Isolation and Pathogenicity of Streptococcus iniae in ...

Journal of Aquatic Animal Health

ISSN: 0899-7659 (Print) 1548-8667 (Online) Journal homepage: http://www.tandfonline.com/loi/uahh20

Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia M. Rahmatullah, M. Ariff, M. Kahieshesfandiari, H. M. Daud, M. Zamri-Saad, M. Y. Sabri, M. N. A. Amal & M. Y. Ina-Salwany To cite this article: M. Rahmatullah, M. Ariff, M. Kahieshesfandiari, H. M. Daud, M. Zamri-Saad, M. Y. Sabri, M. N. A. Amal & M. Y. Ina-Salwany (2017) Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia, Journal of Aquatic Animal Health, 29:4, 208-213, DOI: 10.1080/08997659.2017.1360411 To link to this article: http://dx.doi.org/10.1080/08997659.2017.1360411

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Date: 11 October 2017, At: 19:48

Journal of Aquatic Animal Health 29:208–213, 2017 © American Fisheries Society 2017 ISSN: 0899-7659 print / 1548-8667 online DOI: https://doi.org/10.1080/08997659.2017.1360411

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Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia M. Rahmatullah, M. Ariff, and M. Kahieshesfandiari Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia

H. M. Daud and M. Zamri-Saad Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; and Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia

M. Y. Sabri Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia

M. N. A. Amal Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; and Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia

M. Y. Ina-Salwany* Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; and Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia

Abstract This study describes the isolation and pathogenicity of Streptococcus iniae in cultured red hybrid tilapia (Nile Tilapia Oreochromis niloticus × Mozambique Tilapia O. mossambicus) in Malaysia. The isolated gram-positive S. iniae appeared punctiform, transparently white, catalase and oxidase negative and produced complete β-hemolysis on blood agar, while a PCR assay resulted in the amplification of the 16 S rRNA gene and lactate oxidase encoded genes. The isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin. Pathogenicity trials conducted in local red hybrid tilapia (mean ± SE = 20.00 ± 0.45 g) showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 104, 106, and 108 CFU/mL of the pathogen, respectively. The clinical signs included erratic swimming, lethargy, and inappetance at 6 h postinfection, while mortality was recorded at less than 24 h postinfection in all infected groups. The LD50-336 h of S. iniae against the red hybrid tilapia was 102 CFU/mL. The post mortem examinations revealed congested livers, kidneys, and spleens of the infected fish. This is the first report of S. iniae experimental infection in cultured red hybrid tilapia in Malaysia.

*Corresponding author: [email protected] Received January 20, 2017; accepted July 16, 2017

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Tilapia Oreochromis spp. is one of the most popular fish species in freshwater aquaculture in many Asian countries due to its rapid growth and high adaptability to the environment (El-Sayed 2006). Tilapia production is booming worldwide with a total global production of 5.15 million tons in 2014. However, intensification of aquaculture has led to diseases that affect aquaculture production of tilapia (Shoemaker et al. 2006). The most significant disease causing losses in tilapia culture is streptococcosis, a septicemic disease that occurs following infection by Streptococcus agalactiae or S. iniae (Klesius et al. 2008). Streptococcus iniae was first isolated from a skin lesion of a captive Amazon river dolphin or Boto Inia geoffrensis (Pier and Madin 1976). Since then, it has been isolated from many cultured fish species including European Seabass Dicentrarchus labrax (Zlotkin et al. 1998), Barramundi Lates calcarifer (Bromage et al. 1999), Nile Tilapia O. niloticus (Shoemaker et al. 2001), Japanese Flounder Paralichthys olivaceus (Nguyen et al. 2002), Red Drum Sciaenops ocellatus (Shen et al. 2005), Red Porgy Pagrus pagrus (El Aamri et al. 2010), hybrid tilapia

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(Nile Tilapia × Blue Tilapia O. aureus) (Al-Harbi 2011), and freshwater Asian Seabass L. calcarifer (Kayansamruaj et al. 2017). The affected fishes show either loss of orientation, lethargy, ulcers, exophthalmia, and/or meningoencephalitis (Agnew and Barnes 2007). Streptococcus iniae can be carried asymptomatically but is also associated with sporadic disease outbreaks. Mortality rates of 30% to 50% have been reported with meningoencephalitis in aquaculture farms (CFSPH 2005). These bacteria are also zoonotic with reported infections in humans associated with the handling and preparation of infected fish (Baiano and Barnes 2009). In southeastern Asia, mass mortality associated with S. iniae infections were recorded from cultured tilapia in Indonesia and from cultured tilapia and Asian Seabass in Thailand (Suanyuk et al. 2010; Anshary et al. 2014; Kayansamruaj et al. 2017). However, S. iniae has not been reported in aquaculture systems in Malaysia. This report describes the first isolation, characterization, and pathogenicity of S. iniae isolated from cultured red hybrid tilapia (Nile Tilapia × Mozambique Tilapia O. mossambicus) in Malaysia.

METHODS Bacteria isolation, characterization, and sensitivity to antibiotics.—A total of 120 red hybrid tilapia were sampled from several cages of farmed fish in Kenyir Lake, Terengganu, Malaysia, in 2014. The range of water temperature, ammonia, dissolved oxygen, and pH during the sampling was 27.5–31.0°C, 0.024–0.034 mg/L, 6.8–7.4 mg/L, and 7.31–8.20, respectively. The collected fish showing either abnormal swimming behavior or loss of orientation, isolation from the schooling group, exophthalmia (popeye), and hemorrhages of pectoral, dorsal. and caudal fins. Aseptically, swab samples were collected from the brains, livers, kidneys, and eyeballs of the fish for bacterial isolation. Samples were streaked directly onto Mueller–Hinton agar (Oxoid, Hampshire, UK) supplemented with 5% horse blood and incubated at 37°C for 24 h. The growth bacteria were subcultured on the same medium to obtain pure isolates before they were subjected to gramstain, catalase, and oxidase tests. Several other tests for temperature tolerance, hemolysis, and capability to grow in different media were also conducted on the isolates. Then, the gram-positive, oxidase-negative, and catalase-negative isolates were selected and subjected to API 20 STREP (bioMérieux, Marcy l’Etoile, France). Antibiotic susceptibility tests were conducted with seven antibiotics (Thermo Scientific, Waltham, Massachusetts): ampicillin (25 µg), streptomycin (10 µg), tetracyclin (10 µg), vancomycin (30 µg), bacitracin (10 µg), penicillin (2 U), and chloramphenicol (10 µg), based on Clinical and Laboratory Standards Institute guidelines (CLSI 2006). PCR assays of the isolate.—Polymerase chain reaction assays targeting the 16 S rRNA gene and lactate oxidase encoded genes were carried out to confirm the isolate. Genomic

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DNA was extracted by Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin), according to the manufacturer’s protocol. To detect the target region of the strain, two different primers were used. For the conserved regions in the 16 S rRNA gene (Sin 1/Sin 2), the forward and reverse primers were 5′CTAGAGTACACATGTAGCTAAG-3′ and 5′-GGATTTTCC ACTCCCATTAC-3′, respectively (Zlotkin et al. 1998), while for the lactate oxidase (lctO) gene (Lox-1/Lox-2) the forward and reverse primers were 5′-AAGGGGAAATCGCAAGTGCC3′ and 5′-ATATCTGATTGGGCCGTCTAA-3′, respectively (Mata et al. 2004). The PCR amplifications were performed by using 16 S rRNA and lctO primers in different reaction mixtures containing 5 μL of Go Taq PCR buffer (Promega), 3 μL of 25-mM MgCl2 (Promega), 2 μL of 25-mM MgCI2 (Promega), 0.5 μL of 10-mM dNTPs (Promega), 1 μL of Taq polymerase (Promega), 100 ng of DNA template, and DNase-free water for a total volume of 50 μL. A thermocycler (Eppendorf, Hamburg, Germany) was used to perform PCR reactions with the following parameters: 94°C for 10 min followed by 34 cycles for each at 94°C for 1 min, extension at 72°C for 1 min and 30 s, and postextension at 72°C for 10 min. Annealing temperature was optimized at 50°C for the 16 S rRNA gene and at 54°C for the lctO gene. The amplicons were subjected to electrophoresis in 1% agarose gel in 1× tris–boric acid (borate)–EDTA (TBE) for 1 h at 90 V and stained with ethidium bromide. The gel was viewed under an ultraviolet transilluminator, and the target bands were excised and purified by Wizard SV Gel and PCR Clean-UP System (Promega) following the manufacturer’s protocol. Streptococcus agalactiae strain Millud-II was also included for genotypic comparison (Ali et al. 2010). Sequencing analysis of the isolates.—The PCR products were purified by using the QIAquick PCR Purification kit (Qiagen, Kuala Lumpur, Malaysia) according to the manufacturer’s protocol and followed by sequencing (1st Base, Selangor, Malaysia). The nucleotides sequences were analyzed using the basic local alignment search tool (BLAST) program (http://blast.ncbi.nlm.nih.gov/Blast.cgi of the National Center for Biotechnology Information). Pathogenicity test of the isolate.—In the pathogenicity study, the red hybrid tilapia were selected and obtained from a local hatchery. The approximately 2-month-old fish included both sexes and had an average weight of 20.00 ± 0.45 g (mean ± SE). A preliminary screen for pathogens on 5% of the fish population revealed no detection of any bacteria or parasites. Identified S. iniae TSK2 strain was used in this experimental study. Experimental infection trials consisting of 104, 106, and 108 CFU/mL of the bacterial strain were conducted with four groups of red hybrid tilapia; sterile phosphate-buffered saline was used to provide a noninfected control. Each fish was injected intraperitoneally (i.p.) with final volume of 0.1 mL of the inoculum. For each treatment, three replicate tanks were each randomly stocked with 20 fish. Continuous aeration

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TABLE 1. Comparison of phenotypic characteristics of isolated Streptococcus iniae TSK2 strain from the present and previous studies.

Strain Test

TSK2 (present study)

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Host Gram-staining reaction Cell morphology

Red hybrid tilapia + Cocci punctiform; transparently white; grew in pair and chain like morphology. Catalase – Oxidase – Hemolysis (5% sheep blood) ß Tryptic soy agar + Blood agar + Nutrient agar + Brain heart infusion broth + Temperature, 4°C – Temperature, 12°C – Temperature, 37°C + Temperature, 55°C – Oxidative–fermentative F (OF) test Acetoin production + (Voges–Proskauer test) Hydrolysis (hippuric acid) – ß-Glucosidase hydrolysis + (esculin) Pyrrolidonyl arylamidase + α-Galactosidase + β-Glucuronidase + β-Galactosidase – Alkaline phosphatase + Leucine aminopeptidase + Arginine dihydrolase + Acidification (ribose) + Acidification (arabinose) – Acidification (mannitol) + Acidification (sorbitol) – Acidification (lactose) – Acidification (trehalose) + Acidification (inulin) – Acidification (raffinose) – Acidification (amidon) + Acidification (glycogen) +

was given to each group, while feed was given twice daily. The ranges of water temperature, ammonia, pH, and dissolved oxygen during the experimental trial were 26.05–28.40°C, 0.00–0.19 mg/L, 6.8–7.9, and 5.07–5.95 mg/L, respectively.

SI 1J (Kayansamruaj et al. 2017) Freshwater Asian Seabass + Cocci; small (2–4 mm of colony diameter); transparently white; pair to chain like morphology. – – ß +

S. iniae (Suanyuk et al. 2010) Red tilapia + Cocci – – ß + + +

F –

+

+

+ – + +

– + + + + + + + + + – + – – + – – + +

Clinical signs and mortality were recorded at 24-h postinfection intervals for 14 d. The LD50-336 h of the S. iniae was calculated as previously described (Ramakrishnan 2016). During the experimental period, swabs were aseptically

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collected from the brains, livers, kidneys, and spleens of dead fish and immediately streaked onto blood agar. The DNA of growth bacteria was extracted using One-Tube Bacterial Genomic DNA Extraction Kit (Klee GmbH, Sissach, Switzerland) and confirmed by PCR as described above.

2

1

3

4

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RESULTS Isolate Characterization and Sensitivity to Antibiotics Gram-positive cocci of S. iniae were successfully isolated from 26 (21.7%) of the sampled red hybrid tilapia. A fish was considered positive to S. iniae when the bacteria were detected in at least one of the organs sampled. All of the isolated colonies appeared punctiform, transparently white, catalase and oxidase negative, produced complete β-hemolysis on blood agar, and morphologically grew in pairs and chains. The isolate grew well at 37°C, but failed to grow at 4, 12, and 55°C. The details on the biochemical test results of an isolate, namely S. iniae TSK2 strain, are presented in Table 1. Among the seven antibiotics tested, the isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin.

300 bp

FIGURE 1. Polymerase chain reaction amplification of isolated Streptococcus iniae DNA using the primers, Sin 1 and Sin 2. Lane 1: 1 kb DNA ladder (Thermo Scientific); lane 2: S. iniae (TSK2); lane 3: S. agalactiae (Millud-II); lane 4: negative control (no DNA).

Identification of the Isolate The isolated bacterium was genotypically identified as S. iniae. The PCR assay resulted in the amplification of 300 bp of the 16 S rRNA gene (Figure 1). The lctO gene also showed the expected amplified product of 870 bp (Figure 2). No specific band was amplified in negative control of S. agalactiae Millud-II strain sample. The nucleotide sequence of the 16 S rDNA gene of isolated S. iniae was deposited to GenBank with accession number KT722586. Pathogenicity of the Isolate Following experimental infection, fish mortality started at less than 24 h postinjection in all infected groups (Table 2). The fish showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 104, 106, and 108 CFU/mL of the pathogen, respectively. Most (≤60%) of the challenged fish in each group showed clinical signs such as erratic swimming, lethargy, and inappetence after 6 h postinjection. Gross lesions including congested opercula, pale gills, and congested internal organs were mostly (≤80%) observed in the dead fish in each infected group. The LD50-336 h of the S. iniae strain TSK2 in the red hybrid tilapia was determined to be 3.0 × 102 CFU/mL. Streptococcus iniae was successfully isolated and identified from brain, liver, kidney, and spleen samples of the dead fish. Most of the bacteria (≤50%) were isolated from the brain of the fish. DISCUSSION Streptococcosis caused by S. agalactiae is endemic in many tilapia farms, but S. iniae has never been reported in

1

2

3

4

870 bp

FIGURE 2. Polymerase chain reaction amplification of isolated Streptococcus iniae DNA using the primers, Lox 1 and Lox 2. Lane 1: 1 kb DNA ladder (Thermo Scientific); lane 2: S. agalactiae (Millud-II); lane 3: negative control (no DNA); lane 4: S. iniae (TSK2).

Malaysia (Zamri-Saad et al. 2014). In the present study, we successfully isolated S. iniae from cultured red hybrid tilapia in farms where S. agalactiae had been previously isolated (Amal et al. 2010). Thus, this might be a case of cohabitation of S. agalactiae and S. iniae as has been reported in tilapia farms in Indonesia (Anshary et al.

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TABLE 2. Mortality of red hybrid tilapia during challenge trial with Streptococcus iniae TSK2 strain.

Hour postinfection Bacteria concentration 24 48 72 96 120 144 168 192 216 240 264 288 312 336 Cumulative mortality (%)

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Control 104 CFU/mL 106 CFU/mL 108 CFU/mL

0 0 0 1 5 7 5 12 7 21 20 19

0 6 7 0

0 7 5 0

0 7 8 0

0 5 5 0

0 4 6 0

2014). Moreover, high water temperature (27.5–31.0°C) of the lake during the outbreak might have been a predisposing factor for this disease. Streptococcus iniae isolated in this study showed slightly difference phenotypic characteristics compared with the isolates from Asian Seabass in terms of raffinose acidification (Kayansamruaj et al. 2017) and from red tilapia Oreochromis sp. in terms of acetoin production and β-galactosidase reaction (Suanyuk et al. 2010); fish from both species originated from Thailand. The strain was sensitive to tetracycline, vancomycin, and bacitracin, which is indicative that these antibiotics might be useful in controlling the disease in the future. However, resistant of the isolate to erythromycin is worrisome, since erythromycin and oxytetracycline are frequently incorporated into fish pellets for control against streptococcosis in fish in Malaysia (Najiah et al. 2009). Moreover, a low resistance pattern of isolated S. agalactiae from fish towards selected antibiotics already has been reported in that country (Aisyhah et al. 2015). The S. iniae in this study was proved to be virulent in red hybrid tilapia based on the calculated LD50, which was 102 CFU/mL. Baums et al. (2013) reported that 108 CFU/mL of S. iniae could cause mortality among Nile Tilapia after 3 d of i.p. injection. Another study revealed that S. iniae between 107 and 1010 CFU/mL resulted in high mortality in Redtail Sharkminnow Epalzeorhynchos bicolor within 2 d of infection via i.p. injection (Russo and Yanong 2009). A study by Bromage and Owens (2002) showed that a low-dose i.p. injection of 103 CFU/mL of S. iniae led to 50% mortality in Barramundi within the first 48 h, and the fish had suppurative brain lesions. Experimental S. iniae infection resulted in a variety of clinical signs and post mortem findings such as darkening of the skin, anorexia, lethargy, erratic swimming, enlarged gall bladders, and hemorrhage of spleens, kidneys, and livers. Similarly in the present study, clinical signs such as erratic swimming, lack of appetite causing lethargy, and congestion of internal organs were also observed (Chen et al. 2007; Baums et al. 2013). Thus, our present study indicates that septicemia due to S. iniae infection might be responsible for the mortality of the red hybrid tilapia.

0 4 2 0

0 2 1 0

0 2 0 0

0 1 0 0

0 2 0 0

0 1 0 0

0 54 58 60

(0.0) (90.0) (96.7) (100)

Conclusion Based on phenotypic and genetic characteristics, S. iniae was identified as the causative agent of streptococcosis in the cultured red hybrid tilapia. The bacterium was pathogenic following experimental infection of the fish and led to septicemia, nervous signs, and death.

ACKNOWLEDGMENTS This work was supported by a Higher Institution Centre of Excellence (HICoE) grant from the Ministry of Higher Education, Malaysia (6369100), and the Science Fund grant of the Ministry of Science, Technology and Innovation (MOSTI), Malaysia (5450652). The technical help from staff and students of the Biotechnology Aquatic Laboratory, Faculty of Agriculture, Universiti Putra Malaysia, is gratefully appreciated. ORCID M. Y. Ina-Salwany

http://orcid.org/0000-0003-2905-2915

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