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1.
Artigo em Inglês | MEDLINE | ID: mdl-33802554

RESUMO

Leptospirosis is a worldwide-spread zoonosis causing disease and death in dogs and in humans. A Leptospiral infection has been recorded in several wild carnivore species in Europe, but tissue pathological changes were not commonly described. The Grey wolf (Canis lupus) has been expanding its distribution range in north-eastern Italy during the last decade. A young wolf, representing the first individual handled in the region, was found road-killed and then submitted to necropsy. Pathological changes included erosive lesions of gingival mucosa, mild liver enlargement, and multifocal degenerative-necrotic areas along with hyperemic reactive lesions; multifocal interstitial nephritis and multifocal lung hemorrhages were observed. A Polymerase Chain Reaction (PCR) able to detect pathogenic species of Leptospira performed on a kidney sample was positive. Serological reactions for serogroup Gryppotyphosa (1:6400), Pomona (1:800), and Icterohaemorrhagiae (1:200) were evidenced by MAT. Genotyping by Multilocus Sequence Typing (MLST) performed on detected Leptospira characterized it as belonging to Sequence Type (ST) 117, which refers to L. kirschneri, serogroup Pomona, serovar Mozdok. Regardless of the role of Leptospira infection as an eventual predisposing factor to the road killing of this wolf, to the best of the authors' knowledge, this is the first report of Leptospira-induced pathology in a wolf in Europe. Surveys on Leptospira infection in free-ranging wildlife species should be pursued in order to achieve further epidemiological knowledge on the circulation of the Leptospira strain.


Assuntos
Leptospira , Leptospirose , Lobos , Animais , Cães , Exposição Ambiental , Europa (Continente) , Itália/epidemiologia , Leptospira/genética , Leptospirose/epidemiologia , Leptospirose/veterinária , Tipagem de Sequências Multilocus , Sorogrupo
2.
Front Vet Sci ; 7: 335, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760741

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) affects farmed swine causing heavy direct and indirect losses. The infections sustained by PRRS viruses (PRRSV-1 and PRRSV-2) may give rise to severe clinical cases. This highlights the issue of PRRSV pathogenicity and relevant markers thereof. Since PRRSV strains can be discriminated in terms of immunotypes, we aimed to detect possible correlates of virulence in vitro based on the profile of innate immune responses induced by strains of diverse virulence. To this purpose, 10 field PRRSV isolates were investigated in assays of innate immune response to detect possible features associated with virulence. Tumor necrosis factor-α, interleukin (IL)-1beta, IL-8, IL-10, and caspase-1 were measured in cultures of PRRSV-treated peripheral blood mononuclear cells of PRRS-naive pigs, unable to support PRRSV replication. Two reference PRRSV strains (highly pathogenic and attenuated, respectively), were included in the screening. The PRRSV strains isolated from field cases were shown to vary widely in terms of inflammatory cytokine responses in vitro, which were substantially lacking with some strains including the reference, highly pathogenic one. In particular, neither the field PRRSV isolates nor the reference highly pathogenic strain gave rise to an IL-1beta response, which was consistently induced by the attenuated strain, only. This pattern of response was reversed in an inflammatory environment, in which the attenuated strain reduced the ongoing IL-1beta response. Results indicate that some pathogenic PRRSV strains can prevent a primary inflammatory response of PBMCs, associated with reduced permissiveness of mature macrophages for PRRSV replication in later phases.

3.
Pathogens ; 9(6)2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32570803

RESUMO

Leptospirosis in dogs has been largely described worldwide, and epidemiological studies have been mainly based on serological data. This study aims to detect and genotype leptospires affecting symptomatic dogs in Northeast Italy between 2013 and 2019. Overall, 1631 dogs were tested using real-time PCR, and leptospires from 193 dogs were subjected to Multilocus Sequence Typing and a Multiple Loci Variable-number Tandem Repeat Analysis. Leptospires were successfully isolated from 15 symptomatic dogs. Six distinct Sequence Types (STs) were found for 135 leptospires, with 3 STs characterizing Leptospira interrogans (ST17, ST198 and ST24), 2 STs characterizing Leptospira kirschneri (ST117 and ST289) and 1 ST characterizing Leptospira borgpetersenii (ST155), revealing the circulation of the serogroups Icterohaemorrhagiae, Australis, Sejroe and Pomona. The Multiple Loci Variable-number Tandem Repeat Analysis of 17 samples did not result in any additional discrimination. Genotypes were compared with those of strains present in the historical internal database, and possible transmission chains were identified from rat, mouse, hedgehog and pig. This work highlights the importance of molecular methods in revealing and identifying circulating Leptospira strains, and it also encourages the evaluation of the ability of commercially available vaccines to reduce the disease burden among dogs.

4.
Pathogens ; 9(5)2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32365494

RESUMO

Swine act as both maintenance and incidental hosts of pathogenic Leptospira spp. Here, a serological test was performed on 131,660 pig sera collected between 2002 and 2017 from 4715 farms in Northern Italy. A positivity rate of 13.05% was determined. Australis was the most frequently identified serogroup (77.29%), followed by Pomona (18.47%), Tarassovi (1.51%) and Icterohaemorrhagie (1.40%). Culture isolation and real-time Polymerase chain reaction (PCR) were carried out on 347 kidneys and 470 clinical samples, respectively. Overall, 133 strains were cultured successfully and 43 randomly chosen isolates were identified as serogroup Pomona. Multi-locus sequence typing (MLST) revealed that 41 isolates and 8 DNA extracted from biological samples belonged to sequence type 140. Using a multiple-locus, variable-number tandem repeat analysis, 43 samples produced identical profiles but, after 2014, three new Leptospira interrogans serogroup Pomona genotypes were observed. Interestingly, two isolates showed new MLST profiles and an unclassified identification by monoclonal antibodies. The 16S rRNA gene sequencing clustered them into L. kirschneri species and a core genome MLST analysis revealed an allelic identity of 96% compared with Mozdok strains. Genotyping allowed us to discriminate leptospires and to identify new emerging strains. The accurate identification of infective strains is required for formulating preventive methods and intervention strategies.

5.
Pathogens ; 9(5)2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32384672

RESUMO

Leptospirosis is an infectious disease that causes serious illness in dogs. For this reason, epidemiological and clinical studies focusing on disease characterization are widely advocated. The aim of this study was to characterize the leptospires identified in dogs with confirmed symptomatic acute leptospirosis. Leptospira spp. DNA detected in urine, blood, or both samples from nine infected dogs was analyzed using the multi-locus sequence typing (MLST) technique. Leptospires from two dogs were successfully typed: one was identified as belonging to Sequence Type (ST) 17 and one to ST198, both within the L. interrogans species, serogroups Icterohaemorrhagiae and Australis, respectively. Based on the results of routine serologic tests, antibodies reactive toward these serogroups are commonly revealed in dogs in Italy. This study provides the first molecular analysis that identifies infecting Leptospira directly on DNA from biological samples of dogs, showing that serogroup Australis can lead to a severe clinical presentation of leptospirosis in infected dogs.

6.
J Wildl Dis ; 56(3): 727-729, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32195642

RESUMO

The pathogenic Leptospira species are very widespread in nature, persisting in the renal tubules of many domestic and wild animal reservoirs. We report the isolation of Leptospira interrogans serovar Pomona in a bottlenose dolphin (Tursiops truncatus) stranded along the coast of Sardinia, Italy, in 2016.


Assuntos
Golfinho Nariz-de-Garrafa/microbiologia , Leptospira interrogans/isolamento & purificação , Leptospirose/veterinária , Animais , Leptospirose/epidemiologia , Leptospirose/microbiologia , Mar Mediterrâneo/epidemiologia
7.
Transbound Emerg Dis ; 65(6): 1935-1942, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30094946

RESUMO

Porcine Epidemic Diarrhoea Virus (PEDV) causes watery diarrhoea, dehydration, and a high mortality rate among suckling pigs. Recently, PEDV had a large negative economic impact on the swine industries in Asia and North America. In 2014, PEDV re-emerged in many European countries, but most countries only reported a few sporadic cases. Here, we report the epidemic wave that occurred in Italy from 2015 to 2017. During this time, PEDV was detected by real-time PCR in 438 farms located mainly in the high-density pig production area in Northern Italy. Most of the outbreaks were in farrow-to-finish, farrow-to-wean and finisher farms. Clinical signs were observed mainly in suckling and fattening animals, while mortality rates were higher in piglets, reaching 50%. A sequence analysis showed that a PEDV strain, similar to the OH851 S-INDEL strain isolated in the USA in January 2014, was responsible for the outbreaks in Italy in 2015 and 2016. However, from January 2017, a recombinant variant strain, containing a portion of the Swine Enteric Coronavirus in the S1 gene, spread and almost completely outcompeted the previous nonrecombinant strain. In total, 14.1% of the environmental swabs collected from trucks at slaughterhouses after animals were unloaded tested positive for PEDV before the trucks were cleaned and disinfected, and 46% remained positive after cleaning and disinfection processes were performed. Moreover, environmental swabs indicated that 17.3% of the empty trucks arriving at the farms to load animals were PEDV-positive. This study indicates that trucks can have an important role in the spread of PEDV in Italy.


Assuntos
Infecções por Coronavirus/veterinária , Surtos de Doenças/veterinária , Transmissão de Doença Infecciosa/veterinária , Doenças dos Suínos/transmissão , Transportes , Animais , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Diarreia/veterinária , Itália/epidemiologia , Vírus da Diarreia Epidêmica Suína/genética , Suínos , Doenças dos Suínos/epidemiologia , Desmame
8.
Front Microbiol ; 8: 1174, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28702010

RESUMO

The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs) are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR), has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis, and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad) and the Quant Studio 3D (Applied Biosystems). The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of M. avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was <0.5 Log10, while cultural methods underestimated the number of bacteria by one to two Log10 for Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. On the other hand, cultural and PCRs methods quantified the same amount of bacteria for L. monocytogenes, suggesting for this last pathogen, PCRs approaches can be considered as a valid alternative to the cultural ones.

9.
Front Microbiol ; 7: 2009, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28018330

RESUMO

The porcine epidemic diarrhea virus (PEDV) causes an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, watery diarrhea, and a high mortality rate in seronegative neonatal piglets. In the last few years, PED had a large economic impact on the swine industries in Asia and the US, and in 2014, the PEDV also re-emerged in Europe. Two main PEDV variants circulate worldwide but only the S INDEL variant, considered a mild strain, is spreading in Europe. To gain insights into the pathogenicity of this variant, its viral load and temporal shedding pattern were evaluated in piglets from infected farms. Quantitative real-time PCR (qPCR) targeting the spike gene, was validated according to the minimum information for quantitative real-time PCR experiments guidelines. The qPCR was applied to longitudinal studies conducted in four swine farms naturally infected with the PEDV S INDEL variant. Clinical data, fecal swabs, and blood samples were collected from 103 piglets at 15-30-day intervals for 2-5 months. On all four farms, diarrhea was observed in sows during gestation and in farrowing units, and the mortality rates of piglets were 18, 25, 30, and 35%. Different clinical pictures (0-50% of diarrhea positivity), viral titer levels (mean 5.3-7.2 log10 genome copies/mL), and antibody conditions (30-80% of positivity) were registered among sows on the four farms. The percentage of qPCR positive piglets varied greatly from the beginning (63-100%) to the end (0%) of the infection course. Clinical signs were present in 96% of the qPCR positive animals. Viral loads ranged from 8.5 log10 to 4 log10 genome copies/mL in suckling pigs at 3-6 days of age and were not statistically different among farms, despite the different patterns observed in sows. After 2-3 weeks, only a few piglets still showed detectable viral levels and clinical signs, and they developed antibody responses. Moreover, co-infections with other pathogens and biosecurity procedures limiting the circulation of the virus could have influenced the severity of PED infection. QPCR and clinical data were useful in understanding the dynamics of PEDV infections and, therefore, in implementing appropriate control measures.

10.
Biochem Biophys Res Commun ; 430(1): 38-42, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23200837

RESUMO

Tetanus and botulinum neurotoxins act inside nerve terminals and, therefore, they have to translocate across a membrane to reach their targets. This translocation is driven by a pH gradient, acidic on the cis side and neutral on the cytosol. Recently, a protocol to induce translocation from the plasma membrane was established. Here, we have used this approach to study the temperature dependence and time course of the entry of the L chain of tetanus neurotoxin and of botulinum neurotoxins type C and D across the plasma membrane of cerebellar granular neurons. The time course of translocation of the L chain varies for the three neurotoxins, but it remains in the range of minutes at 37 °C, whilst it takes much longer at 20 °C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates.


Assuntos
Toxinas Botulínicas/metabolismo , Membrana Celular/enzimologia , Metaloendopeptidases/metabolismo , Toxina Tetânica/metabolismo , Animais , Toxinas Botulínicas/toxicidade , Células Cultivadas , Concentração de Íons de Hidrogênio , Metaloendopeptidases/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Biossíntese de Proteínas , Ratos , Proteína 25 Associada a Sinaptossoma/metabolismo , Temperatura , Toxina Tetânica/toxicidade , Fatores de Tempo
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