Haplosporidian Parasite Research Articles

Molecular phylogenetic evidence of a haplosporidian parasite infecting the polychaete Syllis nipponica (Imajima, 1966)

A haplosporidian parasite was detected in the polychaete Syllis nipponica via the amplification and sequencing of the 18S rDNA locus. This provides additional evidence of haplosporidians in polychaetes, the host group from which the first species of the phylum were described about a century ago. In phylogenetic analyses, the parasite was determined to place among mollusk-specific taxa. As such, separate taxonomic groups for haplosporidians infecting different host phyla should not be anticipated. These findings also underscore the as-yet unknown life cycles of commercially important parasites. Attempts to visualize spores of the parasite directly with scanning electron microscopy did not succeed.

Ultrastructural description of new Rickettsia-like organisms in the commercial abalone Haliotis tuberculata (Gastropoda: Haliotidae) from the NW of Spain

Rickettsia-like organisms (RLOs) were found in the commercially farmed abalone Haliotis tuberculata in the northwestern region of the Atlantic Coast of Spain and are described from light and transmission electron microscopy observations. The RLOs measured approximately 1.6 x 0.9 microm and were found in intracytoplasmic, spherical to ellipsoidal vacuoles (up to 8 microm) in the epithelial cells of the digestive diverticulae. The morphological ultrastructure of these organisms was typically prokaryotic, including a plasmalemma and a thin Gram-negative type cell wall. Several ultrastructural changes were observed in the epithelial cells of the host containing the RLOs. The nuclei became pycnotic and several basophilic dense inclusions appeared in the cytoplasm. In addition, the host cell appeared lysed and was ruptured in advanced stages of infection. It was impossible to ascertain whether the RLOs are responsible for this disease, as a haplosporidian infection was also present. We can only conclude that the presence of RLOs simultaneously with a haplosporidian parasite may contribute to the mortality of the abalone host.

Bonamia exitiosa epizootic in Ostrea chilensis from Foveaux Strait, southern New Zealand between 1986 and 1992

Abstract Disease caused by the haplosporidian parasite, Bonamia exitiosa, swept through the dredge oyster (Ostrea chilensis) population of Foveaux Strait between 1986 and 1992, with consequent mortality reducing the population to 9% of the pre-disease level. Dead and dying oysters were first seen by fishers in far western Foveaux Strait in 1985 and more were found further east in 1986. Infection spread slowly through Foveaux Strait so the progress of the epizootic can be described from population surveys. A wave of infection radiated through the oyster population from the epicentre of infection in central western Foveaux Strait, and was followed by a wave of mortality. The epizootic ceased in oyster beds around the margins of oyster distribution in 1992. Infective particles released by diseased oysters spread through the water to infect other oysters directly. The epizootic broadly fitted a simple deterministic epizootic model and suggested that both diffusion and turbulent processes were important in transmission of infection. Bonamia exitiosa was also present in oysters at the end of an epizootic in 1964 and was probably the cause of that epizootic. Bonamiasis appears to be an endemic disease in Foveaux Strait. The high mortality in the 1986–1992 epizootic was like that caused by a newly introduced disease in an immunologically naïve population. We propose that other stressors have increased the susceptibility of oysters to this disease. Mechanical disturbance of oysters by increasingly intense dredging appears to be a major source of stress, as does the increasing scale of modification of benthic habitat by fishing. Recovery of the oyster population after the epizootic is closely linked to regeneration of habitat. The prognosis for the fishery could be improved by mitigating mechanical disturbance during dredging by use of lighter dredges and less damaging towing strategies, as well as pursuing rotational fishing strategies that allow benthic habitat to regenerate in undisturbed areas.

Molecular detection of a haplosporidian parasite in carpet shell clam Ruditapes decussatus from Spain

A protozoan parasite identified by histology as a haplosporidian was detected in carpet shell clam Ruditapes decussatus from Spain during routine samplings. Analysis based on the sequence of the small subunit ribosomal RNA gene revealed that this organism was related to the haplosporidian group, mainly to Urosporidium. A specific probe was used for in situ hybridization studies to show the specificity of the amplified sequence.

Molecular analysis of a haplosporidian parasite from cultured New Zealand abalone Haliotis iris.

In the Austral summer and autumn of 2000 and 2001, mortalities of black-footed abalone Haliotis iris (Martyn, 1784) occurred in a commercial facility in New Zealand. Histological analyses suggested that infection by a haplosporidian parasite was responsible. To confirm identification as a haplosporidian and to help determine if this parasite represented a new, undescribed species, DNA was extracted from infected host tissues scored as positive for infection by histological examination. Small-subunit rRNA (SSU rRNA) gene sequences from both the host abalone and a parasitic organism were amplified by PCR and characterized. Although the sequence for this parasite was novel, not matching any known SSU rRNA gene sequences, phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. Parsimony analyses placed the parasite at the base of the phylum Haplosporidia, ancestral to Urosporidium crescens and the Haplosporidium, Bonamia, and Minchinia species. Sequencing of multiple parasite DNA clones revealed a single polymorphic site in the haplosporidian SSU rRNA gene sequence.

Pathology of cultured paua Haliotis iris infected with a novel haplosporidian parasite, with some observations on the course of disease.

Mortalities among juvenile paua Haliotis iris Martyn 1784 in a commercial culture facility were reported in April 2000. Histology of moribund paua showed heavy systemic infections of a uni- to multi-nucleate stage of a novel organism later confirmed by transmission electron microscopy (TEM) and molecular studies to be a haplosporidian. Multinucleate plasmodia up to 25 microm diameter with up to 17 nuclei were detectable in wet preparations of hemolymph from heavily infected paua. The presence of the haplosporidian in the affected facility was associated with mortalities of slow growing 'runt' paua during the summer months. Total mortalities in affected raceways 6 mo after mortalities began were between 82.5 and 90%. Heavily infected paua exhibited behavioural abnormalities including lethargy, loss of righting reflex, and were easily detached from surfaces. Some heavily infected paua exhibited oedema and pale lesions in the foot and mantle, but no reliable gross signs of disease were noted. Light infections of the haplosporidian were also found in apparently healthy paua from the facility. Histology indicated that the early stages of infection were characterised by small numbers of plasmodia in the connective tissue surrounding the gut, amongst glial cells adjacent to nerves in the mantle and foot and within gill lamellae. In heavy infections, large numbers of small plasmodia (mean size 5.5 x 7 microm in histological sections) were present in the hemolymph, gills, heart, kidneys, mantle, foot, epipodium and connective tissue of the digestive gland. Infections were not transferred horizontally at 14 and 19 degrees C after cohabiting heavily infected paua with uninfected paua for 3 mo in aquaria, or 3 mo after injecting healthy paua with hemolymph containing haplosporidian plasmodia. This may indicate that the prepatent period for disease is longer than 3 mo, that disease is not expressed below 20 degrees C, or that an intermediate host is required for transmission. Spore formation was not observed in juvenile paua but sporocyst-like bodies containing putative spores were observed amongst haplosporidian plasmodia in the right kidney of poorly performing adult paua collected from the wild.

Detection of Haplosporidium nelsoni in Pacific oyster Crassostrea gigas in Japan.

One hundred spat of Crassostrea gigas obtained from a coastal area in north-eastern Japan were examined for the protistan parasite Haplosporidium nelsoni. Haplosporidium-like plasmodia were histologically observed in two spat and positively reacted with a H. nelsoni-specific probe in in situ hybridization. Four spat including the two spat in which the plasmodia were found showed positive reaction in PCR analysis for the detection of H. nelsoni.The small subunit ribosomal RNA sequence amplified from the spat was virtually identical (99.7%) to the sequence of H. nelsoni previously reported. These results demonstrate that H. nelsoni is distributed in Japan.

Ultrastructural description of the spore maturation stages of the clam parasite Minchinia tapetis (Vilela, 1951) (Haplosporida: Haplosporidiidae).

The fine structure of maturing spores of a haplosporidian parasite found in the gill, mantle and foot tissues of Ruditapes decussatus L. (Mollusca, Bivalvia), a species of commercial importance in Portugal, is described. When observed free in suspension, immature spores exhibit one or two epispore cytoplasmic extensions (ECE) which constitute a projection of a portion of the exosporoplasm, sometimes without ultrastructural organisation, surrounded by the plasmalemma. Free spores observed by light microscopy (LM) after 3-5 days of incubation in filtered sea-water exhibit no ECE attached to the spore wall. The mature spore is ovoid to ellipsoid, operculate, uninucleate and measures c. 4.8 microm long and c. 3.9 microm wide. The spore shape and size and the identity of the host living in the same geographical region suggest that this species is the same as previously described using LM observations as Haplosporidium tapetis Vilela, 1951 and later transferred to Minchinia Labbé, 1896.

Increased Virulence in an Introduced Pathogen: Haplosporidium nelsoni (MSX) in the Eastern Oyster Crassostrea virginica.

The protistan parasite Haplosporidium nelsoni has caused extensive mortality in the eastern oyster Crassostrea virginica along the mid-Atlantic coast of the United States since 1957. The origin of H. nelsoni has remained unresolved. Molecular diagnostic tools were used to examine the hypothesis that a haplosporidian parasite in the Pacific oyster C. gigas is H. nelsoni. A DNA probe specific for H. nelsoni reacted positively in in situ hybridizations with haplosporidian plasmodia from C. gigas collected in Korea, Japan, and California. Primers that specifically amplify H. nelsoni DNA in the polymerase chain reaction amplified product from Californian C. gigas infected with the haplosporidian parasite. The DNA sequence of the 565-base pair amplified product was identical to the H. nelsoni sequence except for a single nucleotide transition, a similarity of 99.8%. These results are conclusive evidence that the parasite in C. gigas is H. nelsoni and strongly support previous speculation that the parasite was introduced into Californian populations of C. gigas from Japan. Results also support previous speculation that H. nelsoni was introduced from the Pacific Ocean to C. virginica on the East Coast of the United States, likely with known importations of C. gigas. These results document greatly increased virulence in a naive host-parasite association and reinforce potential dangers of intentional, but improper, introductions of exotic marine organisms for aquaculture or resource restoration.

An unidentified haplosporidian parasite of bay scallop Argopecten irradians cultured in the Shandong and Liaoning provinces of China

Since 1988 growers of bay scallop Argopecten irradians in China have been experiencing mortality in their cultured stocks. Although poorly documented, mortality apparently began near Qingdao and has since spread to other areas of Shandong and Liaoning provinces. Samples of cultured scallops were collected from several growing areas in these provinces and analyzed by histological methods for pathogens. An unidentified haplosporidian parasite was observed in a high proportion of scallops from two of the stocks examined. Most infections were of low intensity, but one heavy infection was also observed. Only plasmodia stages were observed; they occurred intercellularly in connective tissues throughout the scallops. Plasmodia were spherical to oval, varied from 4.0 to 17.0 mu m in diameter and contained from 2 to 18 nuclei. Absence of spores prevented generic assignment of the parasite. The source and pathogenicity of the haplosporidian could not be assessed without additional research. No other microbial parasites (i.e. rickettsia-like, chlamydia-like or kidney coccidia) were observed in any of the scallops examined.

Cellular Responses of Oysters Infected with Haplosporidium nelsoni: Changes in Circulating and Tissue-Infiltrating Hemocytes

The protozoan parasite Haplosporidium nelsoni (MSX) elicits an inflammatory-type response in oysters, Crassostrea virginica. We assayed circulating hemocytes of oysters exposed to H. nelsoni to quantify the effects of parasitism, selection for resistance, and season on total and differential counts. All factors had a significant (P < 0.02) effect on total counts, but explained relatively little of the overall variance [season (12%) > selection (4%) > infection (2%)]. Circulating hemocyte densities increased with infection intensity in resistant animals, but were depressed in susceptible oysters with light infections. Counts in both groups were lowest in August and highest in May, at which times densities in susceptible oysters were reduced (P < 0.02) compared to those in resistant animals. No differences existed in November. Susceptible oysters may be more debilitated by infections than resistant animals, resulting in impaired circulation and depressed cell counts. The number of tissue-infiltrating hemocytes increased with infection intensity (P < 0.0001), but showed no statistically significant (P > 0.05) association with numbers of circulating hemocytes in individual oysters. Size-frequency analysis with a Coulter counter indicated that the proportion of large cells (presumed to be granular hemocytes) was lower (P < 0.0001) in susceptible oysters, which were also heavily infected, compared to resistant oysters, which had very few infections. The loss of granular hemocytes may stem from degranulation associated with tissue damage and inflammation. Present evidence suggests that the principal role of the hemocyte response in MSX disease is to plug lesions, remove debris, and repair tissues and that these functions may help oysters survive infection.

Lectin binding characteristics of haemocytes and parasites in the oyster, Crassostrea virginicd, infected with Haplosporidium nelsoni (MSX)

Lectin-binding surface receptors on haemocytes from host oysters were compared with those on plasmodial stages of the ascetosporan parasite Haplosporidium nelsoni (MSX). Haemocytes were agglutinated, in descending order of strength, by WGA, HPA, LPA, ConA, and CFA. GMA, PHA, and RMA lectins failed to agglutinate at 100 micrograms/ml, the highest concentration tested. These results indicate that haemocytes contain surface receptors resembling N-acetyl-D-glucosamine and alpha-methylmannopyranoside. Fluorescent (FITC) labelled ConA and WGA also bound to H. nelsoni plasmodia, but parasites were commonly excluded from clumps of agglutinated haemocytes, except for those that were apparently trapped passively in large aggregates. Although seasonal variations existed, agglutination titres for all reacting lectins were 2- to 8-fold higher for cells from systemically infected oysters compared to control oysters not manifesting systemic infections. Preincubation of lectins in serum from control animals reduced agglutination titres 6- to 9-fold, whereas incubation in serum from systemically infected oysters reduced titres only 4- to 6-fold. The loss of lectin-like molecules from the serum of systemically infected animals, and the concurrent increase of lectin receptors in the haemocytes population, is probably related to known changes in haemocytes composition and the loss of serum glycoproteins in diseased animals. Antigenic similarities were detected between surface receptors on oyster haemocytes and those on H. nelsoni plasmodia, which may help explain the failure of oyster haemocytes to phagocytose this parasite.

“Panning”, a technique for enrichment of the oyster parasite Haplosporidium nelsoni (MSX)

Plasmodial stages of the parasite Haplosporidium nelsoni (MSX) were enriched an average eight-fold with recovery of 60 to 80% using a process known as “panning” of hemolymph from infected oysters ( Crassostrea virginica). The method takes advantage of the greater adherence of hemocytes, compared to parasites, to inert surfaces. Hemolymph from infected oysters was layered onto a Petri dish and cells were allowed to settle for 30 min. Nonadhering cells were gently washed, decanted, and placed in a second Petri dish. Maximum enrichment and recovery of parasites were obtained after three panning cycles. Viability, estimated by trypan blue dye exclusion, was greater than 95%. Parasite-to-hemocyte ratios before panning were highly correlated to those after panning, but correlation decreased with each incubation. Initial concentration of parasites, which ranged from 1.4 to 5 × 10 5/ml, was also significantly correlated with the percentage recovery. We suggest that other extracellular invertebrate protozoans may also be enriched by the same method, which is much simpler, gentler, and less expensive than most other cell separation procedures.

Influence of selective breeding, geographic origin, and disease on gametogenesis and sex ratios of oysters, Crassostrea virginica, exposed to the parasite Haplosporidium nelsoni (MSX)

The recent spread and intensification of Haplosporidium nelsoni (MSX) disease in oysters, Crassostrea virginica, along the Atlantic coast of the United States has heightened interest in the use of disease-resistant strains in aquaculture and increased the need for understanding all facets of how selective breeding affects oyster performance, including reproduction. Resistance does not prevent infection, but involves an ability to restrict parasite development and to tolerate infections. We examined selected and unselected oysters, from three geographical locations, all exposed to H. nelsoni infection, to determine the influence of selection, strain origin, and parasitism on gametogenesis. Gonadal development was most frequently correlated with geographic origin of oysters: northern strains (from Long Island Sound) ripened and spawned earlier than southern strains (from the James River), even after several generations of selection at a single location in Delaware Bay. Parasitism was associated with impaired gonadal development in May when gametogenesis was occurring, but not in August, when spawning was underway. Selection for disease resistance was associated with improved gametogenic development, but only in oysters with few or no patent infections. Both parasite burden and selection for resistance were correlated with gametogenesis only in strains from the two southern-most locations, the James River and Delaware Bay, and never in those from Long Island Sound. We suggest that our observations resulted from interference by H. nelsoni in energy acquisition, storage, and conversion cycles of the oyster, which, in turn, differed seasonally according to geographic origin. Gonad development appeared to be more sensitive to parasitism when gametes were in the formative stage rather than after they had matured. Thus, southern strains, which were still in the early stages of gametogenesis when they experienced typical high parasite loads in late May, showed significantly greater inhibition than northern strains, which already had well-developed gonads at the same time. In contrast to most reports that environmental stress correlates with an increased ratio of males to females, we found a three-fold increase in the proportion of females among H. nelsoni-infected oysters compared to uninfected oysters. There was no evidence of sex-associated differential infection or mortality rates and we suggest that parasitism inhibits the development of male gametes more than female gametes.

Effects of sublethal infection by the parasite Haplosporidium nelsoni (MSX) on gameto-genesis, spawning, and sex ratios of oysters in Delaware Bay, USA

manuscript. This study was funded by the National Marine Fisheries Service under PL 88-309 (to H. Haskin), by the New Jersey Department of Environmental Protection, and by a fellowship from the Comision Asesora de Investigacion Cientiifica y Tecnica de Espatia to A. J. F. This is New Jersey Agricultural Experiment Station Publication No. D 32504-2-87, supported by state funds.

Infection and Mortality Patterns in Strains of Oysters Crassostrea virginica Selected for Resistance to the Parasite Haplosporidium nelsoni (MSX)

Strains of oysters Crassostrea virginica resistant to mortality caused by the parasite Haplosporidium nelsoni (MSX) were developed and tested through 6 generations. In addition, strains in each generation were followed for up to 6 yr of continuous exposure to the parasite in nature. Selected strains responded to challenge by the parasite with gradually improved survival in successive generations. They were slower to develop patent infections than were unselected groups and were able to delay mortality after infections did develop, but under repeated exposure most oysters eventually died with H. nelsoni parasitism. Many selected strains, however, reached market size before significant mortalities occurred. The data suggest that resistance to H. nelsoni mortality is under the influence of many genes. No clear defense mechanism has been described and we hypothesize that resistance to H. nelsoni may, in part, involve a physiological state in which selected oysters temporarily fail to provide a suitable habitat for the parasite. Temporary insusceptibility would, in this view, be followed by an increased ability to tolerate the parasite when conditions for its development are present. Selection would then favor individuals that are able to prolong periods of insusceptibility and/or to carry out basic life processes while parasitized.

Comparison of hemolymph proteins from resistant and susceptible oysters, Crassostrea virginica, exposed to the parasite Haplosporidium nelsoni (MSX)

Serum protein constituents in laboratory-reared stocks of oysters, Crassostrea virginica, susceptible to mortality caused by the parasite Haplosporidium nelsoni (MSX) were compared with constituents in resistant oysters exposed to the same level of infection activity. Virtually all of the susceptible oysters became infected and 71% died by the end of the 9-month study, while only half of the resistant group had patent H. nelsoni infections and only 7% died. Total serum protein concentrations fell sharply and in approximate proportion to disease intensity in oysters with systemic H. nelsoni infections. Oysters with heavy parasitism had about one-third the protein levels of uninfected individuals. When infections were confined to the gill, however, as was the case with mortality-resistant oysters, there was no depression of protein levels. In all oysters examined, SDS electrophoresis showed three rapidly migrating proteins, with molecular weights between 25,000 and 75,000, that comprised nearly all the total protein. Up to 12 larger proteins, with molecular weights up to 500,000, were also present, but with considerable individual variability in number and concentration. There was no obvious correlation of electrophoresis patterns with H. nelsoni disease or with genetic background. Results of the study suggest that depletion of certain metabolic substrates such as circulating protein and/or interruption of biosynthetic pathways by the parasite may be a primary cause of death in infected oysters.

Cytochemical analysis of the haplosporosomes and vesicle-like droplets of Haplosporidium lusitanicum (Haplosporida, Haplosporidiidae), parasite of Helcion pellucidus (Prosobranchia)

A cytochemical study of the spore of Haplosporidium lusitanicum, a haplosporidian parasite recently found in Helcion pellucidus, is described. Cytochemical analysis with Sudan Black B at the light microscope level revealed that the vesicle-like droplets (VLD) situated in the apical and basal zones of the endosporoplasm in close contact with the external membrane is strongly stained dark blue. These structures are partially digested with lipase. Both reactions suggest the presence of lipoid components. The dense bodies of the exosporoplasm seem to be analogous in chemical composition. On the other hand, these two distinct structures, when subjected to Thiéry's test for glycoproteins, gave positive results. We think that these materials are complex structures simultaneously containing lipids and glycoproteins. They may be involved in the formation of the complex membranous system (“spherule”) that develops during spore maturation in this species. The matrix of haplosporosomes submitted to Thiéry's test for glycoproteins was also positive. A comparative cytochemical analysis has revealed that the external membrane of the haplosporosomes is more glycoproteinaceous than the internal one, which is more lipoidal.

Chronic infections of Haplosporidium nelsoni (MSX) in the oyster Crassostrea virginica

Oysters inhabiting areas enzootic for the parasite Haplosporidium nelsoni (MSX) are exposed to infective particles each summer, and it is often difficult to distinguish newly acquired lesions from older infections. To study long-term parasitism without the complication of new infections, MSX-infected oysters were moved from Delaware Bay to a disease-free area on the New Jersey coast. Because infections seen after the transfer were acquired in Delaware Bay during a known infective period, it was possible to determine how long oysters remained infected and how they were affected by chronic parasitism. Chronic infections displayed the same seasonal cycle (high levels in winter and late spring, low levels in summer and early spring) that occurs in enzootic areas with annual reinfection. Within individual oysters, chronic MSX became localized, relapsed into general infections, and then became localized again in a sequence that was probably controlled by temperature. Some experimental oysters survived with MSX for at least 4 years. Hemocytosis persisted in these oysters, and their poor condition suggested that chronically infected individuals would have lowered resistance to additional stress.

Effects of a haplosporidian parasite, Haplosporidium sp., on species of the molluscan woodborer Teredo in Barnegat Bay, New Jersey

Abundance of the molluscan woodborer, Teredo navalis, in Barnegat Bay, New Jersey, was reduced in breeding seasons following levels of infection of 40% or more by a haplosporidian parasite of the genus Haplosporidium. Infections also occurred in T. bartschi and T. furcifera, subtropical species which have been introduced into Barnegat Bay, and which may have originally introduced the parasite. Infections were not observed in Bankia gouldi, the most common molluscan woodborer in the bay.