Counter-gradient variation in gene expression between fish populations facilitates colonization of low-dissolved oxygen environments.

The role of phenotypic plasticity during colonization remains unclear due to the shifting importance of plasticity across timescales. In the early stages of colonization, plasticity can facilitate persistence in a novel environment; but over evolutionary time, processes such as genetic assimilation may reduce variation in plastic traits such that species with a longer evolutionary history in an environment can show lower levels of plasticity than recent invaders. Therefore, comparing species in the early stages of colonization to long-established species provides a powerful approach for uncovering the role of phenotypic plasticity during different stages of colonization. We compared gene expression between low-dissolved oxygen (DO) and high-DO populations of two cyprinid fish: Enteromius apleurogramma, a species that has undergone a recent range expansion, and E. neumayeri, a long-established native species in the same region. We sampled tissue either immediately after capture from the field or after a 2-week acclimation under high-DO conditions, allowing us to test for both evolved and plastic differences in low-DO vs high-DO populations of each species. We found that most genes showing candidate-evolved differences in gene expression did not overlap with those showing plastic differences in gene expression. However, in the genes that did overlap, there was counter-gradient variation such that plastic and evolved gene expression responses were in opposite directions in both species. Additionally, E. apleurogramma had higher levels of plasticity and evolved divergence in gene expression between field populations. We suggest that the higher level of plasticity and counter-gradient variation may have allowed rapid genetic adaptation in E. apleurogramma and facilitated colonization. This study shows how counter-gradient variation may impact the colonization of divergent oxygen environments.

Development of real-time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3.

In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila.

Hight prevalent of Opisthorchis viverrini infection and coincident Haplorchis metacercariae in cyprinid fishes in upper northeastern region of Thailand.

Cyprinid fishes are commonly acted as second intermediate hosts of Opisthorchis viverrini and Haplorchis spp. This research investigated the prevalence and intensity of both parasites in cyprinid species. This process is regularly used as a guideline for disease surveillance and as a preventive measure in the Upper Northeastern region of Thailand. Fish samples were collected from both lentic and lotic water sources between November 2020 and October 2021, and classified them by their species. Acid pepsin-HCL solution was used to digest the individual samples and create a build-up of precipitate in order to view metacercariae using a stereomicroscope. A total of 3583 fish were caught and classified into 23 species. Out of these fish, 5.49% of them were found O. viverrini, 26.54% Haplorchis taichui, 10.04% Haplorchis pumilio, and 9.29% Haplorchoides sp. Ten species of fish were observed to have been infected with O. viverrini. Amongst the collected fish, M. obtusirostris had the highest prevalence of infection at 52.94%, while B. gonionotus had only 1.71%. The prevalence of O. viverrini infection was highest at 27.41% in Udon Thani Province. No prevalence of O. viverrini infection was observed in Bueng Kan Province. High prevalence of O. viverrini infection in cyprinid fishes was found in both lentic and lotic water sources in almost all provinces in the region. The prevalence of infection and intensity depended on the fish species and fish habitats.

Redescription of Cyprinion muscatense (Teleostei: Cyprinidae) with the first phylogenetic analysis of the genus.

Members of the genus Cyprinion (Cypriniformes: Cyprinidae) are found in the Indus River basin west to the Arabian Peninsula and the Tigris-Euphrates River drainages (Persian Gulf basin). The taxonomic status of Cyprinion including Cyprinion muscatense is poorly understood when compared to other cyprinid genera. C. muscatense has been considered as a member of the Cyprinion watsoni-microphthalmum group and a valid species endemic to the Arabian Peninsula. Here, we redescribe C. muscatense based on an integrative morphological and molecular approach and freshly sampled material from several localities in the Oman Mountains ecoregion. The results showed that C. muscatense is distinguished from the other Cyprinion species in the Arabian Peninsula by having a short, thin, and slightly serrated last unbranched dorsal fin ray; the lower number of circumpeduncular scales; lateral line scales; and also scales between the lateral line and the dorsal-fin origin. Subterminal mouth, presence of one pair of small barbels at the mouth corner, 3-4 unbranched and 9½-10½ dorsal-fin branched rays, 12-14 pectoral-fin rays, 7-8 pelvic-fin rays, 2-3 unbranched and 6½-7½ branched anal-fin rays, and 37-40 lateral line scales are other morphological characteristics of C. muscatense. C. muscatense is also well distinguished by molecular characters among its congeners. The first molecular phylogenetic analysis of the genus, covering all currently recognized Cyprinion species except for C. watsoni, is also presented. C. muscatense is resolved as the sister species to another endemic fish of the Arabian Peninsula Cyprinion mhalense, with a Kimura-2-Parameter model distance of 5.3%.

First molecular confirmation of cyprinid herpesvirus 1 (CyHV1) in diseased carp in Serbia.

Mass mortality of farmed 1 yr old common carp Cyprinus carpio occurred at a carp farm in April 2022. In addition to high mortality, diseased fish exhibited papillomatous growths on the skin and fins, characteristic of carp pox. To investigate a possible viral cause, tissue samples were collected and nucleic acid was extracted using standard procedures. In a pooled sample from the gills and kidneys, carp edema virus (CEV) was detected by real-time PCR. In a skin tissue sample with papillomatous growths, cyprinid herpesvirus 1 (CyHV1) was detected by a conventional PCR targeting a conserved region of the DNA polymerase of cyprinid herpesviruses. PCR products were visualized through agarose gel electrophoresis, and the presence of CyHV1 DNA was confirmed by Sanger sequencing. This represents the first molecular confirmation of CyHV1 in common carp in Serbia.

CyHV-2 infection triggers mitochondrial-mediated apoptosis in GiCF cells by upregulating the pro-apoptotic gene ccBAX.

Apoptosis is a physiological cell death phenomenon, representing one of the fundamental physiological mechanisms for maintaining homeostasis in living organisms. Previous studies have observed typical apoptotic features in Carassius auratus gibelio caudal fin cell (GiCF) infected with Cyprinid herpesvirus 2 (CyHV-2), and found a significant up-regulation of ccBAX expression in these infected cells. However, the specific apoptotic mechanism involved remains unclear. In this study, we utilized the GiCF cell line to investigate the apoptotic mechanism during CyHV-2 infection. Immunofluorescence staining revealed translocation of ccBAX into mitochondria upon CyHV-2 infection. Flow cytometry analysis demonstrated that overexpression of ccBAX expedited virus-induced apoptosis, characterized by heightened mitochondrial depolarization, increased transcriptional levels of Cytochrome c (Cyto c) in both the cytoplasm and mitochondria, and augmented Caspase 3/7 enzyme activity. Bax inhibitor peptide V5 (BIP-V5), an inhibitor interfering with the function of Bax proteins, inhibited Bax-mediated apoptotic events through the mitochondrial pathway and attenuated apoptosis induced by CyHV-2. In this study, it was identified for the first time that CyHV-2 induces apoptosis via the mitochondrial pathway in GiCF cells, bridging an important gap in our understanding regarding cell death mechanisms induced by herpesvirus infections in fish species. These findings provide a theoretical basis for comprehending viral apoptotic regulation mechanisms and the prevention and control of cellular pathologies caused by CyHV-2 infection.

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

A high-quality reference genome for the common creek chub, Semotilus atromaculatus.

Creek chub (Semotilus atromaculatus) is a leuciscid minnow species commonly found in anthropogenically disturbed environments, making it an excellent model organism to study human impacts on aquatic systems. Genomic resources for creek chub and other leuciscid species are currently limited. However, advancements in DNA sequencing now allow us to create genomic resources at a historically low cost. Here, we present a high quality, 239 contig reference genome for the common creek chub, created with PacBio HiFi sequencing. We compared the assembly quality of two pipelines: Pacific Biosciences' Improved Phase Assembly (873 contigs) and Hifiasm (239 contigs). Quality and completeness of this genome is comparable to the zebrafish (Danioninae) and fathead minnow (Leuciscidae) genomes. The creek chub genome is highly syntenic to the zebrafish and fathead minnow genomes, and while our assembly does not resolve into the expected 25 chromosomes, synteny with zebrafish suggests that each creek chub chromosome is likely represented by 1-4 large contigs in our assembly. This reference genome is a valuable resource that will enhance genomic biodiversity studies of creek chub and other nonmodel leuciscid species common to disturbed environments.

Afferent and efferent connections of the secondary general visceral sensory nucleus in goldfish.

The secondary general visceral sensory nucleus (SVN) receives ascending fibers from the commissural nucleus of Cajal (NCC), or the primary general visceral sensoru in the medulla oblongata of teleosts. However, the full set of fiber connections of the SVN have been studied only in the Nile tilapia. We have investigated the connections of the SVN in goldfish by tracer injection experiments to the nucleus. We paid special attention to the possible presence of spinal afferents, since the spinal cord projects to the lateral parabrachial nucleus, or the presumed homologue of SVN, in mammals. We found that the SVN indeed receives spinal projections. Spinal terminals were restricted to a region ventrolaterally adjacent to the terminal zone of NCC fibers, suggesting that the SVN can be subdivided into two subnuclei: the commissural nucleus-recipient (SVNc) and spinal-recipient (SVNsp) subnuclei. Tracer injections to the SVNc and SVNsp as well as reciprocal injections to the diencephalon revealed that both subnuclei project directly to diencephalic structures, such as the posterior thalamic nucleus and nucleus of lateral recess, although diencephalic projections of the SVNsp were rather sparse. The SVNsp appears to send fibers to more wide-spread targets in the preoptic area than the SVNc does. The SVNc projects to the telencephalon, while the SVNsp sends scarce or possibly no fibers to the telencephalon. Another notable difference was that the SVNsp gives rise to massive projections to the dorsal diencephalon (ventromedial thalamic, central posterior thalamic, and periventricular posterior tubercular nuclei). These differential connections of the subnuclei may reflect discrete functional significances of the general visceral sensory information mediated by the medulla oblongata and spinal cord.

Mass mortality events associated with cyprinid herpesvirus 2 (CyHV-2) infection in wild Prussian carp Carassius gibelio in the Netherlands, and molecular biology of virus strains.

In 2011 and 2015, four mass mortalities of Prussian carp (Carassius gibelio) were observed in a recreational freshwater lake and open freshwater in the western part of the Netherlands. Cyprinid herpesvirus 2 (CyHV-2) infection was suspected in these cases, based on presumptive gross diagnosis. To elucidate the cause of the mass mortalities diagnostic PCR assays were performed for CyHV-2, based on the helicase gene. Furthermore, the viral isolates were genotyped by sequencing the enlarged marker A and marker B sequences. Diagnostic PCR revealed that three of four samples were positive for CyHV-2, indicating these three mass mortalities were associated with CyHV-2 infection. The marker A sequence from one of the isolates found in this study was identical to those from different locations such as Asia and Middle East, suggesting a link among the isolates. This is the first detailed report on mass mortalities of Prussian carp associated with CyHV-2 infection in natural aquatic environments in the Netherlands. Since 2015, additionally, in total three CyHV-2 associated outbreaks of Dutch Prussian carp were seen in 2016 and 2020. These outbreaks in Prussian carp from lakes and open water suggest that the virus has been spreading in natural freshwaters in the Netherlands.

The response of stream ecosystem properties to two size classes of herbivorous minnow species.

Losses in freshwater fish diversity might produce a loss in important ecological services provided by fishes in particular habitats. An important gap in our understanding of ecosystem services by fishes is the influence of individuals from different size classes, which is predicted based on known ontogenetic shifts in metabolic demand and diet. I used 20 experimental stream mesocosms located at Konza Prairie Biological Station (KPBS), KS, USA, to assess the influence of fish size on ecosystem properties. Mesocosms included two macrohabitats: one riffle upstream from one pool filled with consistent pebble and gravel substrate. There were four experimental and one control treatment, each replicated four times (N = 20). I used two size classes of central stonerollers (Campostoma anomalum) and southern redbelly dace (Chrosomus erythrogaster). Five ecosystem properties were assessed: algal filament length (cm), benthic chlorophyll a (µg/cm2), benthic organic matter (g/m2), macroinvertebrate biomass (g/m2), and stream metabolism (g O2/m2/day-1). Size structure of fish populations affected some, but not all, ecosystem properties, and these effects were dependent upon species identity. Size structure of both species had effects on algal filament lengths where stonerollers of both size classes reduced algal filaments, but only small redbelly dace kept filaments short. A better understanding of the relationship between these prairie stream minnows and their small stream habitats could be useful to both predict changes in stream properties if species are lost (redbelly dace are a Species In Need of Conservation) or size structure shifts.

Common carp (Cyprinus carpio) CD81 promoting CyHV-3 virus replication via regulating autophagy and RLRs-interferon signaling pathway.

Cyprinid herpesvirus type 3 (CyHV-3), also called Koi herpesvirus (KHV), which leads to mass cyprinid mortality and enormous economic losses. To establish an infection, CyHV-3 needs to counteract host antiviral responses. CD81 belongs to the evolutionary conserved tetraspanin family of proteins. Several studies have shown that different members of the tetraspanin superfamily modulates different virus infectious processes. Here we aimed at analysing the role of CD81 in CyHV-3 infection. In this study, we cloned and characterized the CD81 of Common Carp, the open reading frame of CcCD81 gene was 702 bp, which encoded 234 amino acids with four transmembrane domains (TM1 to TM4), a small extracellular loop (SEL), and a large extracellular loop (LEL). Tissue distribution analysis showed that CcCD81 was widely expressed in all the tested tissues with the highest expression in head kidney, followed by a high expression in brain. Subsequently, expression levels of CcCD81 were significantly increased in CCB cells within the first 3h after infection, meanwhile, the expression of viral gene VP136 was reduced after CcCD81 knockdown in CCB cells post CyHV-3 infection. Furthermore, CcCD81 knockdown can significantly reduce the autophagy process and increase the promoter activity of ISRE and IFN-1 in the CCB cells after viral infection, as well as other genes involved in the IFN signaling pathway, including RIG-1、MDA5、MAVS、TBK1 and IRF3. Taking the data together, we revealed that CcCD81 mediates autophagy and blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN) promoting virus replication. These results reveal a new link between autophagy and four-transmembrane-domain protein superfamily and contribute to elucidate the mechanism of CyHV-3 infection.

Impacts of Some Trace Metals in Cyprinus carpio (Linnaeus, 1758) and Tor soro (Valenciennes, 1842) on Human Health.

Although fish are rich sources of various trace metals in their bodies, when these metals exceed health safety limits, it becomes a global concern. Fish exposed to trace metal pollution in their aquatic ecosystems can lead to bioaccumulation and transfer to consumers. Monitoring trace metal contamination of fish food is crucial because they can reach humans through the aquatic food chain, resulting in health problems. Therefore, a first attempt was made for the analysis of trace metals in two cyprinid fish species, namely Cyprinus carpio (Common carp) and Tor soro (Mahseer), which were abundantly found in Khanozai Dam and assessing their potential health impacts when consumed by humans. A total of 200 fish specimens were collected from this area. These specimens were divided equally between the two carp species, with 100 individuals from each species. The fish specimens were collected over 12 months, from June 2020 to May 2021 to determine the concentration of some trace metals, i.e. Manganese (Mn), Calcium (Ca), Iron (Fe), Potassium (K), and Zinc (Zn), Sodium (Na), Phosphorus (P), Magnesium (Mg), and Chlorine (Cl) using a Flame Atomic Absorption Spectrophotometer with a multi-element hollow cathode lamp. The obtained results from the trace metal analysis of Cyprinus carpio and Tor soro in this study revealed that both calcium and phosphorus were present in higher concentrations. However, iron, manganese, and zinc were not detected in both species. Additionally, magnesium was also not detected in C. carpio. For Cyprinus carpio, the arrangement was Ca > P > K > Na > Cl, while for Tor soro, it was Ca > P > Na > K > Cl > Mg in decreasing order. The health risks associated with fish consumption were assessed using factors like estimated daily intake (EDI), Target Hazard Quotient (THQ), and Total Target Hazard Quotient (TTHQ). The THQ values for all analyzed trace metals in both carp species exceeded the maximum value of 1.0, except for Cl in Tor soro. This suggests the potential for various health-related diseases in regular consumers due to their elevated levels. In both species, the TTHQ factor also surpassed the standard value of 1.0, indicating an acute health risk for humans. Although EDI values did not surpass the recommended daily allowance (RDA), continuous consumption of these contaminated fish from this area could lead to severe health complications. Therefore, these results strongly indicated that the elevated levels of trace metals found in the highly abundant cyprinid species of Khanozai Dam could pose serious health-related issues for the local inhabitants.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of fish.

Vector or reservoir species of five fish diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review (ELR), to support a possible updating of Regulation (EU) 2018/1882. Fish species on or in which highly polymorphic region-deleted infectious salmon anaemia virus (HPR∆ ISAV), Koi herpes virus (KHV), epizootic haematopoietic necrosis virus (EHNV), infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV) were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms or reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected fish was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir fish species during transport was collected from scientific literature. For VHSV, IHNV or HPR∆ ISAV, it was concluded that under transport conditions at temperatures below 25°C, it is likely (66-90%) they will remain infective. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild, aquaculture establishments or through water supply can possibly transmit VHSV, IHNV or HPR∆ ISAV into a non-affected area when transported at a temperature below 25°C. The conclusion was the same for EHN and KHV; however, they are likely to remain infective under all transport temperatures.

Infection of epithelioma papulosum cyprini (EPC) cells with spring viremia of carp virus (SVCV) induces autophagy and apoptosis through endoplasmic reticulum stress.

Spring viremia of carp virus (SVCV) is a lethal freshwater pathogen of cyprinid fish that has caused significant economic losses to aquaculture. To reduce the economic losses caused by SVCV, its pathogenic mechanism needs to be studied more thoroughly. Here, we report for the first time that SVCV infection of Epithelioma papulosum cyprini (EPC) cells can induce cellular autophagy and apoptosis through endoplasmic reticulum stress. The presence of autophagic vesicles in infected EPC cells was shown by transmission electron microscopy. Quantitative fluorescence PCR and Western blot results showed that p62 mRNA expression was decreased, and the expression of Beclin1 and LC3 mRNA was increased. The p62 protein was decreased, and the Beclin1 protein and LC3 were increased in the endoplasmic reticulum stress activation state. To further clarify the mode of death of SVCV-infected EPC cells, we examined caspase3, caspase9, BCL-2, and Bax mRNA, which showed that they were all increased. Apoptosis of SVCV-infected cells increased upon activation of endoplasmic reticulum stress. Our results suggest that endoplasmic reticulum stress can regulate SVCV infection-induced autophagy and apoptosis. The results of this study provide theoretical data for the pathogenesis of SVCV and lay the foundation for future drug development and vaccine construction.

In Vivo Imaging Sheds Light on the Susceptibility and Permissivity of Carassius auratus to Cyprinid Herpesvirus 2 According to Developmental Stage.

Cyprinid herpesvirus 2 (CyHV-2) is a virus that causes mass mortality in economically important Carassius spp. However, there have been no comprehensive studies into host susceptibility or permissivity with respect to developmental stage, and the major portal of viral entry into the host is still unclear. To help bridge these knowledge gaps, we developed the first ever recombinant strain of CyHV-2 expressing bioluminescent and fluorescent reporter genes. Infection of Carassius auratus hosts with this recombinant by immersion facilitated the exploitation of various in vivo imaging techniques to establish the spatiotemporal aspects of CyHV-2 replication at larval, juvenile, and adult developmental stages. While less susceptible than later developmental stages, larvae were most permissive to CyHV-2 replication, leading to rapid systemic infection and high mortality. Permissivity to CyHV-2 decreased with advancing development, with adults being the least permissive and, thus, also exhibiting the least mortality. Across all developmental stages, the skin was the most susceptible and permissive organ to infection at the earliest sampling points post-infection, indicating that it represents the major portal of entry into these hosts. Collectively these findings provide important fundamental insights into CyHV-2 pathogenesis and epidemiology in Carassius auratus with high relevance to other related economically important virus-host models.

Molecular Cloning of Toll-like Receptor 2 and 4 (SpTLR2, 4) and Expression of TLR-Related Genes from Schizothorax prenanti after Poly (I:C) Stimulation.

Toll-like receptor (TLR) signaling is conserved between fish and mammals, except for TLR4, which is absent in most fish. In the present study, we aimed to evaluate whether TLR4 is expressed in Schizothorax prenanti (SpTLR4). The SpTLR2 and SpTLR4 were cloned and identified, and their tissue distribution was examined. The cDNA encoding SpTLR4 and SpTLR2 complete coding sequences (CDS) were identified and cloned. Additionally, we examined the expression levels of seven SpTLRs (SpTLR2, 3, 4, 18, 22-1, 22-2, and 22-3), as well as SpMyD88 and SpIRF3 in the liver, head kidney, hindgut, and spleen of S. prenanti, after intraperitoneal injection of polyinosinic-polycytidylic acid (poly (I:C)). The SpTLR2 and SpTLR4 shared amino acid sequence identity of 42.15-96.21% and 36.21-93.58%, respectively, with sequences from other vertebrates. SpTLR2 and SpTLR4 were expressed in all S. prenanti tissues examined, particularly in immune-related tissues. Poly (I:C) significantly upregulated most of the genes evaluated in the four immune organs compared with the PBS-control (p < 0.05); expression of these different genes was tissue-specific. Our findings demonstrate that TLR2 and TLR4 are expressed in S. prenanti and that poly (I:C) affects the expression of nine TLR-related genes, which are potentially involved in S. prenanti antiviral immunity or mediating pathological processes with differential kinetics. This will contribute to a better understanding of the roles of these TLR-related genes in antiviral immunity.

Bactericidal role of epidermal mucus of freshwater fish treated with Aeromonas hydrophila.

This study explored the bactericidal role of the epidermal mucus (EM) of five freshwater Cyprinid fish species namely Ctenopharyngodon idella, Labeo rohita, Catla catla, Hypophthalmichthys molitrix, and Cirrhinus mrigala after treatment with Aeromonas hydrophila. Extracts of EM (crude and acidic) of each species showed bactericidal activity against various Gram -ve (Pseudomonas aeruginosa, Escherichia coli, Aeromonas hydrophila, Edwardsiella tarda, Salmonella enterica, Klebsiella pneumonia, Serratia marcescens, and Enterobacter cloacae) and Gram +ve (Bacillus wiedmannii and Staphylococcus aureus) bacteria compared with standard antibiotics (Fosfomycin). The zone of inhibition (ZOI) was measured in millimetres against antibiotics (Fosfomycin). Variations in bactericidal activity of EM were observed against bacteria from the same and different fish species. The acidic extract was more effective than the crude extract and showed significantly higher ZOI values against various bacteria and Fosfomycin antibiotics. This result shows that fish EM may perform an important role in fish defence against bacteria. Therefore, this study may hint towards the substitution of synthetic antibiotics with fish EM that may be used as a novel 'bactericidal' in aquaculture as well as in humans against bacterial infections.

Development of a lateral flow immuno-chromatic strip assay for the detection of cyprinid herpesvirus 3 (CyHV-3).

Cyprinid herpesvirus 3 (CyHV-3) is the main pathogen of koi herpesvirus disease (KHVD), which has caused serious damage to the ornamental and food-producing carp industry. Effective and rapid on-site detection methods are needed for early diagnosis of CyHV-3. A lateral flow immuno-chromatographic assay (LFIA) using two specific anti-CyHV-3 monoclonal antibodies has been developed and validated for on-site detection of CyHV-3. MAb 3C9 was used to bio-conjugate CyHV-3 antigen with colloidal gold, and MAb 2A8 was used to capture antigen bound colloidal gold on the test line. The control line was lined with goat anti-mouse IgG to capture unbound colloidal gold to validate performance. The test results can be viewed within 10 min after putting the strip into CyHV-3 virus infection fluid. The lowest limit of detection for the LFIA test was found to be 1.5 × 104 copies/µL and it showed no cross-reactivity with other fish viral pathogens. The specificity of the strip was 100% when spleen and kidney tissues of CyHV-3-infected and healthy koi were validated at the field level. The LFIA strip will be an effective device for the early detection of CyHV-3 in the future.