Histopathology of mortality in cultured rainbow trout fry in some coldwater hatcheries and fish farms of Iran

Causes of the mortality of cultured rainbow trout fry in coldwater hatcheries and fish farms in Iran were investigated. Some 104 tissue specimens of liver, kidney, spleen, pancreas, intestine, and gill from 59 diseased fries from Mazandaran, Fars, Markazy and Kohgiloyely Boyerahmad provinces were collected for histopathological studies. Tissues were immediately fixed in 10% buffered formalin for at least 24 hours. The fixed tissues were processed in an automatic tissue processor using standard procedure. After processing, tissues were embedded into wax and 5 micron sections were prepared using a rotary microtome. The sections were stained using II & E staining method, and examined under compound microscope. The affected fries were dark in color, and showed exophthalmia, ascitis, erratic swimming, faecal casts in the anal area and lethargy. They also gathered near the outlet of the ponds. Microscopic examination of the tissues revealed histopathologic changes including congestion, inflammation of the basal membrane of secondary lamellae, hyperplasia, and fusion of secondary lamellae and clubbing in some cases in the gill. Congestion of blood vessels, degeneration of kidney cells, necrosis of hematopoitic tissue and tubules, increase of melanin pigments and infiltration of inflammatory cells were observed in kidney. In liver congestion of blood vessels, increase of fat in hepatocytes, congestion and dilation of sinusoids with increased monocytes, increase in melano-macrophage number, vacuolation of hepatocyctes and focal necrosis were seen. Bile duct neoplasia (cholangioma) was also present in some cases. Spleen showed congestion, hemosidrosis, increase in melano-macrophage centers and necrosis in some cases. The pancreas tissue showed congestion, degeneration and necrosis of ashlar cells and islets of Langerhans. Congestion of sub-mucusa layer, fusion of mucosa layer, necrosis and detaching of the mucoid columnar epithelium were observed in the intestine tissue. The clinical and histo-pathological examination results suggested that the causative agent of the fry disease is likely to be a virus with the signs similar to UN disease

Aetiologic Agents of Fry Mortality Syndrome in the Rainbow Trout (Oncorhynchus Mykiss) in Iran

An investigation was conducted in order to find out the etiological factors of Fry Mortality Syndrome (FMS) that causes serious economical loss in rainbow trout farms in Iran. In recent years obscure fry mortalities have been observed in many hatchery farms in Iran. It was reported that the rate of fry and juvenile mortality increased dramatically in some provinces e.g. 23 million fry were produced in hatchery centers of Chahar Mohal Bakhtiary province in 2002 but nearly 21 million fry (91.3%) in different stages of growth died before distribution to farmers. Also close to 23 million fry were produced in Mazandaran province, but 12 million fry equivalent to 52.12% of total fry production died mysteriously.This investigation was carried out with objectives of detecting and confirming the main causative agent that contribute to the occurrence of Fry Mortality Syndrome in Iran. During 32 months, from October of 2001 until May of 2004, 52 different hatchery centers and rearing farms of rainbow trout (Oncorhynchus mykiss) which were located in Tehran, Mazandaran, Guilan, Fras, Markazi, Kerman and Kohkiloyeh Boyerahmad provinces, were visited and various samples from affected farms were collected. Collected samples consisted of ovarian fluid, milts, eggs, eyed-eggs, larvae, fry < 1 g and 1-3 g as well as internal organs from adult fishes. A total of 2,107 samples were collected from farms in six provinces and were examined by five methods such as virology (410 samples), bacteriology (899 samples), serology (consisted of IFAT: 392 samples and ELISA: 44 samples), histopathology (160 samples) and hematology (202 samples). Some of the mentioned approaches such as fish cell culture, ELISA and IFAT techniques were set-up and optimized for the first time in Iran. The clinical signs of suspected fishes were darkening, exophthalmia, ascites, abnormal swimming and whirling. From 410 samples that of tissues inoculated on to cell cultures two samples showed CPE in EPC and BF-2 cell lines which were inoculated with ovarian fluid from broodstock obtained from hatchery farms in Mazandaran province. The CPE was similar to IHN virus induced. The CPE foci revealed dying cells congegrated as grape-like clusters (ballony performance with cytolysis).TEM findings in infected cells showed bullet-shaped particles having sizes of 130-180 nm in length and 65-70 nm in diameter. From the virion morphology it was suggested that observed particles were similar to Rhabdovirus. FAT examination revealed that all samples were examined with MAbs and PAbs against IPNV and VHSV were negative. On the other hand, two samples were positive when examined with MAbs and PAbs against IHNV. These smears were originated from samples that had showed CPE in EPC and BF-2 cell lines and bullet shaped particles in electron microscopy. ELISA findings (cut-off value, optical density and detectionlevel percentage) showed that IHNV had higher percentage of detection with 23.25% in comparison with other relevant viral diseases i.e. IPNV with 7.31% and VHSV with 14.29%. Results of histopathological study on the sampled fry revealed that the target tissues in the kidney, liver, spleen, hepatopancreas, intestine and gills showed different degree of tissue changes beginning from cell degeneration to complete necrosis. There were also renal blood vessels congestion, marked degenerative changes in posterior kidney with tubular necrosis and interstitial hematopoeitic tissue degeneration. In addition, interstitial degeneration and oedema in anterior portion of kidney, focal necrosis in the tubular area and several stages of cell necrosis in the hematopoeitic tissue were the most important histopathological changes seen in kidney tissues examined. Hepatopancreatic tissues also revealed marked changes such as congestion, atrophy and necrosis of pancreatic acinar cells and Islets of Langerhans. Spleen samples revealed spleenic congestion, severe necrosis, hemosiderosis and increased presence of melanomacrophage centers (MMC). Gills tissue in sampled fry showed hyperplasia, clubbing and fusion of lamellae. Hematological findings revealed that total white blood cell count, i.e. lymphocyte and neutrophil in investigated fish showed significant increased compared with the control fish (p< 0.05). On the contrary, all the samples showed a decreased in RBC, Hb and HCT values. In addition, MCHC and total protein plasma showed a marked decreased (p<0.05). In the blood serum components analysis, similarly it was revealed LDH and AST showed a significant decreased (p<0.05). In conclusion, with marked clinical signs, cell culture observation and TEM findings, ELISA and IFAT results, histopathology and hematological findings (blood and biochemical parameters) seen in the current investigation lead to possibility of a viral disease agent infection as the cause of fry mortality syndrome in the hatchery and rearing trout farms in Iran. From findings of the current study, it is concluded that IHN-like virus could be most probable etiologic of fry mortality syndrome in Iran. Key words: Fry Mortality Syndrome, Rainbow trout, Cell culture, TEM, ELISA, IFAT, Histopathology, Hematology, IHNV, IPNV, VHSV, Ira

Effect of Zataria multiflora essential oils on rainbow trout (Oncorhynchus mykiss) complement component activity and lysozyme

Influence of dietary administration of Zataria multiflora Bioss was evaluated on complement component activity and lysozyme of rainbow trout (Oncorhynchus mykiss) to determine stimulatory effect of essential oils. Fish weighting 100±10 g were fed with different doses of 10, 20 and 40 ml/kg food for a period of 10 days. Alternative Complement Activity (CH50%) and serum lysozyme activity were measured on days 1, 8, 15 and 29 after the essential oils administration. The obtained results showed that for first and eighth days of sampling, the complement component mean in treatment groups were significant higher than control group, while negligible difference is shown between treatment groups. For fifteenth and twenty ninth days of sampling, no significant difference was seen in the complement component mean among all examined groups. Also, the results of experiment show that for all of days of sampling, essential oils of Zataria multiflora Bioss have no effect on serum lysozyme activity

UV-B impacts on morphology and retina of Oncorhynchus mykiss larvae

The harmful effects of ultraviolet radiation on aquatic animals, due to ozone layer reduction, have been long studied in recent years. Exposure of Rainbow Trout larvae (Oncorhynchus mykiss) to Ultraviolet-B radiation (UV-B) at different doses (68.75 µw/cm^2 and 94.83 µw/cm^2 as the minimum and maximum dose of UV-B in natural environment respectively) for 15 minutes once a day in dark condition in comparison to control group (without any solar or UV) showed a wide variety of body abnormalities and eye damages. Body curvature, yolk sac edema, fin blistering, dwarfism, eye and head abnormalities as morphological malformations were revealed during the experiment, none of the malformations were observed in control group. Histopathological changes in retina such as irregular and discontinues pigmented epithelium, necrosis of photoreceptors and degeneration of nucleus layers confirmed the destructive effects of UV-B radiation in the eyes of Rainbow Trout. Such changes in larvae can be valid as bio-indicator for pollution and UV radiation and also introduce fishes as model for toxicological studies

Study of some hematological and biochemical parameters of rainbow trout (Oncorhynchus mykiss) fry in western part of Mazandaran province, Iran

This study was done to investigate of some hematological and biochemical factors of rainbow trout (Oncorhynchus mykiss ) fry in west of Mazandaran in Iran. About 50 pools of blood samples from diseased fry were collected within 30 months from November 2002 till March 2005 from three hatchery farms in western part of Mazandaran province. In addition 30 pools of blood samples as control group were collected randomly from mentioned farms. Each blood samples were examined for whole blood examination and blood enzymes measurement. It consist of total leukocytes (WBC) and erythrocytes counts (RBC), hemoglobin (Hb) content, hematocrits (PCV), leukocytes differential count and blood indices such as MCV, MCH and MCHC. Also blood serums were analysed for total protein (TP) and blood enzymes. All the calculations were made using the SPSS© and t-test statistical method. In hematological findings nine parameters were revealed significant differences (P<0.05) with control group in t-test. It consisted of total WBC, Lymph, Neut, Hb and HCT, MCHC, AST, DL and total protein plasma. Also in total white blood cell count, Lymph and Neut had significant differences as compared with the control fish (P<0.05). Also blood serum components analysis revealed that only LDH and AST amount showed obvious significant differences (P<0.05). Regarding to results it could be concluded that hematological and biochemical studies could be a valuable tool for prognosis and primary diagnosis in some infectious diseases. So it could be recommended for monitoring and surveillance programs in coldwater hatchery health status in Iran

The effect of different levels of Mentha piperita on some of the hematological, biochemical and immune parameters of Oncorhynchus mykiss

Due to the increased bacterial resistance to common antibiotics, there is a tendency towards using herbal extracts in order to increase the non-specific immune system. This study was conducted to evaluate the effects of different levels of Mentha piperita extract on hematological, biochemical and immune parameters of Oncorhynchus mykiss. For this purpose, fish with mean (±SD) weight of 32.2± 0.12 g were raised for 8 weeks in Vniro tanks (1800 l water), 80 fish to each tank and feeding with different levels of M. piperita (with concentrations of % 0, % 1, % 2.0 and % 3.0, three replicates were used for each concentration). At the end of the trial, blood samples were collected to determine some hematological, biochemical and immunity parameters in different groups and compared to one another. Results showed significant differences in RBC and WBC count, neutrophil percentage, haemoglobin (Hb), haematocrit (HCT) value, total protein (TP), IgM and lysozyme activity in fish fed M. piperita (especially with 2 and 3.0% concentrations) when compared with control group. The results suggest that M. piperita extract may enhance the non-specific immune system of O. mykiss. Thus, using this supplement especially at of 3.0% level as immunostimulants were recommended in O. mykiss diet

Effects of Chlorella vulgaris on blood and immunological parameters of Caspian Sea salmon (Salmo trutta caspius) fry exposed to Viral Nervous Necrosis (VNN) virus

In the present study, the effects of Chlorella vulgaris on blood and immunological parameters of Caspian salmon (Salmo trutta caspius) before and after exposure to Viral Nervous Necrosis (VNN) virus were examined. In this regard, four treatments in triplicate were chosen. Groups included one control and 3 treatments (T1, T2 and T3). Fish in control group, T1, T2 and T3 were fed diets supplemented with 0, 1×10^8, 2×10^7 and 3×10^6 chlorella/450 g of food respectively, for sixty days. In addition, a virus supernatant was prepared from infected wild golden grey mullet (Liza auratus) and used for virus challenge of S. trutta caspius. Virus was injected intraperitoneally and blood samples were collected before and 14 days after the challenge. Immunological (IgM, C3, C4, total protein, respiratory burst, albumin and lysozyme) and changes in blood parameters (RBC, WBC, Htc, Hb, MCH, MCHC and MCV) were also measured. Results showed that C. vulgaris could act as a natural immunestimulant. Also, the alteration trend in hematological and immunological parameters showed that experimental fish could be considered to be resistant to VNN virus after exposure and fish treated with C. vulgaris were more resistant in comparison to those in the control group. The dose used in T1 (1×10^8 chlorella/450 g food) was the most effective approach with significant differences

Pathogenicity of viral nervous necrosis virus for Guppy fish, Poecilia reticulata

The pathogenicity of a Nervous Necrosis Virus isolate obtained from naturally infected Golden grey mullet (Liza auratus) suffering serious mortalities in Iranian coastline water of the Caspian Sea was investigated for first time. An experimental infection has been performed using three groups, two experimental groups and one control group of Guppy (Poecilia reticulata) with mean weight 0.47±0.09 g, at temperature 25ºC. The infectious dosage (50 ml) with TCID50/ml= 10^4.25 for 2 hours in group 1 and 4 hours in group 2 developed the disease with immersion method. Clear clinical signs associated with significant mortality were observed since 15 dpi. Cumulative mortalities rose to 100% at 30 dpi. While in the control group no mortality was recorded. Virus was re-isolated on SSN-1 cell line that showing typical CPE developed after inoculation with tissues filtrate from dead fish. Histopathological examination of exposed fish, showed clear vacuolization in the granular layer of the retina and cerebellum. TEM micrographs revealed intracytoplasmic vacuoles in the retina of infected Guppy. IHC revealed the presence of viral antigens in the brain and retina. These results confirmed the pathogenicity of the NNV isolate obtained from Golden grey mullet suffering high mortality with regard to suggest that the same agent isolated from golden grey mullet is very likely the cause of the mortality observed in the same species

Isolation and phylogenic analysis of emerging new antibiotic resistant bacteria, Acinetobacter lwoffii, associated with mortality in farmed rainbow trout

Whereas it is well documented that the genus Acinetobacter is associated with antibiotic resistant bacteria and human clinical infections, there are very few studies to date that report it as an emerging new pathogen for fish. In the present study, Acinetobacter lwoffii was isolated from diseased rainbow trout (Oncorhynchus mykiss) from Iranian farms, in the Khuzestan Province by both biochemical tests and polymerase chain reaction (PCR)-based methods and further confirmed as the causative agent by infectivity experiment. Uni or bilateral exophthalmia, blackening of the skin, abdominal distension, hemorrhages around the mouth cavity and eyes, basal fine, skin, gills and in internal organs were the main clinical signs in the affected fish. Phylogenetic analysis of the 16S rRNA sequence of MD77 revealed a 99% homology with that of A. lwoffii (GenBank accession no. KR856323.1) from Iran. Histopathological changes of challenged rainbow trout including focal necrosis of liver cells (hepatocyte), pyknotic nucleus and karyolysis of hepatocyte, hyperemia, fatty acid composition change of liver, infiltration of inflammatory cells, sinusoidal dilatation and congestion. The drug resistance of isolates and minimal inhibitory concentration (MIC) was examined and showed that A. lwoffii was multiresistant to 8 of the antimicrobial agents tested

A case report of Enteritis Redmouth Disease in cultured rainbow trout (Oncorhynchus mykiss) in Kohkyloyeh va Boyerahmad Province in Iran

An unknown mortality was occurred in some ponds of a farm in an area of Kohgiloyeh-&- Boyerahmad province. Three ponds showed the highest mortality rate, about 5 to 20 dead fish were reported daily for 45 days. Afflicted fish have darkening in the skin and swimming near surface or at the floor of pond and swam unmatched with flock. Some fish showed hemorrhages around and within the oral cavity and exophthalmia occasionally. About 45 moribund rainbow trout were sampled for dissection and bacteriological examination. Kidney, liver and spleen of the fishes were examined for the detection and identification of bacteria causative agent of disease in rainbow trout. They cultivated aseptically on different media (Blood Agar, Tryptic soy Agar, TCBS Agar and MacConkey Agar). All fish have been infected and suffered septicemia. A type of gram's negative bacteria was responsible of infection and mix infection was not confirmed. The bacterium was being able to grow on Mac Conkey agar. It was a bacterium very similar with Yersinia ruckeri which induced infection. Detection was based on clinical finding and bacteriological laboratory results so using of some confirmation methods such as FAT or PCR would be recommended strongly