Genetic Characterization of Streptococcus iniae in Diseased Farmed Rainbow Trout (Onchorhynchus mykiss) in Iran

Genetic characterization of strains of Streptococcus iniae recovered from morbidity and mortality of farmed rainbow trout in different provinces of Iran were studied. The Gram-positive cocci isolates were obtained from the kidney tissues of diseased rainbow trout on blood agar at 25°C for 72 h. The grown bacteria were then characterized using biochemical and molecular works. The identified 26 isolates of S. iniae producing a 513 bp in PCR procedure were then compared using random amplified polymorphic DNA (RAPD) analysis using 9 random primers. The phylogenetic tree of the RAPD product using UPMGA software included these strains in one genetic group but into two clusters. The results of this study show that S. iniae strains from the diseased rainbow trout in the north part of Iran are genetically similar to those strains in the south and west parts of the country.


Introduction
Streptococcus iniae is not only one of the major causative agents of streptococcosis in aquaculture industry but also is an important zoonotic bacterial disease causing morbidity and mortality in humans [1][2][3][4][5]. The emergence of disease has occurred in a range of aquatic animals including many species of marine and freshwater of both wild and cultured environments [1,2,4,6]. To date, the disease has been identified in almost all continents causing significant losses in several commercial fish species [1,2]. The estimated annual impact of disease outbreaks by S. iniae in aquaculture sector of some countries was reported to be 100 million USD [1,7]. In Iran, since its first report in rainbow trout farming, streptococcosis has caused significant losses in the aquaculture industry. A total annual loss due to this disease in trout farming has been estimated about 15 million USD [8]. Although adequate studies have focused on the immunepathogenesis of the infection, minimal data is available on the genetic characterization particularly on genetic diversity of the isolated strains of this bacterium in fish [3,9]. The importance of this is to provide an effective method of mass vaccination covering a number of isotypes and vaccination is one of the most feasible ways to prevent the losses due to this zoonotic bacterial disease in aquaculture industry [10]. Previoous work showed that it was possible to isolate the bacterium from different parts of Iran and recent attempts resulted in producing a local commercial vaccine inside the country [6,8]. However, because of existing of heterogeneous strains of S. iniae [9], it is important to know the possible genetic diversity of the virulent isolates. Such data will assist to improve the efficacy and potency of the produced vaccines. Therefore, the aim of this study was to compare the recovered isolates of S. iniae at molecular level to determine if intraspecific variants could be found among the isolates from different geographical locations of Iran which is a big land with different climates and environmental conditions.

Bacterial Isolates.
A total of 60 isolates of Grampositive cocci from the affected farmed trout at different geographical regions were used (Table 1). These isolates were recovered from the kidney tissues of diseased trout in states 2 The Scientific World Journal  Typical cycling parameters were: 1 min primary denaturation at 94 • C, 1 min denaturation at 94 • C, 1 min annealing at 45 • C, and 1.5 min extension at 72 • C for 35 cycles. The reaction was started by a denaturation step for 3 min at 94 • C and ended with a 10 min extension step at 72 • C. The PCR products were then electrophoreses using 2% gel agarose stained using Syber green (Sinagen company, Iran). S. iniae Hemolysis (a local strain collection with accession number: AF048773) was included as positive control and Lactococcus garvieae (a local strain collection with accession number X54262) as negative control.

Results
The biochemical profiles of the isolates included them into two groups S. iniae (26 isolates) and L. garvieae (34 isolates) ( Table 3). L. garvieae utilized citrate, nitrate, lactose, and gelatin, while S. iniae isolates were positive for ornithine and mannitol. These 26 isolates were then subjected to PCR for further confirmation and the obtained results showed that all isolates were S. iniae giving a band of 513 bp for PCR products (Figure 1). Therefore, these S. iniae isolates were used for RAPD analysis. The banding patterns of each random primer are shown in Table 4. At most, five different RAPD banding patterns were observed ( Table 4). The largest number of bands (five bands and three patterns) were observed using the primers P14 and1290 (four bands and three patterns) (Figures 2(a) and 2(b)), and the least banding patterns (three bands and one pattern) were seen using primer P4. Also, primers OPS11 and P5 resulted in production of 3-4 bands and two banding patterns (Figures 2(c) and 2(d)). Primers P1, P2, and P3 were able to produce only one band (Table 4), and thus, were not used for banding pattern analysis. The banding patterns were reproducible. The PCR was performed on all S. iniae isolates at two times and no difference was seen in the DNA pattern from one RAPD analysis to the next. The positive S. iniae strain was always included as an internal control for every PCR test to ensure that RAPD always produced the same DNA pattern as before.
When these data were subjected to UPMGA program, they were clustered into one group and two clusters ( Figure 3). Only one strain of the bacterium from Mazandaran region was included in a separated cluster, while other isolates were included in one cluster.

Discussion
Characterization of bacteria into known groups according to phenotypic features and virulence is an important tool to understanding the way for the identification and typing of pathogenic isolates. The morbidity and mortality due to S. iniae in aquaculture sector is now a big obstacle for having a sustainable aquaculture industry worldwide [1,2]. As this bacterial agent is also a zoonotic microganism, this obstacle increases dramatically. The use of biochemical features for differentiation of the virulent strains of gram positive cocci including S. inaie, S. parauberis, S. agalactiae, S. disagalactiae, and L. garvieae is difficult because of the variable results and long time required [1,2]. Therefore, using molecular works for the epidemiological purposes are essential to improve the preventive measures against the disease outbreaks in the fish farms. This is particularly important in case of disease prevention by vaccination methods.
Only a few works focused on the genetic features of S. inaie isolates in fish Eldar et al. (1997) [11] were able to show some genetic differences between some isolates of this bacterium obtained from diseased fish in the United state of America and Middle East using restriction length polymorphism of 16S rDNA of the bacterium. Although Dodson et al. (1999) [3] believed that it is possible to biochemically separate the pathogenic isolates from nonpathogenic isolates of S. iniae in fish, they could not distinguishe between either the invasive and noninvasive isolates in fish as well as between the fish and human isolates of S. iniae using the six different primers and RAPD analysis plus repeated PCR techniques. The S. iniae isolates used in their works were originally recovered from human (7 isolates), dolphin (1 isolate), and fish (38 isolates) in the United State of America (USA), Canada, and Middle East. However, Fuller et al. (2001) [12] reported that S. iniae virulence is associated with distinct genetic profile and demonstrated differences between pathogenic and nonpathogenic isolates. Also, Kvitt and Colorni (2004) [9] were able to separate 35 isolates of this bacterium recovered from the USA and Middle East into two groups using RAPD analysis. They found out that the trout isolates can be separated into one cluster which is different from other isolates recovered from other fish species including Asian sea bass (Lates calcarifer) and European sea bass (Dicentrarchus labrax). In the present study, all bacterial strains of S. iniae showed identical phenotypic features. However, using RAPD analysis, we could find up to 5 profiles/bands for the isolates recovered from diseased trout in 6 states of Iran. The phylogenetic analysis also included all bacterial strains into one group but into two seperated clusters. Therefore, it seems that virulence strains of S. iniae possess a high genetic similarity in trout aquaculture in Iran. If so, then for formulation of a whole inactivated vaccine, it is possible to induce an identical protection in fish using different isolates of this bacterium obtained from different regions of the country. However, more in vivo works are required to evaluate the efficacy of the produced vaccines by isolated bacteria from different parts of the country. Also, more works such as RNA sequencing are required to further genotypic characterization of these bacterial isolates of S. iniae in farmed rainbow trout in Iran.
In conclusion, RAPD analysis of the Iranian isolates of S. iniae obtained from diseased trout in north west and south west of Iran gave high genetic similarity indicating a probable identical protective level against the disease in the studied states of the country. However, more studies are required to isolate and genetically characterize S. iniae strains from other parts of the country because of poor quarantine practice which allow the possible entrance of new and probably genetically different isolates of the bacterium through the frequently importation of large quantity of eyed-eggs of trout as well as several species of ornamental fish into the country.