Busico-Salcedo, Nancy (2004) The role of podovirus-like bacteriophage in the virulence of Vibrio harveyi strain 47666-1. Masters (Research) thesis, James Cook University.

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Abstract

Two studies were conducted to demonstrate the transfer of virulence between the strains infected with phage VHPL and the same strains uninfected with the phage. The first study was to determine if a bacteriophage isolated from the more virulent strain (47666-1) would show the same virulence effects as bacteriophage VHML. The second study was to develop polyclonal and monoclonal antibodies specific to the toxic protein subunits of V. harveyi 47666-1. These antibodies were then used to detect this specific exotoxin from the previously naïve strains of V. harveyi infected with the phage VHPL. SDS-PAGE analysis showed an up regulation and production of extracellular proteins in previously naïve V. harveyi strains receiving this phage compared with uninfected strains. Haemolysin assays indicated a significant increase (P<0.001) in both halo of clearing and colony diameter in bacteriophage-infected strains of V. harveyi compared to the same individual strains uninfected with the phage. However, siderophore production was not significant as all of the inducible strains did not respond positively on Chrome Azurol-S (CAS) agar. Chitin degradation, on the other hand, resulted in significantly greater zones of clearing (P<0.001) in strains infected with bacteriophage from V. harveyi 47666-1 than the same strains receiving no phage. In bath challenge assay, the results indicated that as a group, there was a significant difference in mortality rate among strains infected with bacteriophage from V. harveyi 47666-1 (F=82.824, DF=9,40, P<0.001) than strains of V. harveyi without the bacteriophage. Polyclonal (PAbs) and monoclonal (MAbs) antibodies to the specific toxic subunits 45 and 55 kDa of V. harveyi 47666-1 were produced and used to detect specific toxic subunits from previously naïve strains of V. harveyi challenged with the phage. In western blot assay, PAbs produced only one specific toxin subunit having molecular weight of approximately 55kD in strains 30, 12 and 20 while no bands where obtained from strain 643. Two MAbs (3A1-9 and 5A2-1) were characterised and both detected toxic protein subunits in all naïve strains of V. harveyi infected with the phage. However, cross reactions were observed in naïve strains 30, 12 and 20 uninfected with the phage. In conclusion, the presence of bacteriophage VHPL from V. harveyi 47666-1 probably enhanced the virulence using assays with four naïve strains of V. harveyi as a model. There was significant up-regulation of proteins based on SDS-PAGE, up-regulation of haemolysins, chitinases and greater mortality to larvae of P. monodon. It is therefore suggested that the presence of this bacteriophage may either partly or fully confers virulence to V. harveyi strain 47666-1.

Item ID:	1039
Item Type:	Thesis (Masters (Research))
Keywords:	Phage VHPL, V. harveyi 47666-1, Specific toxic subunits, MAbs, PAbs, Up-regulation, Extracellular proteins, Haemolysins, Chitinases, Mortality of P. monodon larvae, PAbs, MAbs, Virulence
Date Deposited:	13 Oct 2006
FoR Codes:	07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070708 Veterinary Parasitology @ 0% 07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070707 Veterinary Microbiology (excl Virology) @ 0% 07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070712 Veterinary Virology @ 0% 07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070705 Veterinary Immunology @ 0%
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