RESUMO
There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0?×?106 cells/mL, and the population doubling time every 57?h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.
RESUMO
There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0?×?106 cells/mL, and the population doubling time every 57?h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.
RESUMO
Recently, tick and flea-borne pathogens have been detected in wild carnivores maintained in captivity in Brazilian zoos. Since free-roaming cats are frequently found in Brazilian zoos, they could act as reservoirs for arthropod-borne pathogens, which could be transmitted to endangered wild carnivores maintained in captivity in these institutions. On the other hand, stray cats in zoos may play a role as sentinels to pathogens that circulate among wild animals in captivity. The present work aimed to detect the presence of Anaplasmataceae agents, hemoplasmas, Bartonella species, piroplasmas, and Hepatozoon sp. DNA in blood samples of 37 free-roaming cats in a Brazilian zoo. Three (8%) cats were positive for Anaplasma spp. closed related to Anaplasma phagocytophilum; 12 (32%) cats were positive for hemoplasmas [two (5%) for Mycoplasma haemofelis, five (13.5%) for Candidatus Mycoplasma haemominutum, and five (13.5%) for Candidatus Mycoplasma turicensis]; 11 (30%) were positive for Bartonella spp., six (16%) were positive Babesia vogeli and one (3%) for Theileria sp. Coinfection with multiple arthropod-borne agentes was observed in sampled cats. None of sampled cats were positive for Ehrlichia spp., Cytauxzoon spp., or Hepatozoon spp. in PCR. This is the first molecular detection of Babesia vogeli and Theileria sp. in domestic cats in Brazil. The control of the population of free-roaming cats in these conservation institutions is much needed aiming to prevent the potential transmission to endangered wild animals maintained in captivity, such as wild neotropical wild felids, as well as to human beings visiting zoos.
