Ichthyophthirius Research Articles

Chlorella vulgaris extract conjugated magnetic iron nanoparticles in nile tilapia (Oreochromis niloticus): Growth promoting, immunostimulant and antioxidant role and combating against the synergistic infection with Ichthyophthirius multifiliis and Aeromonashydrophila

Nile tilapia reared under intensive conditions was more susceptible for Ichthyophthirius multifilii (I. multifiliis) infection eliciting higher mortality, lower productive rate and further bacterial coinfection with Aeromonas hydrophila (A. hydrophila). The higher potency of magnetic field of iron oxide nanoparticles (NPs) can kill pathogens through inhibiting their viability. Herein, coating of Chlorella vulgaris extract (ChVE) with magnetic iron oxide NPs (Mag iron NPs) can create an external magnetic field that facilitates their release inside the targeted tissues. Thus, the current study is focused on application of new functionalized properties of Mag iron NPs in combination with ChVE and their efficacy to alleviate I. multifiliis and subsequent infection with A. hydrophila in Nile tilapia. Four hundred fingerlings were divided into: control group (with no additives), three groups fed control diet supplemented with ChVE, Mag iron NPs and ChVE@Mag iron NPs for 90 days. At the end of feeding trial fish were challenged with I. multifiliis and at 9 days post challenge was coinfected by A. hydrophila. A remarkable higher growth rate and an improved feed conversion ratio were detected in group fed ChVE@Mag iron-NPs. The maximum expression of antioxidant enzymes in skin and gills tissues (GSH-Px, CAT, and SOD) which came in parallel with higher serum activities of these enzymes was identified in groups received ChVE@Mag iron-NPs. Furthermore, group fed a combination of ChVE and Mag iron-NPs showed a boosted immune response (higher lysozyme, IgM, ACH50, and MPO) prior to challenge with I. multifiliis. In contrast, fish fed ChVE@Mag iron-NPs supplemented diet had lower infection (decreased by 62%) and mortality rates (decreased by 84%), as well as less visible white spots (decreased by 92 % at 12 dpi) on the body surfaces and mucous score. Interestingly, post I. multifiliis the excessive inflammatory response in gill and skin tissues was subsided by feeding on ChVE@Mag iron-NPs as proved by down regulation of IL-1β, TNFα, COX-2 and iNOS and upregulation of IL-10, and IgM, IgT and Muc-2 genes. Notably, group exposed to I. multifiliis-showed higher mortality when exposed to Aeromonas hydrophilia (increased by 43 %) while group fed ChVE@Mag iron-NPs exhibited lower morality (2%). Moreover, the bacterial loads of A. hydrophilia in fish infected by I. multifiliis and fed control diet were higher than those received dietary supplement of ChVE, Mag iron-NPs and the most reduced load was obtained in group fed ChVE@Mag iron-NPs at 7 dpi. In conclusion, ChVE@Mag iron-NPs fed fish had stronger immune barrier and antioxidant functions of skin and gills, and better survival following I. multifiliis and A. hydrophilia infection.

Comparison of Ectoparasites infestation for fresh and saltwater Fishes from Euphrates, and Razzaza Lake, Iraq

Parasitic infestations represent one of the main challenges to fresh water and marine fish all areas of the world. The infections are commonly found on the skin and gills of fish and can cause dangerous problems, as mortality, in heavily parasitized infections. This study was conducted to detect of ectoparasite types in Fishes, 200 of Coptodon zillii from Euphrates and 120 Cyprinus carpio from Razzaza Lake, were used for this study in aquatic environment. The specimens were purchased at 2022 in different periods from market and selected. The collection of fish using gill nets. The collected of fish samples was carried out in lab and used macroscopic inspection, gross viscera, microscopic exam-wet mount of fins, skin, gills and eyes. The results of the both infected fishes revealed the presence of several clinical signs. The prevalence was 65.6%, in Coptodon zillii was 30% and Cyprinus carpio was 100%. Indeed, we observed the appearance of necrotic areas, ulcerations and hemorrhage on the gills. On others body parts, we found Protozoa and Monogeneans parasites. The identification of ectoparasite were obtained in fins, skin, and gills and no infection in eyes. There are 4 types of ectoparasites were infect to Coptodon zillii, that is Trichodina sp., Ichthyophthirius multifiliis sp., Dactylogyrus sp., and Gyrodactylus sp. While, Cyprinus carpio that is I. multifiliis and Gyrodactylus sp. Significantly of results at P≤0.05 of fish type with weight and long but not significant for genders in both types. In future studies should be focusing on modern tools to identification of parasites species in different environments.

The potential mechanism of concentrated mannan-oligosaccharide promoting goldfish’s (Carassius auratus Linnaeus) resistance to Ichthyophthirius multifiliis invasion

Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1β, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.

Investigation of Ichthyophthirius multifiliis infection in fish from natural water bodies in the Lhasa and Nagqu regions of Tibet

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 < 0.05). The study revealed a significantly lower prevalence in Lhasa River than in Cuona Lake (P < 0.05). Notably, our findings revealed instances of I. multifiliis infections even in saline water bodies, thereby emphasizing the potential threat that this parasite poses to the preservation of indigenous fish resources in Tibet. Consequently, immediate and effective countermeasures are imperative. This study represents the first systematic investigation of I. multifiliis infection in natural water bodies in Tibet.

Cold-blooded vertebrates evolved organized germinal center-like structures.

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.

Mucosal immune responses to Ichthyophthirius multifiliis in the ocular mucosa of rainbow trout (Oncorhynchus mykiss, Walbaum), an ancient teleost fish

AbstractThe eye, as a specialized visual organ, is directly exposed to the external environment, and, therefore, it faces constant challenges from external pathogenic organisms and toxins. In the ocular mucosa (OM) of mammals, mucosal-associated lymphoid tissues (MALTs) constitute the primary line of defense. However, the immune defense role of the OM remains unknown in aquatic vertebrates. To gain insights into the immune processes within the OM of teleost fish, we developed an infection model of rainbow trout (Oncorhynchus mykiss) OM using a parasite, Ichthyophthirius multifiliis (Ich). Immunofluorescence, qPCR, and H&amp;E staining revealed that Ich successfully infiltrates the OM of rainbow trout, leading to pathological structural changes, as evidenced by A&amp;B staining. Importantly, the qPCR results indicate an up-regulation of immune-related genes following Ich infection in the OM. Moreover, transcriptome analyses were conducted to detect immune responses and impairments in eye function within the OM of rainbow trout with Ich infection. The results of the transcriptome analysis that Ich infection can cause an extensive immune response in the OM, ultimately affecting ocular function. To the best of our knowledge, our findings represent for the first time that the teleost OM could act as an invasion site for parasites and trigger a strong mucosal immune response to parasitic infection.

Can Chilodonella uncinata induce cross-protection in koi carp (Cyprinus carpio) against Ichthyophthirius multifiliis? Evidence from immune response and challenge experiments

The intraperitoneal injection of koi carp with inactivated Ichthyophthirius multifiliis or Chilodonella uncinata combined with Freund's adjuvant was evaluated for its capacity to protect the fish from I. multifiliis infection. Our results showed that the level of Ig M in serum significantly increased after vaccination, reaching up to 4.09 μg/mL on day 14 in fish vaccinated with C. uncinata, while C3 content also generally increased. In addition, genes related to immunity, including Ig M, IL-1β, TNF-α, TLR 9, TLR 20 and TLR 21, exhibited differential expression, which suggests that the koi carp did indeed produce immune responses after vaccination. In challenge experiments, each fish specimen was challenged with 30,000 theronts on day 14 post vaccination. The relative survival rate of fish vaccinated with 10,000 I. multifiliis theronts was 93.3%, while the survival rate of fish vaccinated with 10,000C. uncinata or with phosphate buffer saline (the latter being the control) was 0% in both cases. However, as the dose of C. uncinata was increased, the survival rate of the fish also increased, with fish vaccinated with 60,000C. uncinata cells achieving a higher survival rate than those vaccinated with 30,000 cells. Above all, we demonstrated that Ig M does not play a major role in protection against ichthyophthiriasis and the protection conferred by inactivated C. uncinata vaccination is related to the dose.

Nanosuspensions as an approach for improved solubility and anti-Ichthyophthirius multifiliis activity of magnolol

Natural products have gained recognition as a valuable resource and have significant implications in the field of aquaculture. Nevertheless, the limited water solubility of drugs, leading to compromised bioavailability, has been a persistent challenge in the realm of pharmaceutical research. Magnolol as a natural product, the low solubility leads to limited activity against Ichthyophthirius multifiliis, which severely limits the application of magnolol. Nanosuspensions are considered to be an important strategy to improve the solubility and bioavailability of insoluble drugs. The purpose of this study is to fabricate nanosuspensions that can effectively improve the solubility and antiparasitic activity of magnolol. Magnolol nanosuspensions (MNs) containg Pluronic F68 as a stabilizer was prepared by the solvent-antisolvent precipitation method. The results of TEM, DSC and FTIR analysis showed that the surfactant attached to the surface of magnolol, and the nanosuspensions process significantly reduced the crystallinity and improved the solubility and dissolution rate of magnolol. The Central Composite Design was adopted to investigate the effects of independent variables viz. amount of temperature (A), the ratio of pluronic F68 to magnolol (w/w) (B), and churning time (C) on particle size (Y), obtaining optimum nanosuspensions with a size of 32.03 nm. Compared with free magnolol, the release rate of magnolol in 119 nm and 35 nm MNs was significantly improved, and the release rate of magnolol was increased from 28.13% to 58.45% and 85.70% within 60 min. Further, the antiparasitic activity of MNs was measured. The results showed the reduction in drug particle size leads to an enhancement in the insecticidal activity of magnolol. Altogether, this study proposes a promising strategy to enhance activity by increasing the solubility of natural products.

A Parasitological Examination on Aquarium Fish Sold in Petshops in Kırıkkale.

The aim of the present study was to determine the prevalence of parasitic infections in aquarium fish sold in local pet shops. Totally 502 fish samples from 8 species were obtained from 7 pet shops. Native preparations were prepared from the gills, fins, skin, intestines, and internal organs of fish and examined under a light microscope. It was detected at the end of the study that 62.7% of the fishes examined were infected with one or more parasite species. Among the fish examined, 28.9% were infected with a single parasite, whereas 33.9% were infected with mixed parasites. Chilodonella sp. identified as the most common species. Other species detected included Trichodina sp., Piscinoodinium sp., Ichthyobodo sp., Ichthyophthirius multifiliis, Apiosoma sp., Epistylis sp., Vorticella sp., Gyrodactylus sp., Dactylogyrus sp., Capillaria sp., Camallanus sp., metacercariae, and nematode larvae, Argulus sp., Philodina sp., Euchlanis sp., Aelosoma sp., and Tetrahymena. The importance of the present study is that highest number of parasite species were detected in aquarium fish sold in pet shops in Türkiye. The aquarium owners should pay attention to the cleanliness of the aquarium water in order to reduce the rate of parasitic infection in fish; when new fish are purchased, they should be taken from reliable sources, and the quarantine process should be observed.

Mass mortality of the Critically Endangered naked characin (Gymnocharacinus bergii): Possible causes and impact

Abstract 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.

Identification of Genes Related to Resistance to Ichthyophthirius multifiliis Based on Co-expression Network Analysis in Grass Carp.

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.

Integrated morphological and transcriptome profiles reveal a highly-developed extrusome system associated to virulence in the notorious fish parasite, Ichthyophthirius multifiliis

ABSTRACT Ichthyophthirius 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.

Non-specific cytotoxic cell receptor protein-1 (NCCRP-1) is involved in anti-parasite innate CD8+ T cell-mediated cytotoxicity in ginbuna crucian carp.

CD8+ cytotoxic T cells (CTLs) are a main cellular component of adaptive immunity. Our previous research has shown that CD8+ cells demonstrate spontaneous cytotoxic activity against the parasite Ichthyophthirius multifiliis in ginbuna crucian carp, suggesting that CD8+ cells play an important role in innate immunity. Herein, we investigated the molecules and cellular signal pathways involved in the cytotoxic response of ginbuna crucian carp. We considered non-specific cytotoxic receptor protein-1 (NCCRP-1) as candidate molecule for parasite recognition. We detected NCCRP-1 protein in CD8+ cells and the thymus as well as in other cells and tissues. CD8+ cells expressed mRNA for NCCRP-1, Jak2, and T cell-related molecules. In addition, treatment with a peptide containing the presumed antigen recognition site of ginbuna NCCRP-1 significantly inhibited the cytotoxic activity of CD8+ cells against the parasites. The cytotoxic activity of CD8+ cells was significantly inhibited by treatment with the JAK1/2 inhibitor baricitinib. These results suggest that teleost CTLs recognize I. multifiliis through NCCRP-1 and are activated by JAK/STAT signaling.

Stationary metal sheets (copper, zinc or brass) in fish tanks prevent Ichthyophthirius multifiliis Fouquet, 1876 infection of rainbow trout: In vivo and in vitro effects

Infections with the parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 challenge health of freshwater fishes worldwide by eliciting white spot disease (ichthyophthiriosis). A series of chemicals, pharmaceuticals and herbal extracts are currently in use to control the infections by regular or continuous administration to aquaculture systems. Recently application of brass (copper/zinc alloys) metal sheets in marine fish tanks was shown to eliminate tomonts of a related ciliate, Cryptocaryon irritans Brown, 1951 causing cryptocaryonosis (marine white spot disease). The present work documents that brass, and each of its pure constituents (copper and zinc), have in vivo and in vitro antiparasitic effects in freshwater. We used an experimental model with I. multifiliis infected rainbow trout (Oncorhynchus mykiss Walbaum, 1792). In the in vivo study metal sheets of either brass (alloy of copper and zinc), pure copper or pure zinc, were placed in fish tanks with trout experimentally infected with I. multifiliis (duplicated trials). All three metals (pure or combined) effectively blocked the life cycle of I. multifiliis and inhibited reinfection and morbidity. In control tanks with infected trout, but no metal sheets, we observed significantly elevated infection levels and development of disease. In vitro assays, exposing three life cycle stages (tomont, tomocyst and theront) to the same metal sheets in Petri dishes, confirmed the antiparasitic effects. Tomonts were sensitive to pure copper and brass (100% and 66% mortality within 15 min, respectively) and zinc (100% mortality within 30 min). Tomocysts, with enclosed tomites, were less sensitive, as even 12 h exposure to brass, pure copper and zinc merely led to mortality rates of 44%, 50% and 60%, respectively. Theronts were killed within 75 min when exposed to brass and pure copper plates, whereas zinc plate exposure eliminated all theronts within 60 min. Microscopical observations suggested that direct contact between the parasite and the metal sheet increased the parasiticidal effect, although the released metal ions may contribute may play a role. The use of stationary metal sheets in aquaculture settings may be a possible way to control ichthyophthiriosis. However, we measured the concentration of free metal ions in the fish tanks with metal sheets, which suggested release of metal to the water. This calls for additional studies on toxicology and environmental effects of metal sheet use before implementation at fish farms level.

Antiparasitic activity of medicinal plants against protozoan fish parasite Ichthyophthirius multifiliis

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&lt;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.

Acute immune responses in zebrafish and evasive behavior of a parasite - who is winning?

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.

Predation of Cyclopoid Copepods on the Theronts of Ichthyophthirius multifiliis: Shedding Light on Biocontrol of White Spot Disease.

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.

Epicatechin gallate: A promising agent isolated from Sargentodoxa cuneata for the treatment of ichthyophthiriasis in goldfish (Carassius auratus)

Ichthyophthirius multifiliis is a pathogenic parasite of freshwater fish that threatens the sustainability of aquaculture industry globally. Our previous studies have identified Sargentodoxa cuneata ethanol extracts exhibited potent antiparasitic activity against I. multifiliis. However, the active ingredients in S. cuneata remain unknown. In this study, we isolated an active compound from S. cuneata by bioassay-guided fractionation and identified it as epicatechin gallate (ECG) by NMR, FT-IR and HR-MS spectroscopy. In vitro assays revealed that ECG was effective against all the free-living stages of I. multifiliis. ECG could cause 100% mortality of theronts at 0.3 mg/L within 30.6 ± 0.4 min, and terminate reproduction of protomonts and tomonts at concentrations of 2.0 and 10.0 mg/L, respectively. Morphologically, 2.0 mg/L ECG treatment for 2 h induced the loss of plasma membrane integrity and leakage of intracellular contents of the protomont. Moreover, an in vivo parasite‑goldfish infection model was applied to evaluate the efficacy of ECG on prevention and treatment of ichthyophthiriasis. The results showed that 2.0 mg/L ECG treatment for 12 days could cure goldfish ichthyophthiriasis and significantly increase the survival rate of the infected goldfish by 86.7%. Acute toxicity test revealed that ECG was relatively safe to goldfish, with a median lethal concentration (LC50) value of 63.7 mg/L, which was >30 times higher than effective concentration against I. multifiliis in vivo. Overall, our data indicated that ECG had the potential as a novel antiparasitic agent for the treatment of ichthyophthiriasis in aquaculture.

Inventory of ectoparasites and molecular identification of pathogenic bacteria on bonylip barb (Osteochilus vittatus) cultivated in Banyumas District

The aims were to do an ectoparasite and pathogenic bacteria inventory of Bonylip barb reared in Banyumas district. Total of 118 fish were randomly taken from Singasari (30 individual), Pabuaran (30 individual), Beji (30 individual) and Jipang (28 individual) villages. This work observed ectoparasite species, prevalence, and intensity as well as pathogenic bacteria species. Ectoparasites were identified based on morphological characteristics observed microscopically and their prevalence and intensity were calculated. Bacterial pathogen was isolated from fish samples showing symptoms of bacterial disease and identified molecularly based on 16s rDNA sequence using BLAST dan phylogenetic analysis. Results showed Trichodina sp. and Monogeneans were found infected fish samples from all location with prevalence was 70-100%. Ichthyophthirius multifiliis was found from all location except Singasari village.  Some of fish samples showed fin necrosis and five bacterial isolates were obtained. 16s rDNA marked gene was successfully amplified and sequenced. Based on sequence analysis, five isolates could be clustered into two groups, the first was found only in Singasari village and the second was found in Singasari and Beji villages.  Similarity value of 16s rDNA gene sequence these two groups was 99,8%. Result of BLAST and phylogenetic analysis showed that the bacterial pathogens were identified as group of Aeromonas hydrophila complex.

Protozoan Parasites of Iranian Freshwater Fishes: Review, Composition, Classification, and Modeling Distribution.

This article investigates the occurrence and distribution of parasitic protozoa of Iranian freshwater fishes (both farmed and wild). Our search shows 26 known parasitic protozoan species were recorded from 52 freshwater fish species across different ecoregions of Iran. Most of these fish are edible. While none of the identified protozoan parasites are of zoonotic importance, our study does not exclude presence of zoonotic species in Iranian fishes. Present data suggest the northern and western regions of the country are the main macrohabitat of protozoa (35 parasitic records reported), with the greatest concentration of parasitic protozoa occurring in the Urmia basin in Iran's northwest. The clustered distribution pattern of protozoa among freshwater fish was also more evident in the northern and western parts of the country. The gills and skin were the most infected microhabitats for parasitic protozoa. The highest number of parasites was observed in the fish family Cyprinidae with nine species found in the native fish, Capoeta capoeta. The most diverse host range was observed in the holotrich ciliate, Ichthyophthirius multifiliis isolated from 46 cyprinid species in 39 different locations. However, due to the great richness of fish and extreme habitat diversity, parts of the parasite fauna of Iranian freshwater fish are still poorly understood. Furthermore, current and future changes in climate and environmental parameters, and anthropogenic interventions are likely to affect fish hosts and their parasites.