Toxoplasma gondii subsp. isolation

Amendoeira, Mrr, Arruda, If, Moreira, Sb, Ubiali, Dg, Barbosa, As, Pena, Hfj, Pereira, Ahb, Raso, Cns, Bonif, Tf, acio, Clemes, Ys, Pissinatti, Ta, Santos, Afa & Pissinatti, A, 2022, Isolation and genetic characterization of Toxoplasma gondii from a captive black-and-gold howler monkey (Alouatta caraya Humboldt, 1812) in Brazil, International Journal for Parasitology: Parasites and Wildlife 19, pp. 187-190 : 188

2.1. Sample collection and T. gondii isolation

Samples were collected from a 10-year-old male black-and-gold howler monkey ( Alouatta caraya ) kept at the Primatology Center of Rio de Janeiro State, Brazil. The aforementioned primate presented fever, prostration, inappetence, abdominal distension and pain, intestinal hypomotility and weight loss, and it was under medical treatment for suspected toxoplasmosis. It was euthanized after 36 days of treatment, when it did not show significant clinical improvement. All descriptions comprising symptoms, anti- T. gondii serology, treatment, disease evolution and post-mortem macroscopic and microscopic lesions were previously reported by Moreira et al. (2022). T. gondii isolation attempts were performed based on using 33g of liver, 19.8g of heart, 13.6g of lungs, 2g of axillary lymph nodes and 1.7g of cerebellum, due to suspected toxoplasmosis in the herein investigated primate. Tissue samples were digested in acid pepsin solution, according to Dubey (1998). Digested samples of each tissue, in separate, and a pooled tissue homogenate (comprising all tissues mentioned above) were subcutaneously inoculated in fourteen female Swiss Webster mice in the age group 25-to-30 days, who were divided into six different groups, as follows: cerebellum and heart (3 mice, each); and liver, lungs, lymph nodes and pooled tissues (2 mice, each). Animals showing clinical signs compatible to acute toxoplasmosis (ruffled coat, hunched behavior, ascites and inactivity) were euthanized for peritoneal washing in order to investigate the presence of tachyzoites. Isolate’ s virulence profile was defined based on Pena et al. (2008). Asymptomatic mice were euthanized 60 days after inoculation in order to investigate the presence of cysts in brain macerates. All procedures involving the animals used in the herein described bioassay were approved by the Ethics Committee on the Use of Animals, IOC/Fiocruz, under license L-041/2019.