February 15-18, 2009 Washington State Convention Center Seattle ...

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RAPID TEST FOR THE DETECTION OF NECROTIZING HEPATOPANCREATITIS
BACTERIUM IN TISSUE EXTRACTS AND FECES OF PENAEID SHRIMP
Raymond L. Houghton*, Carlos Pantoja, Bonnie T. Poulos, Jean Chen, Yvonne Y. Stevens, Stan Morkowski,
Syamal Raychaudhuri and Donald V. Lightner
InBios International Inc.
562 First Avenue South, Suite 600
Seattle, WA 98104 USA
Raymond@inbios.com
A rapid lateral flow dipstick assay was developed for detection of Necrotizing Hepatopancreatitis Bacterium (NHP-B) in
penaeid shrimp. The assay is designed to detect the bacterium in extracts of the shrimp hepatopancreas tissue as well as in feces.
Two monoclonal antibodies were used in assay development that were shown by western blot to detect a 13kDa component in
the bacterium. Experimentally infected Litopenaeus vannamei shrimp were harvested and hepatopancreas and fecal samples
collected and extracted. Dipstick reactivity was observed in both sample types when infection was detected by PCR or immunohistochemistry.
The dipstick assay also reacted with Percoll fractions of hepatopancreas enriched for flagellar components of
the organism as shown by electron microscopy.
The rickettsial like organism resulting in Necrotizing
Hepatopancreatitis (NHP-B) in shrimp has resulted in
the loss of millions of dollars in the shrimp aquaculture
business particularly in the Americas. This raises
the need for a rapid test procedure for application by
farmers on site to detect the infection earlier and also
to monitor spread of the disease. Such a test would
need to be able to detect the organism in either fecal or
hepatopancreas samples (HPs) with sufficient sensitivity
to enable a more rapid response time to containing
infections. The availability of a cost effective test to
routinely monitor shrimp cultures in the field as well as
brood stocks could play an important role in identifying
and containing infections early. Two antibodies designated
3D6 and 4A2 have been used in combination in a
lateral flow immunoassay to detect NHP-B in both PCR
positive (4 strong, 2 weak, 17 negative) hepatopancreas
tissue Table 1 as well as feces of penaeid shrimp Figure
1. It has also been used to detect activity in Percoll fractionated
extracts of HPs enriched for flagellar antigens
(results not shown). This test will facilitate monitoring
of NHP-B infection in the field.