Simplification of complex DNA profiles using front end cell separation and probabilistic modeling.

Forensic samples comprised of cell populations from multiple contributors often yield DNA profiles that can be extremely challenging to interpret. This frequently results in decreased statistical strength of an individual's association to the mixture and the loss of probative data. The purpose of this study was to test a front-end cell separation workflow on complex mixtures containing as many as five contributors. Our approach involved selectively labelling certain cell populations in dried whole blood mixture samples with fluorescently labeled antibody probe targeting the HLA-A*02 allele, separating the mixture using Fluorescence Activated Cell Sorting (FACS) into two fractions that are enriched in A*02 positive and A*02 negative cells, and then generating DNA profiles for each fraction. We then tested whether antibody labelling and cell sorting effectively reduced the complexity of the original cell mixture by analyzing STR profiles quantitatively using the probabilistic modeling software, TrueAllele® Casework. Results showed that antibody labelling and FACS separation of target populations yielded simplified STR profiles that could be more easily interpreted using conventional procedures. Additionally, TrueAllele® analysis of STR profiles from sorted cell fractions increased statistical strength for the association of most of the original contributors interpreted from the original mixtures.

Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

The haplosporidian Bonamia exitiosa is present in Australia, but the identity of the parasite described as Bonamia (formerly Mikrocytos) roughleyi is uncertain.

Protistan oyster parasites in the genus Bonamia have been observed in recent years infecting new hosts on five continents, with most of these parasites genetically similar to austral species Bonamia exitiosa and Bonamia roughleyi. Identification of the newly observed parasites as one or another of these described species has been complicated by the fact that B. exitiosa and B. roughleyi are phylogenetically indistinguishable at the small-subunit ribosomal DNA (SSU rDNA) level, with samples of B. roughleyi type material no longer available for genetic re-analyses using more informative internal transcribed spacer (ITS) region DNA sequences. To resolve this issue, we evaluated B. roughleyi in field collections of hosts Saccostrea glomerata and Ostrea angasi (as well as Crassostrea gigas) in New South Wales, Australia in 2006 and 2007, and re-analyzed histological samples from the original description of this parasite species using in situ hybridization. Despite (1) reports of the oyster disease putatively caused by B. roughleyi during the time of collections, (2) the observation of gross lesions characteristic of the disease, and (3) the observation of B. roughleyi cells in association with the lesions, we detected a Bonamia sp. by PCR in just 1/42 O. angasi (2.4%), and 1/608 S. glomerata (0.2%), the latter oyster of which is the type host. SSU rDNA sequences of the amplicons were nearly identical to those of B. exitiosa and B. roughleyi, and phylogenetic analysis of ITS region sequences placed them on a B. exitiosa clade. A Haplosporidium sp. sequence similar to that of H. costale was PCR-amplified from nearly half the S. glomerata and O. angasi, but no Haplosporidium sp. was observed histologically. Our inability to identify a Bonamia sp. sequence in association with the B. roughleyi observed histologically suggests that this parasite is not a Bonamia sp. at all, and should be regarded as B. roughleyi nomen dubium. We conclude that the Bonamia sp. that we and other investigators detected in southeastern Australian S. glomerata and O. angasi was B. exitiosa.

Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus.

The mangrove oyster Saccostrea palmula coexists with the pleasure oyster Crassostrea corteziensis in coastal lagoons of northwest Mexico. Recent discovery of Perkinsus marinus infecting the pleasure oyster in the region prompted evaluation of S. palmula as an alternative P. marinus host. An analysis to determine the possible presence of P. marinus in natural and cultured populations of S. palmula at four coastal lagoons in Sinaloa, Mexico was carried out during October-November 2010. Tissues from apparently healthy S. palmula were evaluated using Ray's fluid thioglycollate method (RFTM), which revealed a Perkinsus sp. to be present in all four locations at 6.7-20.0% prevalence. Histopathological analysis of these specimens showed tissue alterations and parasite forms consistent with moderate P. marinus infection, which was confirmed by ribosomal non-transcribed spacer (NTS)-based PCR assays on DNA samples from oysters positive by RFTM and histology. DNA sequencing of amplified NTS fragments (307 bp) produced a sequence 98-100% similar to GenBank-deposited sequences of the NTS from P. marinus. Fluorescent in situ hybridization for Perkinsus spp. and P. marinus corroborated the PCR results, showing clear hybridization of P. marinus in host tissues. This is the first record of P. marinus infecting a species from genus Saccostrea and the first record of the parasite from coastal lagoons in Sinaloa, Mexico.

Diseases of oysters Crassostrea ariakensis and C. virginica reared in ambient waters from the Choptank River, Maryland and the Indian River Lagoon, Florida.

To assess potential benefits and liabilities from a proposed introduction of Asian Suminoe oysters, susceptibilities of exotic Crassostrea ariakensis and native C. virginica oysters were compared during exposures to pathogens endemic in temperate, mesohaline waters of Chesapeake Bay and sub-tropical, polyhaline Atlantic waters of southern Florida, USA. Cohorts of diploid, sibling oysters of both species were periodically tested for diseases while reared in mesocosms receiving ambient waters from the Choptank River, Maryland (>3 yr) or the Indian River Lagoon, Florida (10 to 11 mo). Haplosporidium sp. infections (e.g. MSX disease) were not detected in oysters from either site. Perkinsus sp. infections (dermo disease) occurred among members of both oyster species at both sites, but infections were generally of low or moderate intensities. A Bonamia sp. was detected by PCR of DNAs from tissues of both oyster species following exposure to Florida waters, with maximum PCR prevalences of 44 and 15% among C. ariakensis and C. virginica oysters respectively during June 2007. Among C. ariakensis oysters sampled during April to July 2007, a Bonamia sp. was detected in 31% of oysters by PCR (range 11 to 35%) and confirmed histologically in 10% (range 0 to 15%). Among simultaneously sampled C. virginica oysters, a Bonamia sp. was detected in 7% by PCR (range 0 to 15%), but histological lesions were absent. Although this is the first report of a Bonamia sp. from Florida waters, sequences of small subunit (SSU) rDNA and in situ hybridization (ISH) assays both identified the Florida pathogen as Bonamia exitiosa, which also infects oysters in the proximate waters of North Carolina, USA.

Widespread survey finds no evidence of Haplosporidium nelsoni (MSX) in Gulf of Mexico oysters.

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.

Observation of a Bonamia sp. infecting the oyster Ostrea stentina in Tunisia, and a consideration of its phylogenetic affinities.

The small non-commercial oyster Ostrea stentina co-occurs with commercially important Ostrea edulis in the Mediterranean Sea, yet its disposition with respect to the destructive pathogens Bonamia ostreae and Marteilia refringens is unknown. We began an evaluation of the Bonamia spp. infection status of O. stentina from Hammamet, Tunisia, in June 2007 using polymerase chain reaction diagnostics followed by histology and in situ hybridization. Of 85 O. stentina sampled, nine were PCR-positive for a Bonamia sp. using a Bonamia genus-specific assay; of these nine, one displayed the uninucleate microcells associated with oyster hemocytes characteristic of Bonamia spp. There was no associated pathology. DNA sequencing of the parasite from this one infected individual revealed it to be of a member of the Bonamia exitiosa/Bonamia roughleyi clade, an identification supported by positive in situ hybridization results with probes specific for members of this clade, and by the morphology of the parasite cells: nuclei were central, as in B. exitiosa, not eccentric, as in B. ostreae. There is no basis for identifying the Tunisian parasite as either B. exitiosa or B. roughleyi, however, as these species are genetically indistinguishable. Likewise, there is no basis for identifying any of the other Bonamia spp. with affinities to the B. exitiosa/B. roughleyi clade, from Argentina, Australia, Spain, and the eastern USA, as one or the other of these named species. Though they are clearly distinct from Bonamia perspora and B. ostreae, justification for drawing species boundaries among the primarily austral microcells with affinities to B. exitiosa and B. roughleyi remains elusive.

Minchinia mercenariae n. sp. (Haplosporidia) in the hard clam Mercenaria mercenaria: implications of a rare parasite in a commercially important host.

During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria, from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae. No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA.

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.

Experimental cross-infections by Perkinsus marinus and P. chesapeaki in three sympatric species of Chesapeake Bay oysters and clams.

In controlled laboratory transmission experiments, uniform doses of axenic in vitro isolate cultures of Perkinsus marinus from a Crassostrea virginica oyster, and of independent P. chesapeaki isolates from Chesapeake Bay Mya arenaria and Macoma balthica clams, were used to reciprocally challenge Perkinsus sp.-free C. virginica, M. arenaria, and M. balthica experimental hosts. Following mantle cavity inoculations, all 3 experimental hosts acquired high incidences (30 to 100%) of infections by each of the 3 Perkinsus sp. isolates, based on PCR assays of DNAs from experimental host tissues that were collected through 60 d post-inoculation. Lesions containing proliferating pathogen cells were documented histologically in tissues of all experimental host species challenged with all isolates of both Perkinsus species. Experimental Perkinsus sp. challenge isolates were re-isolated and propagated in vitro from infected tissues of host molluscs from most (5 of 9) experimental treatment groups. Koch's postulates were generally satisfied to confirm experimental infections in all bivalve molluscs that were challenged with 3 isolates of 2 Perkinsus spp. These results suggest potential broad and overlapping host specificities for the 2 current Chesapeake Bay-endemic Perkinsus species: P. marinus and P. chesapeaki.

Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei.

Mortalities of Penaeus vannamei, cultured in ponds in Belize, Central America, began during the last part of the grow-out cycle during the cold weather months from September 2004 through February 2005. Tissue squashes of infected hepatopancreata and histological examination of infected shrimp revealed that the mortalities might have been caused by an endoparasite. To confirm the diagnosis, DNA was extracted from ethanol preserved hepatopancreata and the small-subunit rRNA gene was sequenced. The 1838 bp sequence was novel and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as a sister taxon to a clade that includes Bonamia and Minchinia species. In situ hybridization was performed using anti-sense DNA probes that were designed to hybridize specifically with the parasite's nucleic acid. This organism presents similar characteristics to those of a haplosporidian that infected cultured P. vannamei imported from Nicaragua into Cuba, as described by Dyková et al. (1988; Fish Dis 11:15-22).

Bonamia perspora n. sp. (Haplosporidia), a parasite of the oyster Ostreola equestris, is the first Bonamia species known to produce spores.

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 microm) were rarely observed in hemocytes. Spores were 4.3-6.4 microm 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 microm. Parsimony analysis identified B. perspora as a sister species to Bonamia ostreae.

Ultrastructural and molecular characterization of Haplosporidium montforti n. sp., parasite of the European abalone Haliotis tuberculata.

A new member of the parasitic phylum Haplosporidia, which was found infecting the connective tissue, gill, digestive gland, and foot muscle of Haliotis tuberculata imported from Ireland and experimentally grown in Galicia (NW Spain), is described. Scanning electron microscopy, transmission electron microscopy, and molecular characterization of the small subunit ribosomal RNA (SSU rRNA) gene were carried out to confirm the description of this species. The ultrastructural morphology of the spores and their surrounding ornaments attached to the spore wall was described from light, scanning, and transmission electron microscopy observations. Systemic infection with uninucleated and multinucleated plasmodia containing spherical nuclei was observed among several sporocysts containing the different spore maturation stages. The spores were spherical to slightly ellipsoidal (2.42 +/- 0.5 x 2.31 +/- 0.6 microm). The apical zone of the spore wall was modified into a complex opercular system covering a circular orifice that measured about 0.5 microm in diameter. The operculum was connected to the spore wall by a hinge. The spore wall was about 110 nm thick, with 4 filaments (20-28 microm long). The filaments were composed of the same material that formed the wall. The cross-sections through the base of these filaments showed T-like and X-like sections. Internally, the uninucleated endosporoplasm contained typical haplosporidian structures, such as, haplosporosomes, a spherulosome, and mitochondria with vesicular cristae. The SSU rRNA gene sequence was different from previously reported haplosporidian SSU rRNA gene sequences, corroborating morphological data that this was an undescribed species. Based on differences from previously described haplosporidians in ultrastructural characteristics of the spore and SSU rRNA gene sequence, we describe the abalone haplosporidian as Haplosporidium montforti n. sp.

Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes.

Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.

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 thraustochytrid protist isolated from Mercenaria mercenaria: molecular characterization and host defense responses.

A previously undescribed thraustochytrid protist, designated C9G, was isolated from the gills of a clam, Mercenaria mercenaria, collected from the Bay of Fundy, Canada. Sequence data analysis showed C9G to be related to the clam pathogen QPX, quahog parasite unknown; however, it is not enveloped by secreted mucoid material as is the case for QPX. Clam hemocytes recognized and phagocytized C9G in vitro in the absence of plasma recognition factors. Hemocytes were also capable of killing ingested C9G, as shown by the use of a tetrazolium reduction viability assay. The mechanisms underlying intracellular antimicrobial activity are not yet established, but no detectable cytotoxic reactive oxygen species were generated during phagocytosis of C9G. Clam plasma proteins were shown to inhibit C9G growth at concentrations similar to those in unfractionated hemolymph.

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.