Co-occurrence of marine and freshwater phycotoxins in oysters, and analysis of possible predictors for management.

Oysters (Crassostrea virginica) were screened for 12 phycotoxins over two years in nearshore waters to collect baseline phycotoxin data and to determine prevalence of phycotoxin co-occurrence in the commercially and ecologically-relevant species. Trace to low concentrations of azaspiracid-1 and -2 (AZA1, AZA2), domoic acid (DA), okadaic acid (OA), and dinophysistoxin-1 (DTX1) were detected, orders of magnitude below seafood safety action levels. Microcystins (MCs), MC-RR and MC-YR, were also found in oysters (maximum: 7.12 µg MC-RR/kg shellfish meat wet weight), warranting consideration of developing action levels for freshwater phycotoxins in marine shellfish. Oysters contained phycotoxins that impair shellfish health: karlotoxin1-1 and 1-3 (KmTx1-1, KmTx1-3), goniodomin A (GDA), and pectenotoxin-2 (PTX2). Co-occurrence of phycotoxins in oysters was common (54%, n = 81). AZAs and DA co-occurred most frequently of the phycotoxins investigated that are a concern for human health (n = 13) and PTX2 and KmTxs co-occurred most frequently amongst the phycotoxins of concern for shellfish health (n = 9). Various harmful algal bloom (HAB) monitoring methods and tools were assessed for their effectiveness at indicating levels of phycotoxins in oysters. These included co-deployed solid phase adsorption toxin tracking (SPATT) devices, toxin levels in particulate organic matter (POM, >1.5 µm) and whole water samples and cell concentrations from water samples as determined by microscopy and quantitative real-time PCR (qPCR). The dominant phycotoxin varied between SPATTs and all other phycotoxin sample types, and out of the 11 phycotoxins detected in oysters, only four and seven were detected in POM and whole water respectively, indicating phycotoxin profile mismatch between ecosystem compartments. Nevertheless, there were correlations between DA in oysters and whole water (simple linear regression [LR]: R2 = 0.6, p < 0.0001, n = 40), and PTX2 in oysters and SPATTs (LR: R2 = 0.3, p = 0.001, n = 36), providing additional monitoring tools for these phycotoxins, but oyster samples remain the best overall indicators of seafood safety.

The toxin goniodomin, produced by Alexandrium spp., is identical to goniodomin A.

In 1968 Burkholder and associates (J. Antibiot. (Tokyo)1968, 21, 659-664) isolated the antifungal toxin goniodomin from an unidentified Puerto Rican dinoflagellate and partially characterized its structure. Subsequently, a metabolite of Alexandrium hiranoi was isolated by Murakami et al. from a bloom in Japan and its structure was established (Tetrahedron Lett.1988, 29, 1149-1152). The Japanese substance had strong similarities to Burkholder's but due to uncertainty as to whether it was identical or only similar, Murakami named his toxin goniodomin A. A detailed study of this question now provides compelling evidence that Burkholder's goniodomin is identical to goniodomin A. Morphological characterization of the dinoflagellate suggests that it was the genus Alexandrium but insufficient evidence is available to make a definite identification of the species. This is the only report of goniodomin in the Caribbean region.

A novel monoclonal Perkinsus chesapeaki in vitro isolate from an Australian cockle, Anadara trapezia.

A monoclonal Perkinsus chesapeaki isolate was established from 1 of 10 infected Australian Anadara trapezia cockles. Morphological features were similar to those of described P. chesapeaki isolates, and also included a unique vermiform schizont cell-type. Perkinsus olseni-specific PCR primers amplified DNAs from all 10 cockles. Perkinsus chesapeaki-specific primers also amplified DNAs from 4/10 cockles, including DNA from the isolate source cockle. Three different sets of DNA sequences from the monoclonal isolate grouped with the homologous, previously deposited, P. chesapeaki sequences in phylogenetic analyses. In situ hybridization assays detected both P. chesapeaki and P. olseni cells in histological sections from the source cockle for monoclonal isolate ATCC PRA-425.

Perkinsus sp. infections and in vitro isolates from Anadara trapezia (mud arks) of Queensland, Australia.

Perkinsus sp. protists were found infecting Anadara trapezia mud ark cockles at 6 sites in Moreton Bay, Queensland, Australia, at prevalences of 4 to 100% during 2011 as determined by surveys using Ray's fluid thioglycollate medium. Perkinsus sp. lesions were found among gill and visceral connective tissues in histological samples from several cockles, where basophilic, eccentrically vacuolated Perkinsus sp. signet ring trophozoites and proliferating, Perkinsus sp. schizont cells were documented. Two Perkinsus sp. isolates were propagated in vitro during August 2013 from gill tissues of a single infected A. trapezia cockle from Wynnum in Moreton Bay. DNA from those isolate cells amplified universally by a Perkinsus genus-specific PCR assay, and rDNA-internal transcribed spacer sequences respectively grouped them with P. olseni and P. chesapeaki in phylogenetic analyses. This is the first report of P. chesapeaki in Australia, and the first report of a P. chesapeaki in vitro isolate from an Australian mollusc host. Although P. olseni was originally described in 1981 as a pathogen of abalone in South Australia, and has subsequently been identified as a prevalent pathogen of numerous other molluscs worldwide, this is also the first report of a P. olseni-like in vitro isolate from an Australian mollusc host.

Two Perkinsus spp. infect Crassostrea gasar oysters from cultured and wild populations of the Rio São Francisco estuary, Sergipe, northeastern Brazil.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceará state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraíba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio São Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

Irradiated tumor cell vaccine for treatment of an established glioma. II. Expansion of myeloid suppressor cells that promote tumor progression.

These studies report the identification of a population of myeloid suppressor cells (MSC) that are preferentially enriched in the spleens and tumor-infiltrating mononuclear cells (TIMC) from T9.F-vaccinated animals. In this model designed to mimic immunotherapy for an established intracranial (i.c.) glioma, animals were given an i.c. inoculum with 5 x 10(4) T9 glioma cells at day 0, followed by a subcutaneous (s.c.) injection of 5 x 10(6) irradiated T9.F glioma cells 5 days later. Unexpectedly, we found that the survival of these T9.F-vaccinated animals was dramatically shorter than their age-matched counterparts who received only saline injections. Since MSC have previously been demonstrated to be associated with tumor progression, the question arose of whether MSC might play a role in the rapid tumor progression observed in this model. Analysis of the spleen cells and TIMC revealed an increase in the population of myeloid cells expressing granulocytic and monocytic markers. Both the polyclonal and tumor-specific proliferation of splenic T cells and tumor-infiltrating T lymphocytes (T-TIL) from T9.F-vaccinated animals were significantly inhibited in the presence of these myeloid cells. Furthermore, the adoptive transfer of MSC into animals bearing a 5-day T9 glioma caused rapid tumor progression. Reduced survival of the glioma-bearing vaccinated rats was associated with enhanced tumor growth, as well as with an increased density of T-TIL. However, purified T-TIL did not show any discernable signs of inherent defects in terms of their effector functions and T cell receptor (TCR) signal transduction protein levels. Therefore, we believe that an MSC population is responsible for inhibiting the anti-tumor T cell response, resulting in the enhanced growth of the i.c. glioma, and may represent a significant obstacle to immune-based therapies.