Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster.

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR < 0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR < 0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community. HIGHLIGHTS Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds. Differences were found between infected and healthy animals in gut microbiota. Candidatus Arthromitus was closely associated with infected birds. M. ornithogaster can affect intestinal microbiota composition of canaries.

A Set of Multiplex Polymerase Chain Reactions for Genomic Detection of Nine Edible Insect Species in Foods.

On 1 January 2018, a new regulation on 'Novel Food' has come into application in the EU. Insects and insect-based products are therefore included among the categories of food which constitute novel foods. Insects are nutrient-rich, produce fewer greenhouse gases and ammonia than conventional livestock, and have high feed conversion efficiency. Insects may be an alternative food source in the near future, but consideration of insects as a food requires scrutiny due to the risk of allergens. The aim of the present study was to develop a set of multiplex polymerase chain reaction (PCR) to detect nine edible insect species directly in foods. Four sets of mPCRs were designed to detect Locusta migratoria migratorioides (Reiche & Fairmaire, 1849) (Orthoptera: Acrididae), Tenebrio molitor (Linnaeus, 1758) (Coleoptera: Tenebrionidae) (mPCR-I), Acheta domesticus (Linnaeus, 1758) (Orthoptera: Gryllidae), Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae (mPCR-II), Alphitobius diaperinus (Panzer, 1797) (Coleoptera: Tenebrionidae), Schistocerca gregaria (Forskål, 1775) (Orthoptera: Acrididae), Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) (mPCR-III), Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae), and Gryllodes sigillatus (Walker, 1869) (Orthoptera: Gryllidae) (mPCR-IV). Results demonstrate that the panel of mPCRs allowed a rapid genetic identification of the insect species and has proved to be a sensible and highly discriminatory method. The assay is a potential tool in issues related to the labeling of products and food safety, in case of allergic consumers.

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.

Performance Evaluation of a Commercial Real-Time PCR Method for the Detection of Lupin Traces in Food.

In accordance with U.S. FDA Foods Program Regulatory Science Steering Committee guidelines, with this study, we optimized and validated a commercial real-time PCR method for the detection of low amounts of lupin in four classes of food matrices: chocolate cookies, ragù, Olivier salad, and barley and rice flour. DNA extracted from blank (true negative) samples artificially contaminated with lupin (Lupinus albus) flour at 1000 ppm underwent dilutions with the DNA extracted from the true negative samples up to 0.5 ppm. The limit of detection for real-time PCR was 0.5 ppm in the complex matrices (range, Ct 26-34), making this a specific, robust, and rapid method for lupin allergen detection and labeling. Our validation data support the suitability of this commercially available real-time PCR method for this purpose.

Phenotypic Antimicrobial Resistance Profiles of Human Campylobacter Species Isolated in Northwest Italy, 2020-2023.

The spread of antimicrobial resistant Campylobacter strains, linked to antimicrobials use and abuse in humans and food animals, has become a global public health problem. In this study, we determine the prevalence of antimicrobial resistance (AMR) in human Campylobacter isolates (n = 820) collected in Piedmont, Italy, from March 2020 to July 2023. The strains underwent susceptibility testing to determine the minimal inhibitory concentration for erythromycin, ciprofloxacin, gentamicin, streptomycin, and tetracycline: 80.1% of the strains showed resistance to at least one antibiotic. The highest prevalence of AMR was noted for ciprofloxacin and tetracycline (72.1% and 52.9%, respectively) and the lowest for erythromycin and aminoglycosides (streptomycin/gentamicin) (3.2% and 5.4%, respectively). The prevalence of co-resistance against fluoroquinolones and tetracyclines was 41.1%. The prevalence of multidrug resistant strains was 5.7%. Our data support evidence that AMR in human Campylobacter strains is common, particularly against ciprofloxacin and tetracycline, two medically important antimicrobials for humans.

Monitoring of Antimicrobial Resistance of Salmonella Serotypes Isolated from Humans in Northwest Italy, 2012-2021.

Salmonella enterica is among the most common causes of foodborne outbreaks in humans in Europe. The global emergence of resistance to antimicrobials calls for close monitoring of the spread and prevalence of resistant Salmonella strains. In this study, we investigated the occurrence of antimicrobial resistance of Salmonella serotypes isolated from humans between 2012 and 2021 in Piedmont, northwest Italy. A total of 4814 Salmonella strains (168 serotypes) were tested against six classes of antimicrobials. Many strains (83.3%) showed resistance to at least one antibiotic: tetracycline (85.1%), ampicillin (79.2%), quinolones (47.4%), and gentamicin (28.4%). Between the first (2012-2016) and the second study period (2017-2021), a decrease in antimicrobial resistance was noted for tetracycline (from 92.4% to 75.3%), ampicillin (from 85.3% to 71.3%), quinolones (from 49.4% to 44.6%), and cefotaxime (from 34.8% to 4.0%). Many multidrug resistant Salmonella strains (43.6%) belonged to S. ser. Typhimurium, S. ser. Infantis, and S. ser. Typhimurium 1,4,[5],12:i:-. Overall, multidrug resistance decreased from 60.7% to 26.4%, indicating a reduction in the antimicrobial resistance of Salmonella strains in Piedmont and in Europe and demonstrating the effectiveness of the measures that were put in place to reduce antimicrobial resistance.

Food Safety Monitoring of Salmonella spp. in Northern Italy 2019-2021.

Salmonella is the second most frequent bacterial pathogen involved in human gastrointestinal outbreaks in the European Union; it can enter the food-production chain from animal or environmental sources or from asymptomatic food operators. European food legislation has established microbiological criteria to ensure consumer protection. Salmonella is listed under both process hygiene criteria and food safety criteria. Each EU member state designates an agency to organize or perform controls and other official activities. This paper describes the official control plans performed by competent authorities in Northern Italy in the three-year period 2019-2021. A total of 4413 food samples were delivered to the IZS Food Safety laboratories for Salmonella detection, of which 36 (0.8%) tested positive. Salmonella was most frequently detected in poultry meat samples (25/36 positive samples) followed by other meat products and pork products. The official controls for the protection of consumer health apply the EU's farm-to-fork approach: the samples were collected during production (food production plants), from products on the market, and from collective catering (restaurants, cafeterias, canteens). This manuscript will provide information about the presence of Salmonella in foodstuffs that can help competent authorities to set control plans based on risk assessments.

Detection of Peanut Traces in Food by an Official Food Safety Laboratory.

Food safety laboratories rely on validated methods that detect hidden allergens in food to ensure the safety and health of allergic consumers. Here we present test results for the validation and accreditation of a real-time PCR assay for the detection of peanut traces in food products. The method was tested on five classes of food matrices: bakery and pastry products, meats, ready-to-eat and dairy products, and grains and milling products. Blank samples were spiked starting with the peanut samples (Arachis hypogaea) at a concentration of 1000 ppm. Serial dilutions were then prepared with the DNA extracted from the blank samples to a final concentration of 0.5 ppm. The limit of detection in grains and milling products, ready-to-eat, meats, bakery and pastry products was 0.5 ppm (range, Ct 27-34) and 2.5 ppm in dairy products (range, Ct 25-34). In order to determine the exclusivity parameter of the method, the ragù matrix was contaminated with Prunus dulcis (almonds), Glycine max (soy), Sinapis alba (mustard), Apium graveolens (celery), Allium cepa (onion), Pisum sativum (peas), Daucus carota (carrots), and Theobroma cacao (cocoa); no cross-reactions were observed. The method was rated satisfactory for sensitivity (98%), specificity (100%), robustness, and repeatability and it was fully validated and accredited.

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte, Liguria e Valle d'Aosta, Italy.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016-2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

Genetic Characterization of a Listeria monocytogenes Serotype IVb Variant 1 Strain Isolated from Vegetal Matrix in Italy.

We report the chromosome and plasmid sequences of a strain of Listeria monocytogenes IVb variant 1, a recently emerging serotype, isolated in Italy from ready-to-eat vegetables.

Distribution, functional expression, and genetic organization of Cif, a phage-encoded type III-secreted effector from enteropathogenic and enterohemorrhagic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.

Pectin-honey hydrogel: Characterization, antimicrobial activity and biocompatibility.

BACKGROUND: Novel pectin-honey hydrogels have been developed and characterized as medical device. Ideally, a wound dressing should maintain optimal fluid affinity, permit moisture evaporation, protect the wound from microbes, and have shape-conformability, biocompatibility, and antibacterial activity. OBJECTIVE: A novel, simple and fast method to produce pectin-honey wound dressings is described. METHODS: The properties of these pectin-honey hydrogels were investigated, including swelling ability, water vapour transmission rate, hydrogen peroxide production, methylglyoxal content and antibacterial activity. Biocompatibility was assessed by proliferation assays using cultured fibroblast cells and by in vivo study with subcutaneous and intraperitoneal implantation in rats. RESULTS: Hydrogel showed a good water vapour transmission rate, fluid uptake and were not cytotoxic for fibroblasts. The hydrogel demonstrated good antibacterial activity toward clinically relevant pathogens, including S. aureus and E. coli. Biocompatibility was confirmed by the measurement of plasma levels of interleukin (IL)1 beta, IL-6, tumour necrosis factor (TNF) alpha, and prostaglandin (PG)E2. No histological changes were observed. CONCLUSIONS: The presence of a natural active component, conformability, and complete resorbability are the main characteristics of this new biocompatible biomaterial that is well tolerated by the body, possibly improves healing, may be used for surgical complications prevention, with a simple and inexpensive production process.

Cleaning with a wet sterile gauze significantly reduces contamination of sutures, instruments, and surgical gloves in an ex-vivo pelvic flexure enterotomy model in horses.

The objective of this study was to investigate whether cleaning surgical materials used to close pelvic flexure enterotomies with a wet sterile gauze will reduce contamination and whether the use of a full thickness appositional suture pattern (F) or a partial thickness inverting (or Cushing) suture pattern (C) would make a difference in the level of contamination. Large colon specimens were assigned to group F or C and divided into subgroups N and G. In group G, a wet sterile gauze was passed over the suture material, another over the instruments, and another over the gloves. In group N, no treatment was applied. The bacterial concentration was measured by optical density (OD) at 24 h. The OD of subgroup CG was lower than that of subgroup CN (P = 0.019). The OD of subgroup FG was lower than that of subgroup FN (P = 0.02). The OD of subgroups CG, CN, FG, and FN was lower than that of the negative control (P < 0.003, P < 0.001, P < 0.001, and P < 0.00). The use of a sterile wet gauze significantly reduced contamination of suture materials. A partial thickness inverting suture pattern did not produce less contamination than a full thickness appositional suture pattern.

L'objectif de la présente étude était d'examiner si le nettoyage du matériel chirurgical utilisé pour fermer les entérotomies de la courbure pelvienne avec une gaze stérile mouillée réduisait la contamination et si l'utilisation d'un patron de suture d'apposition de la pleine épaisseur (F) ou d'un patron de suture inversé d'une épaisseur partielle (ou Cushing) (C) faisait une différence dans le degré de contamination. Des spécimens du gros côlon ont été assignés au groupe F ou C dans les sous-groupes N et G. Dans le groupe G, une gaze stérile mouillée a été passée par-dessus le matériel de suture, une autre par-dessus les instruments, et une autre par-dessus les gants. Dans le groupe N, aucun traitement ne fut effectué. Les concentrations bactériennes ont été mesurées par densité optique (DO) à 24 h. La DO du sous-groupe CG était inférieure à celle du sous-groupe CN (P = 0,019). La DO du sous-groupe FG était inférieure à celle du sous-groupe FN (P = 0,02). Les DO des sous-groupes CG, CN, FG, et FN étaient inférieures à celles des témoins négatifs (P < 0,003, P < 0,001, P < 0,001, et P < 0,00). L'utilisation d'une gaze stérile mouillée a réduit de manière significative la contamination de matériel de suture. Un patron de suture inversé avec épaisseur partielle n'a pas entrainé moins de contamination qu'un patron de suture par apposition avec pleine épaisseur.(Traduit par Docteur Serge Messier).

Ex vivo antibacterial activity of levofloxacin against Escherichia coli and its pharmacokinetic profile following intravenous and oral administrations in broilers.

The use of antibiotics is necessary to treat bacterial diseases. Determination of optimal dosage in the target animals is increasingly being recognized as vital for maximizing efficacy and minimizing the risk of resistance, so this study aimed to evaluate the pharmacokinetics/pharmacodynamics (PK/PD) of levofloxacin in broilers. Using a parallel study design, each group of animals (n=20) received 5mg/kg of levofloxacin intravenously (IV) and orally (PO). Plasma, serum and tissues were collected for PK and PD studies. Plasma concentrations of levofloxacin were determined by HPLC. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined against E. coli, isolated in clinical broilers. Ex vivo antibacterial activity was evaluated using the time killing method. Mean values of terminal half-life for IV and PO groups were 6.93 and 8.09h, respectively. Following oral administration, the peak plasma concentration was achieved at 0.88h (Tmax). Mean value of oral bioavailability was 123.25%. Levofloxacin residues were found in all the tissues tested (muscle, liver, kidney and lung). Plasma concentration above 8× MIC lead to eradication of E. coli (incubation period of 24h). The results of ex vivo growth inhibition curves were consistent with the in vitro time-kill study. Levofloxacin showed dependent plasma concentration antibacterial activity against a clinical isolate of E. coli. According to the assessment of PK/PD relationship, administration of 5mg/kg of levofloxacin seems to be effective in killing E. coli. Also, simulated optimal dose based on the ex vivo PK/PD approach was 2.9mg/kg/day (bactericidal) to 4.3mg/kg/day (eradication) PO against E. coli (MIC=0.125µg/ml).

Borrelia lusitaniae and spotted fever group rickettsiae in Ixodes ricinus (Acari: Ixodidae) in Tuscany, central Italy.

Prevalence of infection by Borrelia burgdorferi s.l. and spotted fever group (SFG) rickettsiae was estimated in host-seeking ticks in an area in Tuscany, central Italy, where Lyme borreliosis was reported in a forestry worker. B. burgdorferi s.l. was identified by polymerase chain reaction in 16.7% (95% CI = 10.3, 24.8) of Ixodes ricinus (L.) nymphs and 39.6% (95% CI = 26.5, 54.0) of adults. Borrelia lusitaniae accounted for 82.9% of positive samples, followed by Borrelia garinii (9.8%), Borrelia afzelii (2.4%), and Borrelia burgdorferi s.s. (2.4%). One Rhipicephalus spp. adult was infected with B. garinii (prevalence = 8.3%; 95% CI = 0.21, 38.5). Prevalence of infection by SFG rickettsiae was 38.5% (95% CI = 26.7, 51.4) in I. ricinus nymphs, 34.6% (95% CI = 22.0, 49.1) in I. ricinus adults, and 50% (95% CI = 21.1, 78.9) in Rhipicephalus spp. adults. Phylogenetic analysis showed the similarity of B. lusitaniae strains that were identified in this study and of a strain that was previously isolated from a human patient in Portugal. Results of this study confirm the dominance of B. lusitaniae in areas in the Mediterranean basin and the infection by SFG rickettsiae in I. ricinus.

Borrelia burgdorferi sensu lato infection in larval Ixodes ricinus (Acari: Ixodidae) feeding on blackbirds in northwestern Italy.

Birds belonging to 59 species (n = 1,206) were live captured in Piemonte, northwestern Italy, in 2001. Ixodes ricinus (L.) larvae were collected from 59 birds belonging to nine species, and nymphs were recovered on 79 birds belonging to 10 species. Eurasian blackbirds, Turdus merula L., had significantly higher levels of infestation by ticks than other passerine species. Larval I. ricinus of blackbirds peaked in summer, when prevalence was 39% (95% confidence interval 24.2-55.5) and mean number of ticks per host was 3.3 (1.6-7.2), whereas nymphs peaked in spring, when prevalence was 72.2% (54.8-85.8) and mean number of ticks per host was 6.9 (4.4-10.7). Immature I. ricinus were coincidentally aggregated on blackbirds, with 15 blackbirds feeding 67.4% of nymphs and 40.3% of larvae, and coinfestation by both stages was relatively high in summer: Kappa = 0.64 (0.40-0.88). Borrelia burgdorferi sensu lato was identified by polymerase chain reaction (PCR) in 58.3% (35.9-78.5) of larvae with engorgement ratio > or = 3 that were collected from blackbirds. Larvae that were collected from other passerine species gave negative PCR results. Sixteen of 21 PCR-positive samples belonged to B. garinii (76.2%), and five (23.8%) were Borrelia valaisiana. Results of this study suggest that blackbirds play an important role as hosts for immature I. ricinus and as reservoir of Borrelia garinii in northwestern Italy.

Genetic and phenotypic characterisation of Escherichia coli producing cefotaximase-type extended-spectrum ß-lactamases: first evidence of the ST131 clone in cats with urinary infections in Italy.

The incidence of cefotaximase (CTX-M)-type extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli has increased dramatically in humans and animals since the middle of the last century. E coli that produce CTX-M ß-lactamase represent a major cause of urinary tract infections, and pose a significant therapeutic challenge to both human and veterinary medicine. As data on uropathogenic CTX-M-producing strains in cats are limited, the aim of this study was to describe the genetic character and antibiotic resistance phenotypes of CTX-M-producing E coli isolated from cats with cystitis. Seven of 15 E coli bacteria isolated from 138 urine samples had the CTX-M gene and were therefore included in this study. These isolates were screened by polymerase chain reaction for the presence of 14 extra-intestinal virulence factors, class 1 and class 2 integrons, and to identify their phylogenetic groups. Multi-locus sequence typing (MLST) of the strains and susceptibility testing (disc diffusion method) were also performed. Virulence factor iutA was the most frequent determinant identified (86.7%), and the majority of CTX-M-producing strains (n = 5) carried class 1 integrons. MLST allowed us to discriminate four known sequence types (ST131, ST555, ST602, ST155) and three novel sequence types (ST3847, ST3848, ST4181). To the best of our knowledge, this is the first study to report uropathogenic CTX-M-producing E coli ST131 in cats in Italy. Accurate diagnostics and prudent use of antimicrobials are recommended to avoid the spread of multidrug-resistant pathogens in veterinary medicine and to prevent their transmission to humans.

Molecular characterization and antimicrobial resistance of faecal and urinary Escherichia coli isolated from dogs and humans in Italy.

During this study, 109 faecal Escherichia coli samples isolated from 61 dogs and 48 humans were characterised according to phylogenetic group, extraintestinal virulence factors and antibiotic resistance. The isolates from dogs were predominantly distributed within phylogroup B1 (36%), while the majority of human strains belonged to phylogroup B2 (54%). The prevalence of cnf1, hlyA, papC and sfa virulence genes was significantly associated with the group B2. Canine isolates showed multidrug resistance (MDR) more frequently than human strains. Since group B2 contains most of the strains that cause extraintestinal infections, all 46 B2 faecal strains were confronted against an addition population of 57 urinary E. coli strains belonging to the same phylogroup. The comparison shows that there was no significant difference in the occurrence of virulence factors or in the distribution of antibiotic resistance between faecal and urinary E. coli isolates from dogs. At the same time, a highly significant association was detected between multiple resistance and the source of the strains and between MDR and E. coli isolated from urine in human. This study highlighted similar features of E. coli isolated across sources and hosts. The data suggest a high prevalence of antibiotic resistance in faecal strains, which may represent a serious health risk since these strains can function as a reservoir for uropathogenic E. coli.

Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy.

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.

Development of a set of multiplex standard polymerase chain reaction assays for the identification of infectious agents from aborted bovine clinical samples.

The current study describes the development of a set of 5 multiplex polymerase chain reaction (mPCR) assays for the simultaneous detection of abortive infection agents in bovine fetal tissues, including Brucella spp., Leptospira spp., and Campylobacter fetus (mPCR1); Hammondia heydorni, Neospora caninum, and Toxoplasma gondii (mPCR2); Coxiella burnetii and Chlamydophila psittaci (mPCR3); Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum (mPCR4); and Bovine viral diarrhea virus (BVDV) and Bovine herpesvirus-1 (BoHV-1; mPCR5). The protocol was tested on different tissue samples collected from 50 aborted bovine fetuses, and it showed that out of the 50 fetuses, 7 (14%, mPCR2) were PCR-positive for N. caninum, 4 (8%, mPCR5) were PCR-positive for BVDV, and 2 (4%, mPCR4) were PCR-positive for U. diversum. The results obtained by using each multiplex PCR were 100% concordant with those obtained by using the respective PCR assays targeting single genes on the same specimens. Moreover, all multiplex PCR assays on clinical samples were compared with reference methods, obtaining a perfect accordance in all samples and confirming the validity of the set of multiplex PCR assays. The proposed set of multiplex PCR assays is, therefore, suitable for the simultaneous detection of the main infectious agents responsible for bovine abortion.