Eugene M. Burreson

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.

In vitro propagation of the protozoan Perkinsus marinus, a pathogen of the Eastern Oyster, Crassostrea virginica

ABSTRACT. Perkinsus marinus, a pathogen of eastern oysters (Crassostrea virginica), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus‐1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue‐black in Lugols solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus‐1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus‐1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell‐free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.

Phylogenetic analysis of Perkinsus based on actin gene sequences.

Perkinsus species presently are classified within the phylum Apicomplexa. This placement, however, is controversial. Based upon morphological observations and phylogenetic analyses of the small subunit ribosomal RNA gene, it has been suggested that Perkinsus may be more closely related to dinoflagellates. To reevaluate the phylogenetic position of Perkinsus, we obtained nucleotide sequence data for actin genes from Perkinsus marinus and 2 dinoflagellates, Prorocentrum minimum and Amphidinium carterae. Results indicated that there are 2 closely related actin genes in the genome of P. marinus. Phylogenetic comparisons of these actin gene fragments of P. marinus to available actin gene sequences for several ciliates and apicomplexans and to the 2 actin gene sequences from dinoflagellates obtained in this study supported a closer affinity of P. marinus to dinoflagellates than to apicomplexans.

Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

ABSTRACT The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10−2 cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.

Dual disease resistance in a selectively bred eastern oyster, Crassostrea virginica, strain tested in Chesapeake Bay

Selective breeding efforts have yielded oyster strains, Crassostrea virginica, with improved survival and resistance against Haplosporidium nelsoni (MSX); however, because of susceptibility to the oyster pathogen Perkinsus marinus (Dermo), their utility has been limited in areas where the two parasites co-occur. Dual resistance to H. nelsoni and P. marinus was achieved through four generations of artificial selection of wild Delaware Bay oyster progeny at a site in the lower York River, Virginia, USA where both diseases are enzootic. During 1993–1995, survival, growth, and disease susceptibility of third generation Delaware Bay (F3-DEBY) oysters were evaluated at the York River site in comparison to that of similarly selected third generation James River, Virginia oysters (F3-JR), and first generation Louisiana oysters (F1-LA), whose parents were naturally selected in the wild for resistance to P. marinus. During 1997–1999, the performance of F4-DEBY was evaluated at three sites in Virginia in comparison to two groups of first generation oysters whose parents originated from Mobjack Bay and Tangier Sound, Virginia where both P. marinus and H. nelsoni are enzootic. In the presence of high infection pressure from both H. nelsoni and P. marinus, the F3-DEBY stock showed significantly higher survival and growth than either the F3-JR or F1-LA strain. After 15 months of deployment, 79% of F3-DEBY, 11% of F3-JR and 17% of F1-LA oysters were market size (≥76.2 mm) and cumulative mortality was only 16% in F3-DEBY as compared to 42% in F3-JR and F1-LA. At the termination of the study, F3-DEBY oysters exhibited 22% lower mortality than the F1-LA stock, which outperformed the F3-JR stock. Relative performance in respect to disease varied considerably with sample date; however, average H. nelsoni weighted prevalence varied such that F3-DEBY<F1-LA=F3-JR and average P. marinus weighted prevalence varied such that F1-LA<F3-DEBY<F3-JR. In the 1997–1999 trial, F4-DEBY oysters experienced 34–61% lower mortality, greater growth rate, and consistently lower prevalence and intensity of P. marinus than either Mobjack Bay or Tangier Sound oysters. H. nelsoni prevalences were very low (<12%) in all three stocks. This is the first study to demonstrate that reduced susceptibility to both P. marinus and H. nelsoni can be achieved through selective breeding.

Bonamia perspora n. sp. (Haplosporidia), a Parasite of the Oyster Ostreola equestris, is the First Bonamia Species Known to Produce Spores

ABSTRACT. Examination of the oyster Ostreola equestris as a potential reservoir host for a species of Bonamia discovered in Crassostrea ariakensis in North Carolina (NC), USA, revealed a second novel Bonamia sp. Histopathology, electron microscopy, and molecular phylogenetic analysis support the designation of a new parasite species, Bonamia perspora n. sp., which is the first Bonamia species shown to produce a typical haplosporidian spore with an orifice and hinged operculum. Spores were confirmed to be from B. perspora by fluorescent in situ hybridization. Bonamia perspora was found at Morehead City and Wilmington, NC, with an overall prevalence of 1.4% (31/2,144). Uninucleate, plasmodial, and sporogonic stages occurred almost exclusively in connective tissues; uninucleate stages (2–6 μm) were rarely observed in hemocytes. Spores were 4.3–6.4 μm in length. Ultrastructurally, uninucleate, diplokaryotic, and plasmodial stages resembled those of other spore‐forming haplosporidians, but few haplosporosomes were present, and plasmodia were small. Spore ornamentation consisted of spore wall‐derived, thin, flat ribbons that emerged haphazardly around the spore, and which terminated in what appeared to be four‐pronged caps. Number of ribbons per spore ranged from 15 to 30, and their length ranged from 1.0 to 3.4 μm. Parsimony analysis identified B. perspora as a sister species to Bonamia ostreae.

Increased reactive oxygen intermediate production by hemocytes withdrawn from Crassostrea virginica infected with Perkinsus marinus

Perkinsus marinus is a protozoan parasite responsible for a major infectious disease of the Eastern oyster, Crassostrea virginica. Nonspecific immunity was assayed in oysters with known intensities of infection so that the physiological responses of the host elicited by the parasite could be better understood. This report describes the capacity of hemocytes to generate reactive oxygen intermediates during the progression of the disease. The hemocytes constitute the major internal defense effector system of oysters, and cytotoxic oxygen species are thought to play central roles in antimicrobial activities of hemocytes and other phagocytic cells. Production of oxyradicals by both resting and phagocytically stimulated hemocytes was quantified by luminol-augmented chemiluminescence. Hemocytes from oysters with heavy Perkinsus infections produced significantly higher levels of chemiluminescence than their counterparts withdrawn from lightly or moderately infected individuals. Furthermore, in addition to a higher chemiluminescent activity per cell, the total circulating hemocyte count was elevated in the heavily infected animals. Therefore, advanced cases of this disease seem to be characterized by hemocyte activation and recruitment, with concomitant exuberant production of hemocyte-derived reactive oxygen intermediates. The resultant oxidant load may participate in the pathogenesis of the disease.

Molecular, Morphological, and Experimental Evidence Support the Synonymy of Perkinsus chesapeaki and Perkinsus andrewsi

Abstract. Diverse analytical and experimental results confirm that two protistan parasites, Perkinsus chesapeaki and Perkinsus andrewsi, described separately as parasites of Mya arenaria and Macoma balthica clams sympatric in Chesapeake Bay, USA, represent a single species. Ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions, rRNA large subunit (LSU) gene, and actin gene sequences were obtained from clonal Perkinsus spp. cultured in vitro. Although multiple polymorphic sequences were found in DNA from clonal cultures at each locus, identical ITS region and actin gene sequences were found in the P. andrewsi holotype culture and in Perkinsus sp. clonal cultures from M. arenaria and Tagelus plebius. All sequences determined from cultures of P. chesapeaki and P. andrewsi at each locus grouped together in monophyletic clades with high support values in phylogenetic analyses. In vitro isolates of Perkinsus spp. from M. arenaria and M. balthica were reciprocally infective for each others cognate host. Lesions and histozoic parasite cell morphologies were consistent with those described for the original host/parasite interactions. In vitro isolate cell cycles and cell types of both parasites were indistinguishable. In accordance with the International Code of Zoological Nomenclature rules of priority, P. andrewsi is declared a junior synonym of P. chesapeaki.

Molecular phylogeny of the Haplosporidia based on two independent gene sequences.

The phylogenetic position of the Haplosporidia has confounded taxonomists for more than a century because of the unique morphology of these parasites. We collected DNA sequence data for small subunit (SSU) ribosomal RNA and actin genes from haplosporidians and other protists for conducting molecular phylogenetic analyses to help elucidate relationships of taxa within the group, as well as placement of this group among Eukaryota. Analyses were conducted using DNA sequence data from more than 100 eukaryotic taxa with various combinations of data sets including nucleotide sequence data for each gene separately and combined, as well as SSU ribosomal DNA data combined with translated actin amino acids. In almost all analyses, the Haplosporidia was sister to the Cercozoa with moderate bootstrap and jackknife support. Analysis with actin amino acid sequences alone grouped haplosporidians with the foraminiferans and cercozoans. The haplosporidians Minchinia and Urosporidium were found to be monophyletic, whereas Haplosporidium was paraphyletic. “Microcell” parasites, Bonamia spp. and Mikrocytos roughleyi, were sister to Minchinia, the most derived genus, with Haplosporidium falling between the “microcells” and the more basal Urosporidium. Two recently discovered parasites, one from abalone in New Zealand and another from spot prawns in British Columbia, fell at the base of the Haplosporidia with very strong support, indicating a taxonomic affinity to this group.

A Sensitive and Specific DNA Probe for the Oyster Pathogen Haplosporidium nelsoni

ABSTRACT. Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica, along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis, and with SSU rDNA data of C. virginica and various protists in GenBank. A 21‐base oligonucleotide unique to H. nelsoni, designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 μg of genomic DNA from an infected oyster. It did not hybridize with 1 μg of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA‐fixed, paraffin‐embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms (Teredo spp.).