Characterization of tdh gene negative Kanagawa haemolysin of environmental isolates of Vibrio parahaemolyticus

Several isolates of V. parahaemolyticus that produced a very clear beta-haemolysis on Wagatsuma agar, demonstrating  Kanagawa phenomenon like haemolysin (KPLH) were obtained from marine water and seafood samples in around Port Blair. PCR based analysis of these isolates revealed that the isolates were tdh and trh genes negative. This haemolysin was temperature sensitive and unique in the haemolysin family. Further studies on this haemolysin were carried out.

Fig 1. Haemolysis on Wagatsuma agar (VP 1: Test strain, VP 8: Control strain)

Fig 2. Haemolytic activity of Kanagawa phenomenon like haemolysin

All the strains were grown in TDH broth and subjected to partial purification following standard procedure. In brief, overnight culture broth was centrifuged. Supernatant was precipitated by 65%  solid (NH4)2 SO4  at 40 C. The resultant precipitate was dialysed and haemolysis assay was performed on WA spotting 10 ml of such preparation. Antibodies were raised against VP1 (tdh, trh negative ) and VP8 (tdh, trh positive) haemolysins in rabbits. Agarose gel diffusion and  modified Elek test were performed. Haemolytic assay was done using both plate method and tube method. Enterotoxicity was tested using Rabbit Illeal loop assay. Cytotoxicity and adherence assay was done using HeLa cell line.The marine isolates of V. parahaemolyticus belonged to different serotypes. Five tdh trh-ve strains a tdh trh gene positve strain (VP8) were selected. Haemolysin was estimated by Phenol reagent method. It was observed that as low as 5 mg haemolysin could demonstrate Kanagawa like haemolysin (KPLH) activity with an 18 – 20 mm diameter of haemolysis (fig. 1). All the five tdh, trh negetive strains showed almost equal haemolytic activity in comparison to VP8  on WA (1: 50) by plate method and by tube dilution method (Fig. 2).

Antibodies against VP1 and VP8 haemolysins raised in rabbits showed the development of clear  precipitin bands  against homologous antigens  and no bands  against heterologous antigens (Fig. 3).

Haemolysins of different dilutions showed cytopathic effect on different mammalian cell lines (fig. 4). Results showed complete haemolytic fluid accumulation. Fluid accumulation was moderate. For VP1 (ca 0.77) and for VP8 (ca 0.43).  Test strains (VP1 and VP8) did not show any significant adherence (Fig. 5).  For purification of KPLH, the crude haemolysin (ammonium sulphate precipitated, dialysed and concentrated) was passed through DE 52 and was eluted using 0.7M NaCl. The eluted protein(s) were electrophoresed. Three major bands corresponding to 43, 30 and 28 kDa were identified in the electrophoresis.

Fig 4. Cytopathyc effect on HeLa cell lines (A) normal (B) cytopathyc effect

Fig 5. Adherence effect on HeLa cell lines (A) normal (B) adherence