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Histopathological examinations of the gills are very useful for the evaluation of the effect of toxic substances on fish and are also valuable in diagnosing certain fish diseases and the study of their pathogenesis. Gill external location and its intimate contact with water means that they are liable to damage by any irritant materials dissolved and suspended in the water and are favored infection sites for viruses, bacteria and parasites. Gill gross pathology and histopathological changes caused by various factors were presented and analyzed in respect to certain toxic substances and some pathological microorganism.
Studies to identify the koi herpes virus using the PCR are continuously developed and introduced into routine diagnostics. Well-selected ways of storing tissues that are later used for DNA extraction and for the amplification reaction are critical points in methods using molecular biology techniques. The objective of the studies was to determine the impact of the fixating properties of the two most frequently used organic compounds, ethanol 96% and 80%, and isopropanol, on the stability of the nucleic acid isolated and on the preservation of the koi herpes virus in selected tissues and organs of carps. The experimental fish were infected via a bath in a small 20 l aquarium, where 5 ml of a KHV reference strain suspension had been added to the water. The presence and stability of the total DNA isolated from tissues and organs (skin, brain, kidney, gills) was checked using PCR with TK primers designed by Bercovier, and a product of 409 bp was obtained. Primers amplifying a gene fragment of beta-actin, which is found in smooth muscles and non-muscle cells of fish, were used as isolation control in multiplex PCR. On the basis of the studies it was observed that just on the day the samples were taken from the internal organs of fish and a month later, when they were collected from the archived organs, a product of 409 bp and 259 bp was obtained in all the samples analysed. The best organic fixative of organs and tissues sampled for molecular tests to identify KHV is ethanol 96%, even after half a year of archiving. Ethanol 80% and isopropanol also ensure sufficient fixation for up to a month. The most stable virus DNA is isolated from fish skin and gills.
The KHV free carp were experimentally infected with CyHV-3 suspension by immersion at temperatures of 18°C±1 and 25°C±1. Skin, brain, gills, liver, spleen, kidneys, and leukocytes were investigated for the presence of KHV DNA, and sera for the presence of antibodies against the virus. Similar investigations were performed with non-infected carp, designed as control groups. The results of KHV DNA detection in the infected carp kept at 25°C ±1 were compared with the results of the virus isolation onto common carp brain cells using leukocyte co-cultivation method and results of specific antibody detection. The appearance of specific antibodies at various time intervals after detecting the clinical symptoms of KHVD in naturally infected carp was additionally studied. At 18°C water temperature, KHV DNA was not detected up to the 6th d post infection (dpi). Then it was found in samples from skin, gill, liver, spleen, and kidneys and much later from the brain. At 25°C water temperature, KHV DNA was already detected on the 3rd dpi in samples from skin and gills, and from the 5th dpi it was found in all examined internal organs, except the brain where it was found at 6th dpi. At 25°C, isolations of CyHV-3 have succeeded between the 7th and 11th dpi. The first specific antibodies were found no sooner than on the 21st dpi. The serological examination performed in naturally infected carp in 15 carp pond farms showed that first specific antiviral antibodies can be detected 2 weeks after the appearance of clinical symptoms of KHVD.
Epizootic ulcerative syndrome (EUS) is a dangerous fish infection with the water fungus Aphanomyces invadans. It occurs in Asia, Australia and America, in the Florida region, and affects many inland fish species. In accordance with Council Directive 2006/88/EC, EUS is one of the exotic notifiable diseases in Europe. In the paper an attempt was made to answer the question concerning the possibility of Aphanomyces invadans infection occurring in fish reared in Poland. In order to answer this question, the analysis of the fungus biology was performed, the factors predisposing to the infection were evaluated and the EUS-susceptible fish species were presented. If the theory of climate warming is true, a gradual extension of the area inhabited by the stenothermal Aphanomyces invadans seems possible. Even now thermal water conditions in ponds fed with electric plant cooling water in Europe approximate those in Asiatic natural water basins. Many popular Asiatic aquarium fish species are susceptible to Aphanomyces invadans or could be carriers of this fungus. These fishes are a potential source of infection at least to some European fishes. Considering even the slightest possibility of Aphanomyces invadans introduction in Poland followed by EUS occurrence, the authors proposed some necessary preventive measures. Among others, the importation of aquarium fish species which are susceptible to EUS should be prohibited. Also certain legislative regulations of carp (Cyprinus carpio) movement for stocking purposes should be applied.
The aim of the study was to identify the genotype of Polish isolates of salmonid alphaviruses (SAV) and to find the origin of the virus. Samples for virus isolation included the kidneys, spleen, and liver pooled from 10 fish. A typical cytopathic effect was observed after inoculation of samples on cell lines. Total RNA was extracted from cell culture supernatant and submitted to RT-PCR with primers amplifying two informative regions of the genome: a conserved region in the E2 gene and a variable region in the nsP3 gene. The sequences revealed that the strain from Poland belonged to subtype SAV 2, indicating a very strong genetic identity with isolates from Italy and France.
Spring viremia of carp (SVC) is a disease caused by a virus belonging to the genus Vesiculovirus, family Rhabdoviridae. The SVC virus is divided into four genogroups, Ia, Ib, Ic, and Id, due to its geographical distribution. This study aimed to identify the genotype of the SVC virus circulating in Poland. Polish SVC virus isolates were propagated on EPC and FHM cell lines, and genetic material (RNA) was isolated. The virus was detected in test samples by reverse transcription, sequenced and analyzed using MEGA 6.06 software. The phylogenetic tree was constructed by the Neighbor-Joining method. The results of phylogenetic analysis revealed the presence of two genogroups of the SVC virus in Poland. Most of Polish isolates belonged to the genogroup Id, as do isolates AY196200 from the Czech Republic, Z37505 from Belgium and EF593149 from the United States. Only two Polish isolates from Silesian Voivodeship were more closely related to Chinese and US isolates belonging to the genogroup Ia. There were no isolates belonging to the genogroups Ib and Ic. Nucleotide sequence analysis revealed certain point mutations between particular isolates. Knowledge on the genetic variants of the SVC virus circulating in Poland will be useful in epizootic investigations and preventive measures to protect Polish aquaculture from new variants from the neighboring countries.
The purpose of the project was to study the correlation between the presence of the IHN virus and the presence of antibodies against this virus in fish sera. Experimental infection of rainbow trout was performed at temperatures of 12°C±1 and 15°C±1. The trouts’ sera were collected at 7-day intervals. Samples from farms naturally infected with IHNV were also tested. The presence of antibodies against IHNV was determined by seroneutralization and the virus was detected by isolation in cell culture. It was found that in the experimentally infected rainbow trout, antibodies against IHN were found earlier in the higher temperature, after 21 days post infection, whereas the presence of IHNV was observed to last longer in the lower temperature. In the study performed on 30 samples of sera from IHN-infected farms, antibodies were detected in 12 samples. Detection of antibodies against IHNV in fish serum can be a helpful tool supporting recommended techniques.
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