Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei

Abstract

A pathobiological study was conducted using Vibrio parahaemolyticus (V_P) strain isolated from vibriosis affected shrimp (Penaeus vannamei) farms in Kancheepuram and Thiruvallur districts of Tamil Nadu during August 2014 to February 2015. The isolate was identified based on the morphological, physiological, biochemical and molecular characters. LD₅₀ value with intramuscular injection was determined as 2.6 × 10⁴ cfu/shrimp and sequential pathology was studied giving 6.1 × 10³ cfu/shrimp (LD₂₅). Total plate count (TPC) and total Vibrio count (TVC) in water, pond sediment, haemolymph, muscle, HP and gut were found significantly (P < 0.01) higher in natural cases than the experimental set up. Clinical signs and lesions observed in the natural and experimental cases were anorexia, lethargy, cuticle softening, loose shells, abdominal muscle cramp, red discoloration, opaque and whitish abdominal and tail musculature, necrosis of exoskeleton or splinter burns, reddish pleural borders of antennae, uropods and telson, swollen tail fan, ulcers, moribund shrimp sinking to bottom, and mortalities with shrunken discoloured HP with empty gut. Total haemocyte count (THC), small nongranular haemocyte (SNGH), large nongranular haemocyte (LNGH), small granular haemocyte (SGH) and large granular haemocyte (LGH) counts lowered significantly (P < 0.01) at 3, 6, 12, 24, 48, 96 and 192 h post injection (p.i). No LGH were found after 96 h of challenge. The post injection qPCR analyses of haemocytes showed up-regulations of penaeidin-3a, lysozyme, prophenoloxidase I, prophenoloxidase II and serine protein at 3 and 6 h of infection. There was total down-regulation of crustin from 3 to 192 h p.i. There was a remarkable elevation in the level of proPO I with concomitant depletion of proPO II. The pattern of up- and down-regulations in proPO I and SP were similar. The post infection qPCR analyses showed that these immune related genes could be used as markers for assessing the immune status of P. vannamei. Major histopathological manifestations observed were haemocyte infiltration/nodule in the epidermis, skeletal and cardiac muscles, atrophy of the excretory organ, and disrupted HP tubules with diffuse interstitial edema and haemocytic infiltration. Further HP showed that there was thickening of intertubular space, karyomegaly with prominent nucleoli, rounding and sloughing of HP tubular epithelium, many mitotic figures with bacterial colonies and apoptotic bodies, separation of shrunken tubule epithelium from myoepithelial fibers, regeneration of tubules, cystic, dilated and vacuolated appearance of HP tubules, hypoplastic changes in the tubules with no B, R and F cells, granuloma formation, concretions in tubules, calcification, necrosis, and washed out appearance with complete loss of architecture. The progression of the degenerative changes in the HP tubular epithelial cells was from proximal to distal end. In haematopoietic organ, increased mitotic activities with focal to extensive depletion and degeneration were observed. Degeneration of the stromal matrix with spheroid formation in lymphoid organ was observed among the Vp infected natural and experimental animals. Degeneration of glandular structures in the prehensile appendages with bacterial colonies, melanization and loss of epithelial layer in oesophagus, swelling and loss of architecture with mucinous secretion in the stomach, degeneration of peritrophic membrane in the lumen of intestine were observed in field cases but not in the experimental studies. Further, this study established the pathobiology of the Vp isolate to P. vannamei.

Keywords: AHPND; Biochemical and molecular characterizations; EMS -LD(50); Penaeus vannamei; Vibrio parahaemolyticus; Vibriosis.