1,721,010 research outputs found
Salmonella spp. prevalence and antimicrobial resistance pattern (phenotypic profiles and presence of genetic AMR determinants) of serotypes isolated from pet reptiles in Northern Italy
No full textA total of 324 cloacal swabs were collected from reptiles kept at pet animals import centres (48 samples), pet shops (103 samples), zoological park (71 samples) and private owners (102 samples) located in Northern Italy. Samples were collected from healthy snakes (n = 147), lizards (n = 85) and turtles (n = 92). Isolation, typization of Salmonella spp. and the evaluation of sensitivity to different classes of antimicrobial drugs (16 antimicrobial agents) were performed using the classical bacteriological and serological procedures and the agar disc diffusion method (Kirby-Bauer). Isolates showing particular phenotypic AMR profiles were subjected to further molecular characterizations, especially the detection by PCR and sequencing of genetic elements involved in the diffusion and dissemination of AMR among Enterobacteriaceae (i.e. class 1 and class 2 integrons and AMR genes codifying for the resistance to tetracycline, aminoglycosides, beta-lactams, phenicol compounds etc...).
Salmonella spp. was isolated from 205 (63.3 %) cloacal swabs. Prevalence data were different for snakes (76.9 %) and lizards (74.1 %) compared to turtles (31.5 %). Serotyping showed the presence of S. enterica subsp. enterica (53.2 %), S. enterica subsp. diarizonae (29.8 %), S. enterica subsp. houtenae (10.2 %), S. enterica subsp. salamae (6.3 %) and S. enterica subsp. arizonae (0.5 %).
Out of 205 isolates, only 12 (5.8 %) showed full susceptibility to all the drugs tested and 95 (46.3 %) strains showed multidrug-resistance (i. e. resistance to three or more antimicrobials). Out of the 205 isolates, only 1 carried class 1 integron and only few strains showed a correlation between the phenotypic and genotypic patterns of AMR. These data underlines the importance to carry out further genetic investigations to better understand the genetic bases and mechanisms of the diffusion of AMR among these strains
The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances
No full textThe remarkable economic losses due to porcine reproductive and respiratory syndrome (PRRS) have stated the control and eradication of this disease is one of the main issues of swine modern farming. The limited cross-protection of vaccine-induced immunity compelled the adoption of strict biosecurity measures that must be associated with the prompt diagnosis of infection. In our study four RT-PCR methods, a RT-PCR, a SYBR Green I and two hydrolysis probes, were compared to evaluate their respective benefits and disadvantages. One hundred and seventy samples originating from 50 farms located in northern Italy were tested with all assays and performances were evaluated using a Bayesian approach to deal with the absence of a Gold Standard. Sequencing the complete of ORF7, the segment targeted by all methods, allowed a gain of insight into the genetic variability of Italian strains and to investigate the role of mismatches on assay sensitivity. Our study evidenced that methods based only on primers-genome interaction better tolerate PRRSV genetic variability, demonstrating a greater sensitivity (Se): SYBR Green I (Se. = 98.4%) and RT-PCR (Se. = 99%) outperform both in-house (Se. = 71.4%) and commercial (Se. = 91.7%) probe-based methods. On the other hand, probe-based assays allowed an easier genotyping of PRRSV strains and implementation of the internal control system (IC). Phylogenetic analysis allowed demonstration of a presence of two clades circulating continuously in northern Italy since 1996, when their probable ancestors were collecte
Primo isolamento di Bartonella Bovis in ruminanti domestici in Italia.
No full textBlood samples from cattle from herds of the province of Venice, were submitted to microbiological investigation to detect Bartonella spp. after a suspect of infection, based on microscopic observation of blood smears. Blood culture were performed and 10 samples resulted positive. DNA extracted from colonies isolated from each of the 10 culture-positive samples, was subjected to PCR amplifying the16S-23S rRNA intergenic region (ITS), and the genes citrate synthase (gltA), RNA Polymerase β-subunit (rpoB), Riboflavin synthase (ribC) and further sequenced. The sequences obtained by BLAST showed percentages of homology with reference B. bovis sequences ranging from 98% to 100%. This is the first report of B. bovis in Italy
Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus
No full textThe accuracy and rapid diagnosis of PRRSV infection is a major prerequisite for every control and/or eradication strategy. In this study two real time RT-PCR based on different chemistry analysis (TaqMan Probes and SYBR Green) have been developed and validated before comparison to an end point two-step RT-PCR validated previously. All assays were aimed at discrimination between PRRSV genotypes. Furthermore, an exogenous internal control (IC) system had also been implemented in qRT-PCR. A rigorous analytical validation, executed on infected cell cultures and serum, demonstrated good sensitivity, specificity and repeatability. In particular RT-PCR was exceptionally sensitive and could detect a viral titre in the order of a magnitude of 1 copies/mu L, 10-fold lower than other qRT-PCR described in this study. Optimal diagnostic performances have been demonstrated analyzing samples retrieved from an experimental infection, with RT-PCR again outperforming real time RT-PCR assays. All tests, showing substantial agreement between them, were able to detect early stages of viraemia (1 DPI) and some animals were classified as positive until the end of the study (76 DPI). Therefore, this supports the assays usefulness in animals with different clinical conditions and in a broad range of epidemiological scenarios. The benefits and disadvantages of different assays were also considered and discussed
Detection and differentiation of Avian Metapneumovirus by Real Time Rt-PCR
No full textDirect diagnosis of avian Metapneumovirus (AMPV) infections rely on molecular techniques more than on virus isolation due to the fastidious nature of the virus. Six real-time reverse transcription PCR (RRT-PCR) protocols for the detection and differentiation of AMPV subtype A and B were developed in N (two tests), F, SH (two tests) and G genes. Five assays used SYBR Green I as detection system, and one molecular beacon probes. Specificity was evaluated using various AMPV strains, Newcastle disease, Infectious laryngotracheitis and Infectious bronchitis viruses. All tests were able to detect AMPVs and failed to detect non-AMPV viruses, and five out of six of them were also able to discriminate between AMPV A and B subtypes. Sensitivity was determined using serial dilutions of RNA from AMPV of both subtypes. The best results in terms of specificity and sensitivity was given by the RRT-PCR protocol designed in the SH gene
Diagnostica virologica di PRRS: studio comparativo tra metodiche biomolecolari classiche ed innovative con campioni ottenuti da un'infezione sperimentale.
No full textPorcine reproductive and respiratory syndrome
(PRRS) is reported as the most important disease currently affecting the pig industry worldwide.
A comparative evaluation of 4 different molecular methods (RTPCR end-point and Real-time) on samples collected during an experimental infection trial was performed.
TaqMan probe systems may be inefficient in detecting mutant viruses because of their genetic variability leading to a diagnostic failure. In-house assays can be easily adapted to new circulating strains being more flexible than commercial kits if they are not constantly updated
Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B
No full textIn recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian
metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex
quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed
to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and
repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV
subtype A and B strains tested were amplified and specifically detected while no amplification occurred with
other non-target bird respiratory pathogens. The detection limit of the assay was 10 0.41 median infectious
dose/ml and 10 1.15 median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A
strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was
approximately 2 and the error values were 0.2. Standard curves, generated either using the serial dilution
of an RNA suspension or RNA extracted from the serial dilution of titrated viral suspensions as templates,
exhibited good linearity (R2 = 0.9375) between crossing point values and virus quantities, making the assay
herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a
conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls
and from experimentally infected birds. This assay can be effectively used for the detection, identification,
differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry
out rapid surveillance with high levels of sensitivity and specificity
Prevalence of ESBL-producing Escherichia coli from different canine populations in Italy
Full text linkZoonosi emergenti in Italia: indagine sulla presenza di Anaplasma phagocytophilum in zecche del bosco del Cansiglio
No full textAim of the study was to investigate the occurrence of Anaplasma phagocytophilum infection in ticks from a tourist alpine area in North-eastern Italy (Bosco del Cansiglio). Ticks were collected in 5 sites, selected according to altitude, vegetation, presence of water, humans and animals. 65 ticks were collected by dragging, the most of them from the lowest site (350 m asl). Ticks were examined for the Ehrlichia spp. and Anaplasma spp. by a screening one-tube nested PCR. Poisitive samples were sequenced and submitted to PCR using specific primers for A. phagocytophilum. Three samples (5%) proved positive. This was the first confirmation of A. phagocytophilum infection in ticks in the area under stud
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