Ciguatoxin in Hawai'i: Fisheries forecasting using geospatial and environmental analyses for the invasive Cephalopholis argus (Epinephelidae).

Invasive species can precede far-reaching environmental and economic consequences. In the Hawai'ian Archipelago Cephalopholis argus (family Serranidae) is an established invasive species, now recognized as the dominant local reef predator, negatively impacting the native ecosystem and local fishery. In this region, no official C. argus fishery exists, due to its association with Ciguatera seafood poisoning (CP); a severe intoxication in humans occurring after eating (primarily) fish contaminated with ciguatoxins (CTXs). Pre-harvest prediction of CP is currently not possible; partly due to the ubiquitous nature of the microalgae producing CTXs and the diverse bioaccumulation pathways of the toxins. This study investigated the perceived risk of CP in two geographically discrete regions (Leeward and Windward) around the main island of Hawai'i, guided by local fishers. C. argus was collected and investigated for CTXs using the U.S. Food and Drug Administration (FDA) CTX testing protocol (in vitro neuroblastoma N2a-assay and LC-MS/MS). Overall, 76% of fish (87/113) exceeded the FDA guidance value for CTX1B (0.01 ng g-1 tissue equivalents); determined by the N2a-assay. Maximum CTX levels were ≅2× higher at the Leeward vs Windward location and, respectively, 95% (64/67) and 54% (25/46) of fish were positive for CTX-like activity. Fisher persons and environmental understandings, regarding the existence of a geographic predictor (Leeward vs Windward) for harvest, were found to be (mostly) accurate as CTXs were detected in both locations and the local designation of C. argus as a risk for CP was confirmed. This study provides additional evidence that supports the previous conclusions that this species is a severe CP risk in the coastal food web of Hawai'i, and that ocean exposure (wave power) may be a prominent factor influencing the CTX content in fish within a hyperendemic region for CP.

An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management.

Ciguatera Fish Poisoning (CFP) is the most frequently reported seafood-toxin illness in the world. It causes substantial human health, social, and economic impacts. The illness produces a complex array of gastrointestinal, neurological and neuropsychological, and cardiovascular symptoms, which may last days, weeks, or months. This paper is a general review of CFP including the human health effects of exposure to ciguatoxins (CTXs), diagnosis, human pathophysiology of CFP, treatment, detection of CTXs in fish, epidemiology of the illness, global dimensions, prevention, future directions, and recommendations for clinicians and patients. It updates and expands upon the previous review of CFP published by Friedman et al. (2008) and addresses new insights and relevant emerging global themes such as climate and environmental change, international market issues, and socioeconomic impacts of CFP. It also provides a proposed universal case definition for CFP designed to account for the variability in symptom presentation across different geographic regions. Information that is important but unchanged since the previous review has been reiterated. This article is intended for a broad audience, including resource and fishery managers, commercial and recreational fishers, public health officials, medical professionals, and other interested parties.

Screening for petrochemical contamination in seafood by headspace solid-phase microextraction gas chromatography-mass spectrometry.

A headspace solid-phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) method is described, to screen seafood for volatile organic compounds (VOCs) associated with petrochemical taint. VOCs are extracted from the headspace of heated sample homogenates by adsorption onto a SPME fiber and desorbed for analysis by GC-MS. Targeted compounds are determined semi-quantitatively using representative calibration standards for the various classes (alkanes, alkylbenzenes, indanes/tetralins, and naphthalenes) of VOCs analyzed. Sample preparation is minimal, and the analyses are rapid and automated with a capacity of 50 samples per day. The method was optimized in terms of headspace temperature, sample heating time, extraction time, and desorption time using oyster samples fortified with target compounds. Calibrations for hydrocarbon components were linear in the range of 8.3-167 ng/g; the limit of detection ranged between 0.05 and 0.21 ng/g, and the limit of quantitation between 0.16 and 0.69 ng/g. Good precision (RSD < 10 % at 16.7 ng/g for individual VOCs) and accuracy (recovery range 89-118 % at 25 ng/g) were obtained in oyster, crab, shrimp, and finfish matrices. The trueness of the method was demonstrated by quantifying VOCs at 1-2-ppb levels in oyster fortified with certified reference material NIST SRM 1491a. Following single laboratory validation, the method was employed for the determination of VOCs in seafood exposed to oil contaminated seawater and for the determination of background VOC levels in seafood species from the Gulf of Mexico and local food stores. The method as described can be used to supplement human sensory testing for petrochemical taint in seafood.

Federal seafood safety response to the Deepwater Horizon oil spill.

Following the 2010 Deepwater Horizon oil spill, petroleum-related compounds and chemical dispersants were detected in the waters of the Gulf of Mexico. As a result, there was concern about the risk to human health through consumption of contaminated seafood in the region. Federal and Gulf Coast State agencies worked together on a sampling plan and analytical protocols to determine whether seafood was safe to eat and acceptable for sale in the marketplace. Sensory and chemical methods were used to measure polycyclic aromatic hydrocarbons (PAHs) and dispersant in >8,000 seafood specimens collected in federal waters of the Gulf. Overall, individual PAHs and the dispersant component dioctyl sodium sulfosuccinate were found in low concentrations or below the limits of quantitation. When detected, the concentrations were at least two orders of magnitude lower than the level of concern for human health risk. Once an area closed to fishing was free of visibly floating oil and all sensory and chemical results for the seafood species within an area met the criteria for reopening, that area was eligible to be reopened. On April 19, 2011 the area around the wellhead was the last area in federal waters to be reopened nearly 1 y after the spill began. However, as of November 9, 2011, some state waters off the Louisiana coast (Barataria Bay and the Delta region) remain closed to fishing.

Modification and Single-Laboratory Validation of AOAC Official Method 977.13 for Histamine in Seafood to Improve Sample Throughput.

Histamine is the main causative agent in scombrotoxin fish poisoning, the most frequently reported illness related to fish consumption. The AOAC official method for histamine determination in fish is the fluorometric method AOAC 977.13, which is sensitive and reproducible but somewhat labor intensive and time consuming. We investigated multiple modifications to this method in an attempt to reduce assay time and increase sample throughput while maintaining the performance of the original method. Some of the attempted modifications negatively affected the performance characteristics of the method. However, omitting the heating step during extraction and replacing the cuvette style fluorometer with a microplate reader retained method performance while increasing sample throughput. Therefore, we adopted these modifications and conducted a single-laboratory validation. The recovery, precision (RSD), and LOD of the modified method assessed by the single-laboratory validation ranged from 92 to 105%, 1 to 3%, and 0.2 to 0.5 ppm, respectively, in tuna, mahi-mahi, and Spanish mackerel samples. We conclude that the AOAC 977.13 fluorometric method, modified as described, will improve assay time and sample throughput efficiency cumulatively, as the number of sample units analyzed increases. We anticipate that this modified method could be used by regulatory agencies and other laboratories following successful multilaboratory validation.

The microbial carbon pump and climate change.

The ocean has been a regulator of climate change throughout the history of Earth. One key mechanism is the mediation of the carbon reservoir by refractory dissolved organic carbon (RDOC), which can either be stored in the water column for centuries or released back into the atmosphere as CO2 depending on the conditions. The RDOC is produced through a myriad of microbial metabolic and ecological processes known as the microbial carbon pump (MCP). Here, we review recent research advances in processes related to the MCP, including the distribution patterns and molecular composition of RDOC, links between the complexity of RDOC compounds and microbial diversity, MCP-driven carbon cycles across time and space, and responses of the MCP to a changing climate. We identify knowledge gaps and future research directions in the role of the MCP, particularly as a key component in integrated approaches combining the mechanisms of the biological and abiotic carbon pumps for ocean negative carbon emissions.

Characterization of lignin by gas chromatography and mass spectrometry using a simplified CuO oxidation method.

An efficient and high-throughput method to characterize lignin in environmental samples using alkaline CuO oxidation and capillary gas chromatography with mass detection is presented. Monomeric lignin phenols released during oxidation were selectively extracted using a polymer-based solid phase sorbent rather than liquid phase extraction. Sample size and matrix were found to influence lignin phenol yields. Increasing the sample size to an organic carbon content ≥1.5 mg of C minimizes phenol oxidation losses, and the addition of glucose as a sacrificial carbon source helped minimize oxidation losses in samples with <5 mg of organic carbon.

Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus.

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+V. parahaemolyticus.

Subcellular localization of marine bacterial alkaline phosphatases.

Bacterial alkaline phosphatases (APases) are important enzymes in organophosphate utilization in the ocean. The subcellular localization of APases has significant ecological implications for marine biota but is largely unknown. The extensive metagenomic sequence databases from the Global Ocean Sampling Expedition provide an opportunity to address this question. A bioinformatics pipeline was developed to identify marine bacterial APases from the metagenomic databases, and a consensus classification algorithm was designed to predict their subcellular localizations. We identified 3,733 bacterial APase sequences (including PhoA, PhoD, and PhoX) and found that cytoplasmic (41%) and extracellular (30%) APases exceed their periplasmic (17%), outer membrane (12%), and inner membrane (0.9%) counterparts. The unexpectedly high abundance of cytoplasmic APases suggests that the transport and intracellular hydrolysis of small organophosphate molecules is an important mechanism for bacterial acquisition of phosphorus (P) in the surface ocean. On average, each marine bacterium possessed at least one suite of uptake of glycerol phosphate (ugp) genes (e.g., ugpA, ugpB, ugpC, ugpE) for dissolved organic phosphorus (DOP) transport, but only half of them had ugpQ, which hydrolyzes transported DOP, indicating that cytoplasmic APases play a role in hydrolyzing transported DOP. The most abundant heterotrophic marine bacteria, alpha- and gamma-Proteobacteria, might hydrolyze DOP outside the cytoplasmic membrane, but the former could also transport and hydrolyze DOP in the cytoplasm. The abundant extracellular APases could provide bioavailable P for organisms that cannot directly access organophosphates, and thereby increase marine biological productivity and diversity.

Haff disease associated with consumption of buffalofish (Ictiobus spp.) in the United States, 2010-2020, with confirmation of the causative species.

BACKGROUND: In the United States, buffalofish (Ictiobus spp.) are sporadically associated with sudden onset muscle pain and weakness due to rhabdomyolysis within 24 h of fish consumption (Haff disease). Previous genetic analyses of case-associated samples were unable to distinguish the three species of buffalofish that occur in the US, Ictiobus cyprinellus (bigmouth buffalo), Ictiobus bubalus (smallmouth buffalo), and Ictiobus niger (black buffalo). METHODS: Ten events were investigated between 2010 and 2020 and demographic and clinical information was collected for 24 individuals. Meal remnants were collected from 5 of 10 events with additional associated samples (n = 24) collected from another five of 10 events. Low-coverage whole-genome sequencing (genome skimming) was used to identify meal remnants. RESULTS: Patients (26-75 years of age) ranged from 1-4 per event, with 90% involving ≥2 individuals. Reported symptoms included muscle tenderness and weakness, nausea/vomiting, and brown/tea-colored urine. Median incubation period was 8 h. Ninety-six percent of cases were hospitalized with a median duration of four days. The most commonly reported laboratory finding was elevated creatine phosphokinase and liver transaminases. Treatment was supportive including intravenous fluids to prevent renal failure. Events occurred in California (1), Illinois (2), Louisiana (1), New York (1), Mississippi (1), Missouri (2), New Jersey (1), and Texas (1) with location of harvest, when known, being Illinois, Louisiana, Mississippi, Missouri, Texas, and Wisconsin. Meal remnants were identified as I. bubalus (n = 4) and I. niger (n = 1). Associated samples were identified as I. bubalus (n = 16), I. cyprinellus (n = 5), and I. niger (n = 3). DISCUSSION: Time course, presentation of illness, and clinical findings were all consistent with previous domestic cases of buffalofish-associated Haff disease. In contrast to previous reports that I. cyprinellus is the causative species in US cases, data indicate that all three buffalofish species are harvested but I. bubalus is most often associated with illness.

Toxigenic Vibrio cholerae O1 in water and seafood, Haiti.

During the 2010 cholera outbreak in Haiti, water and seafood samples were collected to detect Vibrio cholerae. The outbreak strain of toxigenic V. cholerae O1 serotype Ogawa was isolated from freshwater and seafood samples. The cholera toxin gene was detected in harbor water samples.

Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish.

Prompt detection of bacteria that contribute to scombrotoxin (histamine) fish poisoning can aid in the detection of potentially toxic fish products and prevent the occurrence of illness. We report development of the first real-time PCR method for rapid detection of Gram-negative histamine-producing bacteria (HPB) in fish. The real-time PCR assay was 100% inclusive for detecting high-histamine producing isolates and did not detect any of the low- or non-histamine producing isolates. The efficiency of the assay with/without internal amplification control ranged from 96-104% and in the presence of background flora and inhibitory matrices was 92/100% and 73-96%, respectively. This assay was used to detect HPB from naturally contaminated yellowfin tuna, bluefish, and false albacore samples. Photobacterium damselae (8), Plesiomonas shigelloides (2), Shewanella sp. (1), and Morganella morganii (1) were subsequently isolated from the real-time PCR positive fish samples. These results indicate that the real-time PCR assay developed in this study is a rapid and sensitive method for detecting high-HPB. The assay may be adapted for quantification of HPB, either directly or with an MPN-PCR method.

Quantification of total and specific gram-negative histamine-producing bacteria species in fish using an MPN real-time PCR method.

Quantification of histamine-producing bacteria (HPB) is necessary in order to elucidate the role that HPB play in scombrotoxin (histamine) fish poisoning. We report here the evaluation of a real-time PCR method for the quantification of total and specific Gram-negative HPB species in fish using a most probable number (MPN) format. The species-specific real-time PCR assay was 100% inclusive for independently detecting Morganella morganii, Enterobacter aerogenes, Raoultella planticola/ornithinolytica and Photobacterium damselae and did not cross react with other histamine- or non- histamine-producing bacteria. The efficiency of the reactions in the absence and presence of Spanish mackerel enrichment containing 1 × 10(6) CFU/ml of background microflora were 93-104 and 92-99%, respectively. The MPN-real-time PCR assay accurately quantified total and specific HPB in spiked mahi-mahi (Coryphaena hippurus) and Spanish mackerel (Scomberomorus maculates) samples. These methods were used to quantify total and specific HPB in naturally contaminated, decomposing mahi-mahi, Spanish mackerel and tuna (Thunnus albacares) samples. The results of this study indicate that MPN-real-time PCR assays can be used to accurately enumerate total and specific HPB in fish samples. These assays can be applied to assess the effectiveness of mitigation strategies and understand the relationship between HPB and histamine production in decomposing fish.

An Outbreak Investigation of Scombrotoxin Fish Poisoning Illnesses in the United States Linked to Yellowfin Tuna Imported from Vietnam-2019.

ABSTRACT: Scombrotoxin fish poisoning (SFP) is caused by the ingestion of certain fish species with elevated concentrations of histamine due to decomposition. In fall 2019, the U.S. Food and Drug Administration (FDA) was notified of 51 SFP cases including two hospitalizations from 11 states through the FDA consumer complaint system or directly from state partners. A case patient was defined as an individual who experienced a histamine-type reaction after consumption of tuna imported from Vietnam and an illness onset between 14 August and 24 November 2019. A traceback investigation was initiated at 19 points of service to identify a common tuna source. The FDA and state partners collected 34 product samples throughout the distribution chain, including from a case patient's home, points of service, distributors, and the port of entry. Samples were analyzed for histamine by sensory evaluation and/or chemical testing. Case patients reported exposure to tuna imported from Vietnam. The traceback investigation identified two Vietnamese manufacturers as the sources of the tuna. Twenty-nine samples were confirmed as decomposed by sensory evaluation and/or were positive for elevated histamine concentrations by chemical testing. Both Vietnamese companies were placed on an import alert. Seven U.S. companies and one Vietnamese company initiated voluntary recalls. The FDA released public communication naming the U.S. importers to help suppliers and distributors identify the product and effectuate the foreign company's recall. This SFP outbreak investigation highlights the complexities of the federal outbreak response, specifically related to imported food. Cultural considerations regarding imported foods should be addressed during outbreak responses when timing is critical. Collaboration with countries where confidentiality agreements are not in place can limit information sharing and the speed of public health responses.

Mixing it up in the ocean carbon cycle and the removal of refractory dissolved organic carbon.

A large quantity of reduced carbon is sequestered in the ocean as refractory dissolved molecules that persist through several circuits of global overturning circulation. Key aspects of the cycling of refractory dissolved organic carbon (DOC) remain unknown, making it challenging to predict how this large carbon reservoir will respond to climate change. Herein we investigate mechanisms that remove refractory DOC using bioassay experiments with DOC isolated from surface, mesopelagic and deep waters of the Atlantic Ocean. The isolated DOC was refractory to degradation by native microbial communities, even at elevated concentrations. However, when the refractory DOC was introduced to a series of novel environmental conditions, including addition of a labile substrate, a microbial community from coastal waters and exposure to solar radiation, a substantial fraction (7-13%) was removed within 1.5 years. Our results suggest that while refractory molecules can persist in the ocean for millennia, removal is rapid when they encounter their fate. The observed and projected climate-induced slowdown of global overturning circulation could reduce the exposure of refractory molecules to disparate removal processes. Assuming a constant rate of production, the reservoir size of refractory DOC could increase as overturning circulation slows, providing a negative feedback to rising atmospheric CO2.

Analysis of Chloramphenicol and Two Metabolites in Crab and Shrimp Following Waterborne Exposure.

The use of chloramphenicol (CAP) in aquaculture products is banned in many countries, including the United States, due to human health issues. Very few depletion and metabolism studies of CAP in seafood have been performed. Current detection methods for CAP residues in food are directed toward the parent drug molecule, but rapid elimination following treatment suggests the need for an alternative marker residue. We identified, characterized, and determined the persistence of two CAP metabolites, CAP-base (CAP-B) and CAP-alcohol (CAP-OH), in crab and shrimp. Interday recoveries of CAP, CAP-B, and CAP-OH in muscle fortified ( n = 9) at levels of 0.15 to 0.60 ng/g ranged from 95 to 127% and 101 to 119% for crab and shrimp, respectively, with repeatability ranging from 4 to 19%. The limit of detection for CAP and metabolites in crab and shrimp ranged from 0.05 to 0.11 ng/g. We also monitored the depletion of CAP, CAP-B, and CAP-OH in crab following waterborne exposures. To our knowledge, we present the first CAP depletion and metabolite study following waterborne exposure in crabs, with the aim of identifying alternative marker residues.

Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.

Draft Genome Sequences of Histamine-Producing Morganella psychrotolerans Strains.

Histamine-producing bacteria are responsible for scombrotoxin (histamine) fish poisoning, a leading cause of fish poisoning in the United States. We report here the first draft genomes of three histamine-producing Morganella psychrotolerans strains, isolated from tuna and mahi-mahi.

Draft Genome Sequences of Histamine- and Non-Histamine-Producing Photobacterium Strains.

Histamine-producing bacteria (HPBs) have recently been identified from the marine environment. The identification and characterization of HPBs is important to developing effective mitigation strategies for scombrotoxin fish poisoning. We report here the draft genomes of seven histamine-producing and two non-histamine-producing marine Photobacterium strains.