLP), was carried out. The isolates were cultured, subdivided on MRSA and MSSA and submitted for the genomic DNA extraction that was utilized for AFLP. The data were analysed for genetic similarity using the Dice coefficient. The results of genomic analysis among MRSA and MSSA and within them revealed that the major component of variation was due to variation within strains (82.12%), while variance among strains was lower (17.88%). The low level of genomic similarity found among S. aureus strains implies high level of genetic diversity. Different similarity was found as well in all strains independently of the source.

Detection of antibodies against Rhodococcus equi in Alpaca (Lama pacos) in Italy.

After isolating the two virulent strains of Rhodococcus equi from Alpaca, a serological survey of Rhodococcus equi infection was carried out on 57 blood samples of Alpaca collected in Central Italy. The survey was performed with an ELISA test using a reference R. equi strain as antigen (ATCC 33701). Four (7.0%) sera (OD greater or equal to 0.3) tested positive, while five (8.77%) were considered doubtful (OD between 0.2 and 0.3). This is the first serological survey of R. equi infection in Alpaca in Italy. The results indicate that besides the horse R. equi infection could also affect some ruminant species. The ELISA test was recently introduced as a reliable diagnostic method in horses and was adapted to alpaca.