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Virulence-associated genes profiling of Streptococcus iniae isolated from diseased Nile tilapia ( Oreochromis niloticus )

ABSTRACT


INTRODUCTION
Fish is one of the most important protein sources, particularly in developing and high-population countries (Aboyadak et al., 2015;Ali et al., 2021a). Expansion in aquaculture is a must to meet the nutritional requirements of a rapidly growing human population. In the last two decades, finfish aquaculture has increased by more than four folds from 12.5 to 54.1 million tons (FAO, 2018). Egypt is the leading African country in aquaculture, particularly in tilapia production. Egyptian tilapia production reached 1.22 million tons in 2019 (GAFRD, 2020), and about 88% of this figure comes from aquaculture. Summer mortality syndrome represents a big challenge for expansion in Egyptian tilapia production, in the last few years, these frequent outbreaks resulted in severe economic losses estimated at about one billion Egyptian pounds. The diseased fish show the general signs of septicemia (Ali et al., 2018). Ali et al. (2021b) reported that thermal stress is the main predisposing factor for the summer mortality syndrome associated with disease outbreaks affecting cultured fish, stressed fish became immune-compromised and highly susceptible to bacterial diseases.  (Baiano and Barnes, 2009). simA is a surface protein that protects against phagocytosis and facilitate adherence to fish epithelial cells (Aviles et al., 2013). Pdi gene contributes to bacterial resistance to lysozyme killing as it enhances the adherence and invasion of epithelial cells (Milani et al., 2010). Molloy et al. (2011) reported that Streptolysin S encoded the in sagA gene is a cytolytic toxin-induced β-hemolysis. Buchanan et al. (2005) found that pgmA gene play a role in resistance of S. iniae to innate immune response. This gene is also responsible for normal bacterial cell wall morphology and production of surface capsule, so this gene has a potential role in its virulence. Moreover authors reported that cpsD gene encodes for certain facilitates binding to host tissues, such as epithelial cells. Molecular methods such as PCR is not only enabling rapid and accurate identification of bacterial pathogens but also it is considered a valuable tool for detection of virulence genes which cannot be detected by any other conventional microbiological method ( The aim of the present study was isolation and identification of Streptococcus iniae from cultured diseased O. niloticus and determining the prevalence of virulence genes in the recovered isolates for understanding the clinical picture of the disease.

Study area and fish sampling:
Seven tilapia farms located at Trompat seven, Alriad district, Kafrelsheikh province were studied for determining the cause of increased mortality during the summer season of 2019. One-hundred and forty diseased Nile tilapia ranged between 100 -250 g were collected (20 fish/farm). Each fish sample was preserved in a sterile bag in an ice box and immediately transported to laboratory for further analysis as described by Aboyadak et al., (2017).

Clinical signs and postmortem examination:
Diseased fish were inspected in the affected farms before sample collection to determine any external and behavior abnormalities as well as gross internal lesions then report for each fish was carried out during the microbiological examination as described by Ali et al. (2019).

Bacterial isolation and identification:
The initial bacterial isolation was performed as described by Aboyadak et al. (2016a) on brain heart infusion agar in which isolates were incubated at 35 °C for 24 h. The recovered isolates were subjected to gram staining, and Gram-positive cocci isolates were cultured on modified Edwards Medium enriched with 5% bovine blood and colistin sulphate 5 mg/L for selective isolation of streptococci. Grown colonies were preserved at -86 °C in glycerol for further molecular identification.

Molecular identification of the recovered isolates and virulence genes:
The molecular study was conducted for identification of S. iniae isolates and determining certain virulence genes by PCR. Genomic DNA was extracted using G-spin TM total DNA extraction kit, Intron, Korea. All the PCR reactions were performed in 25 µl reaction volume consisting of 12.5 µl of 2xMaster Mix (Intron, Korea), 3 µl of DNA extract, 1.25 µl of each forward and reverse primer after that 7 µl of nuclease-free water was added. All the streptococcus isolates recovered from modified Edwards Medium were subjected to PCR amplification of 16S rRNA gene to determine S. iniae isolates as described by Zlotkin et al. (1998). PCR identified S. iniae isolates were screened for the presence of scpl, simA, pdi, SagA, pgmA and cpsD virulence genes. Primers used in the molecular study are represented in table (1). PCR reactions were performed in Peltier Thermal Cycler model MG 960T using programs showed in table (2). PCR reaction products were electrophoresed on 1% molecular grade agarose gel supplemented with 0.5 µg/ml of ethidium bromide, 5 µl of DNA marker was used for determining bands size. Electrophoresis was performed at 80 V for 1 h in tris EDTA buffer, after that DNA bands were visualized by gel documentation system.

Clinical and postmortem examination:
Naturally infected fish showed hemorrhagic skin ulcers, scales desquamation, fin, and tail erosions. Internally liver, spleen, and posterior kidney were congested, enlarged with presence of hemorrhagic spots in severely affected fish.
Elementary tract was also congested and partially empty as represented in figure (1).

Initial bacterial isolation:
Thirty Streptococcus isolates were recovered during the initial bacterial isolation on Edward's media, Streptococcus isolates grown as small grayish rounded colonies as showed in figure (2).

DISCUSSION
In the present study, the most frequent clinical signs appeared on infected fish were hemorrhages at fin bases, inflammation, and redness of the abdominal wall and around the anal opening, fin and tail erosions. Few fishes showed corneal opacity and ascites. The behavioral abnormalities include swimming near water surface in a circular movement pattern. Our findings were in harmony with that reported formerly (Suanyuk et