Immunological characterization of a bacterial protein isolated from salmonid fish naturally infected with Piscirickettsia salmonis

Abstract

The Salmon Rickettsia syndrome (SRS) remains a major infectious disease in the Chilean aquaculture. A limited number of Piscirickettsia salmonis proteins have been characterized so far for their use as potential candidates for vaccines studies. In this study, we identified and expressed a highly immunogenic protein of P. salmonis extracted by selective hydrophobicity from crude-cell macerates of naturally infected salmonid fish. One and two-D PAGE gels followed by Western blot analysis with a battery of polyclonal anti-P. salmonis antibodies have allowed the isolation of the target protein. Basic local alignment search (BLAST) done after partial sequencing of the pure protein identified it as a member of the heat-shock protein (HSP) family of prokaryotes. The protein, named ChaPs, was cloned as a single open reading frame encoding 545 amino acid residues with a predicted molecular mass of 57.3 kDa. The amplicon representing the entire novel gene was expressed in vitro in different heterologous systems: the PurePro Caulobacter crescentus expression system from where most of the characterization was attained, and also in the Escherichia coli BL-21 CodonPlus model for commercially potential purposes. The immunologic potential of ChaPs was determined with serum from naturally infected fish.

Introduction

Rickettsial diseases are common and significant in humans and in the veterinary world [1], [2] and fish are not an exception [3], [4]. Salmon Rickettsia Syndrome (SRS), also known as piscirickettsiosis, corresponds to an aggressive infectious disease affecting the world of salmon aquaculture since the late 80s with a notorious impact in southern Chile [5], [6], [7]. The disease is caused by a novel non-motile obligate intracellular Gram-negative bacteria, Piscirickettsia salmonis (P. salmonis) [8] and has been unequivocally identified as the responsible agent for dramatic economic losses suffered by the Chilean salmon industry in the last decade. P. salmonis (type strain LF 89, ATCC VR 1361) survives in salt water better than in fresh water [9], and can be horizontally transmissible by contact and cohabitation in both environments [10], [11]. Suspicious of intermediate vectors in the transmission have also been raised [12], [13].

After the first description of piscirickettsiosis in Chile, other salmonid species have begun experiencing mortalities in saltwater milieu [4]. In time, P. salmonis was identified as the causative agent of diseased salmonids in other latitudes, with special impact in western Canada, Norway and Ireland [14], [15]. Additionally, a piscirickettsiosis-like syndrome was also reported in epizooties affecting farmed salmon in Scotland [16], and similar piscirickettsia-like organisms displaying comparable clinical signs have been reported in several number of different fish species, which in spite of being non-reactive to antibodies against standard P. salmonis, might very well represent antigenic variants to the prototype strain [17]. Notwithstanding, recently a true P. salmonis was isolated in the coast of southern California from a naturally infected white sea bass (Atractoscion nobilis), which was successfully transmitted both in vivo and in vitro, suggesting that the expected spread of the agent to other commercially relevant fish species has already started [18].

Although the causative agent of this disease has been successfully grown in tissue culture cells [19], [4], almost nothing is known about its biology and pathogenesis. Nonetheless, since infected fish are immunoreactive to crude extracts containing the bacteria, the possibility of either purifying the bacteria and/or searching for single antigens appears to be a reasonable way to evaluate potential protective immunity against the disease. Several reports indicate that the use of partially purified-inactivated bacteria (bacterins) does not seem to be an effective vaccination alternative [19], [20], [21]. On the other hand, several potential bacterial antigens have been reported [22], [23], [24], [25], [26]. Though only until recently, the use of a single antigen (OspA) as putative vaccine plus the addition of T cell epitopes has had some stimulating results when the vaccine was administered to coho salmon. A post-challenge relative percent of survival (RPS) as high as 59% was attained, demonstrating the potential efficacy of a putative recombinant vaccine based upon a single antigen [27], [28].

In this study, we report the cloning and partial characterization of the ChaPs gene, which encodes an immunogenic protein of P. salmonis. DNA sequence analysis reveals that the P. salmonis ChaPs gene has an open reading frame encoding 545 amino acid residues. Basic local alignment search tool (BLAST) analysis shows that the putative protein is highly homologous to members of the HSP chaperone-like family of other bacteria (cpn). Finally, we are considering that this protein may very well represent a new target for the development of an efficacious vaccine to control piscirickettsiosis in favor of a sustainable development of the salmon industry both in Chile and worldwide.