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Random Amplified Polymorphic Dna-PCR Typing of Vibrio parahaemolyticus Isolated from Local Cockles (Anadara granosa)

Randomly amplified polymorphic DNA (RAPD) was used in this study to examine the genetic relatedness among the Vibrio parahaemolyticus strains. In the analysis by RAPD-PCR, the size for RAPD fragments ranged from 0.25 to 10.0 kb with average number of ten bands. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested. Hence, this study, demonstrated that the local cockles (Anadara granosa) in the study area are populated by genetically polymorphic strains of V. parahaemolyticus. In addition, RAPD-PCR is simple, robust and sensitive typing methods to differentiate the V. parahaemolyticus strains.


INTRODUCTION
Vibrio parahaemolyticus is a Gram-negative marine bacterium that can cause seafood-borne gastroenteritis and traveler's diarrhea in humans after they consumed contaminated raw or partially cooked fish or shellfish, particularly oysters [1] or exposure to a marine environment. The organism is widely disseminated in estuarine environments throughout the world. Outbreaks of V. parahaemolyticus food poisoning are most common in Japan and Southeast Asia, although they occurred occasionally in other parts of the world [2] , and are associated with diverse serovars. Recent studies [3,4] have shown the emergence of O3:K6 that appears to have the potential to spread and to be associated with infections more than other serovars.
Epidemiological typing of pathogens such as Listeria monocytogenes, V. parahaemolyticus and Salmonella enteriditis has become more and more important in public health control. Molecular typing procedures are applied to show clonal and close relationship between isolates of one species. Thus, it is possible to identify pathogen reservoirs and to follow up the regional and global spread of pathogens. Some typing methods also give an insight into the evolutionary dynamics of the bacterial genome. The classical and microbiological methods, based on the identification of phenotypic markers, while perfectly adequate to identify microorganisms on the level of species, are often not reliable enough when it comes to differentiating further into individual strains. This is where DNA-based procedures like Polymerase Chain Reaction (PCR) or DNA-sequencing approaches helped epidemiological investigations to be conducted more rapidly and thoroughly [5] . RAPD analysis is a commonly used method in PCR. This fingerprint has been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals [6] . Nowadays, molecular typing methods are necessary for proving the similarity between the isolates. The aim of the present investigation was to use RAPD-PCR assay to generate polymorphism in DNA patterns amenable to the differentiation of the V. parahaemolyticus strains isolated from cockles.

MATERIALS AND METHODS
Bacterial strains: The sixty-two isolates of V. parahaemolyticus isolated from local cockles obtained from a harvesting site in Tanjong Karang, Kuala Selangor were examined. These strains were confirmed by using specific-PCR targeting the species-specific ToxR region in V. parahaemolyticus as mentioned by Kim et al. [7] and Dileep et al. [8] .

RESULTS
Of the ten random primers, Gen 1-50-03, Gen 1-50-04 and Gen 1-50-08 were chosen for differentiation of V. parahaemolyticus isolates. The representative RAPD fingerprinting profiles obtained were displayed in Fig. 1-3. The size for RAPD fragments ranged from 0.25 to 10.0 kb with average number of ten bands for each primer used. All results from RAPD-PCR were analyzed with the GelCompare software (version 4.1). The overall achievable patterns were used to construct dendrograms using the UPGMA (unweighted pair group average method) clustering algorithms.

DISCUSSION
The application of PCR-based techniques has had a revolutionary impact on the diagnosis of infectious disease. A rapid typing method based on the random amplification of polymorphic DNA segments (RAPD-PCR) has been described [10,11] . This method has been successfully used in the characterization of several organisms including V. parahaemolyticus and other vibrios [1,[12][13][14] . In this study, a library of 10 primers (50% G+C) was examined for suitability. Of these 10 primers, three (GEN1-50-03, GEN1-50-08 and GEN1-50-08) proved to be useful primers for the RAPD analysis of strains of V. parahaemolyticus. In order to obtain a sufficient number of polymorphic bands that will permit reliable comparison, the three different 10mer primers were used in amplification reactions. Previously, Shangkuan et al. [12] demonstrated the importance of using a minimum of three different primers to discriminate different isolates of toxigenic V. cholerae. The results of using three primers in this study indicate that RAPD provides a high degree of discrimination between V. parahaemolyticus strains, with amplified products ranging from 0.25 to 10.0 kb. To determine the reproducibility of the RAPD analysis procedure, RAPD-PCR was repeated at least three times for DNA from each strain, so that the amplification profiles for each attempt could be compared. We found that profiles for a given strain to be highly reproducible, with very little variation from one RAPD analysis to another.
RAPD-PCR was used as a mean to determine the clonal relatedness of the isolates in this study by their chromosomal polymorphism. The data from the primer GEN1-50-03, GEN1-50-04 and GEN1-50-08 was used to generate a distance matrix. By using Gel Compar version 4.1 software, typable V. parahaemolyticus isolates were placed in 7, 6 and 10 clusters for primer GEN1-50-03, GEN1-50-04 and GEN1-50-08, respectively ( Fig. 1-3). At 24% and 10% similarity, the isolates were classified into 2 major clusters (R1 and R2) and one major cluster (R3) with a single isolate (VP59) using primer GEN1-50-03 and GEN1-50-04, respectively. These main clusters labeled as R1, R2 and R3 were sub-divided into 6, 5 and 9 subclusters, respectively. Our RAPD analysis revealed heterogeneous isolates of V. parahaemolyticus in cockles by using these two primers (GEN1-50-03 and GEN1-50-04). On the other hand, the isolates were observed as homogeneous when typed using primer GEN1-50-08, as the cluster analysis shows that at 52% similarity the isolates were clustered into 1 major cluster and a single isolate (VP25). Our results are supported by others who observed homogeneous or heterogeneous isolates of V. parahaemolyticus or Vibrio spp. in cockles or shellfish by RAPD-PCR assays [15][16][17][18] . The high diversity among the RAPD-types probably means that contamination of cockles by V. parahaemolyticus is derived from wide spectra sources of contamination. It is also likely that different strains of V. parahaemolyticus predominate in cockles in the study area.
The results obtained by RAPD-PCR indicated that multiple clones of V. parahaemolyticus were present in the environment. These observations are in agreement with those of others who demonstrated heterogeneity VP18  VP19  VP50  VP15  VP45  VP10  VP20  VP42  VP7  VP39  VP4  VP46  VP11  VP33  VP34  VP35  VP62  VP58  VP60  VP61  VP59  VP54  VP56  VP55   100  90  80  70  60  50  40  in V. parahaemolyticus or Vibrio spp. [15,19] . Many researches reported the abundance of V. parahaemolyticus during summer in temperate zone when temperature was above 25°C [1,20,21] , whereas the organism is expected to be prevalent throughout the year in the tropical zone like Malaysia [22] . The hot climate in Malaysia and other tropical countries make conditions more favourable for the growth of V. parahaemolyticus, increasing the risk of outbreaks associated with this organism.