Combined effects of ammonia-N exposure and salinity changes on hematological and serum biochemical factors and thyroid hormones in Nile tilapia (Oreochromis niloticus).

The aim of this research was to evaluate the interaction effects of ammonia-N levels and salinity on hematological and serum biochemical parameters in Nile tilapia (Oreochromis niloticus). The fish were randomly divided into 12 treatments including the levels of salinity (0, 4, 8 and 12 ppt) and 0, 50% of LC50-96 h of ammonia-N and 30% of LC50-96 h of ammonia-N in a factorial design (4 salinity levels x 3 ammonia levels). Hemoglobin value in all treatments, except for salinity treatments, namely 2, 3, 4, showed a significant decrease than the control (0 ppt and no poisoning). Also, red blood cells in treatment ammonia-N levels were significantly less than the control. Serum protein concentration, in treatments 9 (50% of LC50-96 h of ammonia-N) and 5 and also with increasing salinity (treatments 2, 3 and 4) had a significant decrease compared to the control. There is a significant increase in serum glucose, cortisol, ammonia and urea levels in 50% and 30% of LC50-96 h of ammonia-N treatments compared to the control, meanwhile these parameters were significantly increased with increasing salinity. Serum thyroid stimulating hormone (TSH), T3 and T4 levels in acute and sub-acute ammonia-N treatments were significantly lower than the control. Moreover, with increasing salinity in 50% and 30% of LC50-96 h of ammonia-N treatments, TSH showed a decreasing pattern. According to the results, fluctuations in blood biochemical factors, increase of stress and decrease of thyroid hormones show that the salinity, ammonia, and their interaction caused adverse effects on fish health during the 96 h of testing.

Effect of killed autogenous polyvalent vaccines against Vibrio harveyi, V. alginolyticus and Streptococcus iniae on survival and immunogenicity of Asian seabass (Latescalcarifer).

Vibriosis and Streptococcosis are the most important bacterial diseases that infect Asian seabass (Lates calcarifer) in various stages of its life cycle. Vaccination is a cost-effective strategy to prevent the occurrence of infectious diseases and increase sustainability in the aquaculture industry. This study was aimed to develop and evaluate a killed polyvalent vaccine against Vibrio harveyi, V. alginolyticus and Streptococcus iniae, delivered by intraperitoneal injection in Asian seabass. The fish were divided into three groups with 60 fish in triplicate: I) a control group injected with phosphate-buffered saline (PBS), II) a group vaccinated by polyvalent vaccine (V. alginolyticus + V. harveyi + S. iniae) and III) a group vaccinated with the same polyvalent vaccine plus an oral booster. Immunological parameters and antibody titer were measured before and at three, five-, and eight-weeks post-vaccination. The efficacy of the killed vaccine was assessed five weeks post-vaccination by challenging with each isolate separately. The vaccinated groups had higher survival rate than control group. The highest relative percentage survival rate, 85.71 ± 3.57 % was observed in group III when challenged with V. harveyi. The vaccinated fish produced significantly higher antibody titers against V. alginolyticus, V. harveyi and S. iniae than the control group (P < 0.05). Non-specific immune parameters were significantly enhanced in the vaccinated groups, especially group III, compared to the control. The results demonstrated that the administration of a killed polyvalent vaccine can effectively protect Asian seabass against V. alginolyticus, V. harveyi and S. iniae.

Detection and distribution of virulence genes in Aeromonas hydrophila isolates causing infection in cultured carps.

Aeromonas hydrophila is a bacterium associated with many diseases and disorders such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. It bears several virulence factors including type III secretion system (T3SS), aerolysin, cytolytic enterotoxin and enzymes (e.g., hemolysins, lipase) that seem to play an important role in its pathogenesis. Detection of virulence markers by polymerase chain reaction (PCR) is a key procedure in defining the patho-genic ability of pathogenic bacteria and preparing a vaccine for its treatment. In this sense, this study was aimed to determine the frequency of virulence genes in isolates obtained from infected cultured carps in Khuzestan province. Out of 200 moribund carps with septicemic symptoms, 125 isolates were belonged to the motile aeromonads and 59 isolates were identified as A. hydrophila by biochemical methods. Finally, using PCR analysis, 31 isolates were identified as A. hydrophila. Five virulence genes were detected in these isolates including hemolysin, aerolysin, cytolytic enterotoxin and T3SS (aopB and ascV) by specific primers. Results showed that 23 (74.19%), 18 (58.06%), 16 (51.61%), 13 (41.63%) and 10 (32.25%) isolates possessed cytolytic enterotoxin, hemolysin, aerolysin, and T3SS genes, respectively. The results of the present study showed that among 31 isolates, only five isolates had all of dominant virulence genes. Thirteen other isolates had genotypes including hlyA +, aerA + , and act + . The remaining isolates had at least one virulence gene. This study showed that determination of the virulence genes by PCR can be a reliable method to identify a potential pathogenic Aeromonad strain.

Severe abdominal distention due to ovarian cysts in a parrot fish (Paraneetroplus synspilus).

In autumn of 2019 a three years old female parrot fish was referred to the veterinary hospital for diagnosis and treatment. The prominent symptom was inappetence and a large mass or unilateral distention in the abdominal cavity. Ultrasonography and radiology imaging with contrast media were done to evaluate the abdominal cavity. According to presumptive diagnosis by imaging, the fluid-filled mass was aspirated by a sterile syringe. No bacteria or other microorganisms were seen in the fluid using microscopy and culturing of the fluid on trypticase soy agar. The parrot fish died after three days because of imbalance and inappetence. Two fluid-filled masses were seen in necropsy. The ovarian structure of the cyst was proved based on the anatomical position, histopathology, ultrasonography and radiology of the cyst tissue. It could be concluded that prompt diagnosis and therapy of ovarian cyst may be necessary for saving the life of the fish.

Achlya infection in an Oscar (Astronotus ocellatus) with typical symptoms of saprolegniosis.

Saprolegniosis is one of the most frequent diseases in the ornamental fish industry that may cause by Achlya spp. There is no report of fungal infection caused by Achlya spp. in Oscar (Astronotus ocellatus) in Iran. An Oscar fish was referred to the Veterinary Hospital, Shahid Chamran University of Ahvaz, Ahvaz, Iran with saprolegniosis symptoms including loss of appetite and yellow to gray cottony mass on the skin of flank and caudal peduncle. The infected area of the fish body was investigated by wet smear. After observing broad aseptate hyphae (approximate width 10-15 µm), the infected skin was inoculated on Sabouraud dextrose agar and incubated at 30 ˚C for seven days. The large fungi colony was appeared on the agar and the isolated fungi were detected as Achlya spp. after examination by wet and stained dry smears according to the morphology of hypha, spores, and zoosporangium. It is the first report of cutaneous saprolegniosis in Oscar caused by Achlya spp. in Iran.

Florfenicol pharmacokinetics following intravenous and oral administrations and its elimination after oral and bath administrations in common carp (Cyprinus carpio).

The aim of this study was to evaluate pharmacokinetic profiles of florfenicol after a single dose of intravenous (5.00 mg kg-1 body weight) and oral (40.00 mg kg-1 body weight) administrations in common carp (Cyprinus carpio). The residue depletion of florfenicol was also investigated after oral administration (10.00 mg kg-1 body weight) and bath treatment (5.00 mg L-1) for 10 consecutive days. Pharmacokinetics of florfenicol in plasma after a single dose administration, at 10 time points (0.50, 1, 2, 4, 8, 12, 24, 72, 120 and 168 hr) and florfenicol concentrations in tissues (plasma, liver and muscle) at three time points (1, 7 and 14 days) after 10 consecutive days, were analyzed by high performance liquid chromatography. The peak concentration of florfenicol was 137.02 ng mL-1 and the time to reach peak concentration in plasma was two hr. The elimination half-lives, the volume of distribution at steady state and total body clearance were estimated as 21.40 hr, 0.30 and 0.03 L hr-1, respectively. After drug administration for 10 days, it's concentration in plasma and muscle in oral treatment was significantly more than bath treatment in all days. Drug concentrations in the liver after bath treatment were significantly higher for a shorter period than the concentration in the oral treatment, indicating that higher levels of florfenicol for a longer period can be achieved in the tissues after oral drug administration. According to pharmacokinetic results, florfenicol may be a suitable candidate for the treatment of common bacterial infections in common carp farming.

Metabolic capacities of common carp (Cyprinus carpio) following combined exposures to copper and environmental hypoxia.

In aquatic ecosystems, a decline in water O2 level is the main factor that can release heavy metal ions from top sediment layer. Therefore, hypoxia in turn, and in association with heavy metals might provide undesirable environment and impairs physiological functions of aquatic animals. To address this, metabolic capacities, including standard metabolic rate (SMR), maximum metabolic rate (MMR), aerobic scope (AS) and factorial aerobic scope (FAS) of common carp were determined following exposures to different levels of water-borne Cu(2+) as well as hypoxia. Treatments for Cu(2+) were included: 100% (acute), 50% (sub-lethal) and 10% (chronic) of LC50-96h for immediately, 24h and 7 days exposures respectively. Hypoxia treatments were assigned as acute for immediately, sub-lethal for 24h and chronic for 7 days. Combined effects of treatments were also considered as acute Cu(2+)+hypoxia, sub-lethal Cu(2+)+hypoxia and chronic Cu(2+)+hypoxia. While SMR of carp was reduced by chronic hypoxia, significant (P<0.05) increase was observed during acute hypoxia, as compared with control. The MMR and AS were significantly reduced (P<0.05) following all hypoxia treatments. The acute and chronic Cu(2+) treatments showed significant (P<0.05) increases in SMR and MMR values. All acute and sub-lethal combined treatments showed significant (P<0.05) reductions in SMR, MMR and AS values, whilst chronic combined treatments showed generally increasing trends for MMR and AS. PCrit was relatively reduced following all treatments except for acute and sub-lethal Cu(2+)-treated fish that showed higher value (P<0.05) and no change respectively. Although all Cu(2+) treatments increased the number of mucus cell, hypoxia treatments did not show any remarkable differences when compared with control group. In general, the results of present study reveal that hypoxia acts as limiting stressor whilst Cu(2+) do act as loading stressors in the case of common carp metabolism. The interactive exposures mostly showing a synergist effect in all metabolic capacities with an exception for chronic treatments.

Occurrence and Intensity of Anisakid Nematode Larvae in Some Commercially Important Fish Species in Persian Gulf.

BACKGROUND: Anisakid nematodes are common parasites of fish, mammals, fish-eating birds, and reptiles with a worldwide distribution, causing diseases in human, fish and important economic losses. METHODS: A preliminary epidemiological study was carried out on Anisakid nematodes larvae in some commercially important fish species to evaluate the anisakid nematode larvae from greater lizardfish, (Saurida tumbil), Japanese thread fin bream (Nemipterus japonicus), crocodile longtom (Tylosurus crocodilus crocodiles) and longfin trevally (Carangoides armatus) from the Persian Gulf of Iran. RESULTS: The collected larvae were identified mainly as the third larval stage (L3) of Hysterothylacium larval type A, B and C, Anisakis sp., Raphidascaris sp., Pseudoterranova sp. and Philometra sp. (Nematoda: Philometridae). The prevalence of Anisakid larvae infection of examined fishes was 97.2% in N. japonicus, 90.3% in S. tumbil, 20.5% in crocodile longtom and 5.5% in longfin trevally. Anisakis type III for the first time was different from Anisakis type I and Anisakis type II. DISCUSSION: Zoonotic anisakids by high prevalence in edible fish could be a health hazard for people. So health practices should be considered in these areas.

Protective and immunogenic effects of Escherichia coli-expressed infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in rainbow trout.

Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae which causes significant losses in the aquaculture industry. To develop a recombinant vaccine for IPNV, a cDNA construct of IPNV VP2-VP3 fusion gene was prepared and cloned into an Escherichia coli (E. coli) expression vector (pET-26b) to obtain recombinant protein products. A study was conducted to determine the antibody responses and protective capacity of this recombinant vaccine expressing VP2-VP3 fusion protein. Subsequently, juvenile rainbow trout were inoculated by injecting purified recombinant IPNV VP2-VP3 proteins, followed by challenge with virulent IPNV in rainbow trout. Our results demonstrate that recombinant E. coli derived VP2-VP3 fusion protein induced a strong and significantly (P < 0.05) higher IgM antibody response in serum samples compared to control groups. Following intraperitoneal challenge, the relative percent survival (RPS) rate of survivors was 83% for the vaccinated group. Statistical analysis of IgM levels indicated that immunogenicity of recombinant VP2-VP3 protein, combined with adjuvant, was much higher than any other groups of rainbow trout challenged with virulent IPNV. This result was confirmed by measuring the viral loads of IPNV in immunized rainbow trout which was drastically reduced, as analyzed by real-time RT-PCR. In summary, we demonstrate that E. coli-expressed IPNV VP2-VP3 injectable vaccine is highly immunogenic and protective against IPNV infection.

Evaluation of the bioaccumulation of heavy metals in white shrimp (Litopenaeus vannamei) along the Persian Gulf coast.

The concentrations of heavy metals in Persian Gulf are low, but petrochemical and refinery activities have caused an increase in heavy metal wastes, especially in coastal regions. The present study was done to determine the bioaccumulation of heavy metals in the muscle of white shrimp (Litopenaeus vannamei) using flame atomic absorption spectrophotometry. The experiment was conducted in four important coastal regions of the Persian Gulf: Bushehr, Deylam, Mahshahr, and Abadan. Amounts of seven heavy metals such as Copper (Cu), Iron (Fe), Lead (Pb), Zinc (Zn), Nickel (Ni), Cadmium (Cd), and Cobalt (Co), were measured as µg/g heavy metal in dry weight in the muscle of white shrimp from the afore-mentioned regions during 2011. This study revealed information that the primary risk for human health and the marine life chain was lead in the muscles of white shrimp in Mahshahr, where intense petrochemical and refinery activities are conducted. Concentrations of other heavy metals were lower than world standards.

Effect of water salinity on total protein and electrophoretic pattern of serum proteins of grass carp, Ctenopharyngodon idella.

In this study the effects of water salinity on serum total protein and its components in grass carp were investigated. The aim of this study was to determine the effect of salinity tolerance of fish on total serum protein level and its components as an indicator of liver and kidney activity. One hundred and twenty grass carp were divided into four groups, randomly. The first three groups were reared in concentration of 4, 8 and 12 g L(-1) of salt solution, respectively, and the fourth group was reared in freshwater and served as control. After 3 weeks, blood samples were collected and after harvesting the blood serum, serum total protein and protein components were measured with Biuret and electrophoresis methods, respectively. Results showed that mean value of serum total protein in the control and three salinities groups were 2.75, 3.28, 2.90 and 3.13 g dL(-1), respectively. Five fractions of serum protein were electrophoretically observed as: albumin (Alb), alpha-1 globulin (α1-glu), alpha-2 globulin (α2-glu), beta globulin (ß-glu) and gamma globulin (γ-glu). There were not any significant differences between the average mean of serum total protein of experimental and control groups (p > 0.05). However, Alb, α1-glu and ß-glu levels in the experimental groups were significantly higher than in the control group (p < 0.05). The average of α2-glu and γ-glu revealed no significant difference between the experimental groups (p > 0.05). In conclusion, our results showed that increasing water salinity could have a significant effect on Alb, α1-glu and ß-glu levels but not on total serum protein in grass carp.

Sequence analysis of infectious pancreatic necrosis virus isolated from Iranian reared rainbow trout (Oncorhynchus mykiss) in 2012.

Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly contagious disease that affects many species of fish and shellfish. This virus causes economically significant diseases of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in Iran, which is often associated with the transmission of pathogens from European resources. In this study, moribund rainbow trout fry samples were collected during an outbreak of IPNV in three different fish farms in north and west provinces of Iran in 2012; and we investigated the full genome sequence of Iranian IPNV and compared it with previously identified IPNV sequences. The sequences of different structural and nonstructural-protein genes were compared to those of other aquatic birnaviruses sequenced to date. Our results show that the Iranian isolate falls within genogroup 5, serotype A2 strain SP, having 99% identity with the strain 1146 from Spain. These results suggest that the Iranian isolate may have originated from Europe.

Study on effect of salinity level of water on electrocardiogram and some of blood serum minerals in grass carp, Ctenopharyngodon idella.

In this study the effects of salinity on the electrocardiogram and some of blood serum minerals in grass carp were investigated. For this purpose, grass carp were exposed to concentrations of 4, 8 and 12 g L(-1) salinities and electrocardiogram of all fish was recorded. Blood samples were also collected from all fish and serum minerals were measured. Our results indicated that mineral level in the studied groups was significantly different. The average of heart rate per minute in control group and 4, 8 and 12 g L(-1) were 10.15, 10.06, 12.17 and 7.79 beat per min, respectively. In 8 g L(-1) group only the average of QT and ST segment decreased significantly in comparison with the control group (p < 0.05). In 12 g L(-1) group the average of heart rate per minute decreased significantly in comparison with 8 g L(-1) group (p < 0.05) and no difference in the average of heart rate per minute was observed in all groups. The average of RR, PT and ST segment in the 12 g L(-1) group were significantly more than the other groups (p < 0.05). The results showed that water salinity level increased to concentrations above 8 g L(-1) can have significant effect on the electrocardiogram and mineral level of blood serum in grass carp. These changes are probably can be considered as one of the causes of impairment of health and death of this fish species in the salinities more than 8 g L(-1) due to ion imbalance and cardiovascular disorders.

Effect of salinity level on TSH and thyroid hormones of grass carp, Ctenophayngodon idella.

Thyroid hormones (T3, T4) have marked effect on body metabolism and in controlling osmoregulation activity in fish. The aim of this study was to determine the effect of water salinity changes on thyroid hormones level and thyroid-stimulating hormone (TSH) of grass carp. For this purpose 120 grass carp were divided randomly in to four groups (10 fish in each group and three replicates per treatment). Three groups were held in three different salinities at concentrations of 4, 8 and 12 g L(-1). The fourth group was reared in fresh water and considered as control. After three weeks blood samples were collected from the caudal peduncle vein. Then serum was separated and serum thyroid hormones and TSH were measured by LISA on Microwell plates. Our results indicated that the average of T3 levels in 4, 8 and 12 g L(-1) groups were 0.43 ± 0.11, 0.22 ± 0.04 and 0.21 ± 0.04 µg dL(-1), respectively. T3 levels in all experimental groups were significantly lower than those of control group (p < 0.05). Serum T4 level in 4, 8 and 12 g L(-1) groups were 0.29 ± 62955/40.06, 0.24 ± 43129/50.05 and 2.85 7958/± 05768/40.55 µg dL(-1), respectively. Thyroxine level was significantly higher only in 12 g L(-1) group in comparison with the control and other experimental groups (p < 0.05). Thyroxine level in other groups had not any significant difference with the control group (p > 0.05). The level of TSH in salinities of 4 and 8 g L(-1) groups was significantly higher than that of control group (p < 0.05). The results showed that increasing water salinity can have significant effect on thyroid activity by decreasing T3 and increasing T4 level in serum of grass carp in experimental condition.