Comparison of light microscopic techniques for the diagnosis of the infection of the European flat oyster Ostrea edulis by the protozoan Bonamia ostreae

A comparison among various histological techniques for the detection of the parasite Bonamia ostreae in oysters Ostrea edulis was performed to evaluate their sensitivity and suitability for different purposes. The comparison involved examination of histological sections, tissue imprints from gills, digestive gland, gonad and heart, and haemolymph cell monolayers, prepared through various protocols. Every technique produced some false negative. The haemolymph cell monolayers were more sensitive than tissue imprints and histological sections. Heart imprints provided the highest sensitivity among tissue imprints. Examination of histological sections was among the least sensitive techniques. Four procedures for estimation of infection intensity were compared. Some differences in accuracy for the estimation of infection intensity between haemolymph cell monolayers and histological sections (HS) were detected: there was a very good agreement when the infection appeared low or heavy in HS but it was not so good in the remaining cases. The results suggest the need for a critical review of the recommendations of the "Office Internationale des Epizooties" and the European Union for diagnosis of bonamiosis.     

Changes in circulating and tissue-infiltrating hemocyte parameters of European flat oysters,Ostrea edulis,naturally infected with Bonamia ostreae

We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.     

Cellular and molecular responses of haemocytes from Ostrea edulis during in vitro infection by the parasite Bonamia ostreae

Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite, B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8 h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1 h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4 h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1 h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite, B. ostreae.     

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy.Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.     

Herpesvirus infection in European flat oysters Ostrea edulis obtained from brood stocks of various geographic origins and grown in Galicia (NW Spain)

We evaluated differences in productive traits and disease susceptibility among Ostrea edulis stocks. We produced 4 to 5 families from each of 4 oyster populations (Irish, Greek and 2 Galician) in a hatchery. Spat corresponding to 19 different families were transferred to a raft in the Ría de Arousa (Galicia, Spain) for grow-out. Samples of each family were histologically processed every month for 2 yr. One of the pathological conditions disclosed by histological examination was characterised by the occurrence of numerous abnormal cells throughout the connective tissue of various organs, showing hypertrophied nuclei with marginated chromatin and a characteristic large intranuclear acidophilic inclusion. Ultrastructural examination showed that the abnormal cells contained herpesvirus-like particles. In situ hybridisation assay using a DNA probe specific for Ostreid herpesvirus 1 (OsHV-1) confirmed that the abnormal cells were infected by OsHV-1 or a closely related herpesvirus. All cases of this pathological condition, except one, were detected during the first year of grow-out; thus it was mostly restricted to juvenile stages. The disease was detected in oysters of each origin but it was not found in all families of each origin, thus suggesting significant parental influence in the susceptibility to this disease or significant influence of the infective status of the parents on the infection of the progeny (vertical transmission). This pathological condition was likely responsible for oyster mortality to some extent during the first year of grow-out.     

Isolation and partial characterization of a calcium-dependent lectin (chiletin) from the haemolymph of the flat oyster, Ostrea chilensis

A calcium-dependent lectin (chiletin) was isolated from oyster haemolymph by mannose elution from Sepharose CL-6B followed by anion exchange chromatography. Chiletin was predominantly composed of 12 and 24 kDa bands when examined with SDS-PAGE under reducing and non-reducing conditions, respectively. Larger molecular weight bands of 36 and 50 kDa were also variably present under reducing conditions. The NH2-terminal sequence of the 24 kDa band was determined and was not homologous to any known protein from the databases searched. Isolated chiletin was composed of multiple isomers approximately 12 kDa in size and ranging in pI from 5.2 to 6.0. Rabbit antiserum was raised to a synthetic peptide coupled to keyhole limpet hemocyanin and the size of the chiletin subunits was confirmed by Western blot. Two and five different conformational aggregates of chiletin were resolved in oyster haemolymph using size exclusion chromatography in 8 M urea and PBS, respectively. The largest aggregate obtained from size exclusion in 8 M urea was estimated to be greater than 640 kDa. The ability of whole haemolymph and isolated chiletin to agglutinate sheep red blood cells was inhibited by galactose and mannose. Chiletin was identified by immunohistochemistry to be most consistently present in the auricle, followed by the digestive gland, however staining was seen sporadically in haemocytes, gastrointestinal epithelium and interstitial connective tissue cells.