Correlation between genetic variability and virulence factors in clinical strains of Malassezia pachydermatis of animal origin.

Malassezia pachydermatis is a yeast belonging to the microbiota of the skin and mucous membranes of dog and cat, but it can also act as pathogen, causing dermatitis. The aim of this work was to evaluate the genetic variability of M. pachydermatis strains isolated from symptomatic dogs and cats and determine a correlation between genotype and phenotype. For this purpose eleven strains of M. pachydermatis were molecularly classified by nested-polymerase chain reaction (nested-PCR) based on ITS-1 and ITS-2 regions, specific for fungal rRNA genes. Furthermore, random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates identifying four different genotypes. Strains belonging to genotype 1 produced the highest amount of biofilm and phospholipase activity. The inflammatory response induced by M. pachydermatis strains in immortalized human keratinocytes (HaCat cells) was significantly different when we compared the results obtained from each strain. In particular, HaCat cells infected with the strains belonging to genotypes 1 and 2 triggered the highest levels of increase in TLR-2, IL-1ß, IL-6, IL-8, COX-2 and MMP-9 expression. By contrast, cells infected with the strains of genotype 3 and those of genotype 4 did not significantly induce TLR-2 and cytokines. The results obtained might suggest a possible association between genotype and virulence factors expressed by M. pachydermatis strains. This highlights the need for a more accurate identification of the yeast to improve the therapeutic approach and to monitor the onset of human infections caused by this emergent zoonotic pathogen.

An update on microbiological causes of canine otitis externa in Campania Region, Italy

Objective: To update the recent knowledge of the microbiological causes of canine otitis externa in Campania Region (Italy) and the antibiotic susceptibility patterns of the iso-lated strains. Methods: A total of 122 dogs were examined by otoscopy, and auricular swab samples were collected from both ears in 74 dogs presenting clinical bilateral otitis and from single ears in 48 dogs displaying clinical unilateral otitis. Cytological examination, bacteriological analysis and antimicrobial susceptibility tests were performed. Results: Thirty-one out of 122 dogs were positive for yeast species (25.4%, 95% con-fidence interval (CI): 18.2%–34.2%) with a higher prevalence of Malassezia pachy-dermatis (21/31 isolates, 67.7%, CI: 48.5%–82.7%), and a total of 91 out of 122 dogs were positive for bacterial species (74.6%;CI:65.8%–81.8%) with a higher prevalence of Staphylococcus pseudintermedius (45/143 isolates, 31.5%, CI: 24.1%–39.8%). These results are the first description of Streptococcus agalactiae-associated otitis. The yeasts isolated showed high levels of susceptibility to all antifungal agents tested; on the con-trary all the isolated bacterial strains were highly resistant to at least four out of ten antimicrobial classes. Both Gram-positive and Gram-negative bacteria showed high resistance to amoxicillin/clavulanate and kanamycin hence they are not recommended as initial empirical therapy for the otitis treatment. Conclusions: This update illustrates an increase in antibiotic resistances providing an insight into the current knowledge of the therapeutic procedures followed on canine otitis externa in Italy. It also emphasizes the importance of considering the results of the microbiological and sensitivity tests to decide on an appropriate antibiotic therapy.

Conjunctival cytological examination, bacteriological culture, and antimicrobial resistance proifles of healthy Mediterranean buffaloes (Bubalus bubalis) from Southern Italy

Objective:To assess normal conjunctival cytological and bacteriological/fungal flora features in the Mediterranean buffalo (Bubalus bubalis). Methods:Swabs were taken from the inferior conjunctival sac of both eyes of 57 healthy female buffaloes aged 24-36 months, with no evidence of ocular disease, farmed in Campania region (Southern Italy), for microbiological analysis. Conjunctival eye specimens of both eyes were subsequently obtained by a cyto-brush, for cytological analysis. The antimicrobial susceptibility of bacterial isolates was also determined using the disk-diffusion method on Mueller Hinton agar plates. Results: Cytological examination of conjunctival swab specimens (114 eyes) revealed epithelial cells (basal, intermediate, columnar and superficial) in all samples, whereas neutrophils, lymphocytes and plasma cells were present in 70%, 10%and 2%of samples, respectively. Microorganisms, for a total of 261 aerobic bacteria and 6 fungi, were isolated from 112/114 conjunctival samples (98.25%;95%confidence interval (CI):93.18–99.70). Only two conjunctival swabs did not yield bacteria and/or fungi (2/114, 1.75%;95% CI:0.30–6.82). Gram-positive aerobes were most commonly cultured (181/261, 69.35%;95%CI: 63.31–74.81), with Enterococcus faecium and Staphylococcus lentus predominating. Escherichia coli was the most frequently isolated as Gram-negative bacteria (80/261, 30.65%;95%CI:25.19–36.69). The antimicrobial resistance patterns of the isolated bacteria showed amoxycillin/clavulanic acid and cephalothin as the least sensitive antibiotics for both Gram-positive and Gram-negative bacteria. Conclusions: These results provided first information on normal conjunctival ocular microflora and cytological features in Mediterranean buffalo.

Occurrence of enteropathogenic bacteria in urban pigeons (Columba livia) in Italy.

This study was aimed at evaluating the prevalence of Campylobacter spp., Escherichia coli O157, Salmonella spp., and related virulence factors (the cdt, stx, and eae genes) in urban pigeons of the coastal area of the Campania region (southern Italy). To achieve this goal, cloacal swab samples from a total of 1800 urban pigeons were collected and subjected to culture methods, PCR, and serotyping. The results of the present study showed a prevalence of 48.3% (870/1800), 7.8% (141/1800), and 0.9% (16/1800), for C. jejuni, E. coli O157, and S. Typhimurium, respectively. All C. jejuni isolates (870/870) carried cdt genes, whereas all E. coli O157 isolates carried stx genes, and 14.9% (21/141) carried the eae gene. These findings clearly show that urban pigeons in the coastal area of the Campania region may constitute an environmental reservoir of these pathogens, thus representing a source of infection for other birds, livestock, and humans.

A comparative evaluation of methicillin-resistant staphylococci isolated from harness racing-horses, breeding mares and riding-horses in Italy.

OBJECTIVE: To investigate the prevalence of methicillin-resistant staphylococci (MRS) which is a potencial risk factor of transmission between animals and humans in different types of horses (harness racing-horses, breeding mares and riding-horses) and to compare the antimicrobial resistance of the isolates. METHODS: A total of 191 healthy horses, housed at different locations of the Campania Region (Italy), were included in the study. Nasal swab samples were collected from each nostril of the horses. The mecA gene was detected by a nested PCR technique. Antibiotic susceptibility was tested for each isolate. RESULTS: MRS was isolated from nasal samples of 68/191 (35.6%; 95% CI: 28.9%-42.9%) healthy horses. All isolates were coagulase-negative with the exception of two coagulase-positive MRS strains, identified as Staphylococcus aureus and Staphylococcus pseudintermedius, 2/83 (2.4%; 95% CI: 0.4%-9.2%). Interestingly, both coagulase-positive MRS isolates were from harness racing-horses. These horses also presented a significantly higher positivity for MRS (53.3%; 95% CI: 40.1%-66.1%) than the breeding mares and riding-horses groups. Antibiotic susceptibility testing showed difference between isolates due to different origins except for an almost common high resistance to aminopenicillins, such as ampicillin and amoxicillin. CONCLUSIONS: It can be concluded that harness racing-horses may act as a significant reservoir of MRS as compared to breeding mares and riding-horses.

A comparative evaluation of methicillin-resistant staphylococci isolated from harness racing-horses, breeding mares and riding-horses in Italy

Objective:To investigate the prevalence of methicillin-resistant staphylococci (MRS) which is a potencial risk factor of transmission between animals and humans in different types of horses (harness racing-horses, breeding mares and riding-horses) and to compare the antimicrobial resistance of the isolates. Methods:A total of 191 healthy horses, housed at different locations of the Campania Region (Italy), were included in the study. Nasal swab samples were collected from each nostril of the horses. The mecA gene was detected by a nested PCR technique. Antibiotic susceptibility was tested for each isolate. Results: MRS was isolated from nasal samples of 68/191 (35.6%; 95% CI: 28.9%-42.9%) healthy horses. All isolates were coagulase-negative with the exception of two coagulase-positive MRS strains, identified as Staphylococcus aureus and Staphylococcus pseudintermedius, 2/83 (2.4%; 95%CI: 0.4%-9.2%). Interestingly, both coagulase-positive MRS isolates were from harness racing-horses. These horses also presented a significantly higher positivity for MRS (53.3%; 95%CI: 40.1%-66.1%) than the breeding mares and riding-horses groups. Antibiotic susceptibility testing showed difference between isolates due to different origins except for an almost common high resistance to aminopenicillins, such as ampicillin and amoxicillin. Conclusions:It can be concluded that harness racing-horses may act as a significant reservoir of MRS as compared to breeding mares and riding-horses.

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares.

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P<0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P<0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P<0.0001). The DGS produced significantly more (P=0.003) intact cells than CB and LVF. Distorted cells were significantly (P=0.001) more frequent in smears by LVF. The CB harvested significantly (P=0.009) more fragmented cells. CB and LVF produced significantly (P<0.0001; P=0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P=0.0062; P=0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P=0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k≤0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.

Methicillin-resistant staphylococci isolated from healthy horses and horse personnel in Italy.

Methicillin-resistant staphylococci (MRS) were isolated from nasal swabs of 56 of 159 (35.2%; 95% confidence interval [CI]: 27.9-43.2%) healthy horses. Two nasal swabs were collected from each horse; 43 of 159 (27%; 95% CI: 20.5-34.8%) of the cohort were colonized by MRS strains in 1 nostril, while in the remaining 13 of 159 (8.2%; 95% CI: 4.6-13.9%), different or identical MRS strains were isolated in both nostrils. Of the 29 humans in close contact with the horses tested, 4 (13.8%; 95% CI: 4.5-32.6%) were found to be carriers of MRS. All isolates were coagulase negative with the exception of 2 coagulase-positive MRS strains, Staphylococcus aureus and Staphylococcus pseudintermedius, both isolated from horses. To assay the methicillin resistance, a susceptibility test to oxacillin with standardized disk diffusion method, a PBP-2a latex agglutination test, and a methicillin resistance gene (mecA) polymerase chain reaction assay were performed. Pulsed-field gel electrophoresis patterns of isolates from horses and humans in close contact with the horses revealed similarity. The results suggest evidence of transmission between animals, from animals to humans, and vice versa.

Shedding of Brucella abortus rough mutant strain RB51 in milk of water buffalo (Bubalus bubalis).

The objective of this study was to determine if Brucella abortus rough mutant strain RB51 (SRB51) is eliminated in buffalo milk. Thirty Brucella-free female buffaloes were used in this study: ten 4-5 years old were inoculated with the triple of the recommended calfhood dose of SRB51 by subcutaneous route, ten 2-3 years old at the first lactation were previously vaccinated twice as calves with triple the recommended calf dose of RB51, while five 4-5 years old and five 2-3 years old not vaccinated Brucella-free female buffaloes served as controls. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on selective media for isolation of SRB51 and incubated for 11 days. Moreover, PCR analysis was also performed directly on milk samples. SRB51 was isolated from milk samples only during the first week post-vaccination while RB51 DNA was detected during the first week till the fourth week post-vaccination only in water buffaloes vaccinated as adults. The identification of Brucella RB51 in milk samples, strongly suggests that this Brucella vaccine could be excreted in milk of buffalo cows vaccinated as adults, while our data demonstrate that the vaccine is safe for use in buffaloes vaccinated as calves in which it was not excreted in milk.