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.

Acute coenurosis in lambs.

Six 100-day-old mixed-breed lambs were examined in a farm with a semi-intensive system due to neurologic signs. Cachexia, bilateral blindness, stupor, severe drowsiness and lethargy with left and right movements of the head and neck were recorded after awakening and stimulation. Lambs died 10 days after the onset of the clinical signs. The lambs were necropsied, and after routine parasitology, bacteriology and histopathology, the occurrence of acute coenurosis was confirmed due to finding multiple cystic structures in the brain tissue. All lambs of the herd were treated with albendazole (orally, 25 mg/kg, two doses with an interval of 14 days). All shepherd dogs were treated with popantel (orally, one tablet/10 kg, two doses with an interval of 14 days). The affected lambs died despite this treatment. No new case of the disease was observed after the initiation of control measures. The present study shows the importance of preventive measure against coenurosis in a semi-intensive sheep farming system that includes implementing consistent parasite control programme in dogs being in contact with sheep.

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.

Acute cysticercosis caused by Cysticercus tenuicollis in lambs.

Twelve 30- to 75-day-old mixed breed lambs were examined in an intensive system because of sudden recumbency and death. Clinical examination revealed sudden recumbency, visceral pain, and auscultation of respiratory crackles. Lambs died shortly (between 30 minutes and 3 hours) after the onset of clinical signs. The lambs were necropsied, and after routine parasitology, bacteriology and histopathology procedures, the occurrence of acute cysticercosis caused by Cysticercus tenuicollis was confirmed. The use of the suspect infested feed (newly purchased starter concentrate) was discontinued and other lambs of the flock were treated with praziquantel (15 mg/kg, single dose, orally). After these actions, no new cases were observed. The present study showed the importance of preventive measures against cysticercosis in intensive sheep farming systems which include proper storage of feed, preventing feed and environmental access by potential definitive hosts, and implementing consistent parasite control programs in dogs that are in contact with sheep.

The effect of subacute co-exposure to carbon tetrachloride and diclofenac on the liver of male wistar rats.

Carbon tetrachloride (CCl4) is a potent liver toxin. Diclofenac (Dic), leading adverse effects on the liver, is used among the employees of the industries that use CCl4. The increased use of CCl4 and Dic in industrial workers has prompted us to investigate their synergistic effect on the liver using male Wistar rats as a model. Male Wistar rats were divided into seven groups (n = 6), and the exposure was by intraperitoneal injection for 14 days as follows. Group 1: Control, 2: Olive oil, 3: CCl4 (0.8 mL/kg/day (3 times per week)), 4: Normal saline, 5: Dic (1.5 mg/kg/day per day), 6: Olive oil with normal saline, 7: CCl4 (0.8 mL/kg/day (3 times per week)) and Dic (1.5 mg/kg/day daily). At the end of day 14, the heart blood was collected to measure the liver enzymes, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), blood alkaline phosphatase (ALP), albumin (ALB), direct bilirubin, and total bilirubin. A pathologist examined the liver tissue. Prism software was used to analyze data using ANOVA and Tukey statistical tests. ALT, AST, ALP, and Total Bilirubin enzymes were increased significantly in the co-administered CCl4 and Dic group, while the ALB levels decreased (p < 0.05). The histological findings reported liver necrosis, focal hemorrhage, adipose tissue change, and lymphocytic portal hepatitis. In conclusion, using Dic while exposed to CCl4 may exacerbate hepatotoxicity in rats. Therefore, it is suggested that more severe restrictions and safety regulations be placed on using CCl4 in the industry, and caution is advised to these industry workers to use Diclofenac.

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.

Histological observations on aural fibrosarcoma in a Holstein cow.

Fibrosarcomas occur as a mesenchymal tumour of malignant fibroblasts in a collagen background and are usually found in the female genital organs and rarely involve the skin. A 5-year-old female Holstein cow with a raised mass at the base of right ear was referred. On gross examination, the mass was approximately 13.00 × 10.00 × 7.00 cm in size. Finally, complete surgical removal was selected. The mass was encapsulated and the dermis was expanded by spindle-shaped to polygonal neoplastic cells. These cells were arranged in interwoven pattern. Mitotic figures were infrequent. Masson's trichrome demonstrated the positive and blue staining of collagen. Immunohistochemically, the sections were uniformly positive for Vimentin and negative for Desmin, SMA and GFAP. A well-differentiated fibrosarcoma was diagnosed based on histopathological features. Surgical excision is the treatment of choice for this neoplasm. In the present case, surgery was also performed successfully and no new growth of the mass was observed 4 months following the surgical procedures. To our knowledge, this is the first report of well-differentiated fibrosarcoma in a Holstein cow.

The immunomodulatory effect of minocycline on gene expression of inflammation related cytokines in lipopolysaccharide-treated human peripheral blood mononuclear cells.

To evaluate the immunomodulatory effect of minocycline, the present study was carried out on the gene expression of toll-like receptor type-4 (TLR4) and some pro-inflammatory (IL-1ß, IL-6) and anti-inflammatory cytokines (IL-10) associated with lipopolysaccharide (LPS) -induced inflammation in human peripheral blood mononuclear cells (PBMCs). The PBMCs were collected and then 5.4 × 106 PBMCs/mL were used in eight groups as follows: control group (only media), LPS group (only LPS), methylprednisolone (Pred) group (LPS plus Pred), meloxicam (Melo) group (LPS plus Melo), three minocycline groups [M1, M5 and M25] (LPS plus 1, 5, and 25 µg/mL minocycline, respectively) and minocycline control (MC) group (5 µg/mL minocycline). After incubation for 24 h, the PBMCs were subjected to quantitative PCR assays. Gene expression levels of TLR4 were not changed in any groups. The IL-1ß levels were increased in the LPS group but the increases were much more intense in the other groups except Pred group. Compared with control group, IL-6 levels increased significantly in Melo, M1 and M25 groups. Significant increases of IL-10 levels were also observed in Melo, M25 and MC groups. It can be concluded that minocycline had dual pro- and anti-inflammatory activities with potential clinical immunomodulatory effects.

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).

Occurrence of myxoma in a bearded dragon (Pogona vitticeps): Surgical and histopathological studies.

Myxoma is a benign mesenchymal tumor of skin and soft connective tissues considered to be of fibroblastic origin. A female bearded dragon (Pogona vitticeps) with a raised mass on the left side of the body was referred to the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. The mass had become evident and grew more extensive within the previous 11-month period. The dragon was anesthetized with 5.00% isoflurane, and the mass was removed surgically. Tissue samples of the mass were fixed in 10.00% neutral buffered formalin, sectioned and stained with Hematoxylin and Eosin (H&E), Alcian blue (pH: 2.50), and periodic acid Schiff (PAS) staining techniques. The patient was administered meloxicam, lincomycin, and marbofloxacin. Histopathologically, the capsulated mass was composed of myxoid structure. Stellate-to-reticular cells distributed in a mucinous stroma containing few tiny blood vessels were seen. Myxoid cells displayed a PAS-positive cytoplasm. The myxomatous matrix was alcianophilic at the pH of 2.50. To our knowledge, this is the first report of surgery, treatment, and histopathological study of myxoma in a bearded dragon (P. vitticeps). The sutures were also removed 12 days after the operation. No new growth of the mass was observed three months following the surgical procedures.

Detection of Carp pox virus (CyHV-1) from koi (Cyprinus carpio L.) in Iran; clinico-pathological and molecular characterization.

Cyprinid herpesvirus 1 (CyHV-1) is the causative agent of carp pox characterized by epidermal papillomas in common carp and other cyprinids. In this study, we identified CyHV-1 in koi (Cyprinus carpio) from Iran in 2017 and 2019, showing clinical signs of the carp pox disease. Histopathology showed severe epidermal hyperplasia and the absence of club and goblet cells. Degenerative changes, including spongiosis and single-cell necrosis, were also observed. Keratinocyte dysplasia and a moderate lymphocytic infiltration were observed within the epidermis. PCR of the extracted DNA from skin lesions of affected koi from both outbreaks showed CyHV-1 specific TK amplicons, with high sequence identity (above 99%) among themselves and with other CyHV-1 isolates belong to Cluster I, as well as show 97% similarity to Cluster II isolates. To the best of our knowledge, this is the first report of Carp pox disease (CyHV-1) of koi in Iran and the Middle East.

Cyprinid herpesvirus 3 (CyHV-3) transmission and outbreaks in Iran: Detection and characterization in farmed common carp.

This study shows the presence of Cyprinid Herpesvirus-3 (CyHV-3) in common carp (Cyprinus carpio) from Iranian carp farms with cumulative mortality up to 80% during 2015-2016. Pathological signs of disease such as gill necrosis, sunken eyes, and increased slime secretion on the skin and fins were observed in affected fish. The extensive fusion of secondary lamellae with necrotic cells, margination of chromatin, and formation of intranuclear inclusion bodies in gill tissues were also observed by histopathological examination. Most tubular epithelial cells and some hematopoietic cells showed intranuclear inclusion bodies in the kidney. The Iranian CyHV-3 isolates showed identity with Asian strains, and displayed the I++ II+ allele of the Asian lineage, as revealed by sequence analysis of the TK gene, Marker I, and Marker II. The detected isolates were also similar to those detected from koi in the same region of Iran, suggesting the probable transmission of CyHV-3 from ornamental to farmed cyprinids. This represents the first report of CyHV-3 from Iranian farmed common carp to the best of our knowledge.

Cutaneous xanthogranuloma associated with Klebsiella pneumoniea in a budgerigar (Melopsittacus undulatus).

Budgerigar is a common name for a colorful Australian native bird belonging to the Melopsittacus undulatus species. It is a very familiar pet around the world and its breeding has been grown in Iran. This study was conducted on a 2-year-old budgerigar with a nodular mass on the left wing. Physical examination revealed a firm, round and well-circumscribed mass approximately 1.70 cm in diameter. Radiographs showed a soft tissue mass with no involvement of bony structures. Fine needle aspiration was performed and the sample was cultured. In cultural examination, Klebsiella spp. were isolated in pure culture. Genus and species of the bacteria were confirmed using multiplex polymerase chain reaction. The mass was surgically excised and it was mainly composed of numerous, large lipid-laden macrophages containing abundant vacuolated cytoplasm, extracellular acicular cholesterol clefts and large number of multinucleated giant cells (especially multinucleated Touton giant cells) in the dermis. Finally, a diagnosis of cutaneous xanthogranuloma was made based on histopathological findings.

Renal Cystic Adenocarcinoma in a Flowerhorn Cichlid with Metastatic Involvement of the Spleen.

A 480-g flowerhorn cichlid (an ornamental hybrid) with severe bilateral abdominal swelling, bulla-like structures on the skin, bilateral exophthalmia, and a prolapsed intestine was presented. Radiographs showed compression of the posterior part of the swim bladder and abdominal distention. Ultrasonography of visceral organs revealed a heterogeneous mass with hypoechoic to anechoic polycystic parenchyma and free fluid in the abdominal cavity. At necropsy, free fluid in the abdominal cavity and a large polycystic mass originating from the posterior kidney were observed. Histologically, the mass was composed of more cystic growth of tubules. The renal architecture was replaced by tubules, often irregular in shape, lined by simple to lightly stratified layers of neoplastic and pleomorphic cuboidal to columnar epithelial cells and the absence of glomeruli. Birefringent crystals were observed with polarized light within the lumen of some tubules. The apical border of the neoplastic cells was periodic acid-Schiff positive. Immunohistochemically, the neoplastic cells were positive for cytokeratin AE1/AE3 and proliferating cell nuclear antigen and were negative for p53 (tumor suppressor protein). Microscopic metastasis was seen in the spleen. The metastatic tumor was classified as a cystic adenocarcinoma of the kidney, originating from the proximal tubules. Received October 7, 2016; accepted June 18, 2017.

Infectious hematopoietic necrosis virus (IHNV) outbreak in farmed rainbow trout in Iran: Viral isolation, pathological findings, molecular confirmation, and genetic analysis.

Infectious hematopoietic necrosis virus (IHNV) is the etiological agent of a contagious disease (IHN) mainly in salmonid fish. In the present study, we isolated and identified IHNV in trout fry from Iranian trout farms with unexplained high mortality in 2016. The affected fry showed cumulative mortality of 90% with the gross pathological signs including exophthalmia and hemorrhage of the eye, skin darkening, abdominal distension, ulceration of the snout, and the visceral pallor and yellowish fluid in the intestine. Histopathological examination revealed marked necrosis in the anterior kidney, liver and spleen with the intracytoplasmic inclusion bodies in the liver sections. Also, intranuclear inclusion body and marginated chromatin were observable in the hematopoietic cells of the kidney. The homogenates tissues of infected fry induced IHNV-positive cytopathic effects (CPE) in EPC cells and confirmed by RT-PCR reactions and sequencing. Phylogenetic analysis revealed the Iranian IHNV isolates belonged to the European (E) genogroup with 100% identity to some Italian isolates. This is the first report of IHNV infection in farmed trout fry in Iran describing the viral isolation, clinical symptoms, histopathological findings, molecular confirmation, and genetic analysis suggestion of the specific country of origin.

Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor.

Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency.