Publicação
Bovine genital campylobacteriosis : new insights into the molecular diagnosis and pathogenesis
| Resumo: | C. 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. |
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| Autores principais: | Silva, Marta Filipa Serra da |
| Assunto: | Campylobacter fetus subsp. venerealis Bovine Genital Campylobacteriosis Campylobacter fetus subespécie venerealis Campilobacteriose Genital Bovina diagnóstico molecular potencial de virulência Campylobacter portucalensis |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | C. 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. |
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