Patrizia Nebbia

Paratuberculosis in red deer (Cervus elaphus hippelaphus) in the western Alps.

During the hunting seasons 1995–96 to 1997–98, 19 red deer from the Upper Susa Valley (Cottian Alps) were examined for paratuberculosis (Johnes disease). Specific DNA amplification on mesenteric lymph nodes detected Mycobacterium avium paratuberculosis in 17 animals. Ten of these red deer were tested for serum antibodies by the AGID and ELISA tests, nine being negative. Three isolates from infected deer were genetically characterized by an arbitrarily primed polymerase chain reaction, and showed similar genetic polymorphism to that of bovine strains isolated in different Italian areas. The study showed that paratuberculosis is present in red deer of the Upper Susa Valley and that serological tests are not an efficient means for monitoring this infection.

Potentiation of thein vitro activity of some antimicrobial agents against selected Gram-negative bacteria by EDTA-tromethamine

Thein vitro synergistic effects of combinations of EDTA-tromethamine and six antimicrobial agents (ampicillin, chloramphenicol, oxytetracycline, streptomycin, nalidixic acid and sulphadimethoxine) on clinically isolated strains ofPseudomonas aeruginosa, Proteus mirabilis andEscherichia coli were investigated. The antibacterial activity was assessed from the minimal inhibitory concentration for the antibiotics alone or in combination with EDTA-tromethamine. EDTA-tromethamine potentiated the antibacterial activity of ampicillin, chloramphenicol, oxytetracycline and streptomycin up to four-fold. There were no significant or consistent synergistic effects with nalidixic acid or sulphadimethoxine.

Phylogenetic background of attaching and effacing Escherichia coli isolates from animals.

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae γ2/θ type, group B1 the eae β1, eae κ, eae ζ, and eae ɛ types, group B2 the eae α1, eae α2 and eae ι types, while the group E possessed the eae γ1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.

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.

Potentiation of antibiotic activity by EDTA-tromethamine against three clinically isolated gram-positive resistant bacteria. An in vitro investigation

Thein vitro synergistic effects of combinations of EDTA-tromethamine and five antimicrobial agents (ampicillin, cephalexin, oxytetracycline, streptomycin and sulphadimethoxine) on three clinically isolated Gram-positive bacteria (Staphylococcus aureus, Staphylococcus hominis andStreptococcus faecium) were investigated. The bacteria had been isolated from three cases of canine otitis resistant to β-lactam antibiotic therapy. The antimicrobial activity was evaluated by measuring the minimal inhibitory concentration for the antibiotics alone or in combination with EDTA-tromethamine. EDTA-tromethamine potentiated the activity of cefalexin againstS. aureus andS. hominis, of oxytetracycline againstS. aureus andS. faecium and of streptomycin againstS. faecium. No significant effects were noted on the activity of oxytetracycline againstS. hominis. The remaining combinations gave a slight synergistic effect. As previously shown for Gram-negative resistant bacteria, these data suggest that the association of EDTA-tromethamine and appropriate antibiotic therapy may be useful to overcome persistent infections of soft tissues in domestic animals.

Corynebacterium urealyticum urinary tract infection in a cat with urethral obstruction

Corynebacterium urealyticum is an uncommon cause of urinary tract infections in cats. However, it is difficult to diagnose and if left untreated it may result in irreversible bladder lesions. C urealyticum is a multiantibiotic-resistant bacterium whose culture requires special care. Risk factors for the occurrence of this infection include urological procedures, foreign bodies, bladder mucosa abnormalities, immuno-suppressed states and antibiotic treatment. This report describes an unusual case of C urealyticum urinary infection in a young cat with pre-existing urethral obstruction. C urealyticum was isolated in pure cultures from two urine samples. Clinical and ultrasound features, results of the urinalysis and urine culture are described as well as therapeutic treatment and eventual favourable outcome to treatment with amoxycillin–clavulanic acid.

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

The incidence of cefotaximase (CTX-M)-type extended-spectrum β-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 β-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 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.

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).

Occurrence and functionality of cycle inhibiting factor, cytotoxic necrotising factors and cytolethal distending toxins in Escherichia coli isolated from calves and dogs in Italy

Escherichia coli isolated from animals up to three months of age, with diarrhea (255 calves and 29 dogs (pups)), without diarrhea (21 calves and 11 pups, used as controls), and 58 adult dogs with cystitis were tested to investigate the occurrence and functional expression of cyclomodulins cycle inhibiting factor (CIF), cytotoxic necrotizing factors (CNFs) and cytolethal distending toxins (CDTs). In cyclomodulin-positive isolates the association was assessed with other virulence genotypes and phylogenetic groups. Of 374 E. coli isolates, 80 (21.4%) were positive for at least one cyclomodulin and 14 of the latter (3.7%) showed different combinations of more than one. cif-positive isolates showed a low number of additional virulence factors, and were commonly associated with phylogroup B1, while cnf- and cdt-positive isolates, harboring many extraintestinal virulence factors, belonged to phylogroups B2 and D. Almost all isolates showed an irreversible cytopathic effect (CPE), displaying functionality of cyclomodulins. Five isolates that presented a mutation of cif were CPE-negative.