Massive mortality associated with Streptococcus iniae infection in cage-cultured red drum (Sciaenops ocellatus) in Eastern China

In August, 2011, an enzootic disease characterized by hemorrhage throughout body surface, enlarged spleen and kidney occurred in cage farmed red drum fish, in Dongtou, Zhejiang of China. The diseased fish weighed between 100 to 200 g, and the cumulative mortality within 60 days was higher than 70%. Several bacterial isolates that exhibited the same phenotypic traits and biochemical characteristics were isolated from the lesions of diseased fish. According to the results obtained from biochemical tests and sequence analysis of the 16S rDNA, the disease’s pathogen (strain WZMH110819) was identified as Streptococcus iniae. In the challenge trials, the LD50 value of the clinical bacterial isolate WZMH110819 was 9.65×10 CFU per fish. Moreover, bath exposure or oral administration by Streptococcus iniae also caused a considerable number of deaths in fish. Antibiotic susceptibility tests showed that strain WZMH110819 was sensitive to most of the antibiotics including ampicillin, erythromycin and gentamicin in vitro. This finding has provided a basis for the control and prevention of further outbreaks of this enzootic disease in red drum farms.


INTRODUCTION
Streptococcal infections in fish have become an increasingly important health problem in modern intensive aquaculture (Eldar et al., 1995a).The main pathogenic species responsible for these streptococcal infections include Streptococcus iniae, Streptococcus agalactiae, Streptococcus parauberis and Streptococcus dysgalactiae (Eldar et al., 1995a;Domenech et al., 1996;Bercovier et al., 1997;Nomoto et al., 2004).S. iniae was firstly isolated from the subcutaneous abscesses in Amazon freshwater dolphins Inia geoffrensis (L.) in aquariums (Pier and Madin, 1976), and then large epidemic diseases in fish associated with this pathogen occurred.The most significant clinical signs of S. iniae infections in fish are septicemia and meningoencephalitis which are quite similar to signs caused by other streptococcal pathogens of fish (Eldar et al., 1994, *Corresponding author.E-mail: wangguoliang@nbu.edu.cn. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1995a, 1995b; Eldar and Ghittino, 1999).More importantly, humans can also be infected by S. iniae and a number of human cases have been reported in North America and Asia (Weinstein et al., 1997;Lau et al., 2003;Koh et al., 2004;Lau et al., 2006).
The red drum (Sciaenops ocellatus) is one of the most important economic fish species and it was introduced in China in 1991 (Shen et al., 2005).In a few decades, bacterial infections in red drum have become an increasingly important health problem because of the modern intensive aquaculture.Streptococcosis is one of the most common bacterial diseases in cultured red drum worldwide (Eldar et al., 1999;Colorni et al., 2002;Shen et al., 2005).Recently, an infectious disease outbreak with high mortality occurred in cage farmed red drum, causing severe economic impact on fisheries in Dongtou, Zhejiang province, China.
The aim of the present study was to isolate, identify and characterize the pathogenic bacteria of enzootic disease in red drum as well as to determine some potential drug candidates.The results obtained upon completion of this research can be taken from the laboratory into the field as a guideline for assisting red drum cage-cultured fish farmers for the control and the prevention of further streptococcal disease outbreak.

Bacterial isolation and biochemical characterization
In August, 2011, an infectious fish disease occurred among the red drum cage farmed fish in Dongtou, Zhejiang province China.More than 80 moribund fish with an approximate weight of 100 to 200 g were subjected to bacteriological examination.Several pure colonies of bacterial isolates (Strain WZMH110819) from liver, spleen and head kidney tissues of the diseased fish were recovered on Brain Heart Infusion Agar (BHIA; HuanKai, China) supplemented with 50% distilled sterile seawater.The sampling was conducted over a conservative two days period of high mortality rate.For the isolation of the etiological pathogen, tissues of liver, spleen and kidney from affected fish were streaked across the Brain Heart Infusion Agar (BHIA; HuanKai, China) supplemented with 50% distilled sterile seawater.The inoculated plates were incubated at 28°C for 24 h.Single colonies from plates with virtually pure culture growth were re-streaked onto the same media to obtain pure isolates.All pure isolates were cultured in liquid BHI and then add 20% glycerol for preservation at -80°C.
Bacterial identification was conducted using a number of biochemical and phenotypical tests by the API20 STREP kits (BioMerieux, France), according to the manufacturer's instructions.For the detection of hemolysis, the bacterial isolate was cultured overnight on trypticase soy agar supplemented with 5% defibrinated sheep blood (Toocle co., Hangzhou, China) at 28°C.The destruction of red blood cells (zones of beta-hemolysis) in the blood agar was detected prior incubation.The gram staining procedure for the bacterium followed the standard protocol.
The amplification products were gel-purified using PCR purification kit (Omega) and sequenced using the same primers as for PCR.Sequence was aligned using Clustal X version 2.1 followed by refinement by eye.Phylogenetic analysis of the 16S rDNA sequence was performed with Molecular Evolutionary Genetics Analysis (MEGA) software version 5.05 with the twoparameter of Kimura model for DNA based on the Neighbour Joining (N to J).

Experimental infection trials
Healthy red drum fish (102.3±15.5 g) were divided into 10 groups (n=10 for each group) and then were acclimatized in tanks (400 l) for 7 days at 30 ± 2°C.Before experimental infection, three fish selected randomly were subjected to bacteriological examination for confirming the fish not to be infected with bacteria.To determine the virulence of the bacterial isolate WZMH110819, four groups were injected intraperitoneally with the bacterium suspension at the doses of 1.93×10 6 , 1.93×10 7 , 1.93×10 8 and 1.93×10 9 CFU per fish respectively and one group of fish was injected intraperitoneally with PBS for negative control.The fish mortalities were recorded daily for 14 days after the challenge, and the LD 50 value was calculated by using the Reed-Muench method (Reed and Muench, 1938).
To investigate the infection routes of this bacterial pathogen, fish were also exposed to bacterium through oral or bath exposure.For oral administration, two group of normal fish (n=10) were challenged by oral admission of 1 ml of bacterial suspension at the doses of 1.93×10 6 and 1.93×10 8 CFU per fish, respectively.For bath exposure, normal fish or fish undergoing epidermal scarification were removed from their tanks and placed into a container of seawater containing 1.93×10 7 CFU/ml bacteria.After an exposure period of 30 min, the fish were removed and placed into their respective tank.Controls used for every method of challenge were subjected to identical handling procedures without being exposed to the isolated bacterium.Bacterial concentrations administered in the artificial experimental trials were evaluated using the indirect viable cell counts method as previously described (Todar, 2009).Briefly, a bacterial suspension was made from bacterial isolate grown on Brain Heart Infusion Agar (BHIA; HuanKai, China) for 24 h at 28°C were serially tenfold diluted with sterile phosphate-buffered saline to 10 8 .Each dilution was then plated to BHIA to determine cells in each dilution.Each viable unit grows and forms of a colony were counted prior incubation.

Antimicrobial susceptibility testing
The antimicrobial susceptibility of the bacterial isolate WZMH110819 was determined using the standard Kirby-Bauer method (Bauer et al., 1966).Briefly, a suspension (0.1 ml) of the bacterial strain WZMH110819 (diluted to a turbidity equivalent of a Macfarland No. 0.5 standard solution) was spread onto Mueller-Hinton agar and antimicrobial agent paper discs (Hangzhou Tianhe Microorganism Reagent) were then added to the surface of the agar.

Bacterial isolation from diseased fish
Diseased fish exhibited emaciation, lethargy, abnormal swimming, exophthalmia and hemorrhages on the base of fins.Internally, congestive kidney and spleen were observed in diseased fish (Figure 1).Multiple small bacterial colonies (strain WZMH110819) were isolated from different lesions of the diseased red drum.

Phenotypic traits and biochemical characteristics of the bacterial isolates
These bacterial isolates formed whitish colonies that were smooth around the edges across the BHIA plates and all strains were positive for β-hemolysis on blood agar plate.These isolates were all Gram-positive streptococcus-shaped bacteria in optical microscope.Moreover, all bacterial isolates and reference S. iniae showed the same biochemical characteristics by using the API 20 STREP kits (Table 1).

16S rDNA sequence analysis
The universal 16S rDNA primers set used in this study yielded the expected 1541 bp amplicon for bacterial strain WZMH110819.Sequence analysis showed that bacterial strain WZMH110819 showed 100% similarity with the reference S. iniae strain ATCC 29178 T , but showed 98% similarity with S. parasuis and 97% similarity with S. agalactiae and S. dysgalactiae, respectively.The phylogenetic analysis based on the 16S rDNA sequence (accession numbers: KF815728) also showed that the bacterial strain WZMH110819 clustered most clearly with S. iniae strain ATCC 29178 T (Figure 2).

Result of experimental infections
In challenge trails, the bacterial isolate WZMH110819 exhibited moderate virulence to the red drum with an intra-peritoneal LD 50 value of 9.65×10 6 CFU per fish.Depending on the amount of doses challenged, mortality rate ranged from 20 to 100% was recorded in our study (Figure 3).The cumulative mortality of each intraperitoneal  challenge group or negative control is showed in Figure 4. Bath exposure with the bacteria isolate WZMH110819 after skin trauma resulted in 40% mortality for 1.93×10 7 CFU per fish, in contrast to 0.0 % mortality for 1.93×10 7 CFU per fish with normal pathogen exposure.For oral administration, the mortality was 10.0% for 1.93×10 6 CFU per fish and 40% for 1.93×10 8 CFU per fish (Figure 4).The typical clinical signs of the moribund fish in experimental trails were hemorrhages on the base of fins, lethargy, abnormal swimming and enlarged and congestive visceral organs which were similar to naturally infected fish.

DISCUSSION
Red drum is one of most important economic fish species mainly farmed in Zhejiang and Fujian provinces, China.During 2011 to 2012, massive mortality in cage cultured red drum occurred in Dongtou fish farms, Wenzhou, Zhejiang province.The outbreak of the disease occurred in breeding fish which were almost one year of age (approximately 100 to 300 g) when the water temperature was up to 29°C.However, the large yellow croaker, another important economic native fish cultured in the same coastal region, was not affected by this epidemic disease.The diseased or moribund red drum exhibited skin lesions, exophthalmia, disorientation and hemorrhage around the tail and anal fin.Moreover, there were some gram-positive streptococcus-like bacteria observed in the tissue smears.
The bacterial strain isolated from the diseased fish was G. positive, cocci-chain and oxidase-negative.On the basis of these results and those of the API20 STREP kits (BioMerieux, France) listed in Table 1, the isolate was identified as S. iniae.The bioche-mical characters of the S. iniae strain WZMH110819 were similar to those results reported previously by other researchers (Pier and Madin, 1976;Shen et al., 2005;Zhou et al., 2008).Meanwhile, this bacterium had ability hydrolyze arginine, suggesting that it may share the same serotype (serotype I) that have been previously reported by other researchers (Pier and Madin, 1976;Shen et al., 2005;Zhou et al., 2008).
Infections associated with S. iniae in red drum in Zhejiang province has been reported early in 2005 (Shen et al., 2005).However, the epidemic reported by Shen occurred from September to December and the cumulative mortality was 20 to30%.Moreover, the S. iniae isolates seemed of low or moderate virulence with the intra-peritoneal LD 50 value of 4.18 × 10 8 CFU per fish and 1.19 ×10 7 CFU per fish (100-120 g), respectively, according to the report (Shen et al., 2005).In the present study, the epidemic broke out from July to September, and the cumulative mortality was up to 70%.Experimental infection trials showed that the clinical isolate WZMH110819 exhibited moderate virulence to the red drum with an intra-peritoneal LD 50 value of 9.65×10 6 CFU per fish (102.3±15.5 g) in the current study.This result suggested that the S. iniae strain WZMH110819 might be more virulent than the strains isolated by Shen et al. (2005).
Previously, the study reported that epidermal scarification did not appeared to enhance the ability of S. iniae to enter the barramundi and increase mortalities over traumatized fish (Bromage and Owens, 2002).However, it showed that the mortalities of the red drum were up to 40% in bath exposure group undergoing epidermal scarification in the current study, compared to normal bath exposure or oral exposure.
Oral administration and bath exposure experiments, which indicated that the red drum were more likely to be infected with S. iniae via skin trauma.The average stocking density of red drum fish in Dongtou cage farms were about 20 to 25 fish per m 3 .High stocking density or close contact between the fish themselves may damage the integrity of their epidermal and protective mucosal layers, consequently predispose them to S. iniae infection (Shoemaker et al., 2001).Thus, the frequent outbreaks of this enzootic disease in this area may be due to high stocking rates and feeding the affected fish meal (Zhou et al., 2008).
Several investigators have reported that some S. iniae strains exhibited resistance to several commonly used antimicrobial drugs.Shen et al. (2005) has reported that the S. iniae isolates from red drum fish developed some resistance to gentamycin, lincomycin and norfloxacin.Similarly, Tran et al. (2013) has reported that S. iniae isolated from in diseased barramundi (Lates calcarifer) cultured in Vietnam was sensitive to several antibiotics including norfloxacin, ciprofloxacin, sulphamethoxazol / trimethoprim, ampicillin, erythromycin and doxycycline but was resistant to gentamicin, and streptomycin.In present study, the bacterial isolate WZMH110819 was sensitive to most of antibiotics tested including gentamicin and norfloxacin in vitro, but showed resistance to sulfamethoxazole compound (SMZ+TMP).This result suggests that antibiotics therapy would be effective in treating fish affected by this pathogen.
The results of the present study demonstrates that the massive mortality in red drum in Dongtou fish farms was due to infection with S. iniae, and the epidermal scarification might enhance the pathogens ability to enter the fish and increase mortalities.This important finding would suggest that reduced handling and decreased the stocking density might be an important means of preventing problems caused by this pathogen.

Figure 1 .
Figure 1.Diseased red drum fish showing (A) enlarged kidney and spleen, (B) cornea opacity or POP eye, (C) hemorrhages on the base of fins and (D) dorsal and tail fin erosion.

Figure 2 .Figure 3 .
Figure 2. Neighbor-joining phylogenetic tree of 16S rDNA sequences of bacterial isolate WZMH110819, reference S. iniae and the most closely related species of streptococci.

Figure 4 .
Figure 4. Mortality rate of Red drum exposed to bacterial strain WZMH110819 in deploys of natural infection trials via bath treatment or oral administration.

Table 1 .
Biochemical Profile of strain WZMH110819 and references S.iniae a S. iniaeSO-2 +, Positive; -, negative; a data was from Pier and Madin 1976; b data was from Shen et al., 2005; N.A, Not available.