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- Interação entre Escherichia coli uteropatogénica e as células epiteliais e do estroma do endométrio caninoPublication . Silva, Marta Filipa Serra da; Mateus, Luísa Maria Freire Leal; Silva, Maria Elisabete Tomé SousaA piómetra é uma doença de diestro comum em cadelas adultas, sendo Escherichia coli o agente mais frequentemente isolado. Neste estudo estabeleceu-se um modelo de cultura primária de células epiteliais e do estroma, para avaliar: a) adesão e internalização de E. coli; b) resposta imunitária inata. Os ensaios de adesão e internalização, com duas estirpes de E. coli de diferente genótipo de virulência, demonstraram que E. coli aderiu às células epiteliais e do estroma. Os resultados de internalização sugerem que este mecanismo não deverá estar envolvido na patogenicidade deste agente na piómetra. Nas células epiteliais, E. coli conduziu a um aumento da transcrição de genes de quimiocinas, citocinas pró-inflamatórias e interferão-. No estroma observou-se principalmente aumento da transcrição de genes de citocinas pró-inflamatórias. Contudo, a transcrição do gene da citocina pró-inflamatória IL-1e da anti-inflamatória IL-10 foi inibida nestas células por uma das estirpes de E. coli. Conclui-se que (1) o sistema de cultura de células implementado é uma ferramenta útil para estudo da fase inicial de infeção por E. coli na piómetra (2) as células epiteliais e do estroma desempenham um papel importante na resposta inflamatória; (3) a resposta imunitária desencadeada parece depender da virulência da estirpe de E. coli.
- Bovine genital campylobacteriosis : new insights into the molecular diagnosis and pathogenesisPublication . Silva, Marta Filipa Serra da; Mateus, Luísa Maria Freire Leal; Silva, Maria Elisabete Tomé Sousa; Duarte, Ana Isabel Simões PereiraC. fetus subsp. venerealis is the pathogen responsible for the Bovine Genital Campylobacteriosis (BGC), a cattle’s venereal disease whose diagnosis relies on the accurate identification of Cfv. Despite its relevance for BCG control and management, the applicability of the molecular methods on the disease diagnosis is controversial whereas the mechanisms underlying the pathogenesis of BGC remain unclear. Therefore, the main objectives of this work were to assess the suitability of different real-time PCR assays to be used in bovine preputial samples for Cfv detection and to further elucidate the virulence potential of Cfv through the genomic characterization of Cfv field isolates. The results showed that the currently used molecular targets for Cfv identification, the parA gene and the insertion sequence ISCfe1, originate a high rate of false-positive results (up to 50 %) in clinical samples. This lack of specificity was also evidenced by the identification of a novel bacterial species in the bovine prepuce, Campylobacter portucalensis, with sequences homologous to parA (98 % identity) and ISCfe1 (94 % identity) sequences of Cfv, hindering the molecular diagnosis of BGC. On the other hand, the absence of the parA gene in a high proportion of Cfv strains (77 %), also invalidate the use of this gene as a diagnostic target for Cfv identification. The identification of three novel Sequence Types (ST) in venerealis subspecies, previously characterized by a high genetic stability, questions the use of Multilocus Sequence Typing (MLST) for subspecies identification. This work additionally evidenced differential features between Cfv and its biovar intermedius, namely in whole-genome Single Nucleotide Polymorphisms (SNPs) and genes encoding genus-specific proteins families. Moreover, the results suggest that a type IV secretion system, previously indicated as involved in Cfv virulence, does not play a crucial role in the pathogenesis. In vitro antimicrobial resistance to streptomycin, penicillin, tetracycline and enrofloxacin was not detected, even in strains encoding two multidrug efflux pumps, CmeABC and YkkCD, revealing that its sole presence in the genome is not enough to provide in vitro antimicrobial resistance. Overall, these results have a significant impact on the molecular diagnosis of BGC and revealed new insights into the Cfv genetic diversity and virulence potential.
- Genomic and phenotypic characterization of Campylobacter fetus subsp. venerealis strainsPublication . Silva, Marta Filipa Serra da; Pereira, Ana L.; Fraqueza, M. J.; Pereira, Gonçalo; Mateus, Luisa; Lopes-da-Costa, Luís; Silva, EThe pathogenesis mechanisms of Campylobacter fetus subsp. venerealis (Cfv), the etiologic agent of Bovine Genital Campylobacteriosis remain elusive. This study evaluated the virulence potential and biovar characteristics of Cfv isolates (n = 13) by PCR screening of putative virulencefactor (VF) genes, Multilocus Sequence Typing (MLST) analysis, antimicrobial susceptibility to tetracycline, penicillin, enrofloxacin and streptomycin testing and whole-genome sequencing (WGS; n = 5), also comparing the latter with 26 other whole-genome sequences of Cfv strains. The putative VF genes encoding type IV secretion system of Cfv (virB2-virB11/virD4) were absent in 92% of isolates, including isolates from aborted foetuses, evidencing that these VF genes are not essential for Cfv pathogenicity. The parA gene, used as a Cfv diagnostic molecular target, was detected in only 3 of 13 isolates, invalidating its use for diagnosis purposes. Three novel sequence types were identified by MLST. Although no in vitro antimicrobial resistance was detected, WGS identified antimicrobial resistance-related genes, including those encoding the multidrug efflux pumps CmeABC and YkkCD, indicating that their presence is not enough to provide antimicrobial resistance. The SNP and accessory protein families analysis segregated the Cfv and Cfv biovar intermedius (Cfvi) strains into different clusters. In conclusion, this study evidenced virulence potential and biovar characteristics of Cfv and Cfvi, which are of relevance for the control of Bovine Genital Campylobacteriosis.