الفهرس 
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Abstract
A contaminated environment could provide a continuing source of H5N1 virus, and restricted access of human and animals to potentially contaminated ponds and lakes should be recommended during and after outbreaks in addition to information regarding the potential risk encountered during collection and consumption of aquatic animals. In particular, bathing or swimming activities in contaminated ponds.
The present study aimed to test the persistence of Low Pathogenic Avian Influenza virus H5N1 (LPAI H5N1) (Egyption isolate) in two differrent water types (Nile water and sea water) with different environmental conditions (temperature and pH) and the possible transmission of virus via aquatic animal Tilipia zillii. For testing the persistence of LPAI H5N1 virus in water, Two types of water (Nile water and sea water) were used with different pH 6.5, 7.0, 7.5 and 8.0 and different temperatures 20 and 34°C. The detection of H5N1 virus in water samples was carried out by using RT-PCR and rt RT-PCR.
For testing the possible transmission of the virus via aquatic animal Tilipia zillii, aquarium with a total capacity of 60 litres were filled with 45 litres water and 9 Tilipia zillii fishes. Then the aquarium was inculated by H5N1 with concentration 108 EID50/ml. The detection of H5N1 virus in water samples and fishes’ organs was carried out by using rt RT-PCR. The survival of the infectious virus in water samples was detected by Plaque and hemagglutination (HA) assays. Immunofluorescence technique was used to detect H5N1 virus in fishes’ tissue samples and the histopathology technique was carried out to detect the histopathological changes in fishes’ tissues.
Our results can be summarized in the following points:
● The results revealed that the LPAI-H5Nl virus was found to persist for extended periods of time in Nile water than in sea water. At 20oC, the longest persistent of the virus in Nile water was 4 weeks at pH 7.0, followed by 3 and 1 weeks at pH 7.5 and 6.5, respectively. While, viral RNA of H5N1 did not persist in Nile water at pH 8.0 and 20oC. The viral RNA persisted in Nile water for a week at pH 7.0 when the experiment temperature was 34oC. The virus appears only at zero time when pH value was 6.5 and 7.5 and temperature condition 34oC. The viral RNA showed no resistance at pH 8.0 and temperature 34oC. In general RT-PCR showed that the most optimum pH was 7.0 and the most optimum temperature was 20°C.
● Concerning sea water, viral RNA of H5N1 was stable for a week at pH 7.0 and temperature 20oC. The virus did not detect by the end of the first week at pH 6.5 and 7.5 when the temperature condition was 20oC. While, viral RNA of H5N1 did not persist at pH 8.0 and 20oC. The H5N1 virus had no duration of persistence in acidic (pH=6.5) and alkaline conditions (pH=8.0) and warmer temperature (34oC). The virus was undetectable by the end of the first week at pH 7.0 and 7.5 when the temperature condition was 34oC.
● In general the real time cycle threshold (CT) revealed that the most optimum pH was 7.0 and the most optimum temperature was 20oC. Also, the CT values showed that the concentration of the virus seemed to be lower in sea water than in Nile water at the same PH values. The infectivity of LPAI-H5Nl virus in water containing fishes Tilapia zillii was stable for 8 days by using plaque assay.
● Viral RNA was extracted from water containing fishes and subjected for real time RT-PCR. The viral RNA persisted 28 days in aquarium water. The CT values revealed that the concentration of the virus seemed to be lowered by time.
● Viral RNA was extracted from fishes’ organs (intestine, gills, muscle and liver) and subjected for real time RT-PCR. The H5N1 virus was found in the intestine of the fishes that was sampled at 4 and 8 days. The viral RNA was detected in gills that were sampled at 4 days only. The H5N1 RNA was not detected in other fishes’ organs (muscle and liver) until the end of the experiment by using real time RT-PCR technique.
● Immunofluorescence assay staining for the fishes’ organs (gills, intestine, liver and muscles) showed that the H5N1 virus found in the intestine of the fishes that were sampled at 4 and 8 days. Also, the virus was found in gills at 4 days. The pervious result support the idea that the fishes possible to be carrier only for H5N1 virus.
● Moreover histopathological examination of the fishes’ organs (gills, intestine, liver and muscles) showed no histological changes in these organs in spite of the detection of the virus in the intestine and gills of the fishes by using real time RT-PCR and immunofluorescence assay.
We concluded from this comprehensive study the following:
a. Real time RT-PCR assay was sensitive and specific for the detection of avian influenza viruses compared with conventional RT-PCR.
b. Avian influenza H5Nl virus was found to persist for extended periods of time in Nile water than in sea water.
c. The temperature is the most important predictor of the virus persistence in water.
d. The neutral pH (7) showed the longest persistence of the LPAI H5Nl virus in water, in contrast the acidic and alkaline conditions showed the shortest persistence of the virus in water.
e. The infectivity of LPAI-H5Nl virus in water containing fishes Tilapia zillii was stable for 8 days by using plaque assay technique.
f. By using real time RT-PCR technique and immunofluorescence assay, the H5N1 virus was found in the intestine of the fishes that was sampled at 4 and 8 days. In addition, the virus was detected in gills that were sampled at 4 days only.
Our future study is aiming to:
Determine transmissibility of AI H5N1 virus from infected aquatic animals to contact persons via naturally contaminated water and to elucidate additional factors affecting the survival and persistence of AI viruses in the environment.
Recommendation:
a. Environmental surveillance is of major relevance for avian influenza control programs.
b. Prevention and control measures can be suggested to minimize, if not eliminate the risk from the consumption of virus-contaminated water.
c. In H5N1 environment, hand hygiene, which includes hand washing and the use of alcohol-based hand rubs, is critical to prevent possible inoculation of the nose and mouth by contaminated hands.
d. Considering a potential risk and knowing that water supplied for drinking water and in health-care facilities, minimum standards are required based on national protocols.
e. Ensuring chlorination or alternative disinfectant residuals be maintained throughout distribution system to prevent the accidental contamination.
f. Elevation of public awareness concern improper disposing of bird carcasses in the Nile water because this might lead to persistence of AI H5N1 in water and surrounding environment increasing the risk of infection transmission into persons who are intensively contacted with contaminated water and environment contaminated with H5N1 virus.
g. Restricted access of human and animals to potentially contaminated ponds and lakes should be recommended during and after outbreaks in addition to information regarding the potential risk encountered during collection and consumption of aquatic animals. In particular, bathing or swimming activities in contaminated.