Development of an immunochromatography assay kit for
rapid detection of Nervous Necrosis Virus (NNV) Jalil Zorriehzahra1*, Fatemeh Hassantabar2, Farid Firouzbakhash2 and Kim D.Thompson 3 1
Department of Aquatic Animal Health and Diseases, Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), Tehran, I.R. Iran. 2
Department of Fisheries Science, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran, I.R. Iran;
3
Aquaculture Research Group, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, UK
28th Sept. 2017 St. Petersburg
Some Important Aquatics in Iran • Coldwater fish: Rainbow trout, Salmo trutta caspius • Warmwater fish: Carp • Bony fish in the Caspian Sea: • Kutum, Mullet, Sturgeon, Kilka,… • and Shrimp.
600kg Beluga Caught from Caspian Sea
Atlas of Marine Fishes in Persian Gulf and Oman Sea Introduce or 465 species belong to 101 family of Marine Fishes in Persian Gulf and Oman Sea Published by Iranian Fisheries Science Research Institute (IFSRI),
Some Important Marine fish in South of Iran ( Persian Gulf and Oman Sea) • • • • • •
Gilthead Sea bream Spottled Sicklefish Silver Pomfret Silver Sea bream Black Pomfret Longtail Tuna
• Common Dolphinfish
Viral Nervous Necrosis (VNN) Disease • Nodavirus may entry the fish via epithelial cells of intestine, skin or nasal, and transport to CNS via the peripheral nerves linked with these epithelium. • NNV can be vertically transmitted by carrier brood stock, or horizontally transmitted from sub-clinically infected fish to other susceptible fish.
7
VNN Morbidity in Golden grey mullet of the Caspian Sea
Detection of Nodavirus by PCR Method Wild Marine fish
RNA extraction (brain)
RT-PCR
Thermal cycler Nested-PCR product
Gel electrophoresis
Gel electrophoresis
Nucleotide sequencing Nested-PCR
RT-PCR product Thermal cycler 9
VNN One-Step Diagnosis kit Ag Sample pad Plastic Backing card
Colloid Goldconjugated NNVAbsorbent pad specific Ab pad NC membrane T
C
C T
T: coated with NNV-specific Ab
C: coated with goat anti-rabbit Ig S
S: sample loading chamber
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One-step diagnosis kit for VNN diagnosis 1
2
3
4
Total procedure including reading result is within 15 min. This kit is able to detect the head homogenate of naturally NNV-affected grouper larvae with titer of 104 TCID50 determined in GF-1 cells. 11
Detection of NNV by one-step kit and RT-PCR Days post NNV challenge
status of fish
One-step kit
RT-PCR
1
Without any clinical sign of VNN
2/4 (+)
4/4 (+)
2
50% fish lost appetite 20% fish swam abnormally
4/4 (+)
4/4 (+)
3
Mortality began to appear (6% fish died).
4/4 (+)
4/4 (+)
13
Mortality stopped, and the surviving fish did not show clinical signs.
2/4 (+)
4/4 (+)
12
(NNV One-Step Rapid Kit )
Thank you for your kind attention
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