SPRING VIRAEMIA OF CARP VIRUS FROM OREOCHROMIS NILOTICUS IN EGYPT
FIRST RECORD OF ISOLATION AND IDENTIFICATION OF
MAGDY K. SOLIMAN1, MOHAMMED M. ABOEISA2 , SAFINAZ G. MOHAMED3, WALID D. SALEH4
1. Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University (Damanhour Branch), Egypt.
2. Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University, Egypt.
3. National Institute of Oceanography and Fisheries, Alexandria, Egypt.
4. Department of Microbiology, Faculty of Agriculture, Cairo University, Egypt.
Abstract
First record of isolation and identification of Spring Viraemia of Carp Virus (SVCV) from Oreochromis niloticus (O.n.) in Egypt is given in the present study. Three hundred samples of Oreochromis niloticus from farms in El-Behera, Alexandria and Kafr El-Sheikh governorates were submitted for examination, from them 3 samples were found to be infected with Aeromonas hydrophila. On the other hand, all examined samples gave negative results for mycological and parasitological examinations except some fish proved to be slightly infested with monogenetic trematodes. Out of examined samples (30 fish which showed severe clinical signs), 13 fish were found to be positive upon examination with immunohistochemistry by using specific monoclonal anti body for SVCV. Moreover, the isolation of the virus was confirmed by using cell line from carp ovary which gave positive Cytopathic Effect (CPE). As well as Electron Microscope (EM) studies. Clinical signs, post mortem lesions (PM) and histopathological changes were examined. Experimental infection, clinical and histopathological studies were carried out. For the best knowledge of the author, this the first time for isolation of SVCV from Oreochromis niloticus not only in Egypt but also in the Middle East area.
Keywords: Spring Viraemia of Carp Virus, Oreochromis niloticus, Egypt
INTRODUCTION
The fisheries still have very big interest, not only for b eing a very important source for food, but also for its valuable incorporation in increasing our ability to understand the wide marine ecological system in all its forms (FAO, 2004). By the year 2030 aquaculture will dominate the fish supplies and less than half of the fish consumed is likely originate in capture fisheries. (FAO, 2000).
One of the main factors affecting fish production and efficiency is the fish diseases especially that resulted from viruses. The fish viruses cause sever losses among different fish especially carp species (Aquatic Animal Health report, 2005).
Viral infections cause significant levels of mortality in intensive fish farming and because of no treatment, and vaccination is not yet feasible, prevention is only possible through fish health control measures (Mourton et al., 1990 ). That makes us in bad need to a further knowledgement about viruses and its importance in fish culture (Habashi, 1980 and Way et al.,2003).
SVCV is considered one of the most important economic threating diseases among the cultured fish because of its severe economic losses especially in the carp species.(Kim et al., 2005).
It is obvious that, the research on the viral fish diseases in Egypt is scanty due to the lack of the tissue cultures and cell lines which are used for viral isolation and identification. And in spite of being difficult, the SVCV was isolated from Common carp, Silver carp Bighead carp and Grass carp for the first time in Egypt (Saad, 2005 and Abo eisa, 2007).
So, to close the circle and to avoid the increase problems occurring by SVCV, the aim of the study was isolation and identification of SVCV from suspected naturally infected Oreochromis niloticus which is usually cultured with the carp fish in polyculture system in Egypt. Moreover, evaluation of the dangerous effects of the isolated virus in fish health status upon experimental infection were also done.
MATERIALS AND METHODS
Fish
Three hundred Oreochromis niloticus (40± 10 gm average weight) showing clinical signs in the form of haemorrhagic patches, fin rot and congestion of internal organs with unilateral exophthalmia in some fish were collected from Behera, Kafr-El-Sheikh and Alexandria governorates cultured in both fish cages and private farms.
-Laboratory examination of collected fishes
According to Buck and Finlay (1979) and Ibrahim, (2002), the fishes were subjected to clinical, bacteriological, mycological and parasitological examination. The fish samples were collected from different organs under aseptic conditions.
-Virological examination
According to Buck and Finlay, (1979),tissue samples from suspected diseased fish were grinded in sterile mortar with sterile sand, then subjected to freezing and thawing for 3 times, after each one the tissues were grinded to liberate the intracellular virus, then centrifuged for 10 minutes at 3000 rpm/min. The supernatant containing the virus was collected under aseptic conditions after addition of three drops from mycostatin, 1000 I.U. /ml, penicillin, 1000 µg/ml, streptomycin and anystatin, 500 I.U./ml and kept at -20 C until used.
-Immunohistochemistery studies
According to Kiernan (2003) and Saad, (2005), samples kept in formalin paraffin by using Spring Viraemia of Carp (SVC) monoclonal antibody (Bio-X Diagnostics Belgium) in addition to anti-mouse Ig.
-Tissue culture
The steps of cell line preparation were made by using the ovaries of sexually mature female Common carp about 1 year old. According to the methods implied by Habashi (1980) and Saad, (2005).
-Histopathological examination
From naturally infected fishes, samples from brain, kidney, liver, musculature, air sac, gills and intestine were fixed in 10% neutral buffered formalin for at least 24 hrs and embedded in paraffin wax at 60 C. Five microns sections were stained by Hematoxyline and Eosin (H. & E.). According to Culling, (1983).
-Electron microscope examination
-Samples were fixed in 3% glutraldehyde and processed for electron microscopical examination according to Jerome et al. (1995).
-Determination of tissue culture infective dose fifty (TCID50)
-The pre-prepared inoculums of samples were centrifuged for 10 minutes then diluted by 1: 10 in TC199.
-After that ten fold serial dilutions from 10-1 to 10-6 for measuring Cytopathic Effect (CPE) was done and inoculated in prepared cell lines.
-After 4 subcultures, primary monolayers were inoculated with the samples inoculums.
-The highest two viruses that gave strong CPE were used for measuring the TCID50 according to the methods implied by Reed and Muench (1938) by the following equation:-
In each sample (Naturally infected) calculate the proportionate distance (PD):
Mortality above 50 % - 50
= -------------------------------------------------------------------------
Mortality above 50 % - Mortality below 50%
The T.C.I.D50 = P.D + Log Dil. Next above 50%.
- Experimental design
Thirty apparently healthy were divided into three groups (ten fish per group). First group was intra peritonially injected (IP.) with the TCID50 virus (1 ml TCID50), according to Saad, (2005). The second group was injected with a mixture of 1 ml from SVCV (10-2.66 TCID50) and 2 ml dexamethasone according to Wiik et al. (1989). The third group was injected with 1 ml of sterile tissue culture 199 (TC 199). The fish were kept under observation for 4 weeks and fed with 25% protein commercial fish feed.
RESULTS
The clinical signs and PM lesions of naturally infected fish
Naturally infected Oreochromis niloticus showed showing congestion of internal organs and gills with unilateral exophthalmia
Fig. 1. Naturally infected Oreochromis niloticus showing congestion of internal organs and gills (white arrow) with unilateral exophthalmia (green arrow).
-Bacteriological, parasitological and mycological examinations
All examined samples were negative for bacteriological and mycological investigations, except 3 fish from which Aeromonas hydrophila was isolated. Moreover, some fish found to be slightly infested with monogenetic trematodes.
-Immunohistochemistry studies
-According to the results of immunohistochemistry to Spring Viraemia of carp (SVCV) for 30 random naturally infected samples, 13 samples were proved to be +ve for the SVCV) by the appearance of brown labeled granules of di amino benzidine (DAB) of immunohistochemistry chromogene.
-Cell line formation and Cytopathic effect (CPE) due to viral infection
The cultured ovarian cells form Common carp formed monolayer sheet after 24 hrs post-incubation. The cells became spindle with oval nuclei. Number of cells highly increased and confluent monolayer sheet formed within 48-72 hrs (fig2).
Fig. 2. Normal confluent monolayer of Common carp ovary 24 hrs post-inc.
The CPE formed within 3 days post infection with suspected samples in which cells changed from spindle to round, detached and formed plaque-like, and then death of cells occurred. After 24 hours, the severity and degree of CPE were increased when the virus concentration increased, also, the increase in concentration of the virus decreased the time of virus to cause CPE and vice versa (fig. 3).
Fig. 3. Sever degree of CPE due to injection of virus appears in the form of rounding and detachment of cells with marked plaque formation (arrows).
-Results of TCID50
After injection of 13 samples which proved to be +ve for SVCV by immunohistochemistry with different dilution ((10-1, 10-2 , 10-3 , 10-4 , 10-5 and 10-6) for each sample, we found that the sample No. (13) gave the highest CPE. The result of TCID 50 for sample No 13 was found to be 10-2.66 (table 1).
Table (1): Results of calculation tissue culture infective dose fifty (TCID50) for the sample No. 13
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Sample No. 13 |
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* |
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6 |
|
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|
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5 |
X |
X |
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|
|
|
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4 |
X |
X |
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|
|
|
|
|
3 |
X |
X |
X |
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|
|
|
|
2 |
X |
X |
X |
X |
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|
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|
1 |
X |
X |
X |
X |
- |
- |
|
|
|
10-1 |
10-2 |
10-3 |
10-4 |
10-5 |
10-6 |
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|
No. of wells with Plaques/6 wells |
Viral titer |
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5/6 |
10-1 |
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5/6 |
10-2 |
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3/6 |
10-3 |
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2/6 |
10-4 |
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0/6 |
10-5 |
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0/6 |
10-6 |
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5/6 X 100 - 50 ** Pd = --------------------------- = 0.66 5/6 X 100 - 2/6 X 100 |
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Virus titer = Log dil. Next above 50 % + P.D = 2 + 0.66 = 2.66 |
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المصدر: كليه الزراعه الاسكندريه مهندس محمود النوبى
نشرت فى 29 يونيو 2010
بواسطة egaselnoby
عدد زيارات الموقع
50,991
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ساحة النقاش