RESUMO
Mycoplasma spp. can cause diseases of the respiratory system as well as urogenital infections, infertility, and anemia. The members of this genus have a low Gâ¯+â¯C content compared to other bacteria. Because primers used in the random amplified polymorphic DNA (RAPD) technique are only 10â¯bp long and have high GC content, this method can be inadequate for genotyping Mycoplasma spp. isolates. The aim of this study was to develop and evaluate multiple-locus variable number tandem repeat analysis (MLVA) and two-primer RAPD (TP-RAPD) procedures for subtyping Mycoplasma cynos isolates. A total of 55â¯M. cynos isolates obtained from 162 bronchoalveolar lavage fluid samples from shelter and pet dogs were used in this study. Seventy-four tandem repeat regions were detected in the M. cynos genome, and two of these loci were determined to be suitable and used for development of the MLVA scheme. The results of variable number tandem repeat (VNTR) analysis and TP-RAPD-PCR were compared with RAPD-PCR. The discriminatory power of TP-RAPD-PCR (Hunter-Gaston diversity index [HGDI]â¯=â¯0.84) was higher than those of RAPD-PCR (HGDIâ¯=â¯0.727), VNTR1 (HGDIâ¯=â¯0.8), and VNTR3 (HGDIâ¯=â¯0.757). We observed that the TP-RAPD-PCR and MLVA methods provide clearer data and are more successful in determining genetic diversity, in contrast to the RAPD-PCR method for this species.
Assuntos
Repetições Minissatélites , Mycoplasma/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Animais , Composição de Bases , Líquido da Lavagem Broncoalveolar/microbiologia , Primers do DNA , DNA Bacteriano/genética , Cães , Variação Genética , Genótipo , Técnicas de Genotipagem , Mycoplasma/isolamento & purificação , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Reação em Cadeia da Polimerase , Sequências de Repetição em TandemRESUMO
Extraction of DNA from Mycoplasma cultured on agar medium is difficult because the plasticity of these microorganisms enables agar penetration. This eventually causes cell loss during harvesting of colonies from the agar surface. Here, we used the GenElute™ gel extraction kit, which is usually used to purify polymerase chain reaction products, for extracting DNA from Mycoplasma. We compared the DNA extraction efficiency of the GenElute™ gel extraction kit from Mycoplasma cynos cultured in agar medium with four other DNA extraction methods. The results were evaluated based on the purity and amount of DNA obtained from one Mycoplasma colony. Eight strains of Mycoplasma cynos isolated from the broncho-alveolar lavage fluid of dogs were used. The GenElute™ gel extraction protocol was the most efficient among all the methods tested in this study as it yielded the highest amount and the purest quality of DNA (199.3±0.744ng/µl) from a single colony. Among the methods tested, the GenElute™ gel extraction method is the most rapid, sensitive, and simple method for DNA extraction from Mycoplasma. This procedure may also prove useful for extracting DNA from other Mycoplasma species.
Assuntos
Técnicas Bacteriológicas/métodos , DNA Bacteriano/isolamento & purificação , Mycoplasma/genética , Ágar , Animais , Técnicas Bacteriológicas/instrumentação , Meios de Cultura , DNA Bacteriano/genética , Doenças do Cão/diagnóstico , Cães , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/veterinária , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
This study aimed to determine the effectiveness of a pregnant mare immunization of a Rhodococcus equi (R. equi) vaccine candidate containing a water-based nanoparticle mineral oil adjuvanted (Montanide IMS 3012) inactive bacterin and virulence-associated protein A (VapA), as well as the administration of anti-R. equi hyperimmune (HI) plasma against R. equi challenge in the mares' foals. The efficacy of passive immunizations (colostral passive immunity by mare vaccination and artificial passive immunity by HI plasma administration) was evaluated based on clinical signs, complete blood count, blood gas analysis, serological response (ELISA), interleukin-4 (IL-4) and interferon gamma (IFN- γ ), total cell count of the bronchoalveolar lavage fluids (BALF) samples, reisolation rate of R. equi from BALF samples (CFU/mL), lung samples (CFU/gr), and lesion scores of the organs and tissue according to pathological findings after necropsy in the foals. The vaccination of pregnant mares and HI plasma administration in the foals reduced the severity of R. equi pneumonia and lesion scores of the organs and tissue by 3.54-fold compared to the control foals. This study thus indicates that immunization of pregnant mares with R. equi vaccine candidate and administration of HI plasma in mares' foals effectively protect foals against R. equi challenge.