High genotypic diversity among methicillin-resistant Staphylococcus pseudintermedius isolated from canine infections in Denmark.

BACKGROUND: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged globally in companion animals in the last decade. In Europe, the multidrug-resistant sequence type (ST)71 is widespread, but recently other clones have appeared. The objective of this study was to examine genotypic diversity and antimicrobial resistance of clinical MRSP isolates obtained from dogs, including dogs sampled on multiple occasions, in Denmark over a six-year period. For that purpose a total of 46 clinical MRSP isolates obtained from 36 dogs between 2009 and 2014 were subjected to antimicrobial susceptibility testing, multilocus-sequence typing (MLST) and SCCmec typing. RESULTS: Twenty-three sequence types were identified with ST71, mostly associated with SCCmec II-III, as the most common occurring in 13 dogs. Among the remaining 33 isolates, 19 belonged to clonal complex (CC)258 comprising ST258-SCCmec IV and its single- and double-locus variants. These were susceptible to 4-7 of the 22 antibiotics tested, whereas CC71 isolates were susceptible to only 2-5 antibiotics. Clone-specific differences were especially pronounced for fluoroquinolones and aminoglycosides with most CC71 isolates being resistant and almost all CC258 isolates being susceptible. Sixteen of the 19 CC258 isolates had oxacillin MICs of 0.5 g/L, whereas MICs for CC71 isolates were consistently above 4 g/L. Four of five dogs representing multiple isolates had distinct STs on different sampling events. CONCLUSIONS: The overall genotypic diversity of MRSP is high in Denmark indicating multiple acquisitions of SCCmec into distinct clones, and mutational evolution, which appears to be particularly rapid for certain ancestral clones such as ST258. ST71-SCCmec II-III is the most common MRSP lineage and is typically multidrug-resistant. CC258-SCCmec IV isolates, which emerged in Denmark since 2012, display susceptibility to a wider range of antimicrobials. The isolation of distinct STs in individual dogs over time suggests repeated exposure or short-term genetic evolution of MRSP clones within patients.

Changes in peripheral blood leucocytes of sheep experimentally infected with Mycoplasma agalactiae.

Contagious agalactia is a serious disease of small ruminants affecting mainly mammary glands, joints and eyes. In sheep, the main aetiological agent is Mycoplasma agalactiae (Ma) whose abilities to persist in the target organs are known. Since there is no information on the effect of acute and chronic Ma infection on circulating leucocytes, the present study was designed to monitor granulocytes, monocytes, T and B lymphocytes, by flow cytometry, in female lactating sheep nasally infected with Ma. A profound depletion of leucocytes was observed from day 5 to day 34 post infection (p.i.). In particular, while the granulocytes returned to baseline levels by day 12 p.i., the monocytes remained significantly low until day 20 p.i. The infection caused a prolonged depletion of peripheral T lymphocytes (both CD4(+) and CD8(+)) while B lymphocytes remained unaltered throughout the study. Mycoplasma agalactiae was detected by real-time PCR in several anatomical sites (ear, nose and milk) from day 2-5 p.i. until the end of the study (i.e., day 50 p.i.) while a transient bacteraemia was observed from day 5 to day 12 p.i. The leucopenia observed following intranasal Ma infection is likely due to leucocyte infiltration within the target organs.

Long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination.

Canine parvovirus (CPV) modified live virus vaccines are able to infect vaccinated dogs replicating in the bloodstream and enteric mucosa. However, the exact duration and extent of CPV vaccine-induced viremia and fecal shedding are not known. With the aim to fill this gap, 26 dogs were administered two commercial vaccines containing a CPV-2 or CPV-2b strain and monitored for 28 days after vaccination. By using real-time PCR, vaccine-induced viremia and shedding were found to be long lasting for both vaccinal strains. Vaccinal CPV-2b shedding was detected for a shorter period than CPV-2 (12 against 19 mean days) but with greater viral loads, whereas viremia occurred for a longer period (22 against 19 mean days) and with higher titers for CPV-2b. Seroconversion appeared as early as 7 and 14 days post-vaccination for CPV-2b and CPV-2 vaccines, respectively. With no vaccine there was any diagnostic interference using in-clinic or hemagglutination test, since positive results were obtained only by fecal real-time PCR testing. The present study adds new insights into the CPV vaccine persistence in the organism and possible interference with diagnostic tests.

Characterisation of a catalase-negative methicillin-resistant Staphylococcus aureus isolate from a dog.

A catalase-negative MRSA strain and a methicillin resistant Staphylococcus pseudintermedius strain (MRSP) were isolated from a dog affected by a severe form of pododermatitis. The catalase negative isolate was typed as SCCmec I, PVL negative, ST5 t002 strain. A deletion at position 487 of the kat gene altered the functionality of the catalase enzyme. This is the first report of a catalase-negative MRSA in animals. As catalase test is a rapid assay routinely employed for the identification of staphylococci in clinical microbiology laboratories, the presence of MRSA with this uncommon phenotype may be underestimated. Moreover, catalase-negative staphylococci should be investigated more in-depth in order to assess their virulence.

Clinical bovine piroplasmosis caused by Babesia occultans in Italy.

A clinical outbreak of bovine piroplasmosis was reported in Italy. The etiological agent was characterized as Babesia occultans, a parasite regarded as apathogenic and never detected before in continental Europe. This report paves the way for further studies to assess the occurrence of this tick-transmitted protozoan in other European regions.

Detection of a canine parvovirus type 2c with a non-coding mutation and its implications for molecular characterisation.

An epidemiological survey for canine parvovirus (CPV) was conducted by collecting 615 faecal samples from dogs with diarrhoea in different European countries. Molecular methods showed that CPV-2a was predominant in most countries, followed by CPV-2c and CPV-2b, whereas 30 strains were not characterised. By sequence analysis of the full-length VP2 gene, 20 of these viruses were characterised as CPV-2c mutants having the synonymous mutation A4061G in the probe-binding region that prevented correct strain characterisation. A real-time polymerase chain reaction (PCR) assay using a minor groove binder probe was able to recognise both mutant and classical CPV-2c strains. These results indicate that the emergence of CPVs with mutations affecting the oligonucleotide-binding region needs a continuous update of molecular diagnostic tools in order to detect efficiently those emerging strains.

Environmental contamination by dog’s faeces: a public health problem?

The risk to public health from the large number of dog stools present on streets of urban areas is cause for concern. Dog faeces may be a serious hazard because they may contain microorganisms that are both pathogenic to humans and resistant to several classes of antibiotics. The aim of this study was to evaluate the potential for zoonotic infections and for the presence of antibiotic resistant bacteria in canine faeces which contaminates the urban environment. A total of 418 canine faecal samples were collected from streets in seven areas of Bari, Southern Italy. We have isolated multi-drug resistant Enterococci and meticillin-resistant Staphylococcus aureus from these dog faecal samples. The presence of the resistant bacteria in an urban environment may represent a public health hazard which requires control measures by competent authorities. No Salmonella, Yersinia or Campylobacter species were isolated. Giardia cysts were detected in 1.9% of the samples. The predominant Enterococcus species were E. faecium (61.6%), E. gallinarum (23.3%) and E. casseliflavus (5.5%). Other species, including E. faecalis were also isolated. These strains were resistant to clindamycin (86.3%), tetracycline (65.7%), erythromycin (60.27%) and ampicillin (47.9%). High-level aminoglycoside resistance (HLAR) was found in 65.7% of enterococci. Resistance to three or more antibiotics and six or more antibiotics were observed in 67.12% and 38.4% of Enterococcus spp., respectively. Resistance to vancomycin and teicoplanin was not detected in any of the Enterococcus spp. isolated. Methicillin-resistant Staphylococcus aureus was isolated in 0.7% of the faecal samples. Canine faeces left on the streets may represent a risk factor for transmission of microorganisms and a reservoir of multidrug- resistant bacteria thus contributing to the spread of resistance genes into an urban area.