Self-assembling ferritin nanoplatform for the development of infectious hematopoietic necrosis virus vaccine.

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

Toxicity Evaluation, Oxidative, and Immune Responses of Mercury on Nile Tilapia: Modulatory Role of Dietary Nannochloropsis oculata.

The current study evaluated the potential ameliorative effect of a dietary immune modulator, Nannochloropsis oculata microalga, on the mercuric chloride (HgCl2)-induced toxicity of Nile tilapia. Nile tilapia (45-50 g) were fed a control diet or exposed to » LC50 of HgCl2 (0.3 mg/L) and fed on a medicated feed supplemented with N. oculata (5% and 10% (50 or 100 g/kg dry feed)) for 21 days. Growth and somatic indices, Hg2+ bioaccumulation in muscles, and serum acetylcholinesterase (AChE) activity were investigated. Antioxidant and stress-related gene expression analyses were carried out in gills and intestines. Histopathological examinations of gills and intestines were performed to monitor the traits associated with Hg2+ toxicity or refer to detoxification. Hg2+ toxicity led to significant musculature bioaccumulation, inhibited AChE activity, downregulated genes related to antioxidants and stress, and elicited histopathological changes in the gills and intestine. Supplementation with N. oculata at 10% was able to upregulate the anti-oxidative-related genes while downregulated the stress apoptotic genes in gills and intestines compared to the unexposed group. In addition, minor to no histopathological traits were detected in the gills and intestines of the N. oculata-supplemented diets. Our data showed the benefit of dietary N. oculata in suppressing Hg2+ toxicity, which might support its efficacy as therapeutic/preventive agent to overcome environmental heavy metal pollution in aquatic habitats.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

A widespread picornavirus affects the hemocytes of the noble pen shell (Pinna nobilis), leading to its immunosuppression.

Introduction: The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations. Methods: We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (four animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome. Results and discussion: In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 104-1.60 × 105 ml-1 cells) and wild animals (14 samples) (1.90-2.42 × 105 ml-1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus-a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean.

Microplastic-Contaminated Feed Interferes with Antioxidant Enzyme and Lysozyme Gene Expression of Pacific White Shrimp (Litopenaeus vannamei) Leading to Hepatopancreas Damage and Increased Mortality.

Microplastic pollution can interfere with aquatic animal health and nonspecific immunity, increasing the potential for pathogen infection in crustaceans. However, the long-term effects of microplastics on crustacean immunity are less understood, especially regarding their toxicity in Pacific white shrimp (Litopenaeus vannamei). Effects of high-density polyethylene microplastics (HDPE-MPs) in feed on the mortality rate, hepatopancreas, and nonspecific immune system gene expression of Pacific white shrimp are presented. The LC50 at day 28 of HDPE-MP exposure was determined as 3.074% HDPE-MP in feed. A significant upregulation of the superoxide dismutase (SOD) and glutathione peroxidase (GPx) genes was observed in shrimp that were fed with 0.1 and 0.5% of HDPE-MP; then, they were downregulated significantly, except for the SOD gene expression of shrimp fed with 0.1% of HDPE-MP. The lysozyme (LYZ) gene was upregulated significantly in shrimp that were fed with 0.5, 1, and 3% HDPE-MP for 7 days and downregulated significantly in HDPE-receiving groups for at least 14 days. Significant histopathological changes in the hepatopancreas were observed in the treatment groups. The histopathological score of each lesion was correlated with the increase in HDPE-MP concentration. This study shows that the ingestion of HDPE microplastics can alter the expression of nonspecific immune system genes and damage the hepatopancreas in Pacific white shrimp.

Estrogen Receptors Mediated Negative Effects of Estrogens and Xenoestrogens in Teleost Fishes-Review.

Estrogen receptors (ERs) play a key role in many biochemical and physiological processes, that are involved in maintaining organism homeostasis. At the most basic level, they can be divided into nuclear estrogen receptors and membrane estrogen receptors that imply their effect in two ways: slower genomic, and faster non-genomic. In these ways, estrogens and xenoestrogens can negatively affect animal health and welfare. Most of the available literature focuses on human and mammalian physiology, and clearly, we can observe a need for further research focusing on complex mutual interactions between different estrogens and xenoestrogens in aquatic animals, primarily fishes. Understanding the mechanisms of action of estrogenic compounds on the ERs in fishes and their negative consequences, may improve efforts in environmental protection of these animals and their environment and benefit society in return. In this review, we have summarized the ER-mediated effects of xenoestrogens and estrogens on teleost fishes metabolism, their carcinogenic potential, immune, circulatory, and reproductive systems.

Molecular Epidemiology of Novirhabdoviruses Emerging in Iranian Trout Farms.

Novirhabdoviruses cause large epizootics and economic losses of farmed trout. In this study, we surveyed Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) through both monitoring and investigation of clinical outbreaks reported by farmers in the regions with major rainbow trout production in Iran from 2015 to 2019. RT-PCR assays of the kidney samples and cell culture (EPC/FHM cells) samples confirmed the presence of the viruses, with 9 VHSV and 4 IHNV isolates, in both endemic and new areas of Iran. Sequence analysis of the G gene revealed that VHSV isolates belonged to genogroup Ia, and IHNV isolates were clustered into genogroup E, both typical for isolates from European countries. A haplotype analysis based on non-homologous amino acids of the G gene supports the emergence of two lineages of IHNV from clade 1 (E-1), as well as VHSV clade 2 (Ia-2) of the European genogroups, confirming that VHSV and IHNV isolates in Iran, have originated from Europe possibly via imported eggs.

Micro- and nano-plastics activation of oxidative and inflammatory adverse outcome pathways.

Microplastics (MPs) and nanoplastics (NPs) have attracted considerable attention in the recent years as potential threats to the ecosystem and public health. This review summarizes current knowledge of pathological events triggered by micro- and nano-plastics (MP/NPs) with focus on oxidative damages at different levels of biological complexity (molecular, cellular, tissue, organ, individual and population). Based on published information, we matched the apical toxicity endpoints induced by MP/NPs with key event (KE) or adverse outcomes (AO) and categorized them according to the Adverse Outcome Pathway (AOP) online knowledgebase. We used existing AOPs and applied them to highlight formal mechanistic links between identified KEs and AOs in two possible scenarios: first from ecological, and second from public health perspective. Ecological perspective AOP based literature analysis revealed that MP/NPs share formation of reactive oxygen species as their molecular initiating event, leading to adverse outcomes such as growth inhibition and behavior alteration through oxidative stress cascades and inflammatory responses. Application of AOP on literature data related to public health perspective of MP/NPs showed that oxidative stress and its responding pathways, including inflammatory responses, could play the role of key events. However insufficient information prevented precise definitions of AOPs at this level. To overcome this knowledge gap, further mammalian model and epidemiological studies are necessary to support development and construction of detailed AOPs with public health focus.

Epidemiology of Noble Pen Shell (Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression.

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such "lower risk" areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

Role of MicroRNAs in regulation of DNA damage in monocytes exposed to polystyrene and TiO2 nanoparticles.

The release of nanoparticles into the environment can interfere with the health of the exposed organisms. MicroRNAs have been suggested as potential toxicology biomarkers. The expression of potential zebrafish nano-toxicity biomarker miRNAs in our previous study was validated in THP-1 human monocytic cell line after exposure to polystyrene (PSNPs) and ARS labeled Titanium dioxide nanoparticles (nano-TiO2-ARS). miRNAs expression post exposure to PLGA nanoparticles and E. coli BioParticles was used to exclude potential activation and engagement of miRNAs through phagocytosis or pro-inflammatory specific responses. miR-155-5p showed the highest potential to be used as biomarker for PSNPs and nano-TiO2-ARS induced toxicity. To determine effects of PSNPs and nano-TiO2-ARS on genotoxicity, time and dose dependent DNA damage profile was established. Severe DNA damage was triggered by both nanoparticles, and expression of DNA damage repairing genes was elevated post nano-TiO2-ARS, but not post PSNPs exposure, questioning the utility of the comet assay as universal assessment tool for genotoxicity induced by nanoparticles in general. Transfection of miR-155-5p mimic influenced the expression of miR-155-5p related, DNA damage responsible genes post both nano-TiO2-ARS and PSNPs exposure. Transfection results suggest significant involvement of miR-155-5p in gene repair mechanisms triggered by adverse effects of PSNPs and nano-TiO2-ARS on monocytes.

In silico prediction of MicroRNA role in regulation of Zebrafish (Danio rerio) responses to nanoparticle exposure.

The release of nanoparticles to the environment can affect health of the exposed organisms. MicroRNAs have been suggested as potential toxicology biomarkers, however the information about use of microRNA in aquatic organisms exposed to nanoparticles (NP) is limited. In silico analysis from publicly available gene expression data was performed. Data selection for the analysis was based on reported biological and pathological outcomes of NP induced toxicity in zebrafish. After identifying relevant genes, we constructed six miRNA-mRNA regulatory networks involved in nanoparticle induced toxicological responses in zebrafish. Based on our prediction and selection criteria we selected six miRNAs that overlapped in constructed networks with remarkable prediction score, and were validated by previous mammalian and zebrafish microRNA profiling studies: dre-miR-124, -144, -148, -155, -19a, -223. The results of this in silico analysis indicate that several highly conserved miRNAs likely have a regulatory role of organismal responses to nanoparticles, and can possibly be used as biomarkers of nanotoxicity in studies using zebrafish as model organism One health approaches.

Acute exposure to chlorpyrifos induces reversible changes in health parameters of Nile tilapia (Oreochromis niloticus).

Chlorpyrifos (CPF) is one of the most common insecticides found in freshwater ecosystems, and has been detected in agricultural and fishery products worldwide. This study focused on comprehensive panel of hematological, immunotoxic and pathology changes in Nile tilapia (Oreochromis niloticus) during and after exposure to CPF at 15 µg/L (0.043 µM) (1/10 LC50, group CPF1), or 75 µg/L (0.21 µM) (1/2 LC50, group CPF2) for 14 days, followed by 2 weeks recovery. Different endpoints were used to determine effects of CPF on fish health: hematological parameters; antioxidant levels in liver and gills; innate immune parameters; expression levels of pro-inflammatory cytokine genes at mRNA level in anterior kidney and spleen; and histopathological assessment of gills, liver, and kidney tissues. RBCs were significantly decreased in CPF1 group compared to other groups only at day 3. Blood packed cell volume (PCV) and mean corpuscular volume (MCV) showed significant increase at day 3 and 14 of CPF exposure. TLC (Total Leukocytic Counts), neutrophil counts were significantly increased in CPF exposed groups at days 3, 7, 14 compared to the control. While, lymphocytes counts were significantly increased at CPF1 group compared to other groups at day 14. Antioxidant enzyme activity in liver and gills showed significant increase of Malondialdehyde (MDA) and glutathione (GSH), and significant decrease in (catalase/CAT/, glutathione S-transferase/GST/, and superoxide dismutase/SOD/); in CPF exposed groups. Serum bactericidal and lysozyme activity was nominally and significantly decreased, respectively, and whole blood respiratory burst was significantly increased in CPF2 group. The cytokine expression levels showed complex changes in expression patterns. In kidney, cytokine interleukin-8 (IL-8) was significantly upregulated at day 1 in both exposed group. Interleukin-1ß (IL-1ß) and Tumor necrosis factor-α (TNFα) were significantly upregulated at day 1 in CPF1 group, and then IL-8 and TNFα downregulated at day 3 in same group. At day 7, only TNFα was up and downregulated in CPF1 and CPF2, respectively compared to control. All gene expression levels in spleen were upregulated on day 7 of exposure in the high exposed group. Histopathology showed dose-dependent changes in CPF treated groups, indicating gill, liver, and posterior kidney changes associated with oxidative stress damages. Following recovery period, all measured parameters showed varying degrees in their reversibility to the control level. These findings provide important insights about the acute toxic effects of CPF on fish and show potential to be used as biomarkers in further toxicological evaluation studies.

Mapping the teaching of aquatic animal veterinary medicine in the European Union and European Free Trade Area.

Aquatic animal production is the fastest growing food sector globally. Aquaculture and fisheries are very dynamic sectors in the EU and the number of ornamental aquarium pets is increasing. Veterinarians have a fundamental role to play by ensuring health and welfare of aquatic species, productivity and profitability of fish farming, public health and ecosystem conservation. This study investigates how the undergraduate curriculum prepares future veterinarians for such roles by analysing data from the 77 European veterinary education establishments based in EU and the European Free Trade Area. Over 95 per cent of these establishments incorporate teaching in aquatic animal veterinary medicine in their curriculum, while the great majority do so within the core curriculum. Almost half of the establishments provide teaching in aquatic animal veterinary medicine as separate subjects. Many establishments (>40 per cent) provide such training as elective option in their undergraduate curricula or as postgraduate opportunities to enhance Day One Competences. The veterinary education establishments integrating adequately aquatic animal veterinary medicine in their curriculum are evenly distributed in all regions of Europe. Veterinarians are trained and empowered by legislation to assess health of aquatic animals, to diagnose, to prescribe medicines, to notify for diseases and to ensure safe food for the consumers. Veterinary education establishments should encourage training of veterinarians to follow a career in aquatic animal veterinary medicine.

Native Prussian carp (Carassius gibelio) health status, biochemical and histological responses to treated wastewaters.

The aim of this study was to assess the impact of treated wastewaters on native wild Prussian carp inhabiting effluent-receiving waters (ERC) receiving municipal and sugar plant treated wastewaters, further downstream waters (DW), and a detached canal unaffected by the WWTP activities. To that end, general fish health status was determined, including plasma biochemical, haematological, oxidative stress and tissue histopathological indices, over three seasons. The greatest tissue alterations were in fall in ERC during sugar beet processing, as hypertrophy of gill epithelial and interlamellar cells, necrosis and lymphocytic infiltration, hyperplasia and hypertrophy of renal tubules, distention of hepatic sinusoids. In fall the lowest leukocytes, lymphocytes and granulocytes (2467 ± 565, 1333 ± 264, 1133 ± 488 cells/µL respectively), as well as highest plasma ALP (52.7 ± 19.39 U/L) were measured. ERC in fall had the highest ammonium (20 mg/L), nitrite (1.48 mg/L), nitrate (13.4 mg/L), and lowest dissolved O2 (1.23 mg/L). Gill, kidney and liver alterations, and the highest plasma cholesterol (9.1 ± 1.98 mmol/L) were noted in DW fish in fall. Tissue morphology during sugar cane processing seems a consequence of cellular and structural tissue integrity loss. Structural heterogeneity of gills and spleen was enhanced with increasing concentrations of heavy metals and correlated with oxidative stress (SOD 392.5 ± 77.28 U/L). Monogenean infestation was moderate in ERC fish in all seasons compared with DW fish. Prussian carp biological responses to multiple stressors, measured by the effects of WWTP on blood and tissue parameters, reached far downstream and were not of localized nature. This study demonstrated that in aquatic environments impacted with complex contaminants acting synergistically, causal relationships between biological responses and environmental stressors should be interpreted. Integrated histopathological, haematological and biochemical findings are valuable biomarkers for native fish adaptive patterns and monitoring of water quality/pollution of freshwater ecosystems.

Polycarbonate and polystyrene nanoplastic particles act as stressors to the innate immune system of fathead minnow (Pimephales promelas).

Water pollution with large-scale and small-scale plastic litter is an area of growing concern. Macro-plastic litter is a well-known threat to aquatic wildlife; however, the effects of micro-sized and nano-sized plastic particles on the health of organisms are not well understood. Small-scale plastic particles can easily be ingested by various aquatic organisms and potentially interfere with their immune system; therefore, the authors used a freshwater fish species as a model organism for nanoplastic exposure. Characterization of polystyrene (41.0 nm) and polycarbonate (158.7 nm) nanoplastic particles (PSNPs and PCNPs, respectively) in plasma was performed, and the effects of PSNPs and PCNPs on the innate immune system of fathead minnow were investigated. In vitro effects of PSNPs and PCNPs on neutrophil function were determined using a battery of neutrophil function assays. Exposure of neutrophils to PSNPs or PCNPs caused significant increases in degranulation of primary granules and neutrophil extracellular trap release compared to a nontreated control, whereas oxidative burst was less affected. The present study outlines the stress response of the cellular component of fish innate immune system to polystyrene and polycarbonate nanoparticles/aggregates and indicates their potential to interfere with disease resistance in fish populations. Environ Toxicol Chem 2016;35:3093-3100. © 2016 SETAC.

Titanium dioxide nanoparticles enhance mortality of fish exposed to bacterial pathogens.

Nano-TiO2 is immunotoxic to fish and reduces the bactericidal function of fish neutrophils. Here, fathead minnows (Pimephales promelas) were exposed to low and high environmentally relevant concentration of nano-TiO2 (2 ng g(-1) and 10 µg g(-1) body weight, respectively), and were challenged with common fish bacterial pathogens, Aeromonas hydrophila or Edwardsiella ictaluri. Pre-exposure to nano-TiO2 significantly increased fish mortality during bacterial challenge. Nano-TiO2 concentrated in the kidney and spleen. Phagocytosis assay demonstrated that nano-TiO2 has the ability to diminish neutrophil phagocytosis of A. hydrophila. Fish injected with TiO2 nanoparticles displayed significant histopathology when compared to control fish. The interplay between nanoparticle exposure, immune system, histopathology, and infectious disease pathogenesis in any animal model has not been described before. By modulating fish immune responses and interfering with resistance to bacterial pathogens, manufactured nano-TiO2 has the potential to affect fish survival in a disease outbreak.

Histopathology of fathead minnow (Pimephales promelas) exposed to hydroxylated fullerenes.

Hydroxylated fullerenes are reported to be very strong antioxidants, acting to quench reactive oxygen species, thus having strong potential for important and widespread applications in innovative therapies for a variety of disease processes. However, their potential for toxicological side effects is still largely controversial and unknown. Effects of hydroxylated fullerenes C60(OH)24 on the fathead minnow (Pimephales promelas) were investigated microscopically after a 72-hour (acute) exposure by intraperitoneal injection of 20 ppm of hydroxylated fullerenes per gram of body mass. Cumulative, semi-quantitative histopathologic evaluation of brain, liver, anterior kidney, posterior kidney, skin, coelom, gills and the vestibuloauditory system revealed significant differences between control and hydroxylated fullerene-treated fish. Fullerene-treated fish had much higher cumulative histopathology scores. Histopathologic changes included loss of cellularity in the interstitium of the kidney, a primary site of haematopoiesis in fish, and loss of intracytoplasmic glycogen in liver. In the coelom, variable numbers of leukocytes, including many macrophages and fewer heterophils and rodlet cells, were admixed with the nanomaterial. These findings raise concern about in vivo administration of hydroxylated fullerenes in experimental drugs and procedures in human medicine, and should be investigated in more detail.

Rac2 expression and its role in neutrophil functions of zebrafish (Danio rerio).

The neutrophil contributes significantly to the immune response. In particular, their phagocytosis and pathogen-killing functions are vital for defense from invading pathogens. Rac2, a Rho small GTPase, is involved in many key neutrophil functions. Loss of Rac2 activity results in severe bacterial infections and neutrophil function deficits in humans and mice. While the genes rac1, 2, and 3 have been identified in the zebrafish genome, their expression has not been well-characterized. We describe rac1, 2, and 3 expression over the first three days of development, as well as the presence and localization of Rac2 protein in adult zebrafish neutrophils. The mRNA for each Rac isoform was detected in zebrafish embryos as early as 12 h post fertilization. Immunocytochemistry and confocal microscopy of adult zebrafish neutrophils confirmed diffuse Rac2 protein within the cytoplasm. Only rac2 was found in sorted neutrophil samples. Armed with knowledge of its presence and exclusive expression, the role of Rac2 in key antimicrobial zebrafish neutrophil responses was examined by small molecule inhibition of Rac during respiratory burst, NET release, and phagocytosis assays. Inhibition of Rac2 during these assays produced a dose-dependent decrease in each function, as was expected due to previous work in mammals. The expression pattern and role of Rac2 in zebrafish neutrophil function allows for comparative studies of innate immune responses in this animal model.

Immunotoxicology of non-functionalized engineered nanoparticles in aquatic organisms with special emphasis on fish--review of current knowledge, gap identification, and call for further research.

The rapid increase in use of nanotechnology products is increasing the presence of metal, metal-oxide and carbon-based nanoparticles in the aquatic environment. These non-functionalized engineered nanoparticles can interact with the immune system of fish and invertebrates, and tip the ecological balance of population sustainability. Most nanoparticle types present in the aquatic environment, such as titanium dioxide, do not exhibit or have very low direct toxicity, but instead display silent or concealed sub-lethal effects on the immune system with serious implications. There is a gap in current available information regarding the immunotoxic potential of engineered nanoparticles toward aquatic organisms. Therefore, there is a critical need to provide the first comprehensive review of the effects of engineered non-functionalized nanoparticles on the immune system of aquatic animals, address the major gaps in current existing information, and recommend the future focus of research. This manuscript identifies cell mediated immunity and the phagocytic cells as the primary target of nanoparticle immunotoxicity. The immunotoxicity is primarily govern by lysosomal destabilization, frustrated phagocytosis, and change in function of the phagocytic cells, which decrease the ability of animals to defend themselves against pathogens and infectious diseases. Humoral immune system is a lesser target of direct immunotoxicity, but plays a critical role in dissemination of the nanoparticles through the body and their presentation to the phagocytic cells. The external innate immunity and the acquired immunity have not been connected with overly important and direct immunotoxic effects, but instead a big gap in current targeted research has been acknowledged.

Heterophil functional responses to dietary immunomodulators vary in genetically distinct chicken lines.

The effect of dietary supplementation of immunomodulators on in vitro chicken heterophil function was investigated using three diverse genetic lines of chickens (broiler, Fayoumi, and Leghorn). Dietary supplementation with ß-glucan, ascorbic acid, and corticosterone was fed from 8 to 11 weeks of age. Heterophil function was evaluated weekly during supplementation using phagocytosis, bacterial killing, and heterophil extracellular traps (HETs)-DNA release. Fayoumis fed the basal diet had significantly higher HETs-DNA release (P=0.002) than Leghorns and broilers. Both genetic line and immunomodulator diet supplementation had significant effects on bacterial killing (line and diet effect: P<0.001) and HETs-DNA release (line: P<0.001; diet: P=0.043). Dietary supplementation with immunomodulators, therefore, shows potential to affect and augment heterophil function in chickens. The current results also suggest the important role of genetics in innate immune responses.