Infected Fish Tissues Research Articles

Fraction and Antibacterial Activity of Bitter Melons (Momordica charantia) in Koi Fish (Cypinus carpio) Infected with Aeromonas salmonicida Bacteria and its Effect on Gills Histopathology

Koi fish (Cyprinus carpio) is a type of freshwater ornamental fish with significant economic value, attracting widespread public interest due to its vibrant colors and agile movements. Therefore, this study introduces a novel approach by evaluating the antibacterial activity of bitter melon extract fraction (Momordica charantia) against A. salmonicida infection in koi fish and its impact on gill histopathology. The novelty of this study lies in the exploration of bitter melon extract fraction as a natural, eco-friendly antibacterial alternative to synthetic antibiotics in aquaculture. This research is crucial as it addresses the growing global concern over antibiotic resistance and the urgent need for sustainable, natural solutions in fish health management. Antibacterial tests were conducted to assess the ability of the bitter melon extract to inhibit bacterial growth, while histopathological analysis aimed to observe the specific changes in gill tissues of infected koi fish. The results revealed that the bitter melon extract fraction significantly reduced bacterial count and improved gill tissue integrity. Histopathological improvements were observed, including reduced hyperplasia, lamellar fusion, edema, and necrosis, indicating tissue recovery. This finding highlights the potential of bitter melon extract fraction as a natural antibacterial agent for controlling bacterial infections in fish, offering an alternative to chemical antibiotics. Further research is needed to determine the optimal dosage and to explore its antibacterial mechanism, ensuring its safe and effective application in aquaculture.

Myxospore density of Kudoa inornata varies significantly within symmetrical white muscle tissue replicates of its fish host, the spotted seatrout, Cynoscion nebulosus

The spotted seatrout, Cynoscion nebulosus, is a popular game fish in the southeastern USA. It is estimated that nearly 90% of the adult population in South Carolina estuaries are infected in their skeletal muscle by the myxosporean, Kudoa inornata. However, little is known about this parasite’s biology, including the distribution and densities of myxospores within tissues of infected fish, which we expect affect the physiology of their hosts. In order to correlate densities with physiological parameters in future studies, we quantified the myxospores density in muscle and characterized the variation among individual fish. Naïve juvenile seatrout was experimentally infected via presumed K. inornata actinospores exposure to raw seawater. A plug of muscle was extracted from two bilaterally symmetrical regions in the epaxial fillet from fresh and frozen carcasses. Variation in density data was calculated both within and among individuals. Within individuals, density counts were compared between left- and right-side biopsies. There was no significant difference between fresh and frozen plugs, and variation among individuals accounted for the greatest proportion of variation at 68.8%, while variation within individuals was substantial at 25.6%. Simulation and correlation tests confirmed that bilaterally symmetrical replicates varied significantly within individuals. When sampled from areas surrounding the initial biopsies, myxospore density estimates were more similar than between sides. Our findings have important implications for sampling design, particularly for studies investigating physiological parameters at the cellular or molecular level in association with parasite infection.

Genome Characterization and Phylogenetic Analysis of Scale Drop Disease Virus Isolated from Asian Seabass (Lates calcarifer).

Scale drop disease virus (SDDV), a double-stranded DNA virus in the family Iridoviridae, has been reported widely in southeast Asian countries as a causative agent of scale drop syndrome (SDS) in Asian seabass. SDS has resulted in high mortality and significant economic losses to the aquaculture industry. This study demonstrated the use of metagenomic methods to investigate bacterial and viral communities present in infected fish tissues and recover a complete genome of the causative agent named SDDV TH7_2019. Characterization of the TH7_2019 genome revealed a genome size of 131 kb with 134 putative ORFs encoding viral proteins potentially associated with host apoptosis manipulation. A comparative genome analysis showed a high degree of amino acid identity across SDDV strains, with variations in number of repeat sequences and mutations within core genes. Phylogenetic analyses indicate a close relationship among SDDV genomes. This research enhances our understanding of the genetic diversity and evolutionary relationship of SDDV, contributing valuable insights for further development of effective control strategies of SDDV.

Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis.

We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc. Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays. The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book. The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc.

Immunological and molecular evaluation of zoonotic metacercarial infection in freshwater fish: A cross-sectional analysis

Improperly cooked fish, carrying active metacercariae (MCs), can pose a significant risk for transmitting fish-borne zoonotic trematodes (FBZTs) to human consumers. This study aimed to enhance our understanding of FBZTs by conducting a comprehensive cross-sectional analysis involving various fish species, such as Nile tilapia (Oreochromis niloticus), African catfish (Clarias gariepinus), and red-belly tilapia (Tilapia zillii). These fish specimens were collected from distinct Egyptian governorates, specifically Giza, Kafr al-Shaykh, and Fayoum. The recovered flukes from experimentally infected domestic pigeons were identified as Prohemistomum vivax, Haplorchis pumilio, and Pygidiopsis genata based on morphological features. Furthermore, the identity of the retrieved adult flukes was confirmed using three species-specific primers for PCR amplification and sequencing analysis of the ITS rDNA region and have been deposited in GenBank with the following accession numbers: P. vivax (OR291421.1 and OR291422.1), P. genata (OP099561.1), and H. pumilio (OM439581.1-OP090510.1).Quantitative real-time PCR targeting the immunological genes Tumor Necrosis Factor-alpha (TNF-alpha) and Interleukin-1 (IL-1Β) was employed to compare the cellular immune response between infected with EMCs and uninfected O. niloticus. The results indicated a significant increase in TNF- and IL-1Β levels in FBZTs-infected vs un-infected fishes. Importantly, the presence of adult flukes and EMCs led to substantial histological alterations in both experimentally infected pigeons and naturally infected fish tissues. These changes included the necrosis of fish muscle bundles and a pronounced inflammatory reaction with muscular necrosis in the digestive tracts of experimentally infected pigeons.

Single-cell transcriptome, phagocytic activity and immunohistochemical analysis of crucian carp (Carassius auratus) in response to Rahnella aquatilis infection.

In teleost fish, kidney is an important immune and hematopoietic organ with multiple physiological functions. However, the immune cells and cellular markers of kidney require further elucidation in crucian carp (C. auratus). Here we report on the single-cell transcriptional landscape in posterior kidney, immunohistochemical and phagocytic features of C. auratus with R. aquatilis infection. The results showed that a total of 18 cell populations were identified for the main immune cells such as monocytes/macrophages (Mo/Mϕ), dendritic cells (DCs), B cells, T cells, granulocytes and hematopoietic progenitor cells (HPCs). Pseudo-time trajectory analysis was reconstructed for the immune cells using Monocle2 to obtain additional insights into their developmental lineage relationships. In the detected tissues (liver, spleen, kidney, intestine, skin, and gills) of infected fish exhibited positive immunohistochemical staining with prepared for antibody to R. aquatilis. Apoptotic cells were fluorescently demonstrated by TUNEL assay, and bacterial phagocytic activity were observed for neutrophils and Mo/Mϕ cells, respectively. Moreover, a similar up-ward/down-ward expression trend of the selected immune and inflammatory genes was found in the kidney against R. aquatilis infection, which were significantly involved in TLR/NLR, ECM adhesion, phago-lysosome, apoptosis, complement and coagulation pathways. To our knowledge, this is the first report on the detailed characterization of immune cells and host-R. aquatilis interaction, which will contribute to understanding on the biology of renal immune cells and repertoire of potential markers in cyprinid fish species.

Isolation and identification of a new strain Micropterus salmoides rhabdovirus (MSRV) from largemouth bass Micropterus salmoides in China

As an important farmed fish worldwide, largemouth bass (Micropterus salmoides) is suffering a severely disease caused by rhabdovirus, which has resulted to a massive economic loss. In the present study, a new Micropterus salmoides rhabdovirus (MSRV) strain named MSRV-NY01 was isolated from largemouth bass juvenile from a fish farm in Nanyang, Henan province, China. Cell culture, histopathological examination, reverse transcription-polymerase chain reaction (RT-PCR), plaque purification, phylogenetic analysis, transmission electron micrograph (TEM) observation, experimental infection, real-time quantitative polymerase chain reaction (RT-qPCR) were used for virus isolation and identification. The diseased fish exhibited abnormal symptoms with anorexia, exophthalmia, bloated abdomen, spiral swimming, hemorrhage on body surfaces, and fluid accumulation in the abdominal cavity. Histopathological examination showed obvious hemorrhage, inflammatory cell infiltration, and widespread cell degeneration and necrosis were presented in the liver, spleen, intestine and stomach of diseased fish. Obvious cytopathic effects (CPE) were induced in epithelioma papillosum cyprini (EPC), grouper spleen (GS) and grouper fin (GF-1) cell lines after inoculation of mixed liver and kidney homogenates from disease fish. RT-PCR, DNA sequencing and phylogenetic tree analysis showed that the virus belongs to rhabdovirus with the highest similar to the MSRV-FJ985 strain. Through five sequential rounds of plaque purification assay, a MSRV plaque isolate was derived and was confirmed by RT-PCR and DNA sequencing. TEM observation showed that numerous virions with typical bullet shape (100–150 nm in length and 70–90 nm in diameter) were present in infected GS cells. Thus, this newly isolated virus from diseased Micropterus salmoides was tentatively named as MSRV-NY01. Regression infection experiment indicated that MSRV-NY01 was highly pathogenic to Micropterus salmoides juvenile and the cumulative mortality of fish injected with 5 × 104, 104, 2 × 103, 4 × 102, 2 × 102, 102 and 50 PFU of virus were 100%, 100%, 100%, 96.7%, 96.7%, 93.3% and 90%, respectively. Moreover, RT-qPCR showed that MSRV was present in all 9 tested tissues of infected fish, and the virus loads of the spleen and heart were significantly higher than that in other tissues tested. In conclusion, we isolated and identified a new MSRV strain from diseased largemouth bass, and our results will provide new material and information for the prevention and control strategy for MSRV.

Biomonitoring of heavy metals using Contracaecum quadripapillatum (Nematoda) in comparison to its fish host, Lates niloticus, from the Nile River, Egypt

Recently, fish parasites have been used as a biomonitoring tool to indicate the health status of ecosystems. Therefore, this research aimed to evaluate the potential capacity of Contracaecum quadripapillatum larvae as accumulation indicators for metal pollution and compare metal concentrations in host tissues of non-infected and infected fish: Lates niloticus from the Nile River. Accumulations of Cd, Cu, Fe, Mn, Ni, Pb, and Zn in larval nematodes and tissues of the liver, kidney, and muscles of both infected and non-infected fish were determined. All metal concentrations exhibit a significantly higher increase in larval nematodes than the muscles of infected fish and vice versa except Cd in the kidney. On the other hand, only Cd, Mn, Pb, and Zn concentrations were significantly higher in the parasite than in the host liver. Therefore, bioaccumulation factors were most obvious and effective in the muscles of infected fish than in the liver and kidney. Contracaecum larvae accumulate Cd and Pb more than other metals. The infrapopulation size of C. quadripapillatum correlated with metal concentrations in different host tissues, especially the kidney, while the correlations between metal levels in the tissues of both parasite and fish organs exhibit different patterns in each organ. The current work revealed that C. quadripapillatum larvae represent environmental monitors for metal pollution in the freshwater ecosystem.

Molecular identification, histopathological analysis and immunohistochemical characterization of non- pigmented Aeromonas salmonicida subsp. salmonicida in Mugil carinatus (Valenciennes, 1836)

Aeromonas salmonicida was isolated for the first time from Mugil carinatus fish sampled from Suez Gulf, Suez governorate, Egypt. Fish samples were identified bacteriologically, molecularly and histopathologically. Biochemical identification of A. salmonicida was performed using API 20 NE kits. The universal 16 S rRNA gene of Aeromonas species was used for the molecular identification of retrieved isolates by producing amplicons at 461 bp. Serine protease (ser) and aerolysin-like protein (act) virulent genes were detected in retrieved isolates at 211 bp and 232 bp, respectively. DNA sequence analysis showed high identity with A. salmonicida subsp. salmonicida strains listed in the GenBank database. Histopathological analysis revealed localization of basophilic bacterial colonies in liver and kidney. Congestion, hepatocellular necrosis and renal tubular necrosis were the marked pathological lesions in liver and kidney tissues of infected fish. An immunohistochemistry technique was used for confirming the presence of A. salmonicida antigen. Immune positive antigen antibody reaction was observed as brown coloration in liver parenchyma and kidney sections of M. carinatus.

Tilapia lake virus immunoglobulin G (TiLV IgG) antibody: Immunohistochemistry application reveals cellular tropism of TiLV infection

Tilapia lake virus (TiLV) is a notable contagious agent that causes massive economic losses in the tilapia industry globally. Evaluations of the histological changes associated with TiLV infection are not only crucial for diagnosis, but also to gain an understanding of the disease. We therefore synthesized a rabbit polyclonal immunoglobulin G antibody against TiLV and developed an immunohistochemical (IHC) procedure to detect TiLV localization in the tissues of infected fish for comparison with in situ hybridization (ISH) testing. A total of four different sample cohorts derived from TiLV-infected fish was used to validate the IHC procedure. The TiLV IHC application was successfully developed and facilitated nuclear and cytoplasmic immunolabelling in the intestines, gills, brain, liver, pancreas, spleen, and kidneys that corresponded with the ISH results. Apart from the ISH results, TiLV-IHC signals were clearly evident in the endothelial cells of various organs, the circulating leukocytes in the blood vessels, and the areas of tissue inflammation. Among the tested sample cohorts, the intestines, gills, and brain had IHC-positive signals, highlighting the possibility of these organs as common TiLV targets. Immunological staining pattern and distribution corresponded with the TiLV viral load but not the inoculation route. The TiLV IHC was also capable of detecting TiLV infection in the experimentally challenged ornamental cichlids, Mozambique tilapia, giant gourami, and naturally infected tilapia, indicating the dynamic range of IHC for TiLV detection. Overall, our study delivers the first IHC platform to detect TiLV infection and provides novel evidence of cellular tropism during TiLV infection. Our findings also reveal the TiLV distribution pattern of infected fish and propose the endotheliotropism and lymphotropism of this virus, which requires further elaboration. Importantly, this new IHC procedure could be applied to study the pathogenesis and interaction of TiLV in future research.

Prevalence, diagnosis and experimental challenge of Dermocystidium sp. infection in Nile tilapia (Oreochromis niloticus) in Egypt

This study confirms the systemic position of Dermocystidium sp. in Nile tilapia (Oreochromis niloticus) internal organs (liver, spleen, kidneys, stomach and intestine). Our work reveals an entire approach for Dermocystidium sp. including prevalence, diagnosis, spores multiplication, experimental infection and histopathological examination. A total of 1388 Nile tilapia (766 cultured fish and 622 wild fish) were collected from May 2012 to May 2014 and screened for Dermocystidium infection. The total prevalence of infection was 35.5% and was higher in wild (45%) than cultured fish (27.8%). The highest rates of infection were recorded in the largest fish and in winter. Several different techniques were employed to detect and identify Dermocystidium sp. from the internal organs of infected hosts including macroscopic examination, microscopic observation of tissue squashes, histological examination and in vitro culture. Infected fish were sluggish and suffered from severe ulceration and black discoloration of the skin. The post-mortem lesion included dark-grayish nodules on internal organs, particularly the liver. Squash preparations from the internal organs revealed spherical spores (signet-ring shape), which is characteristic for Dermocystidium sp. The spores showed multiplication in the freshly dead host tissue. Our study demonstrated new successful attempts for in vitro culture of Dermocystidium either on Eagle's Minimum Essential Medium after adjusting pH of media to 3.5 or on Sabouraud's dextrose agar media enriched with duck decoction 10%. Experimental infection revealed that intra-gastric route was more pathogenic than the immersion route. In histopathological sections from infected fish tissue following natural and experimental infection, spherical spores of Dermocystidium were embedded in the affected tissue. To the best of our knowledge, this study represents the first report of Dermocystidium infection in wild Nile tilapia in Egypt.

High-throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I-based real-time PCR combined with melting curve analysis.

This work describes a primer pair and a high-throughput SYBR Green I-based real-time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034×100 amplicon copies per assay (equivalent to 2×10-11 ng/µl, Cq value of 30.49±1.71). The developed qPCR protocol allowed the detection of V.salmoninarum in non-lethal and lethal fish samples with detection levels of 0.17×100 gene copies in tissues artificially infected and 0.02×100 in tissues of fish experimentally infected with V.salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V.salmoninarum in asymptomatic or carrier fish.

Dietary supplementation of garlic ( Allium sativum ) modulates gut microbiota and health status of tilapia ( Oreochromis niloticus ) against Streptococcus iniae infection

This study was conducted to characterize the causative agent of streptococcosis in tilapia (Oreochromis niloticus) and control of Streptococcus infection by means of garlic (Allium sativum) supplementation. The morphological, biochemical and polymerase chain reaction amplification confirmed 11 isolates belong to Streptococcus iniae from the infected fish eyes and tissue samples. Random screening of 12 well-known medicinal plant parts against S. iniae revealed the garlic extract as the most effective herbal recovery against Streptococcus infection. In vivo challenge test with dietary supplementation of garlic powder significantly improved survival rates of fish against S. iniae infections, and modulate the microbial community and cytokine gene expression profiling in the intestine of the experimental tilapia. Among the two garlic supplemented treatments, 1.0 g garlic supplemented diet significantly increased (p < 0.05) the survival rates of tilapia and the gut bacterial operational transitional units abundance for Proteobacteria and Tenericutes, the phyla associated with healthy intestinal flora. The bacterial diversity index also found high with garlic supplemented diets. Significant upregulations of IL-10 and IL-17F gene expression in the intestinal tissue were observed with 1.0 g garlic supplemented diet where IL-8 and IL-1β expression levels were relatively static. The dietary supplementation of garlic, therefore, could be effective in the prevention of S. iniae infection in fish.

Cloning, characterization and tissue-specific expression of the antimicrobial peptide hepcidin from caspian trout (Salmo caspius) and the antibacterial activity of the synthetic peptide

Antimicrobial peptides have a wide range of antimicrobial activity and widely occur in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine-rich antimicrobial peptides that are active against a variety of pathogens including gram-positive and gram-negative bacteria, as well as viruses. In this study, the hepcidin gene of Caspian trout (CtHep) was identified and characterized. Our results showed that CtHep cDNA has a 267-bp Open Reading Frame (ORF), which is translated to 88 amino acids. The CtHep was classified in the HAMP1 class of hepcidins. Comparison of DNA and cDNA sequences showed that CtHep has 3 exons and 2 introns. The signal, prodomain and mature part of CtHep have 24, 39 and 25 amino acids, respectively. The mature peptide has a molecular weight of 2881.43 Da and a theoretical isoelectric point of 8.53. The expression of CtHep mRNA was detected in different tissues of healthy and infected fish. CtHep expression in the liver, head kidney, spleen and skin was significantly enhanced after bacterial challenge. Expression of CtHep in different embryonic development stages was also substantial. Antibacterial activity of synthetic CtHep peptides was investigated against a number of Gram-positive and Gram-negative bacteria. CtHep inhibited some pathogenic bacteria such as Streptococcus iniae and Aeromonas hydrophila. In the in vivo experiment, CtHep upregulated the cytokines IL-6 and TNF-α in both kidney and spleen tissues after 24 h of the peptide injection. In conclusion, our study showed that CtHep plays an important role in the immune system of Caspian trout and also in the embryonic stages. Moreover, CtHep peptide has a potential to be used as an antimicrobial therapeutic agent as well as an immunostimulant in aquaculture.

Resurgence of Salmonid Herpesvirus-3 Infection (Epizootic Epitheliotropic Disease) in Hatchery-Propagated Lake Trout in Michigan.

Over the past century, populations of Lake Trout Salvelinus namaycush have declined throughout the Great Lakes basin due to overfishing, habitat destruction, introduction of invasive species, and associated recruitment issues from high thiaminase, as well as emerging infectious diseases. To combat these declines, state and federal fishery management agencies undertook substantial stock enhancement efforts, including more stringent regulation of sport and commercial catch limits and increasing hatchery propagation of Lake Trout stocked into Great Lakes basin waterways. One state fish hatchery involved in these rehabilitation efforts experienced mass mortality events in 2012 and 2017. In 2012, following a period of abnormally heavy rain, hatchery staff observed abnormal behavior followed by increased mortalities in two strains of Lake Trout fingerlings, reaching upwards of 20% mortality and totaling a loss of approximately 100,000 fish. In 2017, following another heavy-rain season, 6-8% of 2-year-old Lake Trout experienced morbidity and mortality similar to that observed in 2012. During the 2012 event, Brook Trout Salvelinus fontinalis and splake (Lake Trout×Brook Trout hybrid) reared in flow-through systems receiving water from diseased Lake Trout remained clinically unaffected. Molecular analyses revealed all lots of affected Lake Trout were infected with the salmonid herpesvirus-3 (epizootic epitheliotropic disease virus [EEDV]), a disease that caused complete depopulation of this hatchery in the late 1980s and until 2012 was never again detected in this hatchery or in Michigan. Further sampling detected EEDV in apparently healthy 5-year-old Lake Trout and in wild Mottled Sculpin Cottus bairdii collected in the hatchery source water. The ability of the virus to replicate in tissues of infected fish was verified by exposing naïve Lake Trout to the filtered tissue homogenates of infected fish resulting in similar disease signs. Despite the virus going undetected for many years, these two EEDV episodes clearly demonstrate the continued presence of this deadly herpesvirus in the Great Lakes basin.

Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3)

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

Pathomorphology of experimental Streptococcus iniae infection in tilapia (Oreochromis niloticus)

In the present study, an experimental infection with Streptococcus iniae was conducted with tilapia (Oreochromis niloticus). The bacterial agent was injected to the fish by intraperitoneal inoculation at a dose of 1 × 103 cfu / mL. Grossly, haemorrhages on tail and pectoral fins, hyperaemic and prolapsed anus, darkening and bleeding in the gills and exophthalmos in the eyes were identified in fish. Multifocal whitish foci in the liver, spleen and kidneys was observed with ascites. Histopathological changes of tissues of tilapia were studied and evaluated. Gill tissues of infected fish showed intense lymphocyte and macrophage infiltrations. In addition, epithelial cell separation and lamellar fusion with edema, lamellar curling and aneurysms were detected in gill tissue. Sinusoidal dilatation, lymphocyte infiltrations, congestion and degeneration in liver hepatocyte cells have been identified. In kidney degeneration of glomerulus and tubule cells and dense lymphocyte infiltrations were observed. Dense bacterial clusters were observed in the sinusoids of the spleen, while lymphocytes and macrophage infiltrations were found in the heart and intestinal tissues.

Application of a monoclonal antibody specific for the ORF92 capsid protein of Cyprinid herpesvirus 2

Cyprinid herpesvirus 2 (CyHV-2) infection is a recently discovered viral disease of farmed silver crucian carp (Carassius auratus gibelio) in mainland China that has resulted in massive fish mortality and appears to be spreading cross the world. Various detection methods based on nucleic acids have been developed, but effective immunological diagnostic method has not been reported for CyHV-2. In this study, recombinant ORF92 protein was used as a capture antigen to identify cells and tissues infected with CyHV-2 by immunological methods. Firstly, the open reading frame of CyHV-2 ORF92 was cloned into the PGEX-4T-3 vector and expressed in Escherichia coli. Purified recombinant ORF92 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot-blot assay. The resulting mAb-1B7 was applied to detect CyHV-2 in infected Ryukin goldfish fin (RyuF-2) cells and fish tissues by western blotting and immunofluorescence assays. The monoclonal antibody could specifically identify CyHV-2 in infected RyuF-2 cells and gill, kidney, and spleen tissues, suggesting it could be applied for CyHV-2 detection in crucian carp.

Inclusion of cricket (Gryllus bimaculatus) meal in African catfish (Clarias gariepinus) feed influences disease resistance

In our present study, we observed the effect of cricket meal (CM) on disease resistance of African catfish. Fish were fed diets containing 350 g kg–1 and 400 g kg–1 of CM and 350 g kg–1 of fishmeal as control. The fish were divided into triplicates treatments of ten fish per replicate, weighed 22.5±0.6 g and fed with experimental diets for 40 days before being challenged against Aeromonas hydrophila. Relative percentage of survival (RPS) was recorded over 12 days post-challenge. White blood cell count, total protein, globulin and lysozyme showed significantly increasing levels in those fed with cricket meal diet compared to the control group. Mortalities at 12-day post-challenge significantly decreased to 30% (RPS: 66.7%) and 27% (RPS: 70%) for 35% and 40% CM respectively compared to 90% mortality in control group. Intestines and liver tissues of infected fish were dissected for pathogenic confirmation. The intestines of control diet showed the highest bacterial load (58.2×107 cfu g–1) compared to CM diets. The current study indicates that dietary CM could enhance the innate immune system and disease resistance of African catfish.