KHOA H O C K f THUAT T H O Y TAP XXVI SO 7 - 2019
PHAN LAP VA KIEM TRA MOT SO DAC BIEM COA VBIOO PARAHAEB/UL YTWUS UEN qOAN DEN BENH HOAI TUT GAN TOY CAP TREN TOM ft TRA VINH
Trdn Thi Hdng Tff', Dir Minh Hiep', Nguyen Thi True Linh', Trdn Tlii Phirffng Lan', Hideki Hayashidani'
TOM TAT
Vibrio parahaemolyticus mang gen pir"'' dugc xem la tac nhan gay benh hoai tii gan tuy c§p tr8n tom. Trong nghien ciiu nay, mlu torn, mau ngheu va mlu nuac d vimg nuoi tom, ngheu d Tra Vinh dugc kiem tra su hien di8n ciia V. parahaemolyticus mang gen doc luc pir'"'' lien quan den benh hoai tir gan tuy cap tren tom. 30 mSu tom va 20 mlu nude hi'ao nuoi tom tham canh, 16 mlu ngheu va 15 mau nude tii viing nuoi ngheu d Tra Vinh dugc thu thap de phuc vu cho nghien ciin nay. Mgt s6 dac diem cua V. parahaemolyticus mang gen dgc luc pir''^ nhu typ huyet thanh va sir khang khang sinh cung dugc tim hieu.
Ket qua nghien cim cho thay V. parahaemolyticus mang gen dgc luc pir"" dugc phat hien tir 3/30 (10%) mlu tdm va 1/20 (5%) mau nuac tu ao nuoi tom. Tuy nhien, chiing vi khuan mang gen dgc luc nay khong dugc tim thay trong mau ngheu va mau nude vimg nuoi ngheu. Cac chiing V.
parahaemolyticus mang gen pir''^ dugc tim thay trong nghien cuu nay dugc xac dinh typ huyet thanh dua vao bp kit antisera test kit va tinh khang khang sinh dua vao phuang phap khuech tan tren dTa thach, Ket qua nghien cim cho thay cac chiing mang gen pir'''' dugc phan lap thupc nlidm khang nguyen 01 vai siv da dang ciia khang nguyen K va chiing co tinh kliang cao voi ampicillin (100%), sulfisoxazole (100%) va streptomycin (75%). Nghien cim nay cung cfip them thdng tin giup hieu ro hon ve mgt so dac diem ciia chiing V. parahaemolyticus lien quan den benh hoai tir gan tuy cap trfin torn d Ddng bang Song Cicu Long.
Tir khoa: Benh hoai hi gan hiy cap (AHPND), khang khang sinh, Gen/jzr'P, typ huyet thanb.
Vibrio parahaemolyticus
Isolation and examining several characteristics of Vibrio parahaemolyticus related to acute hepatopancreatic
necrosis disease in shrimp in Tra Vinh province
Tran Thi Hong To, Du Minh Hiep, Nguyen Thi True Linh, Tran Thi Phuong Lan, Hidelii Hayashidani
SUMMARY
Vibrio parahaemolyticus carrying p/r" gene was considered as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, shrimp, clam and water sampies from shrimp ponds and clam farms were examined for the prevalence of V.
parahaemolyticus related to A H P N D in shrimp. 30 shrimp samples and 20 water samples from intensive shrimp ponds and 16 white hard clam and 15 water samples from clam farms were used as materials in this study. Serotypes and antimicrobial resistance of virulent bacteria were also examined.
' Klioa Nong nghiep - Thuy san, Tnrang Dai hoc Tra Vinh ' Khoa Nong nghiep, Tmang Dtii hpc Nong nghiep va cong nglic Tokyo
KHOA HOC KY THUAT THC Y TAP XXVI SO 7 - 2Uiy
The studied results indicated that the pir^ gene positive V. parahaemolyticus strains were detected with 3/30 (10.0%) from shrimp samples and 1/20 (5.0%) from shrimp pond water samples. However, this pathogenic bacterium was not found from the clam and clam farm water samples. All p//^ gene positive V. parahaemolyticus sXrains isolated in this study belonged to O I antigen with several K antigens. They showed high resistance to ampicillin (100%), sulfisoxazole (100%) and streptomycin (75%). This study provides some information on V. parahaemolyticus related to A H P N D in shrimp in the Mekong Delta.
Keywords: A H P N D , antimicrobial resistance, pir^ gene, serotypes, Vibno parahaemolyticus
I. DAT VAN DE
Benh hoai hi gan tuy cap tren torn hay cdn ggi la hgi chiing torn chet sdra dugc bao cao dau tien d Trung Quoc vao nam 2009 va sau do xuat hien 6 mpt so nude Dong Nam A (NACA, 2012), Mexico (Gomez-Gil et al, 2014), Nam My (Restrepo et al, 2016) va My (Dhar et al., 2019). Benh hoai tii gan tuy cap da gay thiet hai dang ke cho nghe nuoi torn tren the gioi, dugc udc tinh la I ty USD h k g nam (FAO, 2013).
Ci Viet Nam, benh hoai tir gan tuy cap tren tom dugc bao cao tir nam 2010. Benh xuat hien chu yeu d cac tinh ven bien cua dong bang song Cuu Long nbu Ben Tre, Tra Vinb, Soc Trang, Bac Lieu va Ca Mau, va gay thiet hai khoang 39.000 ha nuoi tom vao nam 2011 (FAO, 2013) va 50% Iugng torn gidng tha nudi d DBSCL vao nam 2012 (Dang Thi Hoang Oanh va Nguyin Thanh Phuang, 2012). Nam 2015, benh hoai tir gan tuy cap xuat hien a 23 tinh/thanh ciia Viet Nam. Benh gay thiet hai cho nganh nuoi torn Viet Nam udc tinh gan 54 trieu USD hang nam (Hien et ah, 2016).
Benh hoai tic gan Uiy cap c6 the gay chSt tom den 100% trong thang nuoi dau tien. Tom benh CO dau hieu Iam sang nhu gan tuy nhgt nhat.
hi teo va hoai tu, bao tii va rugt khong co thiic an. Nghien ciru mo benh hpc gan tuy ciia tom benb cho thay co su thoai hoa chiic nang cua cac te bao bieu mo B, F va R, su bong troc va roi vao long ong ciia cac te bao ong gan va su tap trung te bao mau xung quanh dng gan tuy (FAO, 2013). 0 giai doan benh mdi xu5t hien, tac nhan gay benh van chua dugc xac dinh. D^n nam 2013, Lpc et al. (2013) xac dinh Vibrio parahaemolyticus (V. parahaemolyticus) la tac
nhan gay benh hoai hi gan niy cap tren tom.
Cac nghien ciiu khac cho tbiy gen dgc lucpir'^
(Photorhabdus insect-related (pir) toxin-like genes) tren plasmid ciia V parahaemolyticus c6 lien quan den benh nay (Lee et a!., 2015). Theo FAO (2013), benb lay nhiem chii yeu tren torn the chan trang {Litopenaeus vannemei), torn sii (Penaeus monodon) va torn he [P. chinensis).
FAO (2016) cho rang mpt trong nhiing moi nguy quan trgng ciia su lay truyen benh hoai tir gan tuy tren tom co the tu mgt so loai nhuyen the. De hieu ro hon ve sir hien dien cua V.
parahaemolyticus mang gen plf'^ trong moi trudng d DBSCL va mgt so dac diem cua chung, nghien cuu nay phan lap V. parahaemolyticus gen dgc luc pir'^ trong moi truong nuoc a Tra Vinh va kiem tra type huyet thanh va su khang khang smh ciia cac chiing V. parahaemolyticus mang g^npif'.
II. VAT LIEU VA PHlTONG PHAP NGHIEN c b U
2.1. Vat lieu nghien ciru
Man torn va mau nude hi cac ao nuoi tom tham canh va mau ngheu va mlu nude vimg nuoi ngheu ven biln tmh Tra Vinh.
2.2. Noi dung
Xacdinhsubiendicncua r • ahaemolyticus mang gen dgc Iuc lien quan den benh hoai tu gan toy cap n-ong mlu torn, ngheu va raau nude d DBSCL.
Xac dinh type huylt thanh va su khang khang smh ciia V parahaemoh:,us lien quan den benh hoai tir gan tuy cap tier ^jni
KHOA HOC KY THUAT THU Y TAP XXVI SO 7 - 2019
2.3. Dia diem nghien ciru
Xii ly mlu va ii mlu dugc thuc hien tai Phong nghiSn ciiu benh hgc, Khoa Nong nghiep-Thuy san, Trudng Dai hgc Tra Vinh.
Phan lap, dinh danh va kiem tra mgt so dac dilm ciia V. parahaemolyticus mang gen dpc Iuc dugc thuc hien tai Phong vi sinh vat, Bg mon IGioa hgc dgng vat, Khoa Nong nghiep, Trudng Dai hpc Nong nghiep va Cong nghe Tokyo.
2.4. Phuong phap nghign ciiu 2.4.L Thu mdu
MIU tom va mau nuoc ao nuoi torn tham canh: 30 mau tom va 20 mau nude dugc thu nglu nhien tir ao nuoi torn sii va torn the chan trSng tham canh d tinh Tra Vinh nam 2016. Doi voi mau torn, moi ao thu mgt mau vdi 5 con torn tren moi mau. Doi voi mau nude, moi ao thu ragt mau vdi 90 ml nude tren moi mau. Mau dugc bao quan trong hii nhua thanh tning, dat trong thung x6p cd da lanh va dugc phan tich ngay sau khi dugc van chuyen den phdng thi nghiem.
Mau ngheu va mau nuac vung nuoi ngheu:
16 mlu ngheu va 15 mau nude dugc thu til bai nuoi ngheu xa Hiep Thanh va xa Trudng Long Hoa, huyen Duyen Hai, tinh Tra Vinh vao nam 2016. Moi mlu ngheu co 10 con va mdi mlu nude chica 90 ml nuoc. Mau dugc bao quan trong tlii nhira thanh trimg, dat trong thung xop CO da lanh va dugc phan tich ngay sau khi dugc van chuyen den phdng thi nghiem
2.4.2. Phdn lap V. parahaemolyticus V. parahaemolyticus tic mau torn, mau ngheu va mau nuoc dugc phan lap dua vao phuang phap dugc mo ta ciia Hara-Kudo et al. (2003) va Alam et al. (2009). D6i vdi mlu tom va ngheu, mlu dugc cho vao bii nhua chiia 225 ml alkaline pepton water (APW, Nissui, Tokyo, Japan). Doi vcri mau nuoc, 90 ml mlu nude dugc tron vai 10 ml APW (10 X APW). Mlu dugc ii ct 37''C trong 24 h. Dung djch sau khi u dugc cay len moi trudng thiosulfate-cih-ate-bile salts-sucrose
(TCBS, Nissui) va CHROMagar^^ Vibrio (CV) (CHROMagar Microbiology, Paris, France).
Khuan lac co mau xanh tren TCBS va mau tim tren CV agar dugc phan lap va trii trong moi tnicmg nutrient broth (NB, Nissui) co bo sung 20% glycerol va bao quan d -80°C.
2.4.3. Ly trich DNA
DNA dugc ly trich bang each dun sdi theo phuang phap dugc mo ta bdi Hara-Kudo et al.
(2003).
2.4.4. Dinh danh V. parahaemolyticus Gen dac trung cho loai (toxR) dugc xac dinh bang phuong phap PCR nhu mo ta ciia Kim et al. (1999). Trinh tu moi va chu trinh nhiet dung de dinh danh V parahaemolyticus dugc mo ta trong bang 1.
2.4.5. Kiem tra gen dpc lire
Gen dgc lucpir"'" dugc kiem tra bang phuang phap PCR theo mo ta cua Tinwongger et al.
(2014). Trinh bJ radi va chu trinb nhiet diing de xac dmh gen dpc luc pir"'' dugc mo ta trong bang 1.
2.4.6. Xdc dinh type liiiyet thanh
Khang nguyen O va K cua V.
parahaemolyticus mang %&npir''" dugc xac dinh theo hudng din trong bp kit andsera test kit (Denka Seiken, Tokyo, Japan).
2.4.7. Xdc dinh sir khdng khdng sinh ciia V.
parahaemolyticus mang gen pir"''
Su khang khang sinli cua cac chung V.
parahaemolyticus dugc xac djnh dua theo phuang phap dugc rao ta cua Clinical and Laboratory Standard Institute (CLSI) ( 2 0 i 2 | Chin Ioai khang sinh dugc kilm tra bao gom ampicillin (10 pg), chloramphenicol (30 pg), gentamiein (10 |ig), kanamycin (30 |.ig) nalidixic acid (30 \xg), ofloxacin (10 pg), oxytetracycline (30 \xg), streptomycin (10 (ig) va sulfisoxazole (250 pg). V. parahaemolyticus dugc nuoi cly tren moi trudng TSA (Trypticase soy agar, Becton Dickinson Company) co bo sung 1% NaCI va li d ^TC trong 24 h. Sau
KHOA HOC K\' THUAT THU Y TAP XX\n SO 7 - 2019
do, vi khuan dugc pha loang trong nude muoi sinh iy voi mat do la 1,5 x 10** CFU/ml (do due dugc so sanh vdi ong chuan 0,5 McFarland).
Tilp tbeo, 0,1 ml dung dich vi khuan dugc tan diu tren dia MHA (Muller Hinton agar, Becton Dickinson Company) cd bo sung 1 % NaCI. Ke
tilp, dTa khang sinh dugc dat len dTa MHA vdi so lugng khong qua 5 dia khang sinli/dia thach.
DTa dugc ii d 37"C trong 24 h. Vong vd trung dugc do va so sanh vdi tieu chuan dugc mo ta trong CLSI (2012) dl xac dinh su khang khang sinh cua vi khuan.
Bang 1. Trinh tir nucleotide va kich thwac phan tu- cua cac cap moi va chu trinh nhlet cua PCR xac dmh gen pirf va toxR
Gen can khuech
dai
Chu trinh nhiet Kich
thu'O'c
phan Cac giai doan tu' (bp)
Tai lieu Chu tham khao
ky
pirAvp
(TUM- 5'-GTGTTGCAT/V\TTTTGTGCA"3' SAT- 5'-TTGTACAGAAACCACGACTA-3' Vp3)
Ti6n bien tinh: 95°C trong 2 phut,
Bi4n tinh 95°C trong 30 giay, bat cap: 56°C trong 30 giay, keo dai: 72°C trong 30 giay Kk thuc: 72°C trong 5 phut
Tinwongger etai. (2014)
Tien bien tinh: 96°C trong 5 phut
1 5'-GTCTTCTGACGCAATCGTTG-3'
5'-ATACGAGTGGTTGCTGTCATG-3'
Biiin tinh: 94<'C trong 1 phut, bat cap: 55°C trong „^
1 phut, keo dai: 72°C trong 1 phut
Kim et al.
(1999)
Ket thuc: 72°C trong 7 philt
1
III. KET QUA VA THAO LUAN 3.1. Su hien dien cua V. parahaemolyticus mang gen/ji'r'f trong mau tom, ngheu va mSu nucrc ff Tra Vinh
Bang 2 cho thay su bien dien ciia V.
parahaemolyticus trong mau torn, ngheu va mau nude d Tra Vinh. Ket qua cho thiy V parahaemolyticus mang gen dgc Iuc pir''' dugc phat hien tu 3/30 (10%) mlu tom va 1/20 (5%) mau nuac tir ao nuoi torn. Tuy nhien, chung vi khuan nay khong dugc tim thay tren mlu ngheu va man nude viing nuoi ngheu. Mot s6 loai nhuyen the co the la vat chii mang mlm
benh do chiing la loai an Igc va co kha nang tich liiy mgt s6 loai vi khuin gay benh. Ngheu dugc nuoi vdi dien tich ldn d ven biln Duyen hai Tra Vinh va vimg nuoi ngheu tiep giap vdi viing nuoi tom tham canh 6 khu vuc nay. Do do, nghien cim nay muon tira hilu su hien dien cua V. parahaemolyticus hen quan din benh hoai hi gan hjy d p tren tdra d \Ting nuoi ngheu dl xac dinh moi nguy lay ian benh nay. Mac dii V parahaemolyticus hen quan din benh hoai hi gan tuy cap tren torn khong dugc tim thiy tren ngheu trong nghien cim nay. cac khao sat tren cac dgng vat thiiy san khac nen duoc thuc hien.
KHOA HOC Kt THUAT THU Y TAP XXVI SO 7 - 2019
Bang 2. S y hien dien cua V. parahaemolyticus mang gen pir^p trong mau t o m , ngheu va mau nu-cfc a Tra Vinh
Loai mau So mau du'O'ng tinh vdi V. parahaemolyticus mang gen pir'P {%) Ao nuoi tom tham canh
MSU tom Mau nu'dc Vung nuoi ngheu
Mau ngheu Miu nu'oc
3(10)=
^(5f
0(0}
0(0)
• V parahaemolyticus mang gen pir"''phdn lap tir mdu torn the : V parahaemolyticus mang gen pir"'' phdn lap tic mdu nu&c ao nuoi tom su
3.2. lype huyet thanh cua cac chiing V. para- haemolyticus mang genj7i>'^
Bang 3 cho thay V. parahaemolyticus mang gen dgc lire pir'''' phan lap tir ao nuoi tdm thugc nhom khang nguyen 0 1 vdi sir da dang ciia khang nguyen K bao gom K68 va Ky.|. Tuong tu vdi nghien cuu nay, Kongrueng et al. (2015) bao cao V parahaemolyticus lien
quan den benh hoai hi gan tuy cap phan lap tren tom nuoi d Thai Lan ciing thugc nhdra khang nguyen 01 voi su da dang ciia khang nguyen K nhu K33, K68 va K^^. Mgt su tien hoa ciia V. parahaemolyticus lien quan den benh hoai tu gan tuy cap tren tom co the xay ra trong tu nhien va dan den su da dang cua type huylt thanh cila V. parahaemolyticus mang gen dgc luc.
Bang 3. Type huyet thanh cua V. parahaemolyticus mang gen pir"'' Chung
vi khuan 1 2 3 4
Type huyet thanh
01 :K^,' 01:K68 01 •K„, 01 K
Gen pif' Gen toxR Nguon goc
Mlu nu'dc tU' ao nuoi torn su tham canh Mlu tdm til' ao nuoi tom the chan tr^ng tham canh Miu tdm til' ao nuoi tdm the chan tr^ng tham canh Mlu torn tLf ao nuoi tom the chan trang tham canh
". UT Untypeable (Khong xdc dinh) 3.3. Sir khang khang sinh cua V. parahaemo- lyticus mang gen pir'P
V. parahaemolyticus mang gen pir'''' cd tinh khang cao voi ampicillin (IOO%o), sulfisoxazole (100%) va streptomycin (75%) (bang 4). Ty 16 khang cao vdi ampicillin va cac khang sinh khac ciing dugc bao cao d chimg V. parahaemolyticus
lien quan den benh hoai tir gan Uiy cap tren tom d cac quoc gia khac. Kongrueng et al. (2015) bao cao 100%) V. parahaemolyticus lien quan din benh hoai hi' gan hay cap tren tora d Thai Lan khang voi ampicillin va erythromycin. Han et al. (2015) bao cao 100% V. parahaemolyticus lien quan den benh hoai tir gan tiiy cap tren tdm d Mexico khang vdi ampicillin va oxytetracycline.
KHOA HOC KY T H U A T THU Y TAP XXVI SO 7 - 2019
Bang 4. S I P khang khang sinh cua V. parahaemolyticus mang gen p/r^^ (n - 4) phan lap 6' Tra Vinh
Khang sinh sa chiing khang (%)
Ampicillin Sulfisoxazole Streptomydn Oxytetracydine Nalidixic acid Chloramphenicol Gentamiein Kanamycin Ofloxacin
4(100) 4(100) 3(75) 1(25) 1(25) 0(0) 0(0) 0(0) 0(0)
Bang 5 cho thay V parahaemolyticus mang gen pu-'^ CO tinh da khang. Chiing khang it nhat vdl 2 loai khang sinh va cao nhat vdi 5 loai khang sinh. Su da khang khang sinh cung dugc bao cao tren cac chimg V. parahaemolyticus Uen quan den hoai tu gan tuy tren torn d Thai Lan va Mexico
(Kongrueng et al., 2015; Han et al., 2015).
Ampicillm, sulfisoxazole va streptomycin dugc sii dung brong nong nghiep va thiiy san trong vai thap ky qua (Done et al., 2015). Vi the, su khang ciia vi khuan vdi cac khang sinh nay co the xuat bien qua ap Iuc chgn Igc.
Bang 5. Kieu khang khang sinh cua V. parahaemolyticus mang gen pir*^
Kieu khang khang sinh So chiing vi khuin AMP-SFX^
AMP-STM-SFX AMP-NA- OTT-SFX-STM
'•.• AMP: Ampicillin. NA: Nalidixic acid. OTT: Oxyteti-acycline, SFX: Sulfisoxazole. STM: Sti-eptomycin
IV. KET LUAN
V. parahaemolyticus mang gen dgc luc pir''' dugc phan lap tren mau tom vdi ty Ie 10%) va mau nuac tir ao nudi torn vdi ty le 5%. Cac chiing vi khuan phan lap thuoc nhom khang nguyen 0 1 . Chimg c6 tinh khang cao vai ampicillin, sulfisoxazole va streptomycin.
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KHOA HOC K t THUAT THU Y TAP XXVI SO 7 - 2019
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and Lighttier, D. V 2015. Plasmid mediated tetracycline resistance of Vibrio pai-ahaemolyticus associated witli acute hepatopancreatic necrosis disease (AHPND) in shrimps. Aquacult. Rep. 2,17-21.
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17. Restrepo, L., Bayot, B., Betancourt, I. and Pinzon, A. 2016. Draft genome sequence of pathogenic bactena Vibrio parahaemolyticus strain Ba94C2, associated with acute hepatopancreatic necrosis disease isolate from south America. Genom. Data 9, 143-144.
18. Tinwongger, S.,Proespraiwong,P., Thawonsuwan, J., Sriwanayos, P., Kongkumnerd, J., Chaweepack, T , Mavichak, R., Unajak, S., Nozaki, R , Kondo, H and Hirono, I. 2014. Development of PCR diagnosis method for shrimp acute liepatopancreatic necrosis disease (AHPND) strain of Vtbno parahaemolyticus Fish Pathol, 49,159-164.
N g a y nhan 2-8-2019 N g a y p h a n bien 2 0 - 8 - 2 0 1 9 N g a y d a n g 1-11-2019
