Improved Detection Methods for Selected Vibrio Species in the Marine Aquatic Environment

Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus are the three most commonly encountered human pathogens in the Vibrio genus. They are frequent agents of foodborne illness contracted from seafood. The work presented here focuses on three aspects of detection of these bacteria. First is a novel agar formulation designed to isolate and directly enumerate V. vulnificus from oyster tissue by using x-gal, a chromogenic analog to lactose, as a carbon source and agent of differential morphological colony growth. Second is a field study on the relative abundance of the three main pathogenic Vibrios following the opening of the Bonnet Carre spillway to prevent the Mississippi River from flooding New Orleans. This influx of fresh water into the Mississippi sound had a significant impact on the salinity and enabled V. cholerae growth to dominate over the other two pathogenic Vibrios during the time the spillway was open. The third and last chapter of work presented here is the development and validation of a novel method for enumeration of V. parahaemolyticus in the viable but nonculturable state. This dormant state prevents the bacteria from being enumerated by standard methods. The work presented here allows enumeration of the bacteria from environmental waters in total, and when coupled with other methods, allows enumeration of the VBNC portion of the population. The work presented in this thesis serves to further characterize these bacteria and presents novel methods for their detection that may be used to mitigate their risk to human health

Exposure to Oil and Hypoxia Results In Alterations of Immune Transcriptional Patterns In Developing Sheepshead Minnows (\u3ci\u3eCyprinodon variegatus\u3c/i\u3e)

The area and timing of the Deepwater Horizon oil spill highlight the need to study oil and hypoxia exposure in early life stage fishes. Though critical to health, little research has targeted the effect of oil and hypoxia exposure on developing immune systems. To this end, we exposed sheepshead minnows (Cyprinodon variegatus) at three early life stages: embryonic; post-hatch; and post-larval, to a high energy water accommodated fraction (HEWAF) of oil, hypoxia, or both for 48 hours. We performed RNAseq to understand how exposures alter expression of immune transcripts and pathways. Under control conditions, the embryonic to post-hatch comparison (first transition) had a greater number of significantly regulated immune pathways than the second transition (post-hatch to post-larval). The addition of oil had little effect in the first transition, however, hypoxia elicited changes in cellular and humoral immune responses. In the second transition, oil exposure significantly altered many immune pathways (43), and while hypoxia altered few pathways, it did induce a unique signature of generally suppressing immune pathways. These data suggest that timing of exposure to oil and/or hypoxia matters, and underscores the need to further investigate the impacts of multiple stressors on immune system development in early life stage fishes

Combined and independent effects of hypoxia and tributyltin on mRNA expression and physiology of the Eastern oyster (Crassostrea virginica)

© 2020, The Author(s). Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tβ-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tβ-4 expression increased, while no effect on hemocytes was observed. In contrast, after 6 and 8 days of laboratory-based hypoxia exposure, both Tβ-4 expression and hemocyte counts declined. After 8 days of exposure to hypoxia + TBT, oysters substantially up-regulated HIF1-α and down-regulated Tβ-4, although hemocyte counts were unaffected. Results suggest that hypoxic exposure induces immunosuppression which could increase vulnerability to pathogens

Combined and Independent Effects On Hypoxia and Tributylin On mRNA Expression and Physiology of the Eastern Oyster (\u3ci\u3eCrassostrea virginica\u3c/i\u3e)

Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tβ-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tβ-4 expression increased, while no effect on hemocytes was observed. In contrast, after 6 and 8 days of laboratory-based hypoxia exposure, both Tβ-4 expression and hemocyte counts declined. After 8 days of exposure to hypoxia + TBT, oysters substantially up-regulated HIF1-α and down-regulated Tβ-4, although hemocyte counts were unaffected. Results suggest that hypoxic exposure induces immunosuppression which could increase vulnerability to pathogens

Dispersed Crude Oil Induces Dysbiosis In the Red Snapper \u3ci\u3eLutjanus campechanus\u3c/i\u3e External Microbiota

The fish external microbiota competitively excludes primary pathogens and prevents the proliferation of opportunists. A shift from healthy microbiota composition, known as dysbiosis, may be triggered by environmental stressors and increases host susceptibility to disease. The Deepwater Horizon (DWH) oil spill was a significant stressor event in the Gulf of Mexico. Despite anecdotal reports of skin lesions on fishes following the oil spill, little information is available on the impact of dispersed oil on the fish external microbiota. In this study, juvenile red snapper (Lutjanus campechanus) were exposed to a chemically enhanced water-accommodated fraction (CEWAF) of Corexit 9500/DWH oil (CEWAF) and/or the bacterial pathogen Vibrio anguillarum in treatments designed to detect changes in and recovery of the external microbiota. In fish chronically exposed to CEWAF, immunoglobulin M (IgM) expression significantly decreased between 2 and 4 weeks of exposure, coinciding with elevated liver total polycyclic aromatic hydrocarbons (PAHs). Dysbiosis was detected on fish chronically exposed to CEWAF compared to seawater controls, and addition of a pathogen challenge altered the final microbiota composition. Dysbiosis was prevented by returning fish to clean seawater for 21 days after 1 week of CEWAF exposure. Four fish exhibited lesions during the trial, all of which were exposed to CEWAF but not all of which were exposed to V. anguillarum. This study indicates that month-long exposure to dispersed oil leads to dysbiosis in the external microbiota. As the microbiota is vital to host health, these effects should be considered when determining the total impacts of pollutants in aquatic ecosystems

Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys

BACKGROUND: Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. METHODOLOGY/PRINCIPAL FINDINGS: Histology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. CONCLUSIONS/SIGNIFICANCE: Congruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other gastropods, indicating that these metals possibly contribute to NS conch reproductive failure

Copper-containing mesoporous bioactive glass promotes angiogenesis in an in vivo zebrafish model

The osteogenic and angiogenic responses of organisms to the ionic products of degradation of bioactive glasses (BGs) are being intensively investigated. The promotion of angiogenesis by copper (Cu) has been known for more than three decades. This element can be incorporated to delivery carriers, such as BGs, and the materials used in biological assays. In this work, Cu-containing mesoporous bioactive glass (MBG) in the SiO2-CaO-P2O5 compositional system was prepared incorporating 5% mol Cu (MBG-5Cu) by replacement of the corresponding amount of Ca. The biological effects of the ionic products of MBG biodegradation were evaluated on a well-known endothelial cell line, the bovine aorta endothelial cells (BAEC), as well as in an in vivo zebrafish (Danio rerio) embryo assay. The results suggest that ionic products of both MBG (Cu free) and MBG-5Cu materials promote angiogenesis. In vitro cell cultures show that the ionic dissolution products of these materials are not toxic and promote BAEC viability and migration. In addition, the in vivo assay indicates that both exposition and microinjection of zebrafish embryos with Cu free MBG material increase vessel number and thickness of the subintestinal venous plexus (SIVP), whereas assays using MBG-5Cu enhance this effect.The authors gratefully acknowledge the financial support provided by the Andalusian Ministry of Economy, Science and Innovation (Proyectos Excelencia Grants no. P10-CTS-6681 and no. P12-CTS-1507) and Spanish Ministry of Economy and Competitivity (BIO2014-56092-R). LBRS acknowledges the CONACYT-Mexico Fellowship PhD Program