Evidence of new sequence types of methicillin-resistant Staphylococcus pseudintermedius in Italy.

Staphylococcus pseudintermedius (SP) and methicillin-resistant SP (MRSP) is one of the most important veterinary pathogens in the dog. Herein, from a total of 126 S. pseudintermediusstrains, 23 MRSP (18%) were identified. Multi-Locus Sequence Typing (MLST) revealed that most of MRSP strains belonged to ST71 (26%), which have been already reported in Italy and other countries. Interestingly, nine new sequence types (39%), from 1053 up to 1061, were described for the first time. Moreover, the isolated MRSP strains showed relevant antibiotic resistance profiles. This report highlights the circulation of new sequence types of MRSP in Italy and underlines the need of a global epidemiological surveillance to limit the increasing spread of multidrug-resistant MRSPstrains worldwide, since they may represent a considerable concern for dog's health.

In vitro virucidal activity of sodium hypochlorite against canine parvovirus type 2.

Canine parvovirosis is a very contagious, severe and often lethal infectious disease of dogs caused by canine parvovirus type 2 (CPV-2). Parvoviruses are very resistant to several disinfectants while are sensitive to halogens such as sodium hypochlorite which is often used for decontamination of veterinary clinics and animal housing facilities due to its broad spectrum of activity. If compliance with vaccination programmes and with proper disinfection plans is ensured, there should be no continuous, nor frequent, CPV-2 outbreaks in kennels and veterinary clinics. However, a continuous spread of CPV-2 infections is observed, even in kennels where an appropriate vaccination programme is applied, and this imposes a re-evaluation of disinfection protocols using sodium hypochlorite. The aim of the present study was to determine the effect of concentration, contact time and presence of organic matter on the virucidal activity of sodium hypochlorite against several CPV-2 strains. A sensitive in vitro assay capable of measuring the infectivity of CPV-2 was employed to determine the efficacy of three different concentrations of sodium hypochlorite. The data indicate that using a 0.75% sodium hypochlorite solution for a short contact time (1 min) can reduce significantly the CPV-2 titres and that even lower concentrations, i.e. 0.37%, can efficiently inactivate the viruses provided that the contact time is extended to 15 min. Results also confirm the importance of cleaning before disinfection since the presence of organic matter totally abrogated the virucidal activity of sodium hypochlorite solutions against the three CPV-2 strains.

Frequency, antimicrobial susceptibility and clonal distribution of methicillin-resistant Staphylococcus pseudintermedius in canine clinical samples submitted to a veterinary diagnostic laboratory in Italy: A 3-year retrospective investigation.

In the last decade there has been a rapid global spread of methicillin-resistant Staphylococcus pseudintermedius (MRSP) clones displaying multidrug resistance in dogs. We investigated prevalence, antimicrobial susceptibility and clonal distribution of MRSP isolated from clinical canine samples between during 2011-2014. Following species identification by nuc PCR, MRSP were confirmed by the presence of mecA and characterized by antimicrobial susceptibility testing, Pulsed Field Gel Electrophoresis (PFGE), SCCmec typing, and Multi-Locus Sequence Typing (MLST) of a few isolates having distinct PFGE profiles. Both the MRSP isolation frequency in the 175 samples tested (12%) and the prevalence of methicillin resistance amongst the 63S. pseudintermedius isolates (33%) were high compared to a previous study in Italy. Sequence type (ST)71 carrying SCCmec type II-III, described as the epidemic European MRSP clone, accounted for approximately half of the isolates. The remaining isolates belonged to ST410-SCCmec type II-III, ST258-SCCmec type IV and other three clones associated with SCCmec type IV (ST261, ST290 and ST477). MRSP were consistently resistant to potentiated sulfonamides, and more frequently to clindamycin, ciprofloxacin and doxycycline than methicillin-susceptible isolates. Gentamicin was the only antibiotic showing good in vitro activity on all MRSP with 20 of the 21 isolates being susceptible. Results confirm a high prevalence of MRSP amongst clinical samples in Italy, revealing the emergence of new clones other than ST71, such as ST258, ST410, ST261, ST290 and ST477, here describe for the first time. Implementation of antimicrobial stewardship and surveillance programmes are required to prevent the emergence of new MRSP clones and reducing transmission in small animal practice.

Pneumonia associated with Salmonella spp. infection in a cat receiving cyclosporine.

Salmonellosis is uncommon in cats, usually affects the gastrointestinal tract or skin, and can be fatal. This report describes a domestic shorthair cat with severe pneumonia caused by Salmonella spp. without accompanying gastrointestinal or skin manifestations, in which previous administration of cyclosporine may have played a permissive role in its development. Clinical and laboratory findings as well as follow-up are described from diagnosis until complete recovery. This unusual presentation serves to alert practitioners to consider Salmonella spp. as a possible cause of lung disease in cats, especially if immunocompromised.

Periodontal disease associated with red complex bacteria in dogs.

OBJECTIVE: Red complex bacteria (Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis) play a major role in the aetiology of periodontal disease in humans. This study was designed to evaluate the association of such bacteria with periodontal disease in dogs. METHODS: Seventy-three subgingival samples taken from dogs ranging from 2 months to 12 years (median age 4 years) were tested for red complex bacteria using a polymerase chain reaction assay. RESULTS: Thirty-six of 73 (49 · 3%) dogs were found to be positive for T. forsythia and P. gingivalis. Dogs with gingivitis or periodontitis were more likely to be infected with T. forsythia and P. gingivalis [odds ratio (OR) 5 · 4 (confidence interval (CI) 1 · 9-15 · 6), P = 0 · 002] than healthy animals. Only 3 (4 · 1%) of 73 samples were positive for red complex bacteria, but the association with periodontal disease was not significant. CONCLUSION AND CLINICAL RELEVANCE: The results indicate that involvement of red complex bacteria in periodontal disease in dogs is similar to that observed in humans. Only the concurrent presence of T. forsythia and P. gingivalis were correlated to periodontal disease in dogs in this study.

Enteric disease in dogs naturally infected by a novel canine astrovirus.

Infection by a novel canine astrovirus was associated with gastroenteritis in two dogs. The virus displayed 70.3 to 73.9% amino acid identity to other canine astroviruses in the full-length capsid. Specific antibodies were detected in the convalescent-phase sera of the dogs, indicating seroconversion. Also, the virus appeared weakly related antigenically to the prototype canine astrovirus isolate ITA/2008/Bari.

Methicillin-resistant staphylococci carriage in the oral cavity: a study conducted in Bari (Italy).

OBJECTIVES: The oral cavity may represent a site of colonization by antibiotic-resistant bacteria, such as methicillin-resistant staphylococci (MRS). To define the prevalence of staphylococci and MRS in the oral cavity, an observational study was carried out in the city of Bari (Italy). METHODS: Sixty subjects were asked to provide oral samples and a questionnaire about risk factors of colonization by MRS. An enrichment medium specific for staphylococci was used for the isolation. RESULTS: Swabs and corresponding questionnaires were available from 36 out of 60 patients. Staphylococci were isolated from seven out of 36 samples (prevalence 19.4%). Among the seven staphylococcal isolates, three were Staphylococcus aureus, and one strain, belonging to S. epidermidis species, was found to be MR (1.7%). No methicillin-resistant S. aureus were isolated. Five out of seven staphylococcal isolates exhibited resistance to more than two classes of non-beta-lactams antimicrobials. None of the risk factors analysed correlated with the status of MRS carriers, except the presence of oral disease. CONCLUSIONS: The results underline the potential role of the oral cavity as a reservoir of staphylococci.

Epizootic abortion related to infections by Chlamydophila abortus and Chlamydophila pecorum in water buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis) are affected by high rates of embryonic mortality and abortion related to infectious diseases and non-infectious factors. A number of viral and bacterial infections have been associated with reproductive failure, but there is limited information on the role of chlamydial infections. In order to investigate the presence and the role of Chlamydiaceae in water buffalo a retrospective study was performed in a herd with a history of reproductive failure. During an 11-month period, the pregnant heifers suffered an abortion rate of 36.8% between the 3rd and 7th month of pregnancy. Antibodies to Chlamydiaceae were detected in 57% of the aborted cows, and in 0% of the overtly healthy cows used as control. By a nested-PCR assay, three of 14 vaginal swabs from aborted animals tested positive for Chlamydophila agents and, additionally, three out of seven aborted fetuses tested positive for Chlamydophila spp., with two being co-infections by Cp. abortus and Cp. pecorum and one being characterised as Cp. abortus. Sequence analysis of the amplicons confirmed the results of the nested-PCR. The presence of anti-Chlamydiaceae antibodies in more than half of the aborting animals (P<0.002) and the detection of Chlamydophila agents in several fetal organs and in the vaginal swabs are consistent with the history of abortions observed in the herd and suggest an abortifacient role by Chlamydophila spp. in water buffalo (B. Bubalis) herds.

Identification of a porcine calicivirus related genetically to human sapoviruses.

Whether animals may act as reservoirs for human caliciviruses is unclear. By sequence analysis of a short fragment of the RNA-dependent RNA polymerase (RdRp) region, porcine sapovirus (SaV) strains that genetically resemble human SaVs have been detected in piglets, but more-informative sequences (capsid gene) were not available for a precise characterization. In this study, the 3' terminus (the 3' end of open reading frame 1 [ORF1], including the polymerase complex and the complete capsid; ORF2; and the 3' untranslated region) of one such human SaV-like strain, 43/06-18p3/2006/It, was determined, revealing that these viruses are more related genetically to human (47.4 to 54.9% amino acid identity) than to animal (35.2 to 44.7% amino acid identity) SaVs in the capsid gene. In addition, the recombination-prone RdRp-capsid junction region was highly conserved with those of human SaVs of genogroup GI. The presence of porcine viruses similar to human SaVs is a significant finding because of the potential for zoonotic infections or generation of porcine/human recombinants.

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products.

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA.

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.

Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003-2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.

Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products.

Staphylococcus aureus is considered to be one of the leading causes of food-borne illnesses. Milk, dairy products and meats are often contaminated with enterotoxigenic strains of this bacterium. Foodstuff contamination may occur directly from infected food-producing animals or may result from poor hygiene during production processes, or the retail and storage of foods, since humans may carry the microorganism. The number of S. aureus strains that exhibits antimicrobial-resistance properties has increased, together with the potential risk of transmitting the same properties to the human microflora via foods or inducing infections hard to be treated. This paper reports the results of a 3-year survey (2003-2005) on the occurrence of S. aureus in meat and dairy products. Of 1634 samples examined, 209 (12.8%) were contaminated with S. aureus. A total of 125 enterotoxigenic S. aureus strains were biotyped and their antimicrobial resistance pattern tested. Most of the isolated strains produced SED (33.6%), followed by SEA (18.4%), SEC (15.2%), SEB (6.4%) and belonged mainly to the Human ecovar (50.4%), followed by Ovine (23.2%), Non-Host-Specific (17.6%), Bovine (7.2%) and Poultry-like (1.6%) ecovars. Finally, the 68.8% analysed strains showed antimicrobial resistance properties at least at one of antibiotics tested. Human biotype showed antimicrobial resistance at more than one antibiotic than the other biotypes (p<0.05). The results provided evidence that the presence of enterotoxigenic and antimicrobial resistant strains of S. aureus has become remarkably widespread in foods. This calls for better control of sources of food contamination and of the spread of antimicrobial-resistance organisms.

Identification of group A porcine rotavirus strains bearing a novel VP4 (P) Genotype in Italian swine herds.

The VP4 gene of a G5 Italian porcine rotavirus strain, 344/04-1, was nontypeable by PCR genotyping. The amino acid sequence of the full-length VP4 protein had low identity (

Influence of free-stall flooring on comfort and hygiene of dairy cows during warm climatic conditions.

An evaluation of behavioral and hygienic conditions was carried out with 4 materials used as free-stall flooring for dairy cows: polyethylene vinyl acetate (EVA) and polypropylene vinyl acetate (PVA) mats, wood shavings, and solid manure. The free-stall type selected by cows was evaluated in response to changes in environmental temperature and humidity. Two tests were used: 1) a preference test, in which 8 cows were housed in a pen with 32 free stalls and 4 types of flooring; and 2) an aversion test, in which 32 cows were placed in 4 pens, each with 8 free stalls. The free stalls in each pen had a single type of bedding material. These tests showed that the comfort of dairy cows was predominantly influenced by environmental conditions. The preference test for lying showed that cows preferred free-stall floors with EVA mats over those with PVA mats, wood shavings, and solid manure (332.4 +/- 24.0 vs. 130.8 +/- 6.2, 160.9 +/- 23.7, and 102.6 +/- 23.2 min/d, respectively), but under conditions of heat stress, with a temperature-humidity index > 80, they chose wood shavings and solid manure lying areas. These results were confirmed by the aversion test. In all experimental and environmental conditions, the PVA mats were the least suitable. The mats contaminated with organic manure and the free stalls bedded with wood shavings and organic solids did not differ in either the coliform load on the lying surfaces (EVA mats: 290 +/- 25; PVA mats: 306 +/- 33; wood shavings: 290 +/- 39; and solid manure: 305 +/- 23 log(10) cfu/mL) or the total bacterial count in the raw milk (EVA mats: 232 +/- 22; PVA mats: 233 + 24; wood shavings: 221 +/- 24; and solid manure: 220 +/- 25 log(10) cfu/mL). These results demonstrate that the comfort of dairy cows housed in barns with free stalls as resting areas does not depend only on the material used, but also on the value of the material in microenvironmental conditions.

Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain.

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep.

Outer membrane proteins (OMPs) of Rev-1 strain of Brucella melitensis were used in a Western blotting assay for the serological diagnosis of brucellosis in ovine sera. Fifty-four sheep sera were tested and divided into the following groups: Group A) n. 9 samples from one sheep that had been experimentally infected with Y. enterocolitica O:9; Group B) n. 10 samples collected from sheep infected with Brucella melitensis and 1 sample from a sheep vaccinated with the Rev 1 strain; Group C) n. 10 samples collected in "officially brucellosis-free" herds; Group D) n. 12 samples classified as "suspicious"; Group E) n. 12 samples classified as "positive". Antibodies were detected by routine tests performed for the diagnosis of brucellosis in serum samples of the sheep infected with Y. enterocolitica O:9 after the 2nd week post infection. In the WB assay, sera of group B recognised a 17 kDa protein, whereas sera of groups A, and D and 9 out of 12 of group E exhibited no reactivity to this protein. The results obtained encourage the use of the WB assay as a confirmatory test for the diagnosis of brucellosis.

Isolation of Salmonella strains from reptile faeces and comparison of different culture media.

AIMS: To provide information on epidemiology and isolation of Salmonella strains from reptiles. METHODS AND RESULTS: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50.5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport-Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58.7%), sulphamethoxazole (55.5%), streptomycin (32.6%), tetracycline (19.6%), ampicillin (17.4%) and nalidixic acid (13.1%). CONCLUSIONS: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.

Methicillin resistance in staphylococci isolated from subclinical mastitis in sheep.

One hundred ovine milk samples were subjected to bacteriological analysis to detect staphylococci. Twenty-four staphylococcal strains isolated were characterised for methicillin resistance with disk diffusion test (DDT) after incubation at 24 and 48 h, oxacillin agar screen test, Minimal Inhibitory Concentration (MIC), nitrocefin test for beta-lactamase production and PCR for the mecA gene. Nine staphylococcal strains resulted resistant in DDT; some differences in the halo diameter at double incubation period were noted; eight of these strains were resistant in MIC test; just one strain was positive to oxacillin agar screen test. All strains were mecA negative by PCR and positive by nitrocefin test. On the basis of these results methicillin-resistant strains can be classified as beta-lactamase hyperproducers.

Inactivated vaccine induces protection against Mycoplasma agalactiae infection in sheep.

The efficacy of an inactivated oil-emulsion vaccine against Mycoplasma agalactiae was evaluated by an experimental infection of sheep. The vaccinated sheep developed high levels of antibodies and, following challenge, they did not develop any clinical signs of disease and the mycoplasmas were not detected, either by isolation trials or PCR assays carried out both on nasal swabs and milk specimens. The unvaccinated-challenged sheep showed typical signs of M. agalactiae infection and bacterial shedding. The results obtained indicate a good efficacy of the vaccine in eliciting protection against M. agalactiae infection.