Protozoan Parasite Perkinsus Olseni Research Articles

First report of Perkinsus olseni infections in blood cockles Tegillarca granosa on the south coast of Korea

The protozoan parasite Perkinsus olseni has become a focus of attention since it has been responsible for mass mortalities and economic losses in a wide range of bivalve hosts globally. The P. olseni host range along the south coast of Korea may extend beyond what was previously understood, and blood cockles in the Family Arcidae are also suggested to be potential hosts of P. olseni. In the present study, we applied histology and molecular techniques to identify Perkinsus sp. infections in the blood cockles Tegillarca granosa, which have been commercially exploited on the south coast of Korea for several decades. Histology and molecular techniques, including genus-specific immunofluorescence assay, species-specific fluorescence in situ hybridization, and phylogeny based on the ribosomal DNA internal transcribed spacer region revealed that T. granosa is infected by P. olseni, although the prevalence was low (0.5%). Histology revealed massive hemocyte infiltrations in the mantle, gill, and digestive gland connective tissues, indicating that the infection exerts negative impacts on the host cockles.

Search for new biomarkers of tolerance to Perkinsus olseni parasite infection in Ruditapes decussatus clams

The grooved carpet shell (Ruditapes decussatus) is a clam species with high economic and social importance in several European and Mediterranean countries. Production of this species suffered a decline caused by biotic (parasite infection) and abiotic factors (environmental factors, stress, poor management methods and intensive culture of the introduced species Ruditapes philippinarum). The protozoan parasite Perkinsus olseni is also responsible for the decline of production, being nowadays one of the major issues for clam culture. Molecular biomarkers that might represent tolerance of R. decussatus to P. olseni have already been uncovered, shedding light in a possible production improvement by selecting those clams with a strongest immune response.In the present study, new tolerance biomarkers to P. olseni infection in R. decussatus were identified. The haemolymph proteomic profiles of naturally non/low-infected (tolerant) and highly-infected (susceptible) clams by the parasite across several heavy affected areas of Europe were characterized through a shotgun proteomics approach. Also, the mechanisms that might be involved in the responses against the disease in chronic infections were explored. Proteins related to energy restoration and balance, metabolic regulation, energy accumulation, ROS production, lysosomal activity, amino acid synthesis, proteolytic activity, iron regulation, iron withholding, and immune response modulation were significantly regulated in susceptible clams. In the tolerant group, proteins related to phagocytosis regulation, control of cell growth and proliferation, gonadal maturation, regulation of apoptosis, growth modulation, response to oxidative stress, iron regulation, shell development and metabolic regulation were significantly expressed.In summary, the protein expression profile of tolerant individuals suggests that an efficient pathogen elimination mechanism coupled to a better metabolic regulation leads to a tolerance to the parasite infection by limiting the spread through the tissues.

Assessment of the environmental distribution of the protozoan parasite Perkinsus olseni by next-generation sequencing, qPCR and histopathology allows the identification of alternative bivalve hosts

Perkinsus olseni is a protozoan parasite that infects a wide variety of molluscs worldwide, causing economic losses in the aquaculture sector. Consequently, Perkinsosis has been catalogued by the World Organization for Animal Health (O.I.E.) as a notifiable disease, and international measures have been established to control it (O.I.E., 2019). In the present study, we analysed the spatial and temporal distribution of transmissible stages of Perkinsus olseni in an endemic area of the parasite from 2016 to 2018. The pathogen was detected using high-throughput sequencing of the 18S rRNA gene and a specific real-time PCR assay (qPCR) in samples of water, sediment and several bivalve species. Histopathological assays were also conducted on bivalve samples.P. olseni was not detected in environmental samples by qPCR; however, eDNA sequencing revealed its presence in both the water and sediment at all sampled points, showing a seasonal pathogen prevalence. As expected, the parasite was detected in clams, but a few cases were also found in mussels and cockles. The presence of the parasite was confirmed in Cerastoderma edule by histology and qPCR using RNA to evaluate the presence of proliferative life stages of the parasite. Therefore, this is the first time that P. olseni has been found in C. edule but with low abundance and infection intensity levels.

Supplementation with lipids enhances zoosporulation of Perkinsus species

The protozoan parasite Perkinsus olseni is a major pathogen of marine mollusks. A large number of zoospores (the most infective stage) is required to experimentally examine the transmission and invasion processes of this parasite, but the conventional zoosporulation method in seawater is inefficient. A recent study found that P. olseni zoosporulation was highly enhanced in a nutrient-rich medium which was developed for trophozoite proliferation in Perkinsus. Here, we examined the effect of each medium component on P. olseni zoosporulation and found that lipids enhanced zoosporulation. Moreover, zoospores failed to develop into trophozoites in seawater supplemented with lipids, indicating that this zoosporulation method using seawater supplemented with lipids is ideal for preparing a large number of P. olseni zoospores for various biological assays. Lipid supplementation also significantly enhanced zoosporulation in P. honshuensis, P. chesapeaki, and P. marinus, although the enhancing effect of the lipid supplementation varied by species. Considering the limited availability of lipids in the sea water column, our findings suggest that Perkinsus zoosporulation likely occurs mainly in the vicinity of hosts (not in the sea water column), such as in the mantle cavity or on the body surface of mollusk hosts.

Are Juvenile Manila Clam Ruditapes philippinarum Free from Perkinsus olseni Infection in Korean Waters?

As a suspension feeder, Manila clam Ruditapes philippinarum (A. Adams and Reeve 1850) plays a crucial role in the coastal soft bottom ecosystem in the temperate region, linking the benthic primary production to the upper trophic level. Manila clam density on tidal flats on the west coast of Korea has been declining for the past decades, and infection by the protozoan parasite Perkinsus olseni (Lester and Davis 1981) is one of the major causes for the decline. Recent studies carried out in Japan revealed that P. olseni induces mortalities of the juveniles in their natural habitats, which may lead to the recruitment failure and subsequent decline in the clam population. In this study, we surveyed P. olseni infection in juvenile Manila clam occurring on two tidal flats on the Taean coast. Ray’s fluid thioglycollate medium assay (RFTM) revealed that P. olseni infection was not limited to the adult clams, and the juvenile and small-sized clams are also infected by P. olseni. As young as four-month-old juveniles from Jugyo tidal flat were infected by P. olseni, with the prevalence (i.e., percentage of the infected individuals) of 75.0% and the intensity of 7.77 × 105 cells g−1 wet tissue weight (WT). The adult Manila clams (SL > 30 mm) from Jugyo tidal flat showed a prevalence of 96.0%, and the intensity as 5.80 × 10 cells g−1 WT. The observed infection prevalence and intensity of the juvenile are somewhat comparable to those of the adult clams, suggesting that a high level of P. olseni infection in the juveniles may lead to mortality and a long term decline in the clam population density.

New insights into the Manila clam – Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum–P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosis.

Experimental Evaluation of the Impact of Perkinsus olseni on the Physiological Activities of Juvenile Manila Clams

High-intensity infection with the protozoan parasite Perkinsus olseni is lethal to the Manila clam Ruditapes philippinarum, especially to juvenile clams; however, how this parasite impacts its host remains unclear. This study was carried out to investigate the effects of P. olseni on the growth, condition index, burrowing behavior, and filtration activity of the Manila clam to elucidate mechanisms by which the infection causes death of the clams. Juvenile Manila clams (5–9 mm in shell length) were challenged with parasitic zoospores and were then reared in seawater for 52 days. Growth and condition index were frequently, but not always, significantly lower in the challenged group compared with the unchallenged group during the challenge experiment. Fewer individuals from the challenged groups compared with the unchallenged groups exhibited lower burrowing activity, when they were placed on sand in wells of 24-well plates. Furthermore, to compare filtration activity, clams from both groups were maintained in separate suspensions of diatoms. Diatom density remained higher in the challenged clams compared with the unchallenged clams, indicating that challenged clams exhibited lower levels of filtration activity. Decreased burrowing activity may cause higher predation rates and make the clams vulnerable to turbulent water and periodic tidal air exposure, resulting in death of Manila clams under natural conditions. In addition, suppression of filtration activity by infection may cause decrease in food and oxygen intake, leading to mortality induced by nutrient loss and hypoxia.

New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

여름철 산란기에 있어 바지락 번식량의 공간적 변이와 기생 원생생물 Perkinsus olseni 감염이 바지락 번식에 미치는 영향

Spatial variation in the reproductive effort of Manila clam Ruditapes philippinarum is often closely associated with variation in the seawater temperature and food availability, which determines gonad maturity and the quantity of gamates produced during spawning. Previous studies also have reported that severe infection by the protozoan parasite Perkinsus olseni exerts a negative impact on clam reproduction, retarding gonad maturation or decreasing the reproductive effort. In the present study, we investigated impacts of P. olseni infection on the reproductive condition of Manila clam during a spawning season. Histology revealed that 54% of female clams in Wando off the south coast were in spawning, while only 10% of the female from Gomso and 0% of the female from Seonjaedo in Gyeonggi bay off the west coast were engaged in spawning at the end of May in 2004. Ray's fluid thioglycollate media (RFTM) assay was applied to assess P. olseni infection and indicated that the infection intensity in Wando (3,608,000 ± 258,000 cells/g wet tissue) was significantly higher than the levels in Gomso (1,305,000 ± 106,000 cells/g wet

Influence of reproductive condition of the Manila clam Ruditapes philippinarum on hemocyte parameters during early post-spawning period

In marine bivalve aquaculture, mortality is a major problem for sustainability of the industry. Mortalities usually result from complex interactions in which the physiological status is suspected to play a major role. In many bivalves, spawning appears as a critical period. Spawning is an energy demanding process potentially resulting in immunodepression and higher susceptibility to pathogenic infection. In an effort to understand the actual impacts of spawning on the cellular defense system (i.e., hemocytes) of the Manila clam Ruditapes philippinarum, we collected clams on a one-time sampling during early post-spawning period. We then applied, for the first time, a histology–flow cytometry combined approach to these animals. Hemocyte parameters, including concentration, sub-populations, mortality, phagocytosis capacity and reactive oxygen species production were analyzed along with gender, condition index as well as the tissue protein and carbohydrate levels. Among the 200 clams analyzed, 60.5% were spent, 24% spawning, 12% resting and 3.5% ripe and ready for spawning. No gender differences could be observed among all parameters and no correlation was determined between the protozoan parasite Perkinsus olseni infection intensity and the immune parameters. CI and tissue protein level of spawning and post-spawning (spent and resting) clams were significantly lower than those of pre-spawning (ripe) clams. The concentration of hemocytes tended to decline from ripe to spent clams, partly because of a concomitant increase of cell mortality and, most of all, a decrease in the quantity of circulating granulocytes. The decline in circulating granulocytes also resulted in a reduced phagocytosis capacity (% of cells able to phagocytise). At the animal scale, these observations support a depression of the global immune defense of spawning and post-spawning clams. However, at the cellular scale, remaining circulating cells did not display signs of stress nor altered metabolism. Indeed, phagocytosis, as the number of beads ingested per cell, was normal and no oxidative stress could be detected. Further studies will be necessary to determine if these hemocytes already reached their limit of stress tolerance, and to potentially help develop immunoprotective food additives for the aquaculture industry.

Physiological responses of Manila clams Venerupis (=Ruditapes) philippinarum with varying parasite Perkinsus olseni burden to toxic algal Alexandrium ostenfeldii exposure

Manila clam stock from Arcachon Bay, France, is declining, as is commercial harvest. To understand the role of environmental biotic interactions in this decrease, effects of a toxic dinoflagellate, Alexandrium ostenfeldii, which blooms regularly in Arcachon bay, and the interaction with perkinsosis on clam physiology were investigated. Manila clams from Arcachon Bay, with variable natural levels of perkinsosis, were exposed for seven days to a mix of the nutritious microalga T-Iso and the toxic dinoflagellate A. ostenfeldii, a producer of spirolides, followed by seven days of depuration fed only T-Iso. Following sacrifice and quantification of protozoan parasite Perkinsus olseni burden, clams were divided into two groups according to intensity of the infection (“Light–Moderate” and “Moderate–Heavy”). Hemocyte and plasma responses, digestive enzyme activities, antioxidant enzyme activities in gills, and histopathological responses were analyzed. Reactive oxygen species (ROS) production in hemocytes and catalase (CAT) activity in gills increased with P. olseni intensity of infection in control clams fed T-Iso, but did not vary among A. ostenfeldii-exposed clams. Exposure to A. ostenfeldii caused tissue alterations associated with an inflammatory response and modifications in hemocyte morphology. In the gills, superoxide dismutase (SOD) activity decreased, and an increase in brown cell occurrence was seen, suggesting oxidative stress. Observations of hemocytes and brown cells in tissues during exposure and depuration suggest involvement of both cell types in detoxication processes. Results suggest that exposure to A. ostenfeldii disrupted the pro-/anti-oxidant response of clams to heavy P. olseni intensity. In addition, depressed mitochondrial membrane potential (MMP) in hemocytes of clams exposed to A. ostenfeldii suggests that mitochondrial functions are regulated to maintain homeostasis of digestive enzyme activity and condition index.

Microsatellite marker development in the protozoan parasite Perkinsus olseni

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 +/- 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 +/- 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease.

First record of Perkinsus olseni, a protozoan parasite infecting the commercial clam Ruditapes decussatus in Spanish Mediterranean waters

We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.

Noble tandem-repeat galectin of Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni

The galectin family of lectins plays crucial roles in the innate immunity systems of vertebrates and invertebrates. Noble galectin (MCGal) was cloned from the marine invertebrate Ruditapes philippinarum and characterized. This protein has an open reading frame of 918 nucleotides, with 309 amino acid residues, and a predicted molecular weight of 33.9kDa. Similar to other galectins, MCGal has neither a signal peptide nor a transmembrane domain, but it contains tandemly repeated carbohydrate recognition domains (CRDs), with typical conserved motifs that are important for carbohydrate recognition. Carbohydrate recognition by the recombinant MCGal (rMCGal), as determined by hapten inhibition of hemagglutination, revealed that rMCGal has features common to the galectin family, i.e., significant affinity for galactose and N-acetylgalactosamine. MCGal mRNA expression was detected mainly in the heart, mantle, foot, adductor, palp, and siphon tissues. Immunohistochemistry (IHC) using an anti-MCGal antibody confirmed MCGal expression in these tissues and in hemocytes. Temporal expression of MCGal mRNA in Manila clams challenged with Perkinsus or Vibrio species was up-regulated as compared with non-challenged healthy clams. rMCGal agglutinated Vibrio tapetis, and agglutination was inhibited by incubation with alpha-lactose. rMCGal also bound to the surface of Perkinsus olseni. MCGal plays a crucial role in Manila clam defense, particularly with respect to pathogen recognition.

Lectin from the Manila Clam Ruditapes philippinarum Is Induced upon Infection with the Protozoan Parasite Perkinsus olseni

Glycan-binding proteins (lectins) are widely expressed in many invertebrates, although the biosynthesis and functions of the lectins are not well understood. Here we report that Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) upon infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a approximately 74-kDa precursor and secreted into hemolymph where it is converted to 30- and 34-kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by one or more factors in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite, and this binding is inhibitable by either EDTA or GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL is concentrated within cyst-like structures. To define the glycan binding specificity of MCL we examined its binding to an array of biotinylated glycans. MCL recognizes terminal non-reducing beta-linked GalNAc as expressed within the LacdiNAc motif GalNAcbeta1-4GlcNAcbeta1-R and glycans with terminal, non-reducing beta-linked Gal residues. Our results show that the synthesis of MCL is specifically up-regulated upon parasite infection of the clams and may serve as an opsonin through recognition of terminal GalNAc/Gal residues on the parasites.

Analysis of EST and lectin expressions in hemocytes of Manila clams ( Ruditapes philippinarum) (Bivalvia: Mollusca) infected with Perkinsus olseni

The hemocytes of invertebrates play key roles in both cellular and humoral immune reactions by phagocytosis or delivering immune factors such as lectin and anti-microbial peptides. Bacterial infection causes changes in components such as lectins, anti-bacterial peptides, and lysosomal enzymes of plasma or hemolymph in molluscs. Previously, we found that infection with the protozoan parasite, Perkinsus, increases lectin synthesis in hemocytes. In order to investigate the patterns of genes expressed in Manila clams ( Ruditapes philippinarum) infected with the protozoan parasite Perkinsus olseni, we constructed a cDNA library and sequenced 1850 clones (expressed sequence tags). A total of 79 ESTs, were related to 29 functional immune genes such as C-type lectin, lysozyme, and cystatin B, in Manila clams. Lectins were the largest group of immune-function ESTs found in our Manila clams library. Among 7 lectin clones, two full length cDNAs of lectins were cloned. MCL-3, which is a simple C-type lectin composed of 151 amino acids, has a relatively short signal sequence of 17aa and single carbohydrate-recognition domain (CRD) of ∼130 residues. It is highly homologous to eel C-type lectin. The sequence of mc-sialic acid-binding lectin consists of 168 amino acid residues with molecular weight of 19.2 and shows high homology to sialic acid-binding lectin from the snail, Cepaea hortensis. The expression of 7 different lectins in hemocytes was analyzed by RT-PCR using gene-specific primers. Hemocytes from Perkinsus-infected clam expressed different sets of lectins than with Vibrio infection. These results demonstrate that several lectins are involved in Manila clam innate immunity and different challenges induce expression of different lectins.

Shikimate and folate pathways in the protozoan parasite, Perkinsus olseni

We have exploited the experimental accessibility of the protozoan parasite Perkinsus olseni and its similarities to apicomplexan parasites to investigate the influence of specific drugs on its proliferation. For this purpose, shikimate and folate pathways present an attractive target for parasitic therapy given their major differences with mammalian pathways. Glyphosate, a potent inhibitor of the shikimate pathway enzyme EPSP synthase inhibited the in vitro proliferation of P. olseni in a dose-dependent manner and this effect was reversed by addition of chorismate, indicating the presence of a shikimate pathway. However, this effect was not antagonised by p-aminobenzoate or folic acid. Furthermore, antagonism was observed, via pyrimethamine to glyphosate inhibitory effect, suggesting that the shikimate pathway is not essential for the biosynthesis of folate precursors and is therefore crucial for another pathway downstream from chorismate. In addition, sulfadiazine, a well known inhibitor of dihydropteorate synthase, an enzyme of the folate biosynthetic pathway,had no inhibitory effect on P. olseni proliferation. In view of these results, the parasite does not appear to require the folate biosynthesis pathway for its survival and is most likely able to use exogenous folate. Even though pyrimethamine was found to inhibit P. atlanticus growth, this inhibitory effect could not be reversed by co-addition of folic acid. Therefore, we propose that the effect of pyrimethamine observed in this study results from the inhibition of a target other than dihydrofolate reductase. Similarly, proguanil target is likely to be separate from DHFR since only its metabolite cycloguanil has been shown to have inhibitory properties on DHFR.

Effect of antiprotozoal drugs on the proliferation of the bivalve parasite Perkinsus olseni

The protozoan parasite Perkinsus olseni causes severe losses among Ruditapes decussatus clams. The development of an in vitro culture of this parasite together with the use of a proliferation assay has provided the opportunity to screen for drug sensitivity of this parasite. Xenobiotics known for their antimalarial and antiprotozoal properties were tested against P. olseni. Only four of these drugs, namely cycloheximide, pyrimethamine, deferoxamine (DFO) and 2,2-bipyridyl (BIP), showed in vitro inhibitory effect on the parasite proliferation. Two in vivo experiments were designed to determine the effect of iron chelators on reducing P. olseni infection in clams. For this purpose, naturally infected clams from the Ria Formosa, Portugal, were exposed to DFO and BIP at various concentrations. In the first experiment, hemolymph samples were taken from each clam before and after treatment to determine the infection intensity and in the second experiment, clams were randomly distributed in groups of five and the parasite burden in treated and untreated groups was determined at the end of the experiment on the whole clam wet tissues. Only DFO was found to be effective in reducing in vivo P. olseni infections. In addition, acute toxicity of DFO and BIP has been determined and no mortality of Perkinsus-free clams was observed.