MAINTENANCE AND PROPAGATION OF EHRLICHIA CANIS STRAIN (DONATIEN, LESTOQUARD, 1935) (RICKETTSIALES ANAPLASMATACEAE) IN IXODES SCAPULARIS CELL LINE

Natália Élida Nascimento Ribeiro; Erica Rodrigues de Matos; Jonathan David Ribas Chagas; João Gabriel Ferreira Cabral; Ana Clara Rodrigues Felix Da Silva; Mirian Cardinot; Maria izabel Fernandes Gouveia Pereira; Matheus Dias Cordeiro; Claudia Bezerra da Silva; Bruna de Azevedo Baêta

Título do Trabalho

Autores

Natália Élida Nascimento Ribeiro
Erica Rodrigues de Matos
Jonathan David Ribas Chagas
João Gabriel Ferreira Cabral
Ana Clara Rodrigues Felix Da Silva
Mirian Cardinot
Maria izabel Fernandes Gouveia Pereira
Matheus Dias Cordeiro
Claudia Bezerra da Silva
Bruna de Azevedo Baêta

Modalidade

Resumo

Área temática

Protozoários e Agentes transmitidos por vetores de importância em Saúde

Data de Publicação

10/03/2025

País da Publicação

Brasil

Idioma da Publicação

Inglês

Página do Trabalho

https://www.even3.com.br/anais/ix-simposio-internacional-em-ciencias-veterinarias-sincvet-ix-international-symposium-in-veterinary-sciences-isvs-499466/1030696-maintenance-and-propagation-of-ehrlichia-canis-strain-(donatien-lestoquard-1935)-(rickettsiales-anaplasmatacea

ISBN

978-65-272-1246-1

Palavras-Chave

In vitro culture, hemoparasite, anaplasmataceae

Resumo

The Canine Monocytic Ehrlichiosis (CME), caused by Ehrlichia canis, is one of the most important canine diseases in Brazil. This bacterium has been cultivated in non-vector tick cells, such as Ixodes scapularis (ISE6), although it is primarily associated with Rhipicephalus sanguineus. The cell culture process involves the isolation and maintenance of cell viability as well as their proliferative capacity in an in vitro system, consisting of essential factors for survival under controlled conditions. The analysis of E. canis interaction with different cell lines is of great importance for studying its life cycle within the tick, in addition to providing insight into studies where E. canis cultivation in certain cell lines was unsuccessful, potentially contributing to the development of new CME control and prevention strategies. The aim of this study was to analyze Ehrlichia canis interaction with embryonic tick cells (ISE6) in vitro to broaden the understanding of its infection and transmissibility. This experiment was conducted using samples from the tick cell line bank at "The Roslin Institute and Royal (Dick) School of Veterinary Studies, BIOBANK" and exclusively in vitro methods. The ISE6 cells used for maintaining E. canis were kept at 32°C in L15-B medium supplemented with 5% fetal bovine serum (FBS), 10% tryptose phosphate broth (TPB), and bovine lipoprotein concentrate (MP, Santa Ana, CA, USA). The E. canis isolate ("E.canisPretinha") was aliquoted in a 1 mL cryotube, frozen in sucrose-phosphate-glutamate buffer (SPG), and stored in liquid nitrogen. The sample was reactivated and transferred to 25 cm² culture flasks (TPP, Trasadingen, Switzerland) at 34°C in a climate-controlled incubator containing 5 mL of supplemented L15-B medium with 0.1% NaHCO3 and 10 mM HEPES solution, without the addition of antibiotics (penicillin or streptomycin), with weekly medium changes. Growth was monitored at 1- to 3-week intervals by microscopic examination of Giemsa-stained smears. The monitoring of the isolate was carried out using an inverted phase-contrast microscope to observe any possible fungal or bacterial contamination. The medium was changed twice a week by removing 3 mL of the supernatant and pipetting an equivalent volume of fresh medium. Slides containing 1 mL of cell suspension sample were subjected to cytocentrifugation (Cytospin, Thermo Fisher Scientific) at a speed of 1,500 rpm for 5 minutes. The smears were fixed in methanol for 5 minutes and stained using the Giemsa method (1:10) for 30 minutes, then observed under an optical microscope (1000x). With the inverted phase-contrast microscope, cell adhesion was observed two days after thawing, with a clear medium and no contamination present. By day 17, cells were well adhered, requiring mechanical detachment using a Pasteur pipette to prepare the slide, where morulae in dense forms were observed. On day 22 post-thawing, a new smear revealed a higher quantity of morulae in dense forms and intact cells. Based on observations made throughout the experiment, it is concluded that E. canis shows good development in the ISE6 cell line. This result suggests that the Ixodes scapularis tick-derived cell line used in this experiment is an effective alternative method for in vitro study of E. canis, allowing advances in the understanding of its biology and potentially contributing to the development of diagnostic tools and control measures, as well as fostering new research initiatives.

Título do Evento: IX Simpósio Internacional em Ciências Veterinárias (SINCVET) / IX International Symposium in Veterinary Sciences (ISVS)
Título dos Anais do Evento: Anais do IX Simpósio Internacional em Ciências Veterinárias (SINCVET) / IX International Symposium in Veterinary Sciences (ISVS)
Nome da Editora: Even3
Meio de Divulgação: Meio Digital

Como citar

RIBEIRO, Natália Élida Nascimento et al.. MAINTENANCE AND PROPAGATION OF EHRLICHIA CANIS STRAIN (DONATIEN, LESTOQUARD, 1935) (RICKETTSIALES ANAPLASMATACEAE) IN IXODES SCAPULARIS CELL LINE.. In: Anais do IX Simpósio Internacional em Ciências Veterinárias (SINCVET) / IX International Symposium in Veterinary Sciences (ISVS). Anais...Seropédica(RJ) UFRRJ, 2025. Disponível em: https//www.even3.com.br/anais/ix-simposio-internacional-em-ciencias-veterinarias-sincvet-ix-international-symposium-in-veterinary-sciences-isvs-499466/1030696-MAINTENANCE-AND-PROPAGATION-OF-EHRLICHIA-CANIS-STRAIN-(DONATIEN-LESTOQUARD-1935)-(RICKETTSIALES-ANAPLASMATACEA. Acesso em: 15/12/2025