SEVERITY OF YELLOW HEAD VIRUS INFECTION IN Litopenaeus vannamei BY EXPERIMENTAL INJECTION AND FEEDING AT DIFFERENT SALINITY LEVELS

Apiruedee Songsok, Chalor Limsuwan and Niti Chuchird

Aquaculture Business Research Center, Faculty of Fisheries Kasetsart University, Bangkok 10900, Thailand

Yellow head disease

First described as an epizootic from black tiger shrimp farms in Thailand (Limsuwan, 1991). and Pattani province.
In 1992, founded the outbreak in Nakornsrithamrath, Songkla

During Year 1992-1993 yellow head disease damage over 40 million U.S. dollars. (Flegel et al., 1997)

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Clinical sign of YHV disease in P. monodon

Increase in feeding for several days Sudden decline in appetite Swimming near the edge of the pond Mortalities soon follow Yellowish colouration of the cephalothorax and gills

Year 2002.

Change to culture of L.vannamei instead of raising P. monodon .

Litopeneaus vannamei
-Genetically improvement -Domesticated brood stock -Can be stocked at higher density -High productivity -Short term growth -More uniform in size

Year 2006.

YHV outbreak reported in Pacific white shrimp farms, especially in the central area culture with low salinity water and poor management system.

* Infected shrimp is found from age 40-60 days.

Objective

Determine the severity of YHV infection in L.vannamei by experimental injection and feeding at different salinity levels

Materials & Methods

300 healthy shrimp (8-10g)

ABRC

Acclimate salinity for 10 days before experiment

300 healthy shrimp

100 shrimp

100 shrimp

100 shrimp

5 psu

15 psu

30 psu

experiment 1 (injection method)

Extract YHV virus
Manual of Diagnostic Tests for Aquatic Animals 2009

Extract the virus

Collect moribund shrimp from a disease outbreak and maintain at 4C. Remove and discard the gill and appendages. shrimp or the retained cephalothorax may be snap-frozen and stored at 80C until required. Thaw stored samples rapidly in a 37C water bath within two snap-seal plastic bags maintain at 4C or on ice during all procedures. Remove the carapace and calciferous mouthparts. Suspend the remaining tissues in six volumes of TN buffer (0.02 M Tris/HCl, pH 7.4, 0.4 M NaCl) homogenise in a tissue grinder to form a smooth suspension. Clarify the homogenate at 1300 g for 20 minutes at 4C. Remove the supernatant fluid below the lipid layer and pass through a 0.45 m filter. Maintain the filtrate at 4C for immediate use or snap-freeze and store in aliquots at 80C. Thaw the filtrate rapidly at 37C and maintain on ice prior to use. Inject at P. vannamei with 0.1 l of filtrate per one shrimp.

Manual of Diagnostic Tests for Aquatic Animals 2009

Separated the specimen in 3 groups

5 psu

15 psu

30 psu

Stocked 10 healthy shrimp in each tank

5 psu

15 psu

30 psu

Injected YHV virus 0.1 ml into each shrimp.

5 psu

15 psu

30 psu

Feeding with commercial pelleted feed.

5 psu

15 psu

30 psu

Experiment 2 (feeding method)

Separated the specimen in 3 groups

5 psu

15 psu

30 psu

Stocked 10 healthy shrimp in each tank

5 psu

15 psu

30 psu

Feeding with infected shrimp for the first meal except control tank

5 psu

15 psu

30 psu

Cumulative mortalities were recorded

Results

Results
12 10 8 6 4 2 0 day1 day2 day3 day4 day5 day6 day7 day8 day9 5psu 15psu 30psu control group

survival rate

Fig.1 survival rate after injected YHV

Results
12

survival rate

10 8 6 4 2 0 day1 day2 day3 5psu day4 15psu day5 30psu day6 day7 day8 day9

control group

Fig.2 Survival rate after fed with infected YHV shrimp

RT-PCR

RT-PCR

Histopathological study

Normal gill

Infected gill

Normal lymphoid organ

Infected lymphoid organ

Normal hepatopancreas

Infected hepatopancreas

Infected haemopoietic tissue

Infected heart

Conclusions

Comparison among 5psu 15psu and 30psu salinity water in experiment 1 (injection method) At 5psu, cumulative mortalities reached 100% within 5 days. At 15psu, cumulative mortalities reached 100% within 6 days. At 30psu, cumulative mortalities reached 100% within 20 days. Histopathological changes Pyknotic nuclei in several organs such as gills,lymphoid organ, haemopoietic tissue. Karyorrhexis in gills and hepatopancreas.

Conclusions

Comparison among 5psu 15psu and 30psu salinity water in experiment 2 (feeding method) At 5psu, cumulative mortalities reached 100% within 7 days. At 15psu, cumulative mortalities reached 100% within 8 days. At 30psu, cumulative mortalities reached 100% within 40 days. Histopathological changes Pyknotic nuclei and Karyorrhexis in several organs such as gills,lymphoid organ, haemopoietic tissue and hepatopancreas.

Acknowledgments
This research was funded by National Research Council of Thailand