Diagnóstico e infecção experimental por Perkinsus beihaiensis em anomalocardia brasiliana (MOLLUSCA:BIVALVIA) no Estado do Ceará

Abstract

Perkinsiosis is a disease caused by protozoa of the genus Perkinsus and causes massive mortality in several species of commercial bivalve mollusks, mainly in Europe and United States. Thus, the present study aimed to verify the occurrence of Perkinsus in the cockle Anomalocardia brasiliana (Veneridae) from a natural population and evaluate the impact of an experimental challenge with Perkinsus beihaiensis in this host. Specimens of A. brasiliana were collected in the Timonha River estuary (West coast of Ceará) in March 2012 (n= 150) and submitted to the diagnosis of Perkinsus spp. by tissues incubation of gills and rectum in Ray’s Fluid Thioglicollate Medium (RFTM). The confirmation of the genus Perkinsus was done by PCR analyzes (ITS) and subsequent sequencing. The RFTM showed the presence of hypnospores of Perkinsus infecting the tissues of the animals, with a prevalence of 14.7% (22/150) and intensity of infection ranging from very mild (n= 18) to mild (n= 4). Analyzes of PCR and sequencing confirmed that this species is Perkinsus beiahiensis. So this is the first record of this species in A. brasiliana. In order to assess the impact of an experimental challenge with P. beiahiensis in cockles from the Jaguaribe River estuary (east coast of Ceará), animals (n= 6) were distributed in 12 tanks (6 controls and 6 treatments). A cell suspension of hypnospores of P. beihaiensis was prepared from tissues of Crassostrea rhizophorae naturally infected. The tissues were incubated in RFTM to obtain a suspension of hypnospores. The animals of the treatment group were challenged by injection of 100μl of cell suspension of the parasite (125 cells) into the paleal cavity and the control animals received 100μl of sterile filtered seawater (AMFE). The challenge lasted 30 days and samples of cockles were collected in the 15th (n= 25) and 30th (n= 41) day after injection for pathological analyzes (RFTM, histology and PCR). The mortality of the animals was recorded daily. Mortalities occurred only during the first 15 days of the experiment (n = 6, of the treatment group), but it was not detected Perkinsus sp. in these animals. From the animals collected in the 15th day, 8 of 17 (control group, 47%) and 2 of 14 (treatment group, 14.3%) had hypnospores of Perkinsus sp. (RFTM) with intensities of infection ranging from very mild to mild, greater in the treatment group than in the control group. From the animals collected in the 30th day, 4 of 19 (control group, 21%) and 4 of 22 (treatment group, 18.2%) were diagnosed as infected with Perkinsus sp., with intensities of infection ranging from very mild to mild, higher in the treatment group than in the control group. The histopathological analyzes confirmed the presence of cells of Perkinsus sp. infecting the organs of the cockles in the two groups, but the treatment group was the only one with cells being phagocytosed The proliferating cells of Perkinsus sp. occurred in both groups. PCR analyzes confirmed the presence of Perkinsus sp. in 4 of 6 animals analyzed (66.7%). This work revealed that the cockle A. brasiliana already is a natural host of Perkinsus beihaiensis, and a challenge with cells isolated from P. beihaiensis causes induction to infection, but the host recovers itself quickly and efficiently activating defense mechanisms.