A Natural Vibrio parahaemolyticus Δ
pirA

Vp

pirB

Vp+

Mutant Kills Shrimp but Produces neither Pir

Vp

Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions
Authors: Phiwsaiya Kornsunee , Charoensapsri Walaiporn , Taengphu Suwimon , Dong Ha T. , Sangsuriya Pakkakul , Nguyen Giang T. T. , Pham Hung Q. , Amparyup Piti , Sritunyalucksana Kallaya , Taengchaiyaphum Suparat , Chaivisuthangkura Parin , Longyant Siwaporn , Sithigorngul Paisarn , Senapin Saengchan 
Published: June 2017

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by
Vibrio parahaemolyticus
isolates (VP
AHPND
isolates) that harbor a pVA plasmid encoding toxins PirA

Vp

and PirB

Vp

. These are released from VP
AHPND
isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that
V. parahaemolyticus
isolate XN87 lacked
pirA

Vp

but carried
pirB

Vp

. Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB

Vp

was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VP
AHPND
isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated
pirA

Vp

gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original
pirA

Vp

gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the
pirA

Vp

gene sequence and in the downstream
pirB

Vp

gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic
pirAB

Vp

mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some
V. parahaemolyticus
isolates carrying mutant pVA plasmids that produce no Pir

Vp

toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.

IMPORTANCE
Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by
Vibrio parahaemolyticus
isolates (VP
AHPND
isolates) that harbor the pVA1 plasmid encoding toxins PirA

Vp

and PirB

Vp

. The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions).
V. parahaemolyticus
isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VP
AHPND
. Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VP
AHPND
. Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.