Bartonella spp.: curva de crescimento em meio Tryptic Soy Broth (TSB) suplementado e avaliação de transmissão transovariana e perpetuação transestadial através de infecção in vitro em Amblyomma sculptum (acari: ixodidae)
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Data
2021-07-12
Autores
Camargo, Jaqueline Valéria
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Universidade Estadual Paulista (Unesp)
Resumo
Bactérias do gênero Bartonella são agentes zoonóticos, podendo causar danos autolimitantes ou fatais aos seus hospedeiros. Essas bactérias infectam diversos animais, e são transmitidas por vetores como pulgas, piolhos, moscas e carrapatos. Além da diversidade de hospedeiros ser um desafio epidemiológico, essas bactérias são fastidiosas e seu isolamento em meio de cultura é uma constante dificuldade. O objetivo do presente estudo foi padronizar a curva de crescimento de Bartonella spp. em meio TSB (Tryptic Soy Broth) suplementado e verificar a ocorrência de transmissão transovariana e perpetuação transestadial em carrapatos da espécie Amblyomma sculptum, com sangue infectado por Bartonella spp., através de alimentação artificial. Para avaliar o crescimento de Bartonella spp. foi utilizado o meio de cultura (TSB), suplementado com soro fetal bovino, sacarose e anfotericina. As contagens de UFC para a formação da curva de crescimento foram realizadas em placas pelo método de superfície. Uma vez estabelecida a fase ótima de crescimento da bactéria, chegando na concentração 107 a mesma foi utilizada para infectar sangue de coelho previamente testado negativo para Bartonella spp. Fêmeas (49) de A. sculptum foram alimentadas artificialmente durante duas horas com o sangue infectado, por meio de tubos de microhematócrito, sendo, em seguida, alimentadas naturalmente em coelhos até o completo ingurgitamento. Ninfas (98) também foram alimentadas artificialmente durante quatro dias, por meio de membrana natural feita de pele de camundongo, para a avaliação da perpetuação transestadial de Bartonella spp. Após oviposição das fêmeas adultas alimentadas e adultos mudados das ninfas alimentadas artificialmente foram testados molecularmente detecção do DNA do patógeno. Os resultados da curva de crescimento da bactéria mostraram que no meio TSB houve um crescimento lento, porém bem delimitado, com a fase de latência se estendendo por 21 horas, fase exponencial ou log de 40 a 100 horas e fase estacionária se mantendo de 100 a 160 horas, e iniciando seu declínio após esse período. Ensaio de qPCR para Bartonella spp., baseado no gene nuoG dos ovos das 30 fêmeas sobreviventes após alimentação artificial, mostraram-se negativos, evidenciando que não houve transmissão transovariana. Uma fêmea resultante de uma ninfa alimentada artificialmente foi positiva, no entanto, a possibilidade de perpetuação transestadial de Bartonella spp. por essa espécie de carrapato não pode ser confirmada, haja vista a presença de sangue residual no referido espécime de carrapato. Conclui-se que o meio TSB se mostrou adequado para a realização da curva de crescimento sendo que conseguiu demonstrar todas as fases necessárias na curva. A alimentação artificial dos adultos demonstrou que não há a transmissão transestadial, porém, a alimentação artificial das ninfas não foi conclusiva devido a uma fêmea positiva na qPCR.
Bacteria of the genus Bartonella are zoonotic agents, being able cause self-damage or fatal to their hosts. These bactéria infec many animals, and are transmitted by vectors like fleas, lice, flies and ticks. In addition to host diversity being na epidemiological challenge, these bactéria are fastidious and their isolation in a culture medium is a constant difficulty. The goal of this study was standardize the growth curve of Bartonella spp. in medium TSB (Tryptic Soy Broth) supplemented and verify the occurrence of transovarian transmission and transstate perpetuation in ticks of the species Amblyomma sculptum with blood infected by Bartonella spp., through artificial feeding. To evaluate the growth of Bartonella spp. Culture medium (TSB) was used, supplemented with fetal bovine serum, sucrose and amphotericin. ScoreS CFU for the formation of the growth curve were performed on plates by the surface method. Once the optimal bacterial growth phase is established, arriving at concentration 107 it was used to infected rabbit blood previously tested negative for Bartonella spp. Females (49) of A. sculptum were artificially fed for two hours with infected blood, through microhematocrit tubes, then being naturally fed to rabbits until complete engorgement. Nymphs (98) were also artificially fed for four days, through a natural membrane made of mouse skin, for the assessment of the transstate perpetuation of Bartonella spp. After oviposition of the fed adult females and changed adults from artifically fed nymphs were tested molecularly the DNA of the pathogen. The results of the bacterial growth curve showed that in the TSB medium there was a slow growth, however well delimited, with the latency phase extending over 21 hours, exponential or log phase iv from 40 to 100 hours and stationary phase remaining from 100 to 160 hours, and starting its decline after that period. qPCR assay for Bartonella spp., based on the nuoG gene in the eggs of 30 surviving females afte artificial feeding, were negative, showing that there was no transovarian transmission. A female resulting from na artificially fed nymph was positive, however, the possibility of transstate perpetuation of Bartonella spp. by this species of tick cannot be confirmed, have a view the presence of residual blood in the above tick specimen. It is concluded that the TSB medium proved to be adequate for the realization of the growth curve being able to demonstrate all the necessary phases in the curve. The artificial feeding of adults demonstrated that there is no transstate transmission, however, the artificial feeding of the nymphs was not conclusive due to a qPCR positive female.
Bacteria of the genus Bartonella are zoonotic agents, being able cause self-damage or fatal to their hosts. These bactéria infec many animals, and are transmitted by vectors like fleas, lice, flies and ticks. In addition to host diversity being na epidemiological challenge, these bactéria are fastidious and their isolation in a culture medium is a constant difficulty. The goal of this study was standardize the growth curve of Bartonella spp. in medium TSB (Tryptic Soy Broth) supplemented and verify the occurrence of transovarian transmission and transstate perpetuation in ticks of the species Amblyomma sculptum with blood infected by Bartonella spp., through artificial feeding. To evaluate the growth of Bartonella spp. Culture medium (TSB) was used, supplemented with fetal bovine serum, sucrose and amphotericin. ScoreS CFU for the formation of the growth curve were performed on plates by the surface method. Once the optimal bacterial growth phase is established, arriving at concentration 107 it was used to infected rabbit blood previously tested negative for Bartonella spp. Females (49) of A. sculptum were artificially fed for two hours with infected blood, through microhematocrit tubes, then being naturally fed to rabbits until complete engorgement. Nymphs (98) were also artificially fed for four days, through a natural membrane made of mouse skin, for the assessment of the transstate perpetuation of Bartonella spp. After oviposition of the fed adult females and changed adults from artifically fed nymphs were tested molecularly the DNA of the pathogen. The results of the bacterial growth curve showed that in the TSB medium there was a slow growth, however well delimited, with the latency phase extending over 21 hours, exponential or log phase iv from 40 to 100 hours and stationary phase remaining from 100 to 160 hours, and starting its decline after that period. qPCR assay for Bartonella spp., based on the nuoG gene in the eggs of 30 surviving females afte artificial feeding, were negative, showing that there was no transovarian transmission. A female resulting from na artificially fed nymph was positive, however, the possibility of transstate perpetuation of Bartonella spp. by this species of tick cannot be confirmed, have a view the presence of residual blood in the above tick specimen. It is concluded that the TSB medium proved to be adequate for the realization of the growth curve being able to demonstrate all the necessary phases in the curve. The artificial feeding of adults demonstrated that there is no transstate transmission, however, the artificial feeding of the nymphs was not conclusive due to a qPCR positive female.
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Palavras-chave
Bartonella spp., Amblyomma sculptum, Medicina veterinária, Carrapato, Bactérias patogênicas