Isolation and characterization of Streptococcus agalactiae from Nile Tilapia Oreochromis niloticus in China

1 Research Center of Fish Disease, Sichuan Agricultural University, Ya’an, Sichuan 625014, People’s Republic of China. 2 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya’an, Sichuan 625014, People’s Republic of China. 3 Department of Aquaculture, Sichuan Agricultural University, Ya’an, Sichuan 625014, People’s Republic of China. 4 Animal Health Research Institute of Tongwei Co., Ltd., Chengdu, Sichuan 610041, People’s Republic of China.


INTRODUCTION
Tilapia is a tropical fish belonging to the family Cichlidae, genus tilapia native to Africa (Shirak et al., 2009).Due to its rapid growth and palatability, this species has been widely introduced in countries with a tropical climate around the world and has become globally important aquatic species produced in nearly 100 countries worldwide (Romana-Eguia et al., 2004).Tilapia production in Asia has grown considerably during the recent ten years, especially in China and Philippines (Keshavanath et al., 2004).With the rapid development of tilapia farming, China has become one of the important tilapia producers in the world (Fitzsimmons, 2000).However, the rapid expansion and intensification of tilapia farming has led to a series of problems including the occurrence of various diseases, which can restrict the sustainable development of the industry.
Streptococcosis has been recognized as one of the most serious bacterial diseases in tilapia culture and usually causes high mortality and lasts a long period of time (Yang and Li, 2009).In China, the main streptococcal pathogen of tilapia has been considered as Streptococcus iniae and its infection in tilapia occurred in main culture regions with high frequency (Chai et al., 2002;Deng et al., 2010;Yu et al., 2008).In recent years, however, the problems of Streptococcus agalactiae infection in tilapia culture have become gradually apparent.Severe streptococcal infection in tilapia caused by S. agalactiae has occurred in China, causing heavy losses (Zhang et al., 2008).In July 2009, there was an outbreak of an infectious disease with high mortality in intensive tilapia cultures in Wenchang and Xinglong villages (Hainan Province, China), which continued at least to October.In order to identify the pathogen of this outbreak, moribund fish were taken to the laboratory.Eight strains which were isolated from diseased tilapias were identified on the basis of 16S rRNA gene sequences phylogenetic analysis, API-20 Strep kits test and characteristics.In this paper, we also studied the etiology and virulence of the strains to provide basic data and information for healthy aquaculture of tilapia.

Samples collection and symptom observation
The naturally infected tilapias (200 to 500 g) were collected from eight different farm ponds in Wenchang and Xinglong villages, Hainan Province and the disease signs were observed and recorded.Ten to thirty fish of each farm showing visible typical clinical signs were transferred alive in plastic bags with an oxygen supply to the laboratory for further study.

Bacterial isolation
For bacterial isolation, samples taken from brain, liver, kidney, eye, and spleen of the moribund fish were directly streaked onto brain heart infusion (BHI, BD Corporation, USA) agar plates with flamed loop and incubated 48 h at 37°C.The dominant colonies were sub cultured on the same media to check the purity of the isolate.Pure culture of eight dominant strains were isolated, and numbered: HN0101, HN0201, HN0202, HN0203, HN0301, HN0302, HN0303 and HN0401.Pure stock isolates were stored at −20°C in 15% glycerol (final concentration) supplied with BHI broth.

Challenge tests
Healthy tilapias (100±5.0g) from an intensive culture farm, with no history of disease, were kept in 120 L aquaria with aeration and fed with commercial pellets twice a day at a daily rate of 2% of their body weight.Water was partly replaced daily and the temperature was maintained at 30±1.0°C.Fish were kept one week before bacterial challenge.Two hundred tilapias were maintained at 30±1.0°C and divided into 20 groups randomly: the groups from NO.1 to NO.9 were used for the animal regression test.Group 1 to group 8 were given intraperitoneal injections with 0.2 ml cell suspension of each isolate per fish (HN0101, HN0201, HN0202, HN0203, HN0301, HN0302, HN0303 and HN0401 respectively) in 0.8% NaCl solution at a concentration of approximately 2×10 8 CFU/ml.Group 9 were injected with sterile 0.8% NaCl solution as a control in the same way.
Among the eight strains, HN0101 and HN0301 were isolated from two moribund tilapias both with apparent clinical signs and symptoms of septicaemia, meningoencephalitis and exophthalmia from Wenchang and Xinglong villages, respectively.The two isolates were selected to do the virulence tests to estimate the median lethal doses (LD 50 ) of the strains using SPSS10.0 for Windows package (SPSS Inc. 1999).Groups from NO.10 to NO.14 were injected intraperitoneally with 0.2 ml/fish cell suspension of the bacteria (HN0101) at a concentration of 1.32×10 9 CFU/fish with tenfold dilution.Groups from NO. 15 to NO.19 were injected intraperitoneally with 0.2 ml/fish cell suspension of the bacteria (HN0301) at a concentration of 1.16×10 9 CFU/fish with ten-fold dilution.The same volume of 0.8% NaCl solution was injected into the group 20 as a control (Zhou et al., 2010).
Mortality was recorded daily for a 7-day period after injection.All dead fish were necropsied and the brain, liver, spleen, kidney, eyes were aseptically streaked on BHIA to confirm the presence of the challenge strain.After incubation at 37°C for 48 h, the isolates were identified by phenotypic characteristics.Mortality was considered only if the challenge strain was re-isolated as a pure culture from the internal organs and biochemically confirmed.

Phenotypic characterization
Phenotypic analysis of the isolates included the following items: colony morphology, hemolytic reactions on 5% sheep blood BHIA, Gram staining, electron microscopic examination, cellular morphology, biochemical test by using the API 20 Strep system (bioMérieux, France) and Lancefield grouping by Streptococcal Grouping Kit (Oxoid, UK).The type culture strain of S.agalactiae (ATCC51487) was tested as a positive control.

16S rDNA sequence analysis and phylogenetic analysis
The DNA of bacterial isolates was extracted from pure cultures of the bacterial isolates with 16S rDNA bacterial extraction Kit (TianGen, China).The universal primers specific for the 16S rRNA gene of bacteria were used to amplify the 16S rDNA gene.The primers are 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5'-TACGGCTACCTTGTTACGAC-3' respectively (Li et al., 2001;Polz and Cavanaugh, 1998).The primers are expected to yield approximately 1.5 kb 16S rRNA gene by PCR.PCR reactions were carried out with a 50 μL reaction volume containing 25 μL PCR premix, 0.5μl of each primer, 1μL DNA template and 23 μL 16s-freeH 2 O.The PCR was performed with one cycle of predenaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1.5min, and a final extension at 72°C for 5 min.The amplification products were analysed with 1% agarose gel electrophoresis followed by ethidium bromide staining and then checked with UVinduced fluorescence.PCR products were sequenced using a 3730 DNA sequencer (Shanghai Invitrogen Biotechnology Co. Ltd., China) and assembled with DNA Star software.
The 16S rDNA sequences were compared to sequences in the GenBank using the BLAST program.Aligned 16S rDNA sequences of the related species were retrieved from the NCBI nucleotide database.Program Clustal X 1.8 was run with default parameters for multiple sequence alignment.Phylogenetic and distance analysis of the aligned sequences was performed by using the program MEGA 4.1.An unrooted evolutionary tree was inferred by using the neighbour-joining (N-J) tree algorithm.The resultant tree topologies were evaluated by boostrap analysis of the N-J method based on 1000 resamplings.

Antibiotic susceptibility assays
Susceptibility to antibiotics of each isolated strain was determined by the disc diffusion method with 18 antibiotics (Hangzhou Taihe Microbiological Reagent, China).The susceptibility and resistance of each isolate were determined according to the manufacturer's instructions and criteria of the NCCLS (2002).The type culture strain of S. agalactiae (ATCC51487) was tested as a positive control.,The injection concentration of HN0101 strain was 1.32×10 9 CFU/fish with ten-fold dilution, while the HN0301 strain was 1.16×10 9 CFU/fish with ten-fold dilution.

Diseased signs
The affected tilapias were usually dark in color and show erratic patterns of swimming, especially near the termination of disease.The external signs of the diseased fish were petechial hemorrhages under the lower jaw, on the operculum and belly, at the base of the fins, and around the anus.Some moribund fish showed exophthalmia (Figure 1A).Internally, hyperemia of meninges (Figure 1B), and grossly enlarged liver, gallbladder and spleen could be seen.Intestine was usually filled with amounts of yellow intestinal mucus.

Challenge tests
During 7 days of the infection experiments, there is neither mortality nor visible changes in the two control groups.The mortality rates from Group 1 to Group 8 were 100% within 7 days after the challenge.Fish show similar clinical signs as naturally infected fish and pure cultures of all inoculated strains were re-isolated from the kidney, brain; liver of all dead fish, which could demonstrated that the eight isolated strains were virulent for the fish.The other challenge test showed an increasing mortalities with the bacteria concentrations gradually increased (Table 1).Affected fish also show similar clinical signs as naturally infected fish within a week after injection challenge.Before dying, some fish show erratic patterns of swimming.External symptoms include petechial hemorrhages under the lower jaw, on the operculum and belly, at the base of the fins, and around the anus and a few tilapias show exophthalmia.The main internal signs were hyperemia of meninges, enlarged liver, gallbladder and spleen and enteritis.The LD 50 of HN0101 and HN0301 from Wenchang and Xinglong villages were 6.8×10 6 CFU/fish and 5.3×10 6 CFU/fish respectively by intraperitoneal injections.

Phenotypic characterization
All isolated strains and type culture strain (ATCC51487) were Gram-positive, oval or spherical-shaped (approxi- mately 0.9 to 1.2 micrometer in diameter) and non-sporeforming bacteria.They usually grew in pairs or chains (Figure 2).The type culture strain and most of the isolates grew poorly on trypticase soy agar (TSA), but growth was enhanced after adding blood.All the strains grew well on brain heart infusion agar (BHIA).After incubation at 37°C for 48 h on 5% sheep blood BHIA, the isolates and the type culture strain produced pinpoint-tosmall colonies, which appeared circular, slightly convex, white with a regular surface and edge and there were no zone of hemolysis around individual colonies.Biochemical characteristics and Lancefield grouping were determined by using API 20 Strep system and Streptococcal Grouping Kit (Oxoid, UK), respectively.The results indicated that all the isolates were Group B Streptococci (GBS) with the same profile number 3663410, which corresponded to good matches to GBS given by API 20 Strep (Table 2).All the isolates were suggested to be S.agalactiae according to the results of phenotypic characterization.

Sequence analysis of 16S rDNA
The 16S rRNA gene sequences of the eight strains isolated in this study were submitted to GenBank with the accession numbers of JF423941-JF423948.A phylogenetic tree was constructed based on the 16S rDNA sequences of the isolated strains and 15 homologous sequences (Figure 3).The eight strains were all with high sequence similarities (99.5 to 99.9%) to S. agalactiae (DQ303183), strongly suggesting that the eight strains were all S. agalactiae.

Sensitive to antibacterial drugs
Drug sensitivity test of these pathogenic bacteria shows that all eight isolates of bacteria were sensitive to cefoxitin, but had quite different sensitiveness to other antibiotics.The type culture strain ATCC51487 show a broad antibiotic susceptibility.It was found to be only resistant to cidomycin, amikacin, streptomycin and sensitive to all other antibiotics (Table 3).

DISCUSSION
In recent years, with a steadily expanding industry of aquaculture in China, Streptococcosis has become an increasingly severe problem, which usually occurs between July and October and spreads widely.It was reported that Streptococcosis had frequently occurred in the south of China (such as Guangdong, Guangxi, Fujian and Hainan Province) during these years (Chai et al., 2002;Zhang et al., 2008).There are several reasons: the first, tilapia aquaculture in China is mainly distributed in southern city, because the warm water is suitable for the growth of tilapia whose optimum growth temperature is 29 to 31°C (Popma and Masser, 1999); the second, tilapia is susceptible to Streptococcus, and during July to October each year the water temperature in main culture regions in South China will usually rise which may increase the tilapia's susceptibility to S. agalactiae (Amal et al., 2008;Evans et al., 2006); the third, in intensive aquaculture, the high-density fish culture would increase the risk of Streptococcosis.In the present study, the eight strains isolated from tilapia in the outbreak of an infectious disease in Hainan were characterized as S. agalactiae by physiological and biochemical tests, and also further confirmed by similarity analysis of the 16S rRNA genes.Challenge experiments demonstrated that all the isolated strains were the pathogenic bacteria and virulent for the fish.S. agalactiae infection on tilapia was also confirmed according to Koch's postulate.The LD 50 of two strains (HN0101 and HN0301) isolated from tilapias from two villages were 6.8×10 6 and 5.3×10 6 CFU/fish, respectively by intraperitoneal injections, a relatively low value, which indicated strong virulence.
Members of the genus Streptococcus are widely distributed in the world.So far, streptococci have become common pathogens of fish.It has been reported that dozens of cultured and wild-ranging marine or freshwater fish are susceptible to Streptococcus such as salmon, mullet, golden shiner, pinfish, eel, sea trout, tilapia, sturgeon, striped bass, rainbow sharks, red-tailed black sharks, danios, some cichlids and several species of tetras (Kitao, 1993;Russo et al., 2006).The major species of Streptococcus which infect fish are S. iniae, S. difficile, S. agalactiae, S. parauberis, S. dysgalactiae and S. Shiloi (Mata et al., 2004;Netto et al., 2011;Eldar et al., 1995).S. agalactiae is a member of group B Streptococcus that can be either haemolytic (Evans et al., 2002) or non-haemolytic (Finch and Martin, 1984).The eight strains of S. agalactiae isolated from tilapias are all  non-haemolytic.S. agalactiae is the only streptococcal species that corresponds to the serogroup B of the Lancefield serotyping (Devriese, 1991).It is common for S. agalactiae to cause diseases in tilapia in foreign countries.However, in China, the pathogenic bacteria of streptococcosis in tilapia are generally considered as S. iniae.After this outbreak of an infectious disease in tilapia culture by S. agalactiae in Hainan province, China may change the previous understanding.Because for two consecutive years (2008 to 2009) severe streptococcal infection in tilapia caused by S. agalactiae had occurred in China, we can assume that S. agalactiae would become the main pathogenic bacteria of streptococcosis in tilapia culture instead of S. iniae in China.
Streptococcosis in fish caused by S. agalactiae is characterized with septicaemia and meningoencephalitis (Mian et al., 2009), which was supported by the clinical signs and symptoms in this outbreak of streptococcosis in tilapia.The affected fish became lethargic or showed erratic patterns of swimming, a classic manifestation of brain infection.There were petechial hemorrhages under the lower jaw, on the operculum and belly, at the base of the fins, and around the anus.The intestine was usually hemorrhagic and filled with pink mucus.The kidney was swollen and the liver congested as well as the brain.In addition, some recently dead or still living but moribund tilapias showed a sign of exophthalmia.It was reported that other pathogenic bacteria might also cause the exophthalmia in tilapia, such as S. iniae (Perera et al., 1994), Francisella sp.(Soto et al., 2009), Pseudomonas fluorescens (Miyazaki et al., 1984) and so on.Therefore, disease signs, isolation and identification of the bacterium are all necessary for definitive diagnosis of S. agalactiae disease in tilapia.
Generally, Gram-positive bacteria, including Streptococcus spp., are susceptible to roxithromycin (Gemmell and McLeod, 1992) amoxicillin (Darwish and Hobbs, 2005).However, as the case in the present study, some isolates were resistant to these antibiotics.All were sensitive only to cefoxitin grouped with the secondgeneration cephalosporins.Compared with the type culture strain of S. agalactiae (ATCC51487), the isolates  show a narrower antibiotic susceptibility, which indicated bacterial resistance to antibiotics was more serious.Consequently, the sensitivity test is necessary to select the most effective antibiotic to use.Besides using of chemotherapeutics, it is advisable to set up proper management procedures to control disease, including improvement of water quality, reduction of stress in fish, and so on.Prevention of disease is always preferable than treatment after its outbreaks.Further studies on rapid diagnostic methods and vaccines against the disease are required for better control of streptococcosis in tilapia.

Figure 3 .
Figure 3. Phylogenetic tree of the bacterial isolates based on 16S rDNA sequences, generated by the neighbor-joining method.

Table 1 .
Dead/total number of HN0101 and HN0301 strains on tilapia by IP injection.
a S. agalactiae a S. dysgalactiae a S. ictaluri b

Table 3 .
Sensitivity of bacterial isolates to different antibiotics.