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.

qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular assays.

Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

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.

Methadone's Resurgence in Bridging the Treatment Gap in the Overdose Crisis: Position Statement of AMERSA, Inc (Association for Multidisciplinary Education, Research, Substance Use, and Addiction).

BACKGROUND: The United States is grappling with an unprecedented overdose crisis, exacerbated by the proliferation of potent synthetic opioids like illicitly manufactured fentanyl. Despite the efficacy of methadone treatment in managing opioid use disorder, regulatory barriers hinder its widespread utilization. This article examines the complex landscape of methadone regulation across federal, state, and local levels, highlighting disparities and opportunities for reform. ISSUE: The COVID-19 public health emergency prompted temporary flexibility in methadone regulations, including expanded take-home doses and telehealth counseling, leading to improved treatment experiences and retention. Permanent revisions to federal guidelines have since been introduced by the Substance Abuse and Mental Health Services Administration, reflecting a progressive shift toward patient-centered care and streamlined access. State regulations, managed by Single State Agencies and State Opioid Treatment Authorities, vary widely, often imposing additional restrictions that impede access to methadone treatment. Local OTP clinics further exacerbate barriers through stringent policies, despite federal and state guidelines advocating for flexibility. RECOMMENDATIONS: Coordinated efforts among policymakers, healthcare providers, and communities are needed to promote the development of accountability measures, incentives, and community involvement to ensure equitable access and quality of care. To truly meet the demand needed to end the existing overdose crisis and enhance accessibility and comprehensive healthcare services, methadone treatment expansion beyond traditional OTP settings into primary care offices and community pharmacies should take place.

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.

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.