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Simple Summary: The aquaculture industry is a major contributor to the supply of animal proteins. Parasitic diseases like the white sport disease caused by Ichthyophthirius multifiliis (ich) is a significant threat to fish production and welfare. Previous studies explored the fish immune response against ich. However, the effects of farming procedures such as fish handling were not studied in the context of white spot disease. Our studies investigated the impact of handling procedures (air exposure, chasing, transfer) on the fish's ability to fight the parasite compared to non-handled fish. The results indicated that the stress caused by handling did not change the amount of parasite detected in the fish, in the tank's water, or the fish's mortality. Nonetheless, several parameters of the fish immune response were significantly altered due to the handling procedures. Overall, our results suggested that the handling procedures investigated did not impair the fish's ability to fight the parasite. Farmed fish are regularly subjected to various stressors due to farming practices, and their effect in the context of a disease outbreak is uncertain. This research evaluated the effects of unpredictable repeated stress in rainbow trout challenged with the ciliate Ichthyophthirius multifiliis, known to cause white spot disease in freshwater fish. Before and after the pathogen exposure, fish were handled with a random rotation of three procedures. At 7 days post-infection (dpi), the parasite burden was evaluated in fish and in the tank's water, and the local and systemic immune responses were investigated in the gill and spleen, respectively. The fish mortality was recorded until 12 dpi, when all the fish from the infected groups died. There was no statistical difference in parasite burden (fish and tank's water) and infection severity between the two infected fish groups. The immune gene expression analysis suggested a differential immune response between the gill and the spleen. In gills, a T helper cell type 2 immune response was initiated, whereas in spleen, a T helper cell type 1 immune response was observed. The stress has induced mainly upregulations of immune genes in the gill (cat-1, hep, il-10) and downregulations in the spleen (il-2, il-4/13a, il-8). Our results suggested that the unpredictable repeated stress protocol employed did not impair the fish immune system. [ABSTRACT FROM AUTHOR]

The fastest-growing animal food-producing agricultural sector in the world is aquaculture, which accounts for almost half of the world's food fish production. However, the expansion of high-density fish populations also brings forth a challenge-the rapid transmission and spread of infectious agents including several viral, bacterial, and parasitic diseases. Expansion and successful development of sustainable aquaculture practice and increasing production largely depends upon the prevention and control of outbreaks of several emerging and re-emerging infectious diseases which can result in economic loss, food safety hazards, and environmental hazards. Vaccination strategies have become highly effective and economical in protecting the health of fish and other aquaculture organisms from various infectious diseases such as edwardsiellosis, motile aeromonas septicemia (MAS), Tilapia Lake Virus (TiLV) disease, infectious salmon anemia (ISA), vibriosis, and white spot disease etc. An ideal vaccine is expected to be safe, effective, economical, and easily administered. Most of the available fish vaccines are empirically designed vaccines based on inactivated or live attenuated bacterin vaccines. Novel advances in the fields of immunology, biotechnology, and molecular biology have led to the development in designing novel and effective fish vaccines and the improvement of the existing vaccines to provide sufficient immune protection against diseases. This review investigates the currently available fish vaccines for use in finfish aquaculture against different infectious diseases different mode of vaccine administration with addressing pros and cons in detail. The information was collected from different secondary sources, and then compiled systematically. [ABSTRACT FROM AUTHOR]

The movement of live aquatic animals crossing international borders is a regular activity for economic, social, and public resource development. A survey was carried out to investigate the prevalence of parasites translocated with imported ornamental fishes. Exotic diseases from imported fish have a potential hazard of biosecurity. A sample of 138 imported ornamental fish originating from Thailand and Sri Lanka were collected from pet shops around Muscat governorate and examined for ectoparasites. Parasitological examination was based on a visual macro and microscopic examination of fish's exterior. The findings of the current survey of imported ornamental fish revealed that 50.72% of the imported ornamental fish were infected by the one or more of ectoparasites species including Ichthyophthirius multifiliis, Dactylogyrus sp., Gyrodactylus sp., digenean metacercaria and Trichodina sp. Parasitic infection of the gills (55.795) was higher than the skin (42.75%). The most prevalent parasite in skin infection was Gyrodactylus sp. (14.49%) but this was absent from the gills which was dominated by digenean metacercaria (26.08%). The existence of such parasitic organisms could pose a threat to native fish populations and the aquaculture sector. It is recommended that a risk analysis method based on the detection and assessment of fish diseases found in live ornamental fish be established and used. Also, exotic pathogenic parasites, as well as their hosts, must be closely monitored to reduce the likelihood of infections being introduced into new areas. [ABSTRACT FROM AUTHOR]

Ichthyophthirius multifiliis , the causative agent of white spot disease, is a ciliated protozoan parasite that infects freshwater fish and induces high mortality. Outbreaks occur both in natural and production sites. The aim of the present study was to describe the lesions caused by chronic infection by I. multifiliis in goldfish (Carassius auratus) from an ornamental fish farm, highlighting important ultrastructural aspects of this protozoan. Damaged skin and gills, collected from fish with white or ulcerative skin lesions, were routinely processed for histological analysis and transmission electron microscopy. The parasitic forms present in the skin were associated with an inflammatory infiltrate consisting of macrophages, lymphocytes and other polymorphonuclear cells. The lesions associated with the presence of the parasite were organized in the form of granulomas, with macrophages in the layers closest to the parasites. A trophont-thickened membrane and induction of granulomatous inflammation were identified in this study as mechanisms for evasion of the immune response. We concluded that the presence of I. multifiliis trophonts resulted in the formation of granulomatous inflammation, whether associated or not with pathogen lysis, suggesting that the parasite can use an inflammatory response to evade the immune response. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate chemical characteristics and biological activity of progenies from the first cycle of recurrent selection of Varronia curassavica in order to identify promising progenies for the next stages of the breeding program. The seeds were collected from the parental accession VCUR-503 (chemotype E-caryophyllene/viridiflorol) and subsequently sown in seedling containers and transplanted to the field. The essential oil was extracted 300 days after transplanting, and the levels of E-caryophyllene and viridiflorol were analyzed, as well as their activity against the protozoan Ichthyophthirius multifiliis. Application of the recurrent selection method resulted in increased levels of both compounds. The levels of E-caryophyllene (0.00-23.61%) and viridiflorol (0.00-64.74%) exhibited significant variation within the population under study. The essential oil rich in viridiflorol from progenies led to high mortality (72%) of the protozoan. Promising progenies for the second cycle of recurrent selection were identified based on the variables analyzed. [ABSTRACT FROM AUTHOR]

Farmed fish are regularly subjected to various stressors due to farming practices, and their effect in the context of a disease outbreak is uncertain. This research evaluated the effects of unpredictable repeated stress in rainbow trout challenged with the ciliate Ichthyophthirius multifiliis, known to cause white spot disease in freshwater fish. Before and after the pathogen exposure, fish were handled with a random rotation of three procedures. At 7 days post-infection (dpi), the parasite burden was evaluated in fish and in the tank’s water, and the local and systemic immune responses were investigated in the gill and spleen, respectively. The fish mortality was recorded until 12 dpi, when all the fish from the infected groups died. There was no statistical difference in parasite burden (fish and tank’s water) and infection severity between the two infected fish groups. The immune gene expression analysis suggested a differential immune response between the gill and the spleen. In gills, a T helper cell type 2 immune response was initiated, whereas in spleen, a T helper cell type 1 immune response was observed. The stress has induced mainly upregulations of immune genes in the gill (cat-1, hep, il-10) and downregulations in the spleen (il-2, il-4/13a, il-8). Our results suggested that the unpredictable repeated stress protocol employed did not impair the fish immune system.

To investigate the relationship between the polymorphism of the nlrc3 gene in grass carp （Ctenopharyngodon idella） and resistance to Ichthyophthirius multifiliis, susceptible and resistant groups were selected from grass carp populations infected with I. multifiliis. The single nucleotide polymorphism （SNP） sites of the nlrc3 gene in the two groups were analyzed and compared. High-resolution melting （HRM） and Sanger sequencing techniques were used to validate the genotypes of the SNP sites, and the obtained SNP sites were analyzed for their association with the disease resistance traits in grass carp. The results showed that four SNPs sites were identified from the nlrc3 gene in grass carp, and three SNP sites （SNP1, SNP3, and SNP4） were successfully genotyped. Among them, SNP1 and SNP3 exhibited two genotypes each （AA and AG for SNP1, AA and GG for SNP3） and were significantly associated with resistance to I. multifiliis. The analysis of haplotype combinations and their correlation with disease resistance revealed that the genotype composed of SNP1 AG and SNP3 AA （D1） showed 100% disease resistance, while the genotype composed of SNP1 AG and SNP3 GG （D2） showed 95% disease resistance, both representing advantageous genotypes for disease resistance. The study demonstrated the existence of nlrc3 gene polymorphism in the entire population of I. multifiliis resistant grass carp, and different genotypes were significantly correlated with the ability to resist I. multifiliis infection. Among them, the D1 genotype with SNP1 AG and SNP3 AA combination exhibited the strongest resistance. These findings provide important theoretical and technical support for further breeding and prevention of I. multifiliis disease in grass carp. [ABSTRACT FROM AUTHOR]

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)–specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non–M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA–associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function. [ABSTRACT FROM AUTHOR]

This study aimed to examine the prevalence of Ichthyophthirius multifiliis in fish inhabiting natural water bodies in the Lhasa and Nagqu regions of Tibet in September 2020 and August 2021. The results showed that Schizopygopsis selincuoensis had the highest prevalence of I. multifiliis at 33.73% (56/166), followed by Triplophysa tibetana at 30.00% (6/20), Triplophysa brevicauda at 27.91% (12/43) and Schizopygopsis thermalis at 23.66% (31/131). No infection with I. multifiliis was observed in exotic fish species. In addition, the prevalence of I. multifiliis in Boqu Zangbo (river), Selincuo Lake and Cuona Lake in the Nagqu region was found to be significantly higher than that in Lalu Wetland and Chabalang Wetland in the Lhasa region (P

Pathogen surveillance must be part of any population supplementation or reintroduction program for the conservation of threatened and endangered species. The unintended transmission of pathogens can have devastating effects on these already at-risk populations or the natural ecosystem at large. In the San Francisco Estuary (estuary), abundance of the endemic Delta Smelt (Hypomesus transpacificus) has declined to the point where regulatory managers are preparing to augment the wild population using fish propagated in a hatchery to prevent species extinction. Although disease is not an overt cause of population decline, comprehensive pathogen presence and prevalence data are lacking. Here, we performed a pilot study that applied molecular assays originally developed in salmonids to assess the presence of a wide variety of pathogens in the gill tissue of cultured and wild Delta Smelt—as well as cultured fish—deployed in enclosures in the estuary. We found the assays to be highly sensitive, and observed positive detections of a single pathogen, Ichthyophthirius multifiliis, in 13% of cultured Delta Smelt. We also detected ten other pathogens at very low levels in cultured, enclosure-deployed, and wild Delta Smelt that likely represent the ambient pathogen composition in the estuary (as opposed to actual infection). Our results corroborate previous work that cultured Delta Smelt do not appear to present a high risk for pathogen transmission during population supplementation or reintroduction. However, the molecular pathogen screening assays tested here have great utility as an early warning system indicator of when further diagnostic testing might be necessary to limit the extent and frequency of disease outbreaks; their utility will be further increased once they are customized for Delta Smelt.

Abstract Background The beneficial effects of Bacillus subtilis on growth, immune response, and disease resistance against various diseases in different fish species have been proved. However, there are no data concerning this probiotic effect on skin mucosal immunity in fish infected with Ichthyophthirius multifiliis (Ich). Ich has a high mortality rate in both edible and ornamental fish and consequently is concerned with heavy economic losses. Objectives Thus, we assessed the efficacy of live and heat‐killed B. subtilis on skin immunity and histopathology in goldfish (Carassius auratus) infected with Ich. Methods Goldfish (144 fish, 2.38 g average weight) were stocked in nine glass tanks each in three replicates. Fish were fed 109 CFU g−1 live or heat‐killed B. subtilis for 80 days. Results Probiotic administration in both viable and non‐viable forms could enhance the growth performance in goldfish. Probiotic therapy also reduced the density of the parasite and histopathological level on skin and gill tissues of the treated fish. Real‐time polymerase chain reaction analysis showed a higher expression of lysozyme and tumour necrosis factor‐α in the treated groups compared to the control group. Conclusions These data demonstrated the beneficial effect of B. subtilis as probiotic and paraprobiotic on growth performance and disease resistance to Ich infestation in goldfish.

Abstract Background : Parasitic infestations are one of the most significant problems in global aquaculture. In addition to direct economic losses due to significant mortalities, parasites may have a significant negative impact on the fish's behaviour, energetic demands, trophic position, interspecific competition, growth rate, and reproductive function. Objectives : We conducted the present study to evaluate the infection status of parasitic pathogens in freshwater sutchi catfish (Pangasianodon hypophthalmus) and silver dollar (Metynnis hypsauchen) farmed in Alborz province of Iran. Methods : Between January and February 2021, 140 ornamental fish, including 70 sutchi catfish (P. hypophthalmus) and 70 silver dollar (M. hypsauchen), were delivered from various ornamental fish farms for parasitological analysis. Thorough macroscopic and microscopic examinations were performed to detect parasitic infections in delivered freshwater ornamental fish. Results : A total of six parasite species, including five protozoan species (Nyctotherus piscicola, Trichodina heterodentata, Ichthyophthirius multifiliis, Protoopalina sp., and Hexamita sp.) and one monogenean species (Ancyrocephalus sp.), were identified in the examined fish. The overall prevalence of the recovered parasites was 46.43% (65/140) of the fish. Conclusions : In the current study, several parasites, including Hexamita sp., Ancyrocephalus sp., Protoopalina sp., and N. piscicola, were identified as the first records of the sutchi catfish (P. hypophthalmus) and/or silver dollar (M. hypsauchen), and these two fish species are known as new hosts for the isolated parasites in ornamental fish farms in Iran. Assessing the parasitic fauna of ornamental fish is therefore critical for preventing parasite introduction into adjacent provinces as well as neighbouring countries and promoting fish health.

Reviewing literature concerning the occurrence of the causative agent of the white spot disease; Ichthyophthirius multifiliis as well as unidentified Ichthyophthirius species, infecting the skin, fins and gills of fishes in Iraq until the end of 2022, showed that 40 fish species are so far known as hosts for I. multifiliis and five fish species for Ichthyophthirius spp. The infected fishes were distributed in different localities of rivers of Iraq, their branches, some lakes, depressions, marshes, drainage networks, fish hatcheries and fish farms in addition to some fishes from fish markets and fish aquaria. These fishes included 24 endemic freshwater fishes, seven exotic freshwater fishes, one marine fish and seven aquarium fishes in addition to one unspecified aquarium fish species.

ABSTRACTIchthyophthirius multifiliis is an obligate parasitic ciliate that causes severe economic damage in aquaculture. The parasite contains numerous extrusive organelles (extrusomes) that assist in its pathogenesis and reproduction. However, the structure of these extrusomes and the molecular profiles involved in exocytosis remain unclear. In the present study, through comparative ultrastructural observations across the life cycle of I. multifiliis, we demonstrated that all three of its life stages (theront, trophont, and tomont) exhibited an abundance of extrusomes. In addition, two different types of extrusomes were identified according to their unique structures. Type I extrusomes (mucocysts) are crystalline, oval-shaped, 0.7–1.4 × 0.6–1.1 μm, and distributed as “rosettes” below the trophont membrane. Type II extrusomes, 2.0–3.0 × 0.2–0.3 μm, are rod-shaped with tubular cores and identified as toxicysts, the aggregation of which in the anterior part of the theront and cortex of the trophont revealed their potential roles in I. multifiliis invasion. This was confirmed by our transcriptome investigations of the three stages of I. multifiliis, which revealed that a set of genes involved in proteolysis and DNA/protein biogenesis was highly expressed in the theront and trophont. Furthermore, to map the molecular mechanisms of extrusome release, we characterized 25 Rab family genes in I. multifiliis and determined their expression profiles across the life cycle, reflecting the distribution patterns of the two extrusomes. Collectively, our data revealed that a highly developed extrusome system could play a potential role in the virulence of I. multifiliis, which facilitates a better understanding of the parasite’s development.

Research on the ichthyoparasitic fauna of fish from the Cyprinidae family, taken from the ten dam lakes in the lower sector of Jiu, lakes that are part of the “Preajba-Făcăi Lacustrine Complex” site, revealed the following aspects: 9 species of parasites were identified: 2 cilophores (Chilodonella cyprini, Ichthyophthirius multifiliis), 1 oomycete Saprolegnia sp., 1 monogenous Dactylogyrus sp., 1 myxosporidia Myxobolus sp., 1 acanthocephalus Pomphorhynchus laevis, 2 crustaceans (Lernaea cyprinacea, Argulus foliaceus) and 1 annelid Piscicola geometra, in five of the seven host species studied. (6) species of parasites have been identified in crucian carp (Saprolegnia sp., Ichthyophthirius multifiliis, Myxobolus sp., Dactylogyrus sp., Lernaea cyprinacea, Piscicola geometra), in carp (5) (Chilodonella cyprini, Dactylogyrus sp., Pomphorhynchus laevis, Lernaea cyprinacea, Argulus foliaceus), bream (1) (Saprolegnia parasitica), bream (1) (Myxobolus sp.) and spotted murgoi (1) (Saprolegnia parasitica), followed by slugs and tomatoes in which no parasites were identified. Of the 9 species of parasites identified, 7 species are ectoparasitic. Clinical signs of ectoparasitosis have been identified in both ichthyophthyrosis of the crucian carp where hypoxia reactions and the presence of white punctate colonies have been found, as well as in the case of saprolegniosis due to the appearance of fine wool hyphae and scoliosis, argulosis and piscicolosis highlighted in crucian carp and carp by haemorrhagic wounds and obvious ulcerations but also by the presence of parasites. [ABSTRACT FROM AUTHOR]

The ciliate protozoan Ichthyophthirius multifiliis is an essential parasite causing white spot disease in grass carp, leading to significant economic losses. Understanding the molecular basis of grass carp's response to I. multifiliis has important scientific and environmental values. The transcriptional network analysis offers a valuable strategy to decipher the changes in gene expression in grass carp infected with I. multifiliis. Our goal was to screen the genes and pathways involved in resistance to I. multifiliis in grass carp. The different traits exhibited by grass carp infected with I. multifiliis may be caused by the differences in gene expression among grass carp individuals. Herein, to reveal those resistance-associated genes against I. multifiliis infection, we performed RNA sequencing using weighted gene co-expression network analysis (WGCNA). The biological function analysis and hub gene annotation for highly relevant modules revealed that different pathogen recognition and clearance responses resulted in different resistance to I. multifiliis infection. Furthermore, gene enrichment analysis revealed that I. multifiliis invasion in the disease-resistant group mainly activated immune pathways, including scavenger receptor activity and kappa B kinase/NF-kappa B signaling. By the annotation of the highly correlated module of the hub gene, we revealed that the apoptosis and ribosome biogenesis-related genes were enriched in the disease-resistant grass carp. The results of the dark grey module showed that several genes were mainly enriched in the two-component system (ko02020) and steroid biosynthesis (ko00100), suggesting that they are resistance-associated and energy metabolism-associated genes. In the disease resistance group, hub genes mainly included Nlrc3, fos, AAP8, HAP2, HAX, cho2, and zgc:113,036. This study revealed the gene network associated with disease resistance after I. multifiliis infection. The disease resistance–related pathways and central genes identified in this study are candidate references for breeders breeding disease-resistant. The results of this study may also provide some references for the development of drugs to antagonize I. multifiliis infection. [ABSTRACT FROM AUTHOR]

Snakehead, Channa striata, has a long history of invasion and associated negative impacts on the biodiversity of native aquatic organisms in the Mekong Delta. However, internal white spot disease caused by Aeromonas schubertii has a massive effect on the production of snakehead fish but also can spread to wild populations and to consumers. The aim of the study was to investigate the prevalence of multi‐antibiotic resistance (MAR) of A. schubertii isolated from snakehead fish cultured in several Mekong Delta fish farms, as well as its potential to spread throughout the infection season. Based on morphological and biochemical tests, as well as phylogenetic analysis of three housekeeping genes (16S rRNA, gyrB, and rpoD), isolates recovered from white nodules were strongly identified as A. schubertii. The antibiotic susceptibility testing revealed that all of the isolates (n = 25) were phenotypically multidrug‐resistant, with resistance to erythromycin (84%), rifampicin (84%), flumequine (88%), amoxicillin (96%), ciprofloxacin (92%), and florfenicol (92%). The multiple antibiotic resistance indices ranged from 0.33 to 0.92, with A. schubertii isolate As28 being resistant against 11 out of the 12 tested antibiotics. In particular, 15 MAR phenotypes (MARPs) were found in 25 isolates with 8 isolates (32%) commonly displaying MAR phenotypes against patterns of Ery–Rif–Flq–Aml–Cip–Flo, and four isolates (16%) expressing patterns of Ery–Rif–Nov–Flq–Aml–Cip–Flo. A hierarchical clustering analysis of their antibiotic‐resistant bacteria may indicate cross‐invasion between ponds/fish farms and its ability to survive in aquatic environments for long periods (at least 3 months). Our results suggest that the diversity of MARPs bacteria in snakehead represents a potential hazard to modifying the composition of native aquatic organisms, especially to consumers. To successfully manage C. striata in the future, several eco‐friendly techniques should be used to restrict the spread of A. schubertii inside the invaded habitat of the Mekong Delta river. [ABSTRACT FROM AUTHOR]

The naked characin (Gymnocharacinus bergii) is the only endemic fish of the arid Patagonia of Argentina, living only in the thermal headwaters of the Valcheta Stream in the Somuncura Plateau. This species is listed as Critically Endangered on the IUCN Red List.In April 2018, a massive mortality event was observed in the species. Parasitological studies identified the cause as an epidemic of white spot disease, caused by the parasite Ichthyophthirius multifiliis (Ich).Ich is a common disease in aquaculture settings, but it has also been reported as an epizootic in wild fish populations across tropical and temperate regions worldwide. This occurrence is associated with the introduction of fish into non‐native environments.This article presents the first recorded instance of a mass mortality event and Ich outbreak in the naked characin. It includes a mapping of the disease in the headwaters of the stream, an estimation of the impact on the species' population size, and an analysis of potential causes associated with this epidemic.The results indicate that the epizootic produced mortality of 82% of the global population of the naked characin and was linked to the presence and high abundances of the invasive fish species Uruguay tetra (Cheirodon interruptus). The disease occurred in stream sectors where both characid species coexist, particularly in areas with the highest water temperatures.The Ich outbreak represents the most recent and severe threat recorded for the Critically Endangered naked characin. Implementing control measures against the invasive Uruguay tetra is imperative to prevent future outbreaks that could push the species to the brink of extinction in the near future.This study provides crucial information for assessing current management interventions and developing new strategies within the framework of the action plan for naked characin conservation. [ABSTRACT FROM AUTHOR]

In this study, the effect of two plant extracts, garlic and thyme, on the theront and tomont stages of the parasitic protozoa Ichthyophthirius multifiliis was evaluated. For this purpose, first, hydroalcoholic extracts of these plants were prepared. In the next step, both stages of the parasite were exposed. The parasite survival rate at each concentration, every 3 hours and for 12 hours were recorded and LD50 of each extract in this time period was calculated using Probite method. Malachite green was used as a positive control. The results showed that the LD50 of garlic and thyme on tomont were 1 and 2.5 mg/L, respectively. While this amount was 0.24 mg/L for malachite green. LD50 of garlic and thyme extracts on theront were 1.66 and 0.57 mg/L, respectively, and in the case of malachite green was 0.121 mg/L. According to the results, the antiparasitic effect of garlic and thyme extracts is insignificant compared to the effects of malachite green and it is not possible to replace malachite green with these extracts. Although these extracts can be used as adjunctive therapy along with chemical treatments. [ABSTRACT FROM AUTHOR]

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation. [ABSTRACT FROM AUTHOR]

The parasitic ciliate Ichthyophthirius multifiliis infests all freshwater fish species and can cause severe economic losses in aquaculture. The present study aims to evaluate the antiparasitic activity of onion (Allium cepa), sage (Salvia officinalis), menthe (Menthe spicata), garlic (Allium sativum), lavender (Lavandula officinalis) and oregano (Origanum onites) essential oils against I. multifiliis trophonts. Gas Chromatography-Mass Spectrometry (GC-MS) analyses determined these essential oils' chemical components. The major components were dipropyl disulfide (46.15%) in onion, alpha-thujone (46.80%) in sage, carvone (48.00%) in menthe, diallyl disulfide (32.70%) in garlic, 1,8-cineole (32.65%) in lavender; carvacrol (73.00%) in oregano. I. multifilis trophonts were isolated from infected European catfish's skin and gill arches (Silurus glanis). Then in vitro, antiparasitic assays obtained those essential oils of sage (0.50 mL L-1), lavender (0.25-, and 0.50-mL L-1), and oregano (0.1, 0.25- and 0.50-mL L-1) have been found as 100% effective against trophonts at the different concentration of after 60 min of exposure, respectively (p<0.05). In addition, onion, menthe, and garlic essential oil had effectiveness with 94%, 94%, and 92% mortalities after 60 min of exposure, respectively. These results suggested that sage, lavender, and oregano essential oils can be eco-friendly in controlling I. multifiliis trophonts. [ABSTRACT FROM AUTHOR]

Reviewing literature concerning the occurrence of the causative agent of the white spot disease; Ichthyophthirius multifiliis as well as unidentified Ichthyophthirius species, infecting the skin, fins and gills of fishes in Iraq until the end of 2022, showed that 40 fish species are so far known as hosts for I. multifiliis and five fish species for Ichthyophthirius spp. The infected fishes were distributed in different localities of rivers of Iraq, their branches, some lakes, depressions, marshes, drainage networks, fish hatcheries and fish farms in addition to some fishes from fish markets and fish aquaria. These fishes included 24 endemic freshwater fishes, seven exotic freshwater fishes, one marine fish and seven aquarium fishes in addition to one unspecified aquarium fish species. [ABSTRACT FROM AUTHOR]

Background: The beneficial effects of Bacillus subtilis on growth, immune response, and disease resistance against various diseases in different fish species have been proved. However, there are no data concerning this probiotic effect on skin mucosal immunity in fish infected with Ichthyophthirius multifiliis (Ich). Ich has a high mortality rate in both edible and ornamental fish and consequently is concerned with heavy economic losses. Objectives: Thus, we assessed the efficacy of live and heat‐killed B. subtilis on skin immunity and histopathology in goldfish (Carassius auratus) infected with Ich. Methods: Goldfish (144 fish, 2.38 g average weight) were stocked in nine glass tanks each in three replicates. Fish were fed 109 CFU g−1 live or heat‐killed B. subtilis for 80 days. Results: Probiotic administration in both viable and non‐viable forms could enhance the growth performance in goldfish. Probiotic therapy also reduced the density of the parasite and histopathological level on skin and gill tissues of the treated fish. Real‐time polymerase chain reaction analysis showed a higher expression of lysozyme and tumour necrosis factor‐α in the treated groups compared to the control group. Conclusions: These data demonstrated the beneficial effect of B. subtilis as probiotic and paraprobiotic on growth performance and disease resistance to Ich infestation in goldfish. [ABSTRACT FROM AUTHOR]

Background: : Parasitic infestations are one of the most significant problems in global aquaculture. In addition to direct economic losses due to significant mortalities, parasites may have a significant negative impact on the fish's behaviour, energetic demands, trophic position, interspecific competition, growth rate, and reproductive function. Objectives: : We conducted the present study to evaluate the infection status of parasitic pathogens in freshwater sutchi catfish (Pangasianodon hypophthalmus) and silver dollar (Metynnis hypsauchen) farmed in Alborz province of Iran. Methods: : Between January and February 2021, 140 ornamental fish, including 70 sutchi catfish (P. hypophthalmus) and 70 silver dollar (M. hypsauchen), were delivered from various ornamental fish farms for parasitological analysis. Thorough macroscopic and microscopic examinations were performed to detect parasitic infections in delivered freshwater ornamental fish. Results: : A total of six parasite species, including five protozoan species (Nyctotherus piscicola, Trichodina heterodentata, Ichthyophthirius multifiliis, Protoopalina sp., and Hexamita sp.) and one monogenean species (Ancyrocephalus sp.), were identified in the examined fish. The overall prevalence of the recovered parasites was 46.43% (65/140) of the fish. Conclusions: : In the current study, several parasites, including Hexamita sp., Ancyrocephalus sp., Protoopalina sp., and N. piscicola, were identified as the first records of the sutchi catfish (P. hypophthalmus) and/or silver dollar (M. hypsauchen), and these two fish species are known as new hosts for the isolated parasites in ornamental fish farms in Iran. Assessing the parasitic fauna of ornamental fish is therefore critical for preventing parasite introduction into adjacent provinces as well as neighbouring countries and promoting fish health. [ABSTRACT FROM AUTHOR]

White spot disease, caused by the parasitic ciliate Ichthyophthirius multifiliis, is a significant threat to the freshwater fish farming industry worldwide, resulting in massive mortality and economic losses. Eliminating the free-swimming theronts from the culture environment is considered crucial for the control of I. multifiliis infection. It is well-documented that planktonic ciliates are valuable food resources for macro-zooplankton in aquatic ecosystems. In this study, we developed a fluorescence labeling method for alive theronts and found that cyclopoid copepods Thermocyclops taihokuensis, Mesocyclops spp., Macrocyclops sp., and Paracyclopina sp. present predation on the theronts in co-culture experiments. Laboratory challenge tests further confirmed that the presence of zooplankton in the culture water body significantly reduced the infection of I. multifiliis in goldfish (p < 0.01). Results from this study revealed that cyclopoid copepods have the potential to be used as biological control agents against white spot disease in aquaculture. [ABSTRACT FROM AUTHOR]

Ichthyophthiriasis, caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich), is considered one of the most harmful diseases affecting freshwater fish globally. It can cause mass mortalities of fish in intensive farming systems. In such systems, it is thus necessary to detect and quantify the number of Ich in the water so that control measures can be implemented before Ichthyophthiriasis breaks out. In recent years, molecular diagnostic methods have become increasingly important in aquaculture. Real‐time quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) have become robust assays for detecting pathogens. In this study, a set of specific primers and a TaqMan‐minor groove binder probe targeting the small‐subunit rDNA (SSU rDNA) of Ich were developed. They were used in qPCR and ddPCR assays to compare the performance of these two different methods in quantitatively detecting Ich. After optimizing the reaction conditions, both qPCR and ddPCR assays were found to have high linearity and quantitative correlations for standard plasmid DNA. When used for the detection of Ich eDNA in water samples, the qPCR assay had a wider detection range, making it a suitable method to screen for the prevalence of Ichthyophthiriasis. However, the ddPCR approach had higher sensitivity, which would help provide advance notice of the disease in complex water environmental samples. [ABSTRACT FROM AUTHOR]

The current study was aimed to investigate ciliated protozoa that can infest Procambarus clarkii in Assiut governorate, Egypt. Hymenostomatida (Ichthyophthirius multifiliis), Peritrich ciliates (Epistylis sp., Vorticella sp. and Trichodina sp.), Suctorians ciliates (Tokophrya cyclopum, Tokophrya infusionum, Tokophrya quadripartite and Podophrya sp.), Ichthyophonida (Psorospermium haeckeli) and Cyrtophorids (Chilodonella sp.) were all identified from gills, branchiostegite and pleopods of infested individuals. Gills melanization was recorded in 11.5% of infested individuals associated with granulomas or encapsulation reaction filled with hemocytes aggregations (3.84%). Abdomen, pleopods and tail (telson and uropod) were also melanized and covered with brown slime like material in 38.46% of examined samples. Histopathological examination showed lamellar disorganization and distortion of the tip of gills lamellae with vacuoles formation. Hyperplasia and hypertrophied, strongly basophilic nuclei of lamellar epithelial cells and damaged gill lamellae with cuticle lysis were recorded. Lesions of epithelial cells varied from degeneration and necrosis to complete epithelial cell lysis. Melanization and granuloma formation were also recorded. It is obvious from the results that infestation of external ciliated protozoa induced pathogenic lesions in tissue of Procambarus clarkii. [ABSTRACT FROM AUTHOR]

Ichthyophthirius multifiliis is a parasite that poses a considerable threat to aquaculture and the ornamental fish industry, but with limited effective treatment options available. This study employed RT-qPCR to detect and analyze the expression changes of partial toll-like receptor (TLR) genes (TLR1 and TLR21), adapter protein and signal transduction molecule genes (MyD88 , TRIF , NF-κB , IRAK4 , and IRF3), and cytokines (IL-6 , IL-8 , IL-13 , CXC-α and CXCR1), as well as complement C3 , in the skin, gill, fin, liver, head kidney and spleen of Rhinogobio ventralis under different infection conditions. Additionally, tissue sections and scanning electron microscopy were utilized to observe the pathological changes in the gills and fins of R. ventralis after infection with I. multifiliis. The expression patterns of TLR-related DEGs (differentially expressed genes) in diseased wild fish were analyzed, revealing upregulation of TLR1 , TLR21 , MyD88 , NF-κB , IRAK4 , TRIF , IRF3 , IL-6 , IL-8 , IL-13 , CXC-α , CXCR1 , and C3 genes in various tissues, indicating that these genes may be involved in the immune response of R. ventralis to I. multifiliis infection. To further analyze the gene expression of sampled from the field, an artificial infection model of R. ventralis was established under laboratory conditions, with additional sampling from the skin and fins. These genes continued to show varying degrees of upregulation, but the results were not entirely consistent with those from Wudongde samples, which may be due to the more complex environment in the wild or differences in the degree of I. multifiliis infection in wild fish. The infection of I. multifiliis caused severe damage to the gills and fins of R. ventralis , characterized by extensive secretions on the gill and fin surfaces, with the presence of attached I. multifiliis trophonts, including damage and loss of gill filaments, swollen gill lamellae, and deformed gill plates, as well as cell proliferation and necrosis of gill epithelial cells. This study sheds light on the role of the TLR signaling pathway in resisting I. multifiliis infection and its associated histopathological changes in R. ventralis , providing valuable insights for the prevention and treatment of I. multifiliis infection in R. ventralis. • An artificial infection system for Rhinogobio ventralis - Ichthyophthirius multifiliis was established. • Infection by I. multifiliis triggered alterations in the expression of part immune genes associated with the TLR signaling pathway in R. ventralis. • The infection led to pathological changes in the gills and fins tissues of R. ventralis. • The complex environment in the wild led to a greater inflammatory response in fish. [ABSTRACT FROM AUTHOR]

Currently, in Romanian aquaculture, the rearing of the European catfish into intensive production systems raises a growing interest determined by its higher technological performances. In comparison with pond traditional aquaculture, the new farming systems are characterized by modified environmental conditions that may lead, in case of exceeding thresholds of tolerability, to occurrence of stress factors, thus setting the disease conditions. From this perspective, the purpose of this paper is to present the association circumstances of the predisposed environmental conditions that have weakened the fish body resistance promoting the pathological action of Ichthyophthirius multifiliis after the transfer of European catfish juveniles (G=33.6±1.7 g, L=17.5±2.9 cm) from a pond system into a tank culture system with partially reused water. During a 21-day period, some restrictive parameters of water quality have registered values far beyond the optimum interval for wells juveniles growth (t≤16oC, CmaxN-NH4+=3.62mg/l, CmaxNO2-=9.18mg/l, CmaxCl2=0.1mg/l) which caused the appearance of some characteristic symptoms, such as the gills brown coloration, masking the first symptoms of ichthyophthiriasis and contributing to the subclinical evolution of the disease. The concentrations of water chemical parameters were determined by spectrophotometer methods using Merck kits, analogous to standard methods. Subsequently, the “ich” diagnosis was confirmed by microscopic examination of the gills and skin scrapings highlighting the presence of parasites. In order to alleviate methaemoglobinaemia and parasite treatment, sodium chloride was gradually added in water till 1 ppt concentration. Maintaining the salinity of the system led to a reduction of gills parasitic intensity, while mature trophonts encapsulated in the skin were protected by mucus hypersecretion. Finally, the destruction of gill epithelium seriously affected the osmoregulation, resulting in fish death by asphyxia which led to a loss of 90% of the population.

Marker-assisted selective breeding of fish with higher levels of resistance towards specific pathogens may improve fish health, but the impact of host genotype on susceptibility to multiple pathogen infections is still poorly investigated. This study examined the resistance in rainbow trout Oncorhynchus mykiss towards infection with the eye fluke Diplostomum pseudospathaceum. We used genetically selected rainbow trout, carrying SNPs associated with resistance towards the parasitic ciliate Ichthyophthirius multifiliis, and exposed the fish to eye fluke cercariae. We showed that fish partly resistant to I. multifiliis were more susceptible to eye fluke invasion. The expression of immune relevant genes (encoding innate and adaptive factors) was also affected as these genotypes responded less strongly to a secondary fluke infection. The complexity of genome architecture in disease resistance towards multiple pathogens is discussed. [ABSTRACT FROM AUTHOR]

Ichthyophthirius multifiliis is a pathogenic ciliate parasite, which infects almost all freshwater teleost fish and leads to significant economic losses. The present study aimed to evaluate the acute toxicity of berberine to the free-living stages of I. multifiliis, that is, theronts and tomonts. Our results indicated that 99.30% of I. multifiliis theronts were killed by a concentration of 15 mg/L berberine during the 4 h exposure time, while berberine had no effect on protomonts. Nevertheless, berberine at a concentration of 5 mg/L could effectively reduce the release of theronts from tomonts treated for 4 h. Additionally, according to the transmission electron microscopy results, berberine at 15 mg/L could strongly change the shape of protomonts, destroy their organelles, and significantly decrease the number of ribosomes. The median lethal concentration (LC50) of berberine for goldfish at 96 h was 528.44 mg/L, which was almost 67 times the median effective concentration (EC50) of berberine for killing theronts (7.86 mg/L). The results demonstrated that berberine could be an effective and safe potential parasiticide for killing I. multifiliis. [ABSTRACT FROM AUTHOR]

Ichthyophthiriasis, which is also known as white spot or Ich, is caused by a parasitic ciliate called Ichthyophthirius multifiliis and it is one of the most important and common parasitic diseases. It may cause high mortality in large scale in ornamental and edible fresh water fish. To control this disease Malachite green, formalin, chloramine-T and other chemical compounds can be used, but each of these compounds may cause serious harm to human population and the environment. Therefore, introducing natural alternative compounds to use against this disease seems necessary. The aim of this study was to evaluate the alcoholic extract of Chelidonium majus L. in concentrations (0/1-6/4 gr/l) and exposure time of 1 to 3 hours on Ichthyophthirius multifiliis in vitro. The obtained data were statistically compared with the results obtained from the positive control sample (15 ppm formalin) and the control treatment. The results implicated that the mortality rate of parasitic teronts has a direct and significant relationship with increasing the concentration of the extract and the time of exposure and the mortality of the parasites increased significantly (p<0.05) with increasing the concentration of alcoholic extract from 0/1 to 6/4 gr/l. dosage 6/4 with 100% lethality had the best performance and is the most appropriate dose. There is no significant difference (p>0.05) between obtained data in comparison with formalin (control positive group), although the possibility of using this dose to treat live fish requires clinical examination. [ABSTRACT FROM AUTHOR]

Ichthyophthirius multifiliis, a ciliated parasite causing ichthyophthiriasis (white spot disease) in freshwater fishes, results in significant economic loss to the aquaculture sector. One of the important predisposing factors for ichthyophthiriasis is low water temperature (i.e., below 20°C), which affects the health and makes freshwater fishes more susceptible to parasitic infections. During ichthyophthiriasis, fishes are stressed and acute immune reactions are compromised, which enables the aquatic bacterial pathogens to simultaneously infect the host and increase the severity of disease. In the present work, we aimed to understand the parasite-bacteria co-infection mechanism in fish. Later, Curcuma longa (turmeric) essential oil was used as a promising management strategy to improve immunity and control coinfections in fish. A natural outbreak of I. multifiliis was reported (validated by 16S rRNA PCR and sequencing method) in Pangasianodon hypophthalmus from a culture facility of ICAR-CIFRI, India. The fish showed clinical signs including hemorrhage, ulcer, discoloration, and redness in the body surface. Further microbiological analysis revealed that Aeromonas hydrophila was associated (validated by 16S rRNA PCR and sequencing method) with the infection and mortality of P. hypophthalmus, confirmed by hemolysin and survival assay. This created a scenario of co-infections, where both infectious agents are active together, causing ichthyophthiriasis and motile Aeromonas septicemia (MAS) in P. hypophthalmus. Interestingly, turmeric oil supplementation induced protective immunity in P. hypophthalmus against the co-infection condition. The study showed that P. hypophthalmus fingerlings supplemented with turmeric oil, at an optimum concentration (10 ppm), exhibited significantly increased survival against co-infection. The optimum concentration induced anti-stress and antioxidative response in fingerlings, marked by a significant decrease in cortisol and elevated levels of superoxide dismutase (SOD) and catalase (CAT) in treated animals as compared with the controls. Furthermore, the study indicated that supplementation of turmeric oil increases both non-specific and specific immune response, and significantly higher values of immune genes (interleukin-1b, transferrin, and C3), HSP70, HSP90, and IgM were observed in P. hypophthalmus treatment groups. Our findings suggest that C. longa (turmeric) oil modulates stress, antioxidant, and immunological responses, probably contributing to enhanced protection in P. hypophthalmus. Hence, the application of turmeric oil treatment in aquaculture might become a management strategy to control co-infections in fishes. However, this hypothesis needs further validation. [ABSTRACT FROM AUTHOR]

Ichthyophthiriasis (Ich), also known as white spot disease, causes significant economic losses to fish farmers once an outbreak occurs. For fish survival, it is therefore crucial to understand the pathogenic mechanism and find effective prevention methods. In this study, we obtained data for four stages (theront, trophont, protomont and tomont) of Ichthyophthirius multifiliis by single-cell RNA sequencing (scRNA-seq). We found that the invasion-related proteins encoded by highly expressed genes in the theront stage mainly belong to the leishmanolysin family proteins, heat shock proteins, transmembrane proteins and cysteine proteases (CPs). Additionally, the exosome pathway appears to play a significant role in the invasion process of the theront. Since cysteine proteases are expressed at all stages of the I. multifiliis, and five CP-related genes were significantly upregulated at the theront stage of its life cycle - two of which are enriched in the exosome pathway - we incubated I. multifiliis theronts and protomonts with cysteine protease inhibitor (E-64). Our findings revealed that E-64 could kill both stages of the parasite in vitro and affected tomont division and subsequent release. Furthermore, infection experiment showed that E-64 could significantly inhibit the invasion of theronts. Based on our preliminary analysis from the transcriptomic and E-64 experiments, we have confirmed that CPs play a crucial role in I. multifiliis. This research establishes a foundation for future strategies in the prevention and control of Ich., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

White spot disease, caused by the parasitic ciliate Ichthyophthirius multifiliis, is a significant threat to the freshwater fish farming industry worldwide, resulting in massive mortality and economic losses. Eliminating the free-swimming theronts from the culture environment is considered crucial for the control of I. multifiliis infection. It is well-documented that planktonic ciliates are valuable food resources for macro-zooplankton in aquatic ecosystems. In this study, we developed a fluorescence labeling method for alive theronts and found that cyclopoid copepods Thermocyclops taihokuensis, Mesocyclops spp., Macrocyclops sp., and Paracyclopina sp. present predation on the theronts in co-culture experiments. Laboratory challenge tests further confirmed that the presence of zooplankton in the culture water body significantly reduced the infection of I. multifiliis in goldfish (p < 0.01). Results from this study revealed that cyclopoid copepods have the potential to be used as biological control agents against white spot disease in aquaculture.

of supporting mucosal immune barrier integrity and prevention of some pathogenic infections in aquatic species, are key areas of active study, often focusing on feed additives. The objectives of this study were to explore the effects of feeding cMOS (concentrated mannan oligosaccharide) on the gill and skin mucosal barriers of goldfish (Carassius auratus Linnaeus) and evaluate health status during Ichthyophthirius multifiliis infection. After feeding the cMOS-containing diet for 60 days, Hematoxylin and eosin (H&E) staining showed greater length of gill lamella and thicker dermal dense layer, while Alcian Blue and Periodic acid–Schiff (AB-PAS) staining showed higher numbers of mucin cells in cMOS fed fish. Chemical analysis showed that fish fed cMOS had greater enzyme activity of lysozyme (LZM) and alkaline phosphatase (AKP) in gill and skin tissues, while qRT-PCR revealed higher expression of Muc-2 and IL-1β , as well as lower expression of IL-10. After Ichthyophthirius multifiliis challenge, goldfish fed the cMOS diet had lower mortality and infection rates, as well as fewer visible white spots on the body surfaces. Histologically, the gill and skin of these fish presented less tissue damage and fewer parasites, and had a greater number of mucus cells. In addition, the expression of Muc-2 and IL-10 were notably higher while the expression of IL-1β was significantly lower in cMOS fed goldfish than control fed fish. In this study, cMOS fed goldfish had stronger immune barrier function of skin and gill mucous, and better survival following Ichthyophthirius multifiliis infection. • Feeding cMOS increased the length of gill lamella and thickened the dermal dense layer of goldfish. • CMOS supported gill and skin mucosal barriers. • Feeding cMOS promoted the secretion of mucous cells. • CMOS fed goldfish had better survival following Ichthyophthirius multifiliis infection. [ABSTRACT FROM AUTHOR]

A survey of gill parasites was conducted in July 2019 on a small sample of cypress minnow, Hybognathus hayi Jordan, from Calion Lake, Union County, Arkansas (Ouachita River drainage). Six of 9 (67%) specimens harbored parasites, including ciliates Ichthyophthirius multifiliisFouquet, 1876, and an undetermined Trichodina sp., a myxozoan, Myxobolus sp., and a monogenean, Dactylogyrus nuchalisWood and Mizelle, 1957, in 2 (22%). Dactylogyrus hankinsoniHanek, Molnar, and Fernando, 1975, was found in 3 (33%) of the host specimens. All 5 taxa of these gill-inhabiting parasites are reported for the first time from H. hayi, and the monogeneans are documented as new geographic distribution records in Arkansas. [ABSTRACT FROM AUTHOR]

The Japanese puffer, Takifugu rubripes , is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1β (IL-1β), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1β (TrIL-1β) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1β protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1β may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1β promoted the expression of endogenous IL-1β, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1β group had less tissue damage compared with control groups of fish. IL-1β-small interfering RNA and IL-1β overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1β regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1β also promoted the differential expression of IgM , showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1β could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1β. They also suggested that recombinant TrIL-1β could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes. • The incubation experiments revealed that TrIL-1β may reduce trophont activity by destroying membranes. • Immunofluorescence experiments showed that recombinant TrIL-1β penetrated and disrupted the cell membranes of trophonts. • Transmission electron microscopy showed that the IL-1β group had less tissue damage compared with control groups of fish. • Quantitative RT-PCR results showed that TrIL-1β regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. • The cumulative mortality experiment show that TrIL-1β could protect fish against C. irritans infection. [ABSTRACT FROM AUTHOR]

Ichthyophthirius multifiliis is a protozoan ciliate that causes white spot disease (also known as ichthyophthiriasis) in freshwater fish. Holland's spinibarbel (Spinibarbus hollandi) was less susceptible to white spot disease than grass carp (Ctenopharyngodon Idella). In this study, grass carp and Holland's spinibarbel are infected by I. multifiliis and the amount of infection is 10,000 theronts per fish. All grass carp died within 12 days after infection, and the survival rate of Holland's spinibarbel was more than 80%. In order to study the difference in sensitivity of these two fish species to I. multifiliis , transcriptome analysis was conducted using gill, skin, liver, spleen and head kidney of Holland's spinibarbel and grass carp at 48 h post-infection with I. multifiliis. A total of 489,296,696 clean reads were obtained by sequencing. A total of 105 significantly up-regulated immune-related genes were obtained by Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in grass carp. Cluster of differentiation 40 (CD40), cluster of differentiation 80 (CD 80), tumor necrosis factor-alpha (TNF-α), toll-like receptor 4 (TLR-4), interleukin 1 beta (IL-1β) and other inflammatory-related genes in grass carp were enriched in the cytokine-cytokine receptor interaction pathway and toll-like receptor pathway. In Holland's spinibarbel, a total of 46 significantly up-regulated immune-related genes were obtained by GO classification and KEGG pathway enrichment analysis. Immune-related genes, such as Immunoglobin heavy chain (IgH), cathepsin S (CTSS), complement C1q A chain (C1qA), complement component 3 (C3) and complement component (C9) were enriched in phagosome pathway, lysosome pathway and complement and coagulation concatenation pathway. C3 was significantly up-regulated in gill and head kidney. Fluorescence in situ hybridization (FISH) showed that the C3 gene was highly expressed in gill tissue of Holland's spinibarbel infected with I. multifiliis. A small amount of C3 gene was expressed in the gill arch of grass carp after infected with I. multifiliis. In conclusion, the severe inflammatory response in vivo after infecting grass carp with I. multifiliis might be the main cause of the death of grass carp. The extrahepatic expression of the gene of Holland's spinibarbel might play an important role in the immune defense against I. multifiliis. • We evaluated transcriptomic alterations in grass carp and Holland's spinibarbel induced by I. multifiliis infection. • 12185 DEGs and 15386 DEGs were found in the transcriptomic of grass carp and Holland's spinibarbel, respectively. • Inflammatory storm after infecting grass carp with I. multifiliis might be the main cause of the death of grass carp. • The expression of C3 gene outside the liver may be the reason for the resistance to I. multifiliis in Holland's spinibarbel. [ABSTRACT FROM AUTHOR]

We have successfully designed and constructed a RAEV vector system with regulated-delayed attenuation in vivo attributes that synthesizes Ichthyophthirius multifiliis (Ich) protective antigen IAG52B to enable vaccination of fish susceptible to edwardsiellosis and white spot disease. The first feature of this vaccine delivery system is an Edwardsiella piscicida strain carrying genomic deletions of asdA. AsdA is an enzyme necessary for the synthesis of diaminopimelic acid (DAP), which is an essential component of the peptidoglycan layer of the cell wall of Gram-negative bacteria. asdA mutant strains have obligate growth requirements for DAP in the medium or a plasmid vector with the wild-type asdA gene enabling synthesis of DAP. This balanced-lethal plasmid vector-host system in E. piscicida enables as a second feature the synthesis of recombinant antigens to induce protective immunity against fish pathogens. Recombinant protective antigen IAG52B from the fish pathogen I. multifiliis was synthesized by RAEV strains harboring the AsdA+ plasmid pG8R8029. The third feature of this vaccine strain is a regulated-delayed attenuation in vivo phenotype that is based on the replacement of an arabinose-regulated araC ParaBAD cassette for the promoters of the fur and crp genes of E. piscicida such that the expression of these genes is dependent on arabinose provided during growth. Thus, following colonization, the Fur and Crp proteins stop being synthesized due to the lack of arabinose and attenuation is progressively achieved in vivo to prevent generation of diseases symptoms. Our vaccine strain χ16022 with the genotype Δ asdA10 ΔPfur170::TT araC ParaBAD fur ΔPcrp68::TT araC ParaBAD crp contains the AsdA+ plasmid, pG8R8029, which encodes the IAG52B antigen. Vaccine strain χ16022(pG8R8029) is attenuated and induces systemic and mucosal IgM titer against E. piscicida and Ich in zebrafish. In addition, transcript levels of tnf-α , il-1β , il-6 and il-8 were significantly increased in different tissues of vaccinated zebrafish compared to unimmunized fish. Zebrafish vaccinated with χ16022(pG8R8029) showed 60% survival upon intracoelomic (i.c.) challenge with a lethal dose of virulent E. piscicida strain J118. Our RAEV system could be used as a generalized vaccine-vector system to protect teleost fish against multiple bacterial, viral and parasitic infectious diseases. [ABSTRACT FROM AUTHOR]

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG -RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG -RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG -RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG -RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides. • fliG gene of Pseudomonas plecoglossicida was silenced by RNAi. • The silence of fliG significantly reduced pathogenicity of P. plecoglossicida to E. coioides. • The silence of fliG significantly affected host-pathogen interactions of P. plecoglossicida with E. coioides. [ABSTRACT FROM AUTHOR]

CD79a and CD79b heterodimers are important components that consist of B cell receptor compound, which play a crucial role in transduction activation signal of the antigen binding BCR, and B cell development and antibody production. In order to investigate the characters and potential functions of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss), we firstly cloned and analyzed the expression of CD79a and CD79b and found that the cDNA sequences of CD79a and CD79b both contained open reading frame of 711 and 645 bp in length for encoding the protein of 237 and 215 amino acid residues, respectively. The predicted amino acid sequences from trout were highly conserved with those of other teleost fishes in structure. Phylogenetic tree was constructed to analyze the evolutionary relationship between the trout and other known species, the result indicated that CD79a and CD79b of trout clustered at high bootstrap values with Salmo salar. Moreover, three trout infection models with F. columnare G 4 , I. multifiliis and infectious hematopoietic necrosis virus (IHNV) were constructed, which resulted in morphological changes and serious lesions in skin and gills. Importantly, the high expression of CD79a and CD79b occurred in skin, gills, and followed by head kidney in response to bacterial, parasitic, and viral infection, as its expression was closely related to that of Igs. Our findings indicated that CD79a and CD79b play vital roles in both systemic and mucosal immune responses of rainbow trout during bacterial, parasitic, and viral infection, which will contribute to explore the roles of CD79 subunits in B cell signaling during ontogeny and disease. • The CD79a and CD79b full-length sequences of Rainbow trout (Oncorhynchus mykiss) were firstly identified. • The expression of CD79a and CD79b were analyzed and compared at three infected models. • CD79a and CD79b may play vital roles in both systemic and mucosal immune responses. [ABSTRACT FROM AUTHOR]

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Ichthyophthirius multifiliis is a pathogenic ciliate parasite, which infects almost all freshwater teleost fish and leads to significant economic losses. The present study aimed to evaluate the acute toxicity of berberine to the free-living stages of I. multifiliis, that is, theronts and tomonts. Our results indicated that 99.30% of I. multifiliis theronts were killed by a concentration of 15 mg/L berberine during the 4 h exposure time, while berberine had no effect on protomonts. Nevertheless, berberine at a concentration of 5 mg/L could effectively reduce the release of theronts from tomonts treated for 4 h. Additionally, according to the transmission electron microscopy results, berberine at 15 mg/L could strongly change the shape of protomonts, destroy their organelles, and significantly decrease the number of ribosomes. The median lethal concentration (LC50) of berberine for goldfish at 96 h was 528.44 mg/L, which was almost 67 times the median effective concentration (EC50) of berberine for killing theronts (7.86 mg/L). The results demonstrated that berberine could be an effective and safe potential parasiticide for killing I. multifiliis.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish. [ABSTRACT FROM AUTHOR]