Histopathological, morphological, and molecular characterization of fish-borne zoonotic parasite Eustrongylides Excisus infecting Northern pike (Esox lucius) in Iran.

Eustrongylides excisus is a fish-borne zoonotic parasite known to infect various fish species, including Northern pike (Esox Lucius). This nematode, belonging to the family Dioctophymatidae, has a complex life cycle involving multiple hosts. This study aimed to investigate the occurrence of Eustrongylides nematodes in Northern pike (E. Lucius) collected from Mijran Dam (Ramsar, Iran). Between June and October 2023, an investigation was conducted on Northern pike from Mijran Dam in Ramsar, Iran, following reports of reddish parasites in their muscle tissues. Sixty fish were examined at the University of Tehran, revealing live parasites in the muscles, which were then analyzed microscopically and preserved for a multidisciplinary study. The skeletal muscle tissues of 85% (51/60) of fish specimens were infected by grossly visible larvae which were microscopically identified as Eustrongylides spp. In histopathological examination, the lesion was composed of encapsulated parasitic granulomatous myositis. Microscopically, the cystic parasitic granulomas compressed the adjacent muscle fibers, leading to their atrophy and Zenker's necrosis. Moreover, epithelioid macrophages, giant cells and mononuclear inflammatory cells were present around the larvae and between the muscle fibers. Finally, a molecular analysis by examining the ITS gene region, revealed that they belong to the species E. excisus. Eustrongylidiasis in northern Iran necessitates further research into the biology, epidemiology, and control of Eustrongylides nematodes, focusing on various hosts. This study is the first to comprehensively characterize E. excisus in Northern pike in Ramsar, Iran, raising concerns about possible zoonotic transmission.

Growth performance, mucosal immunity and disease resistance in goldfish (Carassius auratus) orally administered with Escherichia coli Strain Nissle 1917.

The current research aimed to shed light on the efficacy of Escherichia coli strain Nissle 1917 (EcN) on goldfish (……) growth, gut immunity, morphology, bacterial nutritional enzyme activity and resistance to Aeromonas hydrophila infection. Fish fed with EcN at 106, 107 and 108 CFU/g feed for 80 days showed an enhancement in growth better than control fish. The gut innate immunity in terms of lysozyme activity, immunoglobulin and total protein levels was increased in the treatment fish with the best result being observed in fish fed EcN at 108 CFU/ g. In addition, an increase was noted in the upregulation of immune-relevant genes, namely lysozyme, interleukin-1ß, inducible nitric oxide synthase and tumor necrosis factor α of fish intestine. A marked surge in the number of proteolytic and heterotrophic bacteria was noted in the gut of fish nourished with the probiotic. Histological studies exhibited an improvement in the intestinal absorption surface area, intraepithelial lymphocyte count and goblet cell density. Significantly higher survival rate was obtained in fish fed EcN at 108 CFU/g compared with the fish fed with the basal diet. These data exhibited the beneficial effect of EcN on goldfish growth, digestive enzymes, intestine heterotrophic bacteria and resistance against Aeromonas hydrophila challenge. This study confirmed the favorable outcomes resulting from the administration of EcN at108 CFU/g.

Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020-2022.

Free-living amoebae (FLA) are capable of inhabiting diverse reservoirs independently, without relying on a host organism, hence their designation as "free-living". The majority of amoebae that infect freshwater or marine fish are amphizoic, or free-living forms that may colonize fish under particular circumstances. Symphysodon aequifasciatus, commonly referred to as the discus, is widely recognized as a popular ornamental fish species. The primary objective of the present study was to determine the presence of pathogenic free-living amoebae (FLA) in samples of discus fish. Fish exhibiting clinical signs, sourced from various fish farms, were transferred to the ornamental fish clinic. The skin, gills, and intestinal mucosa of the fish were collected and subjected to culturing on plates containing a 1% non-nutrient agar medium. The detection of FLA was conducted through morphological, histopathological and molecular methods. The construction of the phylogenetic tree for Acanthamoeba genotypes was achieved using the maximum likelihood approach. The molecular sequence analysis revealed that all cultures that tested positive for FLA were T4 genotype of Acanthamoeba and Acanthamoeba sp. The examination of gill samples using histopathological methods demonstrated the presence of lamellar epithelial hyperplasia, significant fusion of secondary lamellae, and infiltration of inflammatory cells. A multitude of cysts, varying in shape from circular to elliptical, were observed within the gills. The occurrence of interlamellar vesicles and amoeboid organisms could be observed within the epithelial tissue of the gills. In the current study, presence of the Acanthamoeba T4 genotype on the skin and gills of discus fish exhibiting signs of illness in freshwater ornamental fish farms was identified. This observation suggests the potential of a transmission of amoebic infection from ornamental fish to humans, thereby highlighting the need for further investigation into this infection among ornamental fish maintained as pets, as well as individuals who interact with them and their environment.

Clinico-histopathological and phylogenetic analysis of protozoan epibiont Epistylis wuhanensis associated with crustacean parasite Lernaea cyprinacea from ornamental fish in Iran.

Parasitic infestations are one of the most concerning problems limiting ornamental fish farming. In addition to the direct economic losses due to the major mortalities, parasites may significantly negatively impact the body shape, coloration, growth rate, and health condition of the fish. The results of the study highlight the importance of comprehensive parasitological analysis in the diagnosis and treatment of parasitic infections in ornamental fish farms. The presence of multiple parasites in the affected fish emphasizes the need for effective biosecurity measures, such as extending the quarantine period for newly imported fish, closely monitoring fish populations, and implementing isolation units to prevent the spread of infections. By implementing these preventative measures, ornamental fish farmers can reduce the risk of parasitic infections and ensure the health and well-being of their fish populations. This, in turn, can lead to increased profitability and sustainability for their business. Overall, the current study aimed to conduct a clinical, histopathological, and phylogenetic analysis of the epibiont ciliated protozoan Epistylis wuhanensis and the copepod crustacean Lernaea cyprinacea in a freshwater ornamental fish farm in Iran. Furthermore, it provides valuable insights into the prevalence and impact of parasitic infections in ornamental fish farms and underscores the need for continued research and the development of effective preventative measures to address this issue. A total of 60 symptomatic freshwater ornamental fish, including 30 guppy (Poecilia reticulata) and 30 sailfin molly (Poecilia latipinna), were packed in polyethylene bags filled with oxygenated pond water and transported to the Ornamental Fish Clinic, Faculty of Veterinary Medicine, University of Tehran, for parasitological analysis. Following the clinical examination, histopathological analysis was performed on 10% NBF (neutral buffered formalin)-fixed samples from affected tissues, including the skin, skeletal muscle, and liver, to identify any pathological changes associated with the parasitic infections. Furthermore, the DNA was extracted from the 99% ethanol-fixed samples using a commercial DNA extraction tissue kit (SinaPure DNA, Iran), and PCR was performed using Peri18S-F1 (5'-ACC TGG TTG ATC CTG CCA GT-3') and Peri18S-R1 (5'-TGC AGG TTC ACC TAC GGA AA-3') (first reaction), and Peri18S-F2 (5'-CCG CGG TAA TTC CAG CTC-3') and Peri18S-R2 (5'-GAT CCC CTA ACT TTC GTT CTT GA-3') (second round) primers for the identified parasites. Finally, the PCR products were sequenced using Sanger dideoxy sequencing methods, and the resulting sequences were compared to sequences in the BLAST search program to provide a comprehensive picture of the current parasite-based disorder. The crustacean L. cyprinacea and the epibiont sessilid E. wuhanensis were identified in the examined ornamental guppy (6/30) and sailfin molly (6/30), with an overall parasitic prevalence of 20.00% (12/60). Ciliates were found in all tissue lesions but not in fish without lesions. A great number of the ciliated protozoan E. wuhanensis were found attached to the integumentary area of L. cyprinacea. Microscopically, oval to round granulomatous lesions were observed in cutaneous and skeletal muscles. Lymphoplasmacytic dermatitis and myositis were also observed. The crustacean L. cyprinacea serves as a mechanical vector for E. wuhanensis infection and spreads the disease in ornamental fish farming operations. For the first time in Iran, we successfully presented diagnostic morphological and molecular data on sessilids isolated from L. cyprinacea. Based on the findings of the current study, such parasitic infections may cause significant economic losses following invasion of the integument area of fish, eventually leading to death, if treatment is neglected or inadequate. Furthermore, the findings of the analysis were used to develop effective diagnostic approaches for the affected fish, as well as recommendations for improved health conditions to prevent future outbreaks of parasitic infections. However, further research is needed to determine the precise mechanisms of crustacean attachment and host-crustacean-peritrich protozoan interactions. Furthermore, the direct and indirect effects of various environmental factors on the emergence and spread of the current disease should be considered.

Effects of Escherichia coli strain Nissle 1917 on arsenic-challenged goldfish (Carassius auratus): histological evidence.

Arsenic (As) contamination in natural water resources has become a great disaster throughout the world posing serious health problems. The current study was performed to evaluate the protective effects of Escherichia coli strain Nissle 1917 (EcN) against As exposure in goldfish (Carassius auratus). Fish were fed three times a day with 4.00% of body weight of diet with different doses (0.00, 1.00 × 106, 1.00 × 107 and 1.00 × 108 CFU g-1) of EcN for 80 days and then, challenged with 20.00 mg L-1 As for 96 hr under stagnant flow. Physicochemical characteristics of the inlet water were temperature of 25.10 ± 0.70 ˚C, pH of 7.30 ± 0.20 and dissolved oxygen of 7.30 ± 0.30 mg L-1 and 50.00% of water was exchanged once a week. Afterwards, fish were euthanized with a clove oil solution (50.00 µL L-1) and tissues were dissected from each fish and immediately fixed in 10.00% buffered formalin. The histopathological results indicated that the supplemented EcN did not have any side effects on various organs. It was also observed that the damages to kidney, liver, gill and skin were pronounced in fish exposed to As. However, the histopathological damages induced by As in fish tissues were less pronounced in the EcN-treated groups compared to the fish fed with the basal diet. Lamellar blood congestion in gills and epidermal cells detachment from the skin surface as well as hepatocytes, enterocytes and tubular necrosis were reduced in treated groups. These findings indicate that EcN has the potential to ameliorate the As-induced organ toxicity.

Efficacy of dietary live or heat-killed Bacillus subtilis in goldfish (Carassius auratus) infected with Ichthyophthirius multifiliis.

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.

A rare case of ocular and testicular T-cell lymphoma in a hermaphrodite koi carp (Cyprinus carpio Linnaeus 1758): clinical, histopathological, and immunohistochemical study.

BACKGROUND: Lymphatic neoplasia may occur in various types, such as lymphoma, lymphosarcoma, lympholeukemia, and plasmacytoid leukemia. Lymphoma, defined as a malignant tumour of lymphoid tissue, has been found in a number of fish families including Esocidae and Salmonidae. However, the occurrence of lymphoma is rare in those belonging to the Cyprinidae. A final diagnosis of ocular and testicular T-cell lymphoma in the present study was based on the clinical signs, morphology, and texture of the tumour masses in the macroscopic and microscopic examinations. In addition, histopathological and immunohistochemical findings corresponded to T-cell lymphoma characteristics. CASE PRESENTATION: A 2-year-old hermaphrodite koi carp (Cyprinus carpio Linnaeus 1758) with a large ocular mass and severe exophthalmia in the right eye was referred to the Ornamental Fish Clinic in October 2020. Under anesthesia, enucleation was performed. 57 days after enucleation of the right eye, exophthalmia in the left eye was discovered. 221 days after surgery, the fish was discovered to be dead. At necropsy, a large soft tissue mass attached to the left testis was discovered. There were also small whitish nodules on the surface of the liver. Histopathology revealed a hypercellular ocular mass with scant connective tissue. The sections also revealed multifocal hemorrhages, round to ovoid neoplastic cells, mild-to-moderate anisokaryosis and anisocytosis, and mitotic figures. Basophilic neoplastic cells were found in blood vessels within the testicular mass, raising the possibility of systemic spread. The liver showed microscopic metastasis with morphologic similarities to the ocular and testicular tumors. The neoplastic cells infiltrating the left and right eyes as well as the testicular mass were immunohistochemically positive for CD3 but negative for CD20. The masses were diagnosed as T-cell lymphoma based on histopathological and immunohistochemical findings. CONCLUSIONS: This case report provides the first evidence of clinical, histopathological, morphological, and immunohistochemical findings of an ocular and testicular T-cell lymphoma in a hermaphrodite koi carp (Cyprinus carpio) in Iran.

Parasitic fauna of farmed freshwater ornamental sutchi catfish (Pangasiandon hypophthalmus) and silver dollar (Metynnis hypsauchen) in Alborz province, Iran.

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.

Phylogenetic analysis and genotyping of Iranian infectious haematopoietic necrosis virus (IHNV) of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene.

BACKGROUND: Infectious haematopoietic necrosis (IHN) is known as one of the most contagious systemic viral diseases in salmonids which can lead to significant mortality rates and negative impacts on the salmonid farming industry. Infectious haematopoietic necrosis virus (IHNV) was first detected in rainbow trout (Oncorhynchus mykiss) farms in Iran in 2003. OBJECTIVES: We conducted the present study to determine the detection of IHN genotypes in rainbow trout (O. mykiss) in farms in the central parts of Iran, using molecular and phylogenetic techniques. METHODS: Samples were collected from fries exhibiting clinical signs such as darkening of the skin, abdominal swelling, and loss of appetite. Phylogenetic analysis was performed by the neighbour-joining method, using MEGA 5.1 software. For phylogenetic analysis and genotyping of IHNV from central parts of Iran, the sequences of the glycoprotein gene were determined for two Iranian isolates (Jahad-UT1 and Jahad-UT2). RESULTS: Phylogenetic analysis revealed that the detected strains (Jahad-UT1 and Jahad-UT2 isolates) are closely related (97.23%-100%) to European isolates within genogroup 'E'. CONCLUSIONS: This finding indicates that Jahad-UT1 and Jahad-UT2 isolates have been widely transferred to Iran from European countries. Moreover, the nucleotide diversity of these Iranian isolates showed a close relationship with the North American and Asian isolates, although the Iranian isolates were collected from a smaller geographical area and within a shorter time period between 2014 and 2015.

Basal cell carcinoma in an albino pindani (Chindongo socolofi) and a cobalt-zebra (Maylandia callainos): Diagnostic imaging, clinical and histopathological study.

A female albino pindani (Chindongo socolofi) and female cobalt-zebra (Maylandia callainos) were referred to the Ornamental Fish Clinic with a soft tissue mass on their upper jaw. Also, during clinical examination, an unencapsulated intraoral soft tissue mass was observed in the cobalt-zebra cichlid. Digital radiography showed soft tissue masses and osteolytic lesions. Computed tomography (CT) imaging confirmed that osteolytic lesions occurred in skull bones of cobalt-zebra. Also, soft tissue mass invaded to the intraoral space. Histopathologically, a non-encapsulated, multilobulated and densely cellular neoplastic mass was identified. Extensive infiltration of neoplastic cells to the dermis and subcutaneous tissue was observed in both cases. The masses were composed of multiple coalescing nests, sheets, lobules and trabeculae of neoplastic cells within a fibrovascular stroma. The palisading basaloid neoplastic cells were markedly extended from the basal cells of the epidermis into the dermis and subcutis. The majority of the neoplastic cells had scant amounts of eosinophilic cytoplasm with indistinct margins, round to ovoid nuclei and typically a single small basophilic nucleolus. There was invasion of basaloid neoplastic cells into the bone tissue that caused osteolytic lesions. Mitotic figures were three to seven per high-power field. Epidermal basement membrane was periodic acid-Schiff (PAS) positive and neoplastic cell extension into the dermis was identified by the PAS staining. Fibrovascular stroma of the mass was also stained blue with Masson's trichrome. Based on histopathological and histochemical findings, the masses were diagnosed as infiltrative type of basal cell carcinoma (BCC).