Aquaculture Science 54(3), 269-274 (2006)

Multiplex PCR for Detection of garvieae, and S. dysgalactiae in Cultured Yellowtail

Mortada M. A. HUSSEIN1,2 and Kishio HATAI1,*

Abstract: Streptococcosisis one of serious disease in culturedyellowtail, Seriola quinqueradiata, amberjack, S. dumerili and salmonids, and economically is problematic. Streptococcusiniae, is known as a of the marine and freshwaterfishes. On the other hand, Lactococcusgarvieae, and recently S. dysgalactiaeare known as the of the yellowtailand amberjak. Mixed infectiondue to the pathogens, however, occurs very often in yellowtail.Therefore, the correct diagnosisis sometimes confused. In an attempt to elucidatethe main pathogen, a multiplexPCR was newly designed in this study.The new multiplex PCR assays involvesamplifying the three multiplegene products in a singlereaction based on primers designed from the 16S rRNA,16S-23S rDNA intergenicspacer region and lactateoxidase(lctO)genes of L.garvieae, S. dysgalactiaeand S. iniae,respectively. The specificityof the multiplexPCR using the primer setswas confirmed by the factthat specificbands were only amplifiedequivalent to 1,100,870 and 259-bp for L. gayvieae, S. iniae,and S. dysgalactiae,respectively. In addition,the specificpositive amplifications in used alltemplates were consistentlyobserved only for each corresponding pathogen. Multiplex PCR did not produce any non-specificamplification products when tested againstpure DNAs, bacterial suspensions or tissuehomogenates from ,Oncorhynchus mykiss artificiallyinfected with the three pathogens. Multiplex PCR was an effectivetool for the rapid and specificdetection of L.gravieae, S. dysgalactiaeand S. iniaefrom fishtissues.

Key words: Lactococcus garvieae; Multiplex PCR; Streptococcusdysgalactiae; Streptococcus iniae

Streptococcusinfection of fisheshas become fore, a definitive diagnosis of the etiological

a major problem with the development of agent was based on the microbiological charac- intensive aquaculture in Japan, particularly, teristics of isolated from diseased fish yellowtail,Seriola quinqueradiata, amberjack, (Mata et al.2004a). S. dumerili and salmonids. These fishes are Recently, molecular methods based on DNA of importance because of theirhigh economic probes or PCR have overcome problems associ- value.Streptococcus iniae, Lactococcus garvieae, ated with culture-based techniques, enabling and recentlyS. dysgalactiaesubsp. dysgalactiae the detection of microorganisms directly from are known as pathogens of the fishes, and clinical samples without the isolation (Gonz�lez sometimes cause mixed (Kusuda et al. et al. 2004). However, a large number of indi-

1991;Kitao 1993;Nomoto et al.2004).Yellowtail vidual PCR assays would be necessary if single with streptococcosis exhibits very similar primer sets are used on a large number of clini- symptoms and clinicalsigns regardless of the cal samples and that can be a relatively costly etiologicalagents (Zlotkinet al.1998;Eldar and and time-consuming process. The detection

Ghittino 1999; Kusuda and Salati1999), there- of several pathogens using a multiplex PCR

Received December 17,2005:Accepted May 9,2006. 1Laboratory of Fish Diseases , Nippon Veterinary and Life Science University,1-7-1Kyonan-cho, Musashino, Tokyo 180-8602,Japan.2 Department of Fish Diseases and Managements, Faculty of VeterinaryMedicine, Beni-SuefUniversity, Beni-Suef 62511, Egypt. *Corresponding author: Tel.& Fax:+81-422-31-6796. E-mail:[email protected] 270 M. M. A. Hussein and K. Hatai

(m-PCR) would be rapid and cost-effective acclimatization for lo days, fish were divided (Osorio et al. 2002). into 8 groups and kept separately in 101aquaria In this present study we attempted to develop at 15℃. Two groups were designated as A and a m-PCR for the concurrent detection of B (n=3 each),2groups as C and D (n=2each) Streptococcus iniae, Lactococcus garvieae, and and 4 control groups as E, F, G, and H (n=

S. dysgalactiae, the most prevalent gram posi- 2each). Each fish of the group A was injected tive cocci (GPC) pathogens incriminated in fish with single dose of 0.1 ml of either L. garvieae strepococcosis incidence in Japan, from the NJB E2, S. iniae NJB 02-011, or S. dysgalactiae pure cultures as well as tissues of fish artifi- NJB 0442 with bacterial suspension of 3×107 cially infected with the pathogens. cells/ml. Group B was treated as group A but

with bacterial suspensions at concentrations of

Materials and Methods 3×102 cells/ml. On the other hand, fish in the

group C were injected with 0.1 ml mixed bacte- Bacterial strains and the preparations rial suspensions of the three selected isolates

Eight strains used in this study were iso- at a concentration of 3×107 cell/ml while fish lated from diseased fishes with clinical signs in the group D were injected with 0.1 ml of the of streptococcosis (Table 1). All strains were same bacterial mixtures but at a concentration inoculated in Todd Hewitt Broth and incubated of 3×102 cell/ml. Control groups were injected at 25•Ž for 48 h (THB, Sigma, Missouri, USA), with 0.1 ml sterile bacterial free phosphate- and then the bacterial pellets were collected by buffered saline. Fish in all groups were moni- centrifugation at 3000 rpm at 4•Ž for 10 min. tored for 10 d post injection and tissue samples

Subsequently, the obtained pellets were 10-fold including , , brain, and muscles

serially diluted using sterile phosphate-buffered were collected from moribund fish, and homog-

saline starting with 3•~108 (McFarland No. 1) enized for DNA extraction. till 3•~10 cells/ml. Bacterial suspensions were In parallel, a loopful of each tissue homog- used for either m-PCR (with and/or without enates was streaked onto the TH agar supple-

DNA extraction) or artificial infection. mented with 3% sheep and incubated at

25℃ for 48 h. Live fish were scarified at the end

Artificial infection and tissue homogenates of the experimental period(10 d)and the same

Eighteen apparently healthy rainbow trout tissues were sampled. Fish used as control

used in this study were 70•}5g in body weight were processed in the same way with the exper-

and 20•}2 cm in body length, which were imentally infected ones.

obtained at a private farm in Tokyo, Japan. After

Table 1. Isolatesused for m-PCR assay Multiplex PCR for Streptococcosis in Yellowtail 271

Table 2. Oligonucleotide primer sets used for m-PCR amplification and the expected amplicon sizes

Design of mulitiplex PCR(m-PCR) Table 3. Recovery of the three streptococcal pathogens The new multiplexPCR assay involvesampli- and their detection by m-PCR in different tissues from artificiallyinfected fish Eying the three multiple gene products in a singlereaction based on primers deduced from the regions carrying the 16S rRNA (Zoltkin et al.1998), 165-23S rDNA intergenic spacer region (Hassan et al.2003;Nomoto et al.2004) and lactateoxidase (lctO)genes (Mata et al.

2004a) of L. garvieae, S. dysgalactiaeand S. a: Recovery of the three streptococcal pathogens . b: Recovery of one or two out of the three streptococcal path iniae,respectively. ogens.

DNA extractionand m-PCR amplification

DNA was extracted using DNAzol〓 reagent

(Invitrogen Life Technologies, California, USA) simultaneously analyzed by electrophoresis of from bacterial suspensions and fish tissue 5μl of each amplification mixture in 2% agarose homogenates. The target gene and species- in Tris-borate acid EDTA buffer(100 V,30 min) specific oligonucleotide primer sets used in the gels containing ethidum bromide. m-PCR are indicated in Table 2.

The m-PCR amplifications were performed Results in 25μl reaction mixtures with Ready-to-go

PCR beads (Amersham Pharmacia Biotech, Recovery of the bacteria from fish artificially infected with the three pathogens

Ltd., Piscataway, USA) by adding 1 pg of each primer, 1μl of template (extracted DNA, bac- Tissues of artificiallyinfected fish were terial suspension or fish tissue homogenate) attempted to recover the injected pathogens and 18μl distillated water (DW). Samples were with TH agar containing3% sheep blood (Table subjected to a designated regime of amplifica- 3).The injectedthree pathogens were recov- tion in PCR thermal cycler (Takara Biomedical, ered in pure culturesfrom fishin groups A and Tokyo, Japan) with the following parameters: B. On the other hand, re-isolationof the three an initial denaturation at 94℃ for 1 min, 30 injectedpathogens from groups C and D was serial cycles of a denaturation at 92℃ for l min, varied considerably. In contrast,one or two annealing at 52℃ for 1 min, extension at 72℃ pathogen (s) were recovered from selected for 90 s, and a final extension at 72℃ for 5 min. organ homogenates except that of kidneys and Negative control was included in each batch of they could be differentiatedin theirpure form m-PCR reaction. PCR-generated products were after several subcultures, however, the three 272 M.M. A. Hussein and K. Hatai

injected pathogens were recovered purely from kidney homogenates. Discussion

Development of multiplex PCR Identification based on biochemical and

The specificity of the m-PCR assay was evalu- antigenic characteristics can be barely dif-

ated by testing the three primer sets with the ferentiating among L. garvieae, S. iniae and

pure DNA templates either from pure bacte- S. dysgalactiae, however, they are limited by

rial suspensions and/or tissue homogenates the length of time required to complete the

of the artificially infected fish. Specific positive assays (Zlotkin et al.1998). In addition, even

PCR amplifications in all used samples includ- isolation of those pathogens, particularly in a

ing bacterial suspensions, tissue homogenates case of mixed infection, is considered as time

from different organs, and extracted DNAs and effort consuming and sometimes lead to

from either bacteria or tissues were consistently misdiagnosis. The results represented herein

observed for each corresponding pathogen. In showed that in a case of mixed infection with

contrast, positive PCR amplifications of DNA the three pathogens, they could be re-isolated

templates produced a single specific fragment in pure after several subcultures and not from

bands for each pathogen with a size of 1,100, all selected tissues (Table 3). That is prob-

870,and 259 by for L. garvieae, S. iniae, and ably due to special requirements by such fas-

S. dysgalactiae, respectively (Fig.1). Similarly, tidious microorganisms(Shoemaker et al.2001;

single specific positive PCR amplifications Hassan et al.2003).Therefore, a rapid diagno-

bands were obtained when m-PCR assay was sis is necessary to initiate prompt therapeutic

performed with DNA templates including tis- and prophylactic measures in order to limit the

sues homogenates and/or extracted DNA. In economic losses caused by such pathogens.

addition, the specificity of the m-PCR was clear The molecular techniques have been the most

not only with infected tissue samples obtained effective methods for diagnosis because they are

from groups A and B but also with groups C more specific and sensitive detection than other

and D. The detection limit of m-PCR assay in conventional or commercial assays (Gonz�lez et

fish tissue was equivalent to 3×102 cells/ml al.2004).Alternatively, m-PCR assays using bac-

(Fig.2).The m-PCR assays neither produce terium-specific gene can be used as targets for

non-specific amplification products of used sam- PCR amplification to permit more specific detec-

ples nor false positive ones of the uninfected tion as well as subspecies and strain differen-

controls. tiation in a single reaction. Williams et al.(1999)

previously developed m-PCR for the detection of

Fig. 1.Amplification products obtained using the m-PCR developed forthe concurrent detectionof L. garvieae (1,100bp), S. iniae (870 bp) and S. dysgalactiaesubsp. dysgalactiae(259 bp). Lanes M, 100 (right)and 500 (left)by DNA ladder (Bio- Rad, Marnes-la-Coquette,France); lane 1, negativecontrol;lanes 2-5, L. garvieaeKGLA 0415, NJB B1, NJB K2, NJB E2;lane 6,S. iniaeNJB 02-011;lanes7-9, S. dysgalactiaesubsp. dysgalactiaeKdys 0432,Kdys 0440, Kdys 0442. Multiplex PCR for Streptococcosis in Yellowtail 273

Fig.2. Sensidvity of detection of L. garvieae(1,100 bp), S. iniae (870 bp) and S.dygalactiae subsp. dysgalactiae (259 bp)

by m-PCR of experimentally infected fish tissues homogenates. Lanes M,100 (left)and 500 (right)by DNA ladder (Bio-

Rad, Marnes-la-Coquette, France); lanes 1 and 10, negative control; lanes 2 and 8,(3×107)and(3×102) of S. dysgalactiae

subsp. dysgalactiae; lanes 3 and 7,(3×107)and(3×102)of L. garvieae; lanes 4 and 9,(3×107)and(3×102) of S. iniae;

lanes 5 and 6,(3×107)and(3×102)of bacterial mixture of the three pathogens used.

fish viruses. In addition, a m-PCR for detection than that reported by Mata et al.(2004b), who of Aeromonas salmonicida, Yersinia ruckeri,and demonstrated that their m-PCR could detected

Flavobacterium psychrophilum was established L. gayvieae and S. iniae in tissue at a concentra- by del Cerro et al.(2002). tions of 2.5×103 and 5×103, respectively.

In this work, a m-PCR was developed for the From these data, it can be concluded that the

concurrent detection of L. garvieae, S. iniae, proposed m-PCR assay can be useful not only

and S. dysgalactiae in their pure cultures and as a diagnostic tool but also for epidemiologi-

the tissues from fish artificially infected with cal surveys and could be efficientto establish

the pathogens. The specificity of our m-PCR preventive measures on time. The m-PCR that

using the above-mentioned primer sets was we have developed is sensitive,fast, and simple

confirmed by the fact that only specific bands technique that can be accomplished in 5 h for

were amplified equivalent to 1,100,870 and rapid and specific detection of L. garvieae, S.

259 bp, which are characteristics for L. garvieae, iniae and S. dysgalactiae,the main GPC patho-

S. iniae and S. dysgalactiae, respectively. These gens responsible for fish streptococcosis in results are in agreement with those obtained Japan.

by Zlotkin et al.(1998)for L. garvieae, Mata et

al.(2004a)for S. iniae and Hassan et al.(2003) Acknowledgments

for S. dysgalactiae subsp. dysgalactiae when

they used single PCR. In addition, same results This work was financiallysupported by Japan

were reported by Mata et al.(2004b)for L. Societyfor The Promotion of Science (JSPS).

garvieae and S. iniae but not for S. dysgalactiae because the later was not included in their References

m-PCR. Moreover, our m-PCR could detect

the three pathogens in the tissues of fish artifi- del Cerro, A., I.Marquez and J. A. Guijarro (2002) Simultaneous detection of Aeromonas salmonicida, cially infected with them at low concentrations Yersiniaruckeri, and Flavobacterium psychrophilum, of 3×102 indicating its specificity as well as three major fishpathogens, by Multiplex PCR. Appl. sensitivity. Interestingly, this concentration in Environ.Microbiol.,68,5177-5180.

the artificially infected tissue samples is lower Eldar, A. and C. Ghittino(1999)Lactococcus garvieae and 274 M.M. A. Hussein and K. Hatai

Streptococcus iniae in rainbow trout Mata, A.I., A. Gibello, A. Casamayor, M. M. Blanco, L.

Oncorhynchus mykiss: similar, but different diseases. Dom�nguez and J. F. Fern�ndez-Garayz�bal (2004b)

Dis. Aquat. Org.,36,227-231. Multiplex PCR assay for detection of bacterial patho-

Gonz�lez, S. F., M. J. Krug, M. E. Nielson, Y. Santos and D. gens associated with warm-water streptococcosis in

R.Call (2004) Simultaneous detection of marine fish fish. App l. Environ. Microbiol.,70,3183-3187.

pathogens by using multiplex PCR and DNA microar- Nomoto, R.,L.I. Munasinghe, D-H. Jin,Y. Shimahara, H. ray. J. Clin. Microbiol., 42,1414-1419. Yasuda, A. Nakamura, N. Misawa, T. Itami and T. Hassan, A. A.,I. U. Khan and C. Lammler (2003) Ybshida (2004)Lancefield group C Streptococcus dys Identificationof Streptococcus dysgalactiaestrains of ga-lactiaeinfection responsible for fish mortalitiesin Lancefield'sgroup C, G and L by polymerase chain Japan.J. Fish Dis.,27,679-686. reaction.J. Vet.Med. B,50,161-165. Osorio, C. R.,A. E. Torazo, J. L. Romalde and J. L. Barja Kitao, T. (1993) Streptococcal infection. In“Bacterial (2002) Multiplex PCR assay for ureC and 16S rRNA

Diseases of Fish”(ed. by V. Inglis, R. J. Roberts and genes clearlydiscriminates between both subspecies

N.R. Bromage), Blackwell Scientific Publications, of Photobacterium damselae. Dis. Aquat. Org.,40,

Oxford, pp.196-210. 177-183. Kusuda, R.,K. Kawai, F. Salati,C. R. Banner and J. L. Fryer Shoemaker, C. A., P. H. Klesius and J. J. Evan (2001) (1991) Enterococcusseriolicida sp. Nov., a fish patho- Prevalence of Streptococcusiniae in tilapia,hybrid gen. Int. J. Sys.Bacteriol.,41,406-409. stripedbass, and channel catfishon commercial fish Kusuda, R. and F. Salati (1999) seriolicida and farms in the United States.Am. J. Vet. Res.,62,

Streptococcus iniae. In“Fish Diseases and Disorders 174-177.

Volume 3, Viral, Bacterial and Fungal Infections” (ed. Williams,K., S. Blake, A. Sweeney, J. T. Singer and B. L.

by P. T. K. Woo and D. Bruno), CAB International, Nicholson (1999) Multiplexreverse transcriptase PCR

Wallingford, UK, pp.303-317. assay forsimultaneous detectionof three fishviruses. Mata, A.I., M. M. Blanco, L. Dom�nguez, J. F. Fern�ndez- J. Clin.Microbiol.,37,4139-4141. Garayz�bal and A. Gibello (2004a) Development of a Zlotkin,A., A. Eldar,C. Ghittingand H. Bercovier (1998)

PCR assay for Streptococcus iniae based on the lactate Identificationof Lactococcusgravieae by PCR. J. Clin. oxidase (lctO)gene with potential diagnostic value. Microbiol.,36,983-985.

V et. Microbiol.,101,109-116.

養 殖 ブ リ のLactococcus garvieae, Streptococcus iniaeお よ び S.dysgalactiae検 出 の た め の 複 合PCR

モ ル タ ダM.A.フ セ イ ン ・畑 井 喜 司 雄

レ ン サ 球 菌 症 は,日 本 の 養 殖 ブ リ,カ ン パ チ お よ び サ ケ 科 魚 類 の 重 要 な 病 気 の 一 つ で あ り,経 済

的 に 問 題 と な る 。Streptococcus iniaeは 海 産 魚 と 淡 水 魚 の 病 原 体 と し て 知 ら れ て い る 。 い っ ぽ う, Lactococcus garvieaeとStreptococcus dysgalactiaeは ブ リ お よ び カ ン パ チ の 病 原 体 と し て 知 ら れ て い る 。

し か し な が ら,こ れ ら の 病 原 体 の 混 合 感 染 症 が ブ リ で 発 生 し て い る た め,正 確 な 診 断 は 時 に 困 難 で あ

る 。 主 た る 病 原 体 が 何 で あ る の か を 特 定 す る た め に,こ の 研 究 の 中 で 複 合PCRに よ る 診 断 の 有 効 性

に つ い て 検 討 を 行 っ た 。PCRは, L. garvieaeで は16S rRNAか ら, S. dysgalactiaeで は16S-23S rDNA

のITS領 域 か ら,ま たS. iniaeで はlactate oxidaseか ら 設 計 さ れ た プ ラ イ マ ー を 使 用 し て 行 っ た 。 こ

れ ら を 使 用 し た 複 合PCRに よ り,培 養 し たL. garvaeae, S. dysgalactiaeお よ びS. iniaeか ら 各 々 明 瞭 な

1,100,259お よ び870bpの 増 幅 産 物 が 得 ら れ,非 特 異 的 な 増 幅 産 物 は 得 ら れ な か っ た 。 ま た,ニ ジ マ

ス に こ れ ら3種 の 細 菌 を 接 種 し,各 組 織 か ら 複 合PCRに よ り各 細 菌 の 検 出 を 試 み た 結 果,迅 速 で 正

確 に 各 細 菌 の 増 幅 産 物 を 得 る こ と が で き た 。 こ の こ と か ら 本 複 合PCRは ブ リ の レ ン サ 球 菌 症 の 診 断

に 有 効 で あ る と 判 断 さ れ た 。