Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Abstract

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-Paque TM Plus, Amershan Biociences ® . The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles ® , Alexa Fluor ® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.Keywords: Chelonia mydas, green turtle, leukocytes, flow cytometry.A citometria de fluxo como ferramenta para avaliar a função celular de leucócitos sangüíneos de Chelonia mydas (Testudines, Cheloniidae) ResumoChelonia mydas é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e uma doença denominada fibropapilomatose é uma das mais importantes ameaças à sobrevivência dessa espécie. Desta forma, a definição de uma metodologia que permita analisar a função dos leucócitos sangüíneos torna-se extremamente necessária. Foram utilizadas amostras sangüíneas de C. mydas com e sem fibropapilomas capturadas no litoral norte do estado de São Paulo. As amostras sangüíneas foram colocadas em tubos contendo heparina sódica e transportadas em meio de cultura celular RPMI 1640 estéril e sob refrigeração.

Biometria corporal e parâmetros hematológicos de Trachemys scripta elegans e Trachemys dorbignyi (Testudines: Emydidae) criadas em cativeiro em Petrolina, Pernambuco

“…Os valores obtidos neste estudo foram semelhantes também aos de Chelonia mydas (Rossi et al 2009, Santos et al 2009) e Chelonia caretta (Cubas & Baptistotte 2006, Pires et al 2009); inferiores aos de Phrynops geoffroanus de áreas urbanas (Silva 2011) e ligeiramente superiores aos de T. callirostris (Velásquez et al 2014). Brites (2002) observou influência do sexo e da área geográfica nas concentrações de hemoglobina, pois o maior tamanho das fêmeas seria o responsável pelas maiores concentrações de hemoglobina, fato que não pôde ser confirmado neste estudo, pois não houve distinção entre os sexos na realização dos exames.…”

“…(Alves 2013), onde os valores foram semelhantes aos de P. geoffroanus (Zago et al 2010) e T. callirostris (Velásquez et al 2014) e superiores aos de C. caretta (Cubas & Baptistotte 2006, Pires et al 2009); C. mydas (Rossi et al 2009, Santos et al 2009) e P. geoffroanus (Silva 2011). Correlação negativa entre a contagem total de eritrócitos e VCM e HCM foi observada em ambas as espécies estudadas, todavia a ausência de correlação com a CHCM discordou de P. geoffroanus (Brites 2002).…”

“…(Alves 2013); T. scripta elegans (Medeiros et al 2012); C. mydas (Rossi et al 2009, Santos et al 2009); C. caretta (Cubas & Batistotte 2006, Pires et al 2009), P. geoffroanus (Brites 2002) e T. callirostris (Velásquez et al 2014). Embora os resultados possam ser altamente variáveis em répteis, até mesmo quando se utiliza métodos consistentes de aná-lise, a contagem global de leucócitos é um parâmetro muito útil quando se realizam estudos comparativos ou o monitoramento da evolução do quadro clínico (Work et al 1998).…”

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

“…The phagocytosis is considered the first cellular innate immune response against pathogens and is the end point of several humoral responses such as complement activity and antibody opsonization (Secombes and Fletcher, 1992;Tizard, 2014). In fish, as in other vertebrates, the phagocytosis is a complex processes in which the phagocytes, usually granulocytes, monocytes and macrophages, recognize and engulf microorganisms or foreign particles, destroying them with endogenously produced antimicrobial substances (Neumann et al, 2001;Marques et al, 2003;Rossi et al, 2009). Phagocytosis is also important in rejection processes, participating in the destruction of the exert, in the antigen presentation and in the production of interleukins (Abbas et al, 2014;Tizard, 2014).…”

Host stress physiology and Trypanosoma haemoparasite infection influence innate immunity in the woylie ( Bettongia penicillata )

“…Phagocytosis flow cytometry is a high throughput, quantitative assay, which assesses leukocytes' capacity to phagocytose fluorescently labelled bacteria [25,27]. It has numerous advantages for use in comparative immunology and has proven to be a rapid, reliable tool to evaluate phagocytosis in different species ranging from teleosts [28], mice and macaques [27] to salamanders [29], seals [30], camels [31], sea turtles [22,32], beluga whales [33] and Tasmanian devils [34]. The ex vivo nature of the phagocytosis flow cytometry assay also has advantages over in vivo immunologic tests such as allograft rejection, which may be ethically challenging to carry out in critically endangered species.…”