Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

The growth performance and parasite load of angelfish juveniles Pterophyllum scalare kept at different stocking densities in two rearing systems.

This study evaluated the growth performance and parasite load of angelfish juveniles Pterophyllum scalare kept at different stocking densities using two rearing systems. The experiment was conducted in a factorial design (4x2) with four stocking densities (0.1, 0.4, 0.7, and 1.0 g/L), two type of aquarium tanks (glass and ceramic aquariums), and four replicates. The experiment lasted 60 days using 148 juvenile fish (3.05 ± 0.09 g) randomly placed in 32 aquariums (50 L) equipped with filters and aeration. All fish received two meals a day ad libitum (8:00 and 16:00). Water quality parameters such as temperature, dissolved oxygen, pH, and total ammonia were measured. At the end of the experiment, all fish were measured and weighed to determine growth performance and then subjected to parasitological analysis. The data were analyzed with a two-way ANOVA with post-hoc Tukey test (p<0.05). No effects on growth performance at different stocking densities were observed. However, there was an increase in Capillaria pterophylli infestation in the high stocking density within ceramic aquariums. Thus, this study recommends the use of 1.0 g/L for the intensive aquaculture system of freshwater angelfish, and applying cleaning management to avoid parasite infestation, particularly in ceramic aquariums.

Structure-activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus.

BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host's immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure-activity relationships of its intramolecular disulphide bonds. RESULTS: The predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa. CONCLUSIONS: Our study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Novel insights into immune stress markers associated with myxosporeans gill infection in Nile tilapia (molecular and immunohistochemical studies).

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1ßeta (IL-1ß), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage.

Molecular characterization of lymphocystis disease virus in Indian glass fish: first report from the Andaman Islands.

Here, we report the first detection of lymphocystis disease virus (LCDV) in Indian glass fish in the Andaman Islands, India. Microscopic examination revealed the presence of whitish clusters of nodules on the fish's skin, fins, and eyes. The histopathology of the nodules revealed typical hypertrophied fibroblasts. Molecular characterization of the major capsid protein (MCP) gene of the virus showed a significant resemblance to known LCDV sequences from Korea and Iran, with 98.92% and 97.85% sequence identity, respectively. Phylogenetic analysis confirmed that the MCP gene sequence of the virus belonged to genotype V. This study represents the first documented case of LCDV in finfish from the Andaman Islands, emphasizing the necessity for continued monitoring and research on the health of aquatic species in this fragile ecosystem.

MOLECULAR PHYLOGENY OF THE LEECH GENUS PONTOBDELLA (HIRUDINIDA: PISCICOLIDAE) WITH NOTES ON PONTOBDELLA CALIFORNIANA AND PONTOBDELLA MACROTHELA.

Leech specimens of the genus Pontobdella (Hirudinida: Piscicolidae) were found off the coast of the state of Oaxaca (Pacific) as well as in Veracruz and Tabasco (Gulf of Mexico), Mexico. Based on the specimens collected in Oaxaca, a redescription of Pontobdella californiana is provided, with emphasis on the differences in the reproductive organs with the original description of the species. In addition, leech cocoons assigned to P. californiana were found attached to items hauled by gillnets and studied using scanning electron microscopy and molecular approaches. Samples of Pontobdella macrothela were found in both Pacific and Atlantic oceans, representing new geographic records. The phylogenetic position of P. californiana is investigated for the first time, and with the addition of Mexican samples of both species, the phylogenetic relationships within Pontobdella are reinvestigated. Parsimony and maximum-likelihood phylogenetic analysis were based on mitochondrial (cytochrome oxidase subunit I [COI] and 12S rRNA) and nuclear (18S rRNA and 28S rRNA) DNA sequences. Based on our results, we confirm the monophyly of Pontobdella and the pantropical distribution of P. macrothela with a new record in the Tropical Eastern Pacific.

Microbe transmission from pet shop to lab-reared zebrafish reveals a pathogenic birnavirus.

Zebrafish are popular research organisms selected for laboratory use due in part to widespread availability from the pet trade. Many contemporary colonies of laboratory zebrafish are maintained in aquaculture facilities that monitor and aim to curb infections that can negatively affect colony health and confound experiments. The impact of laboratory control on the microbial constituents associated with zebrafish in research environments compared to the pet trade are unclear. Diseases of unknown causes are common in both environments. We conducted a metatranscriptomic survey to broadly compare the zebrafish-associated microbes in pet trade and laboratory environments. We detected many microbes in animals from the pet trade that were not found in laboratory animals. Cohousing experiments revealed several transmissible microbes including a newly described non-enveloped, double-stranded RNA virus in the Birnaviridae family we name Rocky Mountain birnavirus (RMBV). Infections were detected in asymptomatic animals from the pet trade, but when transmitted to laboratory animals RMBV was associated with pronounced antiviral responses and hemorrhagic disease. These experiments highlight the pet trade as a distinct source of diverse microbes that associate with zebrafish and establish a paradigm for the discovery of newly described pathogenic viruses and other infectious microbes that can be developed for study in the laboratory.

Genetic relatedness and diversity of Capillaria species infecting bayad (Bagrus bajad) in upper Egypt.

BACKGROUND: This study investigates the genetic characteristics of Capillaria isolates from the infected fish, Bagrus bajad, and their relation to human Capillaria philippinensis using Random Amplified Polymorphic DNA (RAPD-PCR) analysis. Fifteen fish Capillaria were isolated and compared to identified human C. philippinensis using six primers: M-are, M-1, G-7, G-11, G-15, and G-18. RESULTS: All six primers successfully amplified DNA, highlighting their efficacy in distinguishing between human and fish Capillaria isolates. The analysis revealed distinctive banding patterns between fish and human isolates, with variations in size and number of DNA fragments. Additionally, genetic similarity analysis showed intriguing patterns of relatedness, with certain pairs exhibiting high similarity percentages. Comparative assessment of RAPD polymorphism demonstrated consistent findings of 100% polymorphism across all primers. The Unweighted Pair Group Method with Arithmetic Mean Algorithm (UPGMA) evaluated the closest relationship between human and fish isolates. These results underscore the utility of RAPD analysis in delineating the genetic diversity among Capillaria isolates from different hosts. CONCLUSION: Overall, this study contributes to our understanding of the genetic variability and relatedness among Capillaria isolates, shedding light on their evolutionary dynamics and zoonotic potential.

Pathological caudal skeleton of an ichthyodectiform fish from the Upper Cretaceous Niobrara Formation of western Kansas, USA.

A series of 12 contiguous caudal vertebrae of an ichthyodectiform fish from the Smoky Hill Chalk Member of the Niobrara Formation is described. The vertebral centra exhibit extensive overgrowth of pathological bone and there is additional pathological bone within the centra and intervertebral spaces, which together resulted in the coossification of most centra. The extent of the pathology is greatest on preural vertebrae 1-3 and decreases anteriorly, which suggests that the pathology began posteriorly and progressed anteriorly. In addition to the pathological overgrowth on bones, the specimen preserves features interpreted as calcified and/or ossified soft tissues associated with the neural and haemal canals. The pathologies are unlike previously described examples of bony pathologies in fish, and it is suggested that they resulted from combined bacterial and fungal infections. As the pathologies developed, they would have adversely impacted the fish's swimming and feeding abilities, and presumably eventually led to the fish's death.

Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Associating with kin selects for disease resistance and against tolerance.

Behavioural and physiological resistance are key to slowing epidemic spread. We explore the evolutionary and epidemic consequences of their different costs for the evolution of tolerance that trades off with resistance. Behavioural resistance affects social cohesion, with associated group-level costs, while the cost of physiological resistance accrues only to the individual. Further, resistance, and the associated reduction in transmission, benefit susceptible hosts directly, whereas infected hosts only benefit indirectly, by reducing transmission to kin. We therefore model the coevolution of transmission-reducing resistance expressed in susceptible hosts with resistance expressed in infected hosts, as a function of kin association, and analyse the effect on population-level outcomes. Using parameter values for guppies, Poecilia reticulata, and their gyrodactylid parasites, we find that: (1) either susceptible or infected hosts should invest heavily in resistance, but not both; (2) kin association drives investment in physiological resistance more strongly than in behavioural resistance; and (3) even weak levels of kin association can favour altruistic infected hosts that invest heavily in resistance (versus selfish tolerance), eliminating parasites. Overall, our finding that weak kin association affects the coevolution of infected and susceptible investment in both behavioural and physiological resistance suggests that kin selection may affect disease dynamics across systems.

Ornamental fish mortality reveals an old parasite introduction: A case study of Koi carp and fish louse.

Koi carp are globally known for their colors and cultural significance. The introduction of these fish to new environments poses a threat to local biodiversity, in addition to releasing parasites, such as argulid ectoparasites. This study presents a record of Argulus japonicus infecting carp in an artificial lake in Southern Brazil using morphological and molecular methods, with a 100% prevalence (n = 3) and a mean intensity of 21.6 parasites per host, distributed over the body surface. The invasion history of hosts in the study locality indicates that the introduction of A. japonicus occurred decades before its first formal record in Brazil.

Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

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.

Phylogenomic Characterization of Ranavirus Isolated from Wild Smallmouth Bass (Micropterus dolomieu).

In September 2021, 14 smallmouth bass (SMB; Micropterus dolomieu) with skin lesions were collected from Green Bay waters of Lake Michigan and submitted for diagnostic evaluation. All the skin samples tested positive for largemouth bass virus (LMBV) by conventional PCR. The complete genome of the LMBV (99,328 bp) isolated from a homogenized skin sample was determined using an Illumina MiSeq sequencer. A maximum likelihood (ML) phylogenetic analysis based on the 21 core iridovirus genes supported the LMBV isolated from SMB (LMBV-WVL21117) as a member of the species Santee-Cooper ranavirus. Pairwise nucleotide comparison of the major capsid protein (MCP) gene showed that LMBV-WVL21117 is identical to other LMBV reported from the United States and nearly identical to doctor fish virus and guppy virus 6 (99.2%) from Southeast Asia, as well as LMBV isolates from China and Thailand (99.1%). In addition, ML phylogenetic analysis based on the MCP gene suggests three genotypes of LMBV separated by region: genotype one from the United States, genotype two from Southeast Asia, and genotype three from China and Thailand. Additional research is needed to understand the prevalence and genetic diversity of LMBV strains circulating in wild and managed fish populations from different regions.

The myxozoans Myxobolus cerebralis and Tetracapsuloides bryosalmonae modulate rainbow trout immune responses: quantitative shotgun proteomics at the portals of entry after single and co-infections.

Introduction: Little is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae. Methods: One group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection. Results: In the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated. Discussion: The results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.