Annalisa Bietta

Occurrence of Giardia duodenalis infection in chinchillas (Chincilla lanigera) from Italian breeding facilities.

The present work investigated the occurrence of Giardia infection in Chinchilla lanigera reared in three Italian breeding facilities and determined their role as potential zoonotic reservoir. One hundred and four fecal samples were tested for the presence of Giardia spp. cysts using a Direct Fluorescent Assay (DFA). A high positivity rate (39.4%) was found despite all animals were asymptomatic at the time of sampling. Thirty-one positive samples were genetically characterized by sequence analysis of the ITS1-5.8S-ITS2 region of the Giardia ribosomal DNA. Assemblages B (29 isolates) and C (two isolates) were identified. These results showed that Giardia infection can be common in chinchillas, thus spurring further molecular epizootiological studies of the infection to assess the zoonotic potential or host specificity of their isolates, to determine the source of infections, to identify the routes of transmission, and to control the infection among animal populations.

Detection of Helicobacter spp. in gastric, fecal and saliva samples from swine affected by gastric ulceration.

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.

Insertion sequence IS256 in canine pyoderma isolates of Staphylococcus pseudintermedius associated with antibiotic resistance

Staphylococcus pseudintermedius is the most frequent staphylococcal species isolated from canine pyoderma. The control of S. pseudintermedius infection is often difficult due to the expanded antimicrobial resistance phenotypes. Antibiotic resistance in staphylococcal pathogens is often associated to mobile genetic elements such as the insertion sequence IS256 that was first described as a part of the transposon Tn4001, which confers aminoglycoside resistance in Staphylococcus aureus and in Staphylococcus epidermidis. In this study a collection of 70 S. pseudintermedius isolates from canine pyoderma was used to investigate antimicrobial susceptibility to 15 antibiotics and the presence of IS256, not revealed in S. pseudintermedius yet. Antibiotic resistance profiling demonstrated that all S. pseudintermedius isolates had a multi-drug resistance phenotype, exhibiting simultaneous resistance to at least five antibiotics; indeed methicillin resistant S. pseudintermedius isolates were simultaneously resistant to at least nine antibiotics and all were also gentamicin resistant. PCR analyses revealed the presence of IS256 in 43/70 S. pseudintemedius isolates. The association between the presence of IS256 and the resistance was particularly significant for certain antibiotics: cefovecin, amikacin, gentamicin and oxacillin (χ(2)p-value<0.05). However, there was a striking result in frequency of strains resistant to gentamicin and oxacillin, suggesting a specific association between the presence of the IS256 element and the determinants for the resistance to these antibiotics. To the best of our knowledge, this is the first report showing the detection of IS256 in S. pseudintermedius isolates and its association with antibiotic resistance. Our findings suggest that S. pseudintermedius may acquire antibiotic resistance genes through mobile genetic elements which may play a predominant role in the dissemination of multi-drug resistance.

Cloning and expression of pigeon IFN-γ gene

This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.

Isolation and characterization of β-haemolytic-Streptococci from endometritis in mares.

The objective of this manuscript was to validate published PCR-based methods for detection of β-haemolytic Streptococci by comparison with established bacteriological techniques using 85 clinical isolates recovered from uterine swabs of mares with clinical signs of endometritis and to determine the distribution of SeeL/SeeM and SzeL/SzeM superantigens in isolates of Streptococcus equi subsp. equi (S. equi) and S. equi subsp. zooepidemicus (S. zooepidemicus). The conventional bacteriological techniques showed the vast majority of these isolates (78) were S. zooepidemicus with just 5 Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis) and 2 S. equi strains detected. The PCR analyses confirmed the bacteriological results demonstrating the reliability of the 16S rRNA PCR assay for detecting Streptococci, the multiplex PCR for differentiating between S. zooepidemicus, and S. equi, and PCR assays based on streptokinase genes for identification of S. equisimilis. PCRs for genes encoding superantigens revealed seeL and seeM specific amplicons with size of approximately 800 and 810 bp respectively for the S. equi strains and for 2 S. zooepidemicus strains. To our knowledge, this is the first report of szeL and szeM possession by S. zooepidemicus isolates derived from endometritis in mares.

Is the horse a reservoir or an indicator of Coxiella burnetii infection? Systematic review and biomolecular investigation

The role of the horse in Coxiella burnetii infection has not been defined. Accordingly, a twofold approach was taken to further our knowledge on this topic: (1) conduct a systematic review of the literature to establish available evidence of C. burnetii infection in the horse; (2) undertake a biomolecular investigation of 122 cases of equine abortion, stillbirth and neonatal foal death, for the presence of C. burnetii using a PCR test targeting the IS1111 gene of C. burnetii. A review of the literature turned up seven studies that identified C. burnetii DNA in equine specimens, especially aborted fetuses, while an additional 34 studies sought to determine seroprevalence of the infection in the horse. A meta-analytical approach was taken to calculate a pooled mean seroprevalence in equines based on published studies. A seroprevalence of 15.8% (95% confidence interval: 9.6-23.0%) was obtained. This figure is comparable to those previously reported in other species, especially ruminants. None of the 122 cases of equine abortion, stillbirth or neonatal foal death were positive for C. burnetii DNA. C. burnetii has rarely been looked for in equine specimens in previous studies. Cases of equine abortion should be comprehensively investigated to assess the risk of abortion in a pregnant mare infected with C. burnetii. Consideration should also be given to the possible role of the horse as a source of the organism for other animal species including humans.

Phenotypic and genotypic characterization of canine pyoderma isolates of Staphylococcus pseudintermedius for biofilm formation

Biofilm-forming ability is increasingly being recognized as an important virulence factor in several Staphylococcus species. This study evaluated the biofilm-forming ability of sixty canine derived clinical isolates of S. pseudintermedius, using three phenotypic methods, microtiter plate test (MtP), Congo red agar method (CRA) and tube adherence test, and the presence and impact of biofilm-associated genes (icaA and icaD). The results showed that icaA and icaD genes were detected concomitantly in 55 (91.7%) of 60 isolates. A majority (88.3%) of the strains screened had matching results by the tube adherence test, MtP and PCR analysis. Better agreement (95%) was found between the PCR-based analysis and the CRA. Results of the icaA and icaD gene PCRs showed good agreement with CRA results, with a kappa of 0.7. Comparing the phenotypic methods, the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good. Considering two classes (biofilm producer and biofilm non-producer), the percentage of matching results between the CRA method and the tube adherence test and between the CRA method and the MtP was 93.3%. A concordance of 100% was revealed between the MtP and the tube adherence test. The results indicate a high prevalence of the ica genes within S. pseudintermedius isolates, and their presence is associated with in vitro formation of a biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. pseudintermedius.

Influence of different rearing systems on natural immune parameters in broiler turkeys

The aim of this study was to determine serological values of lysozyme, hemolytic complement levels (alternative pathway), and bactericidal activity of serum in turkeys kept in different rearing systems (industrial, backyard, and experimental). Results showed that the values for serum bactericidal activity and hemolytic complement levels increased with age, and their values were higher in experimental and in industrial turkeys than in turkeys reared in backyard. Lysozyme concentration showed a similar pattern; its value was higher in the industrial and experimental groups than in the backyard group. Data obtained suggest that rearing system can have an influence on the natural immune parameters considered; experimental and industrial groups showed a similar trend, differentiated from that observed in the backyard group. In the backyard group, the values observed may suggest that hybrid turkeys, selected for high production, have difficulty with being reared outside where predators (foxes and weasels) and weather conditions could be responsible for a stress situation.

Indagine su antibiotico-resistenza e formazione di biofilm in ceppi di Staphylococcus pseudintermedius isolati da piodermiti canine

The aim of the present study was to investigate the antibiotic resistance and bio lm formation among a collection of 51 clinical isolates of Staphylococcus pseudintermedius collected from canine pyoderma. All isolates were tested for the susceptibility to a panel of 14 antimicrobial agents by the disk di usion method in Müeller-Hinton agar. Oxacillin resistance was detected by subculture on oxacillin screening agar base. Bio lm formation was investigated by the Microtitre Plate test (MtP) and for some strains by transmission electron microscopy (TEM). Antibiotic resistance pro ling demonstrated that 45/51 S. pseudintermedius isolates had a multi drug resistant (MDR) phenotype, exhibiting simultaneous resistance to at least 3 antibiotics categories; whereas 6 isolates showed a non-MDR phenotype. Thirty strains (59%) were resistant in oxacillin resistant screening agar, the same strains were also positive for mecA by PCR assay. All S. pseudintermedius isolates showed bio lm production by MtP method. Seventeen out of 51 isolates were classi ed as weakly adherent, 26 as moderately adherent, and 8 as strongly adherent. Moreover, no di erence in bio lm formation between meticillin-resistant S. pseudintermedius (MRSP) and meticillin-suscebtible S. pseudintermedius (MSSP) (P value > 0.05) was noted. The antimicrobial resistance mechanisms and bio lm formation could explain the di culty in treating S. pseudintermedius canine infections, chemotherapeutic failure, and consequently persistent infections.