In vitro effects of different ozone preparations on microorganisms responsible for endometritis in the mare.

Infectious endometritis is considered one of the major causes of infertility and it can affect up to 60% of barren mares. It is characterized by the presence of one or more microorganisms in the reproductive tract and it is treated with the administration of antibiotics, ecbolic agents and uterine lavages. Ozone, thanks to its antimicrobial properties that are based on its high oxidative potential, could represent an effective alternative treatment for endometritis. The aim of this study was to test in vitro the bactericidal and fungicidal properties of different ozone formulations, either as gas (experiment 1) or dissolved in two liquid matrices (experiment 2), specifically distilled water or oil (Neozone 4000, Cosmoproject, Parma, Italy), onto 6 different species of microorganisms isolated from mares with clinical endometritis, namely Escherichia coli, Staphylococcus aureus, Streptococcus equi subsp. Zooepidemicus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans. In the first experiment, 3 clinical antibiotic-resistant strains per each species were exposed to different conditions: to O2O3 gas mixtures (15 and 40 µg/ml for 1, 3 and 5 min), to 100 % O2 or left untreated. The results showed a reduction of the microbial count of over 99,9% for every pathogen, time and concentration of O2O3 gas mixtures tested. Furthermore, gaseous ozone showed both a time-dependant effect (5 vs 3 vs 1 min of exposure) and a concentration-dependant effect (40 vs 15 µg/ml) at 1 and 3 min, while after 5 min no differences were observed. In the second experiment, minimum inhibitory concentration (MIC), and minimum bactericidal/fungicidal concentration (MBC, MFC) of ozonated distilled water and ozonated oil were evaluated. Ozonated oil showed a bactericidal/fungicidal activity against all the strains tested (MIC range 12.5-25 % v/v, MBC/MFC range 12.5-50 % v/v) while ozonated distilled water didn't show an observable antimicrobial effect, discouraging its use as an antimicrobial agent for the treatment of endometritis. The results of this in vitro study indicate that both gaseous ozone and ozonated oil exerted remarkable antimicrobial activities and are promising alternative treatments for infectious endometritis, even when caused by antibiotic-resistant bacteria, and encourage further experiments in an effort to scale down or even prevent the use of antibiotics in equine reproduction.

Biosecurity and Hygiene Procedures in Pig Farms: Effects of a Tailor-Made Approach as Monitored by Environmental Samples.

In livestock, the importance of hygiene management is gaining importance within the context of biosecurity. The aim of this study was to monitor the implementation of biosecurity and hygiene procedures in 20 swine herds over a 12-month period, as driven by tailor-made plans, including training on-farm. The measure of adenosine triphosphate (ATP) environmental contents was used as an output biomarker. The presence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and extended-spectrum ß-lactamase producing Escherichia coli (ESBL-E. coli) was also investigated as sentinels of antibiotic resistance. A significant biosecurity improvement (p = 0.006) and a reduction in the ATP content in the sanitised environment (p = 0.039) were observed. A cluster including 6/20 farms greatly improved both biosecurity and ATP contents, while the remaining 14/20 farms ameliorated them only slightly. Even if the ESBL-E. coli prevalence (30.0%) after the hygiene procedures significantly decreased, the prevalence of LA-MRSA (22.5%) was unaffected. Despite the promising results supporting the adoption of tailor-made biosecurity plans and the measure of environmental ATP as an output biomarker, the high LA-MRSA prevalence still detected at the end of the study underlines the importance of improving even more biosecurity and farm hygiene in a one-health approach aimed to preserve also the pig workers health.

Antibiotic resistant Escherichia coli in wild birds hospitalised in a wildlife rescue centre.

The aim of this work was to evaluate the consequence of a hospitalisation period on antimicrobial resistance in Escherichia coli isolated from wild bird species admitted in the wildlife rescue centre of the Department of Veterinary Sciences (Turin University, Italy). Samples were collected from 121 raptors and 51 synanthropic animals, at the time of arrival as well as 5 and 10 days afterwards for a total of 372 faecal samples, and the susceptibility of E. coli strains was tested to a panel of seven antibacterials. Of the total, 109 animals (63.37 %) presented at least one sample positive for E. coli, 36 strains (39.6 %) were multi-drug resistant (MDR) and 12 (13.2 %) were extended spectrum beta-lactamase (ESBL)-producing E. coli. During the first 10 days of hospitalisation E. coli strains increased the number of resistances towards each antimicrobial principle, the number of ESBL E. coli and the therapy with fluoroquinolones developed resistance towards ceftriaxone, marbofloxacin, sulfamethoxazole-trimethoprim and tetracycline. Our results suggest that wild birds act as reservoirs of MDR bacteria, being potential sources for their spreading in the environment and to other species.

Feasibility of 16S rRNA sequencing for cerebrospinal fluid microbiome analysis in cattle with neurological disorders: a pilot study.

Bacterial infection of the central nervous system (CNS) in cattle requires prompt and adequate antimicrobial treatment. The current gold standard for antemortem etiological diagnosis is cerebrospinal fluid (CSF) culture, which often yields false negative results. CSF has long been considered a sterile district in healthy patients, but this notion has been recently challenged. For this pilot study, we used 16S rRNA gene sequencing to investigate the microbial composition of CSF of cattle presenting with CNS disorders and to compare it between subjects with CNS infections and with CNS disorders of other nature. The study sample was 10 animals: 4 presenting with CNS infectious-inflammatory diseases and 6 with other CNS disorders, based on definitive diagnosis. Since the initial round of a standard 16S rRNA PCR did not yield sufficient genetic material for sequencing in any of the samples, the protocol was modified to increase its sensitivity. Bacterial genetic material was identified in 6 animals and 2 groups were formed: an infectious inflammatory (n = 3) and a noninfectious inflammatory group (n = 3). The most frequently expressed bacterial families were Pseudomonadaceae (44.61%), Moraxellaceae (19.54%), Mycobacteriaceae (11.80%); the genera were Pseudomonas (45.42%), Acinetobacter (19.91%), Mycobacterium (12.01%). There were no detectable differences in the CSF microbial composition of the samples from the two groups. Sequencing of bacterial DNA present in the CSF was possible only after increasing PCR sensitivity. The results of 16S rRNA sequencing showed the presence of a microbial community in the CSF in cattle with neurological disorders. Further studies, in which CSF samples from healthy animals and samples from the environment are included as controls, are needed.

Staphylococci isolated from cats in Italy with superficial pyoderma and allergic dermatitis: Characterisation of isolates and their resistance to antimicrobials.

BACKGROUND: In cats, superficial pyoderma traditionally is considered rare and few reports are available. There is a particular lack of studies concerning Staphylococcus species associated with pyoderma in subjects affected by allergic skin diseases. HYPOTHESIS/OBJECTIVES: (i) To evaluate the association between Staphylococcus spp. and superficial pyoderma in allergic cats and (ii) to characterise isolated staphylococci and analyse their antimicrobial resistance patterns. ANIMALS: Forty-one cats with allergic dermatitis and superficial pyoderma in Italy. MATERIALS AND METHODS: Skin swabs were cultured for the isolation of Staphylococcus spp. Species identification was performed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry and 16S-rRNA sequencing. Staphylococcus aureus isolates were further characterised by staphylococcal protein A gene-typing. Antimicrobial susceptibility was performed by the disk diffusion method. RESULTS: Staphylococci were isolated from 36/41 cats sampled and 39 different isolates were identified. Uneven distribution of staphylococcal species was observed among different body locations. The 39 isolates were S. aureus (n = 15), S. felis (n = 10), S. pseudintermedius (n = 8) and other staphylococci (n = 6). Eight different S. aureus spa-types associated with human clonal complexes were identified. Antimicrobial resistance was observed to penicillin (56.4%), tetracycline (46.2%), enrofloxacin (33.3%), erythromycin (28.2%), amikacin (25.6%), clindamycin (23.1%), marbofloxacin (15.4%), gentamicin (12.8%), trimethoprim-sulfamethoxazole (10.3%), chloramphenicol (7.7%) and cefoxitin/oxacillin (5.1%). Fifteen isolates (38.4%) were multidrug-resistant while meticillin resistance was associated only with S. pseudintermedius. CONCLUSIONS AND CLINICAL RELEVANCE: These results confirm that S. aureus, S. pseudintermedius, and S. felis are frequently associated with superficial pyoderma in allergic cats. Semi-synthetic penicillins remain a suitable first-line treatment in this study, yet the high prevalence of antimicrobial resistant isolates suggests that antimicrobial susceptibility testing should be performed routinely.

Resistance to Critical Important Antibacterials in Staphylococcus pseudintermedius Strains of Veterinary Origin.

Staphylococcal infections represent a challenge in companion animals and hospitalized patients. This study aimed to assess the resistance of Staphylococcus pseudintermedius isolates, against a broad panel of antibacterials, including exclusive to human medicine. A total of 40 S. pseudintermedius were collected from clinical specimens of dogs (n = 31) and cats (n = 5). All strains were tested for 20 antibacterials, namely 14 Critical Important and eight Highly Important Antibacterials (CIA and HIA, respectively), indicative for 18 antimicrobial classes. All strains were susceptible to seven antibiotics (daptomycin, fosfomycin, fusidic acid, linezolid, quinupristin-dalfopristin, teicoplanin/vancomycin, tigecycline). The highest resistance was against penicillin (97.5% Confidence Interval [CI]: 83.8-100.0), whereas the lowest against telavancin (2.5%, CI: 0.0-16.2). Resistance versus Highest Priority CIA was observed, namely against macrolides (70.0, CI: 52.1-84.3), quinolones (62.5, CI: 44.5-78.3), 5th generation cephalosporins (7.5, CI: 1.3-21.6), and glycopeptides (2.5%, CI: 0.0-14.2). Among High Priority CIA, strains were resistant only to aminoglycosides (65.0, CI: 47.0-80.4) and ansamycins (12.5, CI: 3.8-28.1). We observed the highest resistance against veterinary medicine antibacterials, but there was also resistance against antibacterials exclusive to human medicine, namely ceftaroline (7.5, CI: 1.0-23.8) and telavancin. S. pseudintermedius zoonotic potential and its rate of acquisition of new resistance should encourage surveillance on a broad spectrum of antibacterials.

Homozygous CADPS2 Mutations Cause Neurodegenerative Disease with Lewy Body-like Pathology in Parrots.

BACKGROUND: Several genetic models that recapitulate neurodegenerative features of Parkinson's disease (PD) exist, which have been largely based on genes discovered in monogenic PD families. However, spontaneous genetic mutations have not been linked to the pathological hallmarks of PD in non-human vertebrates. OBJECTIVE: To describe the genetic and pathological findings of three Yellow-crowned parrot (Amazona ochrocepahala) siblings with a severe and rapidly progressive neurological phenotype. METHODS: The phenotype of the three parrots included severe ataxia, rigidity, and tremor, while their parents were phenotypically normal. Tests to identify avian viral infections and brain imaging studies were all negative. Due to their severe impairment, they were all euthanized at age 3 months and their brains underwent neuropathological examination and proteasome activity assays. Whole genome sequencing (WGS) was performed on the three affected parrots and their parents. RESULTS: The brains of affected parrots exhibited neuronal loss, spongiosis, and widespread Lewy body-like inclusions in many regions including the midbrain, basal ganglia, and neocortex. Proteasome activity was significantly reduced in these animals compared to a control (P < 0.05). WGS identified a single homozygous missense mutation (p.V559L) in a highly conserved amino acid within the pleckstrin homology (PH) domain of the calcium-dependent secretion activator 2 (CADPS2) gene. CONCLUSIONS: Our data suggest that a homozygous mutation in the CADPS2 gene causes a severe neurodegenerative phenotype with Lewy body-like pathology in parrots. Although CADPS2 variants have not been reported to cause PD, further investigation of the gene might provide important insights into the pathophysiology of Lewy body disorders. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Occurrence of Methicillin-Resistant Coagulase-Negative Staphylococci (MRCoNS) and Methicillin-Resistant Staphylococcus aureus (MRSA) from Pigs and Farm Environment in Northwestern Italy.

Swine farming as a source of methicillin-resistant Staphylococcus aureus (MRSA) has been well documented. Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have been less studied, but their importance as pathogens is increasing. MRCoNS are indeed considered relevant nosocomial pathogens; identifying putative sources of MRCoNS is thus gaining importance to prevent human health hazards. In the present study, we investigated MRSA and MRCoNS in animals and environment in five pigsties in a high farm-density area of northwestern Italy. Farms were three intensive, one intensive with antibiotic-free finishing, and one organic. We tested nasal swabs from 195 animals and 26 environmental samples from three production phases: post-weaning, finishing and female breeders. Phenotypic tests, including MALDI-TOF MS, were used for the identification of Staphylococcus species; PCR and nucleotide sequencing confirmed resistance and bacterial species. MRCoNS were recovered in 64.5% of nasal swabs, in all farms and animal categories, while MRSA was detected only in one post-weaning sample in one farm. The lowest prevalence of MRCoNS was detected in pigs from the organic farm and in the finishing of the antibiotic-free farm. MRCoNS were mainly Staphylococcus sciuri, but we also recovered S. pasteuri, S. haemolyticus, S. cohnii, S. equorum and S. xylosus. Fifteen environmental samples were positive for MRCoNS, which were mainly S. sciuri; no MRSA was found in the farms' environment. The analyses of the mecA gene and the PBP2-a protein highlighted the same mecA fragment in strains of S. aureus, S. sciuri and S. haemolyticus. Our results show the emergence of MRCoNS carrying the mecA gene in swine farms. Moreover, they suggest that this gene might be horizontally transferred from MRCoNS to bacterial species more relevant for human health, such as S. aureus.

Borrelia burgdorferi sensu lato and spotted fever group rickettsiae in small rodents and attached ticks in the Northern Apennines, Italy.

Ticks and ear biopsies were collected from wild small rodents in 2011 and 2012 in the northern Apennines (Italy), up to 1650 m above sea level. Apodemus spp. (n = 83) and Myodes glareolus (n = 22) were infested by Ixodes ricinus (192 larvae and two nymphs), Dermacentor marginatus (179 larvae and 29 nymphs), and Ixodes trianguliceps (three larvae and two nymphs). We detected several Borrelia burgdorferi sensu lato (s.l.) genospecies (B. afzelii, B. burgdorferi sensu stricto, B. garinii, B. lusitaniae, B. valaisiana) in I. ricinus and skin biopsies. The most common genospecies found in I. ricinus was B. valaisiana, while it was B. lusitaniae in tissues. Spotted fever group (SFG) rickettsiae (Rickettsia monacensis, R. slovaca and R. raoultii) infected I. ricinus, D. marginatus and rodent tissues. Rickettsia slovaca was the Rickettsia species most frequently found in our samples. Coinfections by B. burgdorferi s.l. and SFG rickettsiae indicate an overlap of transmission cycles and potential risk for humans to be infected by multiple pathogens, resulting in more severe symptoms. The findings of B. lusitaniae and R. slovaca in bank voles, and of B. valaisiana in small rodents, open new questions about host-pathogen interactions. In addition, our results highlight the importance of small rodents as data sources for studying tick-borne pathogens.

Molecular analysis and associated pathology of beak and feather disease virus isolated in Italy from young Congo African grey parrots (Psittacus erithacus) with an "atypical peracute form" of the disease.

This study is the first report on the genetic and pathogenic characterization of beak and feather disease virus (BFDV) occurring in Italy. Twenty BFDV strains isolated in Italy from juvenile Congo African grey parrots (Psittacus erithacus) were investigated. Seventeen strains showed an "atypical peracute form" (aPF) of the disease, and three a chronic form (CF). The birds with aPF had been weaned, were independent as far as food and protection were concerned and apparently were without lesions. The gene coding for the putative coat protein was amplified in all isolates while the BFDV genome was sequenced completely in 10 samples, eight of them belonging to aPF affected birds and two from CF of the disease. All full genomes clustered into the J strain of BFDV, where two new subtypes were identified. Recombination analyses showed evidence of genetic exchanges in two BFDV genomes. In addition, a correlation between viral isolate and origin of the breeding material was shown, while an association between the genetic features of the virus and the clinical form was not observed. Histologically, apoptosis was detected frequently in aPF samples and sporadically in CF samples. Interestingly, BFDV antigens were detected in the nuclei and cytoplasm of such apoptotic cells. The data presented here support the hypothesis that, in the absence of a defined BFDV genetic variant accountable for a specific clinical form of psittacine beak and feather disease, differences in the apoptotic rate between aPF and CF are strictly host related.

Rickettsia slovaca in immature Dermacentor marginatus and tissues from Apodemus spp. in the northern Apennines, Italy.

Immature Dermacentor marginatus ticks and tissues from small rodents were tested for infection with Rickettsia slovaca in the northern Apennines, Lucca Province, where tick-borne lymphadenopathy (TIBOLA) was previously reported in people. Prevalence of infestation with D. marginatus was 30.5% (n=131, 95% CI: 22.8-39.2%) in Apodemus spp. and 26.5% (n=34, 95% CI: 12.9-44.4%) in Myodes glareolus, which were captured during 1980 trap nights in 2009 and 2010. Rickettsia slovaca was identified by polymerase chain reaction, targeting the gltA and OmpA genes, in ear biopsies from 8 out of 37 tested Apodemus (22%, 95% CI: 9.8-38.2%), but not from 9 M. glareolus. The prevalence of R. slovaca in D. marginatus feeding on Apodemus spp. was 53% in larvae (n=51, 95% CI: 38.5-67.1%) and 47.5% in nymphs (n=59, 95% CI: 34.3-60.9%). No larvae (0.0%, 95% CI: 0-36.9%), but one nymph removed from M. glareolus was positive (10%, 95% CI: 0.3-44.5%). Prevalence of R. slovaca in host-seeking D. marginatus larvae, collected in the same area, was 42% (n=38; 95% CI: 26.3-59.2%). Prevalence of R. slovaca was greater in larvae feeding on PCR-positive Apodemus than in those feeding on negative mice (78.6% vs. 37.1%). Furthermore, levels of infestation with D. marginatus larvae were greater for R. slovaca-positive mice. The infection of Apodemus spp. was probably the result of repeated bites by transovarially infected larvae. On the other hand, the finding of R. slovaca in mice tissues would be compatible with transmission from these hosts to feeding D. marginatus. Based on such a hypothesis, the most heavily infested Apodemus might play a role as amplifiers of the infection.

The Slit/Robo system suppresses hepatocyte growth factor-dependent invasion and morphogenesis.

The Slit protein acts through the Roundabout receptor as a paracrine chemorepellent in axon guidance and as an inhibitor in leukocyte chemotaxis, but its role in epithelial cell motility and morphogenesis remains largely unexplored. We report that nontransformed epithelial cells and cancerous cells empower the Slit-2/Robo1 signaling system to limit outward migration in response to motogenic attractants and to remain positionally confined within their primitive location. Short hairpin RNA-mediated depletion of SLIT-2 or ectopic expression of a soluble decoy Robo enhance hepatocyte growth factor (HGF)-induced migration, matrix invasion, and tubulogenesis, concomitantly with the up-regulation of Cdc-42 and the down-modulation of Rac-1 activities. Accordingly, autocrine overexpression or exogenous administration of Slit-2 prevent HGF-triggered motile responses, reduce Cdc-42 activation, and stimulate Rac-1. This antimigratory activity of Slit-2 derives from the inhibition of actin-based protrusive forces and from an increased adhesive strength of cadherin-mediated intercellular contacts. These results disclose a novel function for Slit and Robo in the inhibition of growth factor-mediated epithelial cell motility and morphogenesis, invoking a critical role for both molecules as natural antagonists of neoplastic invasive growth.