Samples and study design
A total of 1.265 serum samples from domestic (n=682), synanthropic (n=133), and wild animals (n=450) were taken from different animal species to investigate the presence of anti-Leptospira spp. agglutinins. Twelve animal species were sampled, including 100 chickens, 220 cats, 146 dogs, 105 cattle, 83 sheep, 12 goats, 16 horses, 108 black rats (Rattus rattus ), 25 Norway rats (Rattus norvegicus ), 435 cattle egrets (Bubulcus ibis ), 13 tegu lizards (Salvator merianae ), and two rock cavies (Kerodon rupestris ).
Since no reliable estimates for the wild and domestic animal populations were available prior the study period, a convenience sampling was performed. The samples were obtained between 2007 and 2014. All samples were obtained exclusively in the main island of Fernando de Noronha, although the cattle egrets only used the main island during the day for foraging, while nesting in multiple islets of the archipelago. A total of 12 sampling sites were systematically used in the main island, all located at the APA territory.
Rodents and rock cavies were captured using Tomahawk live-traps daily baited with pineapple slices at five pm and checked in the next morning. Cattle egrets were captured at the waste treatment station unit and were physically restrained by using nets and gloves. Tegu lizards were captured using tunnel traps made with a 1-meter segment of plastic pipe with one end closed and the funnel traps were installed in shaded places with an inclination of 20º, preventing that the captured lizard could escape. Additionally, tomahawk live-traps daily baited with one piece of raw chicken or fish early in the morning were used. The domestic animals were contained with the help of their owners.
Blood samples from all domestic species were collected from the jugular, cephalic or saphenous vein, and drawn into BD Vacutainer tubes (BD Diagnostics, New Jersey, USA) to obtain serum samples. Rodents were anaesthetized using ketamine hydrochloride (40–90mg/kg) and xylazine hydrochloride (2–5mg/kg) and blood samples (3mL) were collected by cardiac puncture. Additionally, 12 brown rats and eight black rats were euthanized in a hermetically sealed box containing a cotton wool soaked with isoflurane. Rock cavies were anaesthetized using ketamine hydrochloride (10mg/Kg) and xylazine hydrochloride (2 mg/kg), and blood samples (0.5mL) were collected from the saphenous vein. Cattle-egrets were anaesthetized using ketamine hydrochloride (20-30 mg/kg) and xylazine hydrochloride (2-4mg/kg), and blood samples were collected from the jugular vein or by cardiac puncture. Tegus were physically contained and blood samples were collected by caudal vein puncture.