KHOA HOC CONG NGHE
PHAIM L A P V A X A C DflVH D A C DIEIVI VI K H U A M Streptococus agalactiae EiAY BEIXIH TREM IECH THAI LAIV MUOI TAI T H U A THIEIM - H U E
N g u y e n N g p c P h u 6 c S N g u y § n T h i H u ^ U n h S N g u y g n TTii Xu^n H d n g
TOMTAT
Ket qua pban lap vi khuan tir nao ciia 20 mau ech Thai Lan {Rana tigerina) bi benh due m i t tai hai huy^n Huong Tra va Phu Loc, tinb Thiia Thien - Hue thu duoc 20 chung vi khuan binb cau. Gram duong. khong di dOng, CO phan ung oxidase, catalase va bile-esculin am tinh. Vi khuIn duoc dinh danh la Streptococus agalaetiaeheu huyet thanh lb va III bang phuong phap Lancefield, kit API 20 strep va phuong phap ngung ket mign dich. Ket qua thi nghiem gay benh tbuc ngbiem b i n g phuung phap tiem vao co diu ech hai chung vi khuan 5. agalactiae TTHUTOOl (kieu buyfet tbanb lb) va 5. agalactiae TTUPIOOS (kieu huyet tbanh III) cho thay ca hai chiing vi khuan nay co kba nang gay benb tren ech thi nghiem voi dau hieu benh ly giong benh xay ra ngoai tu nhien. Lieu gay chet 60% ODsJ ciia hai chiing vi khuan 5, agalactiaeTTHRTtiOl va S.
agalactiae TTHPL008 doi voi ech tbi nghiem lln luot la 2 x 10^ cfii/mL va 6,3 x 10^ cfu/mL. Ket qua thii nghiem voi khang sinh cho thay cac chung vi khuan nhay cam voi ofloxacin va 3 khang sinh thuoc nhom p- lactam (oxacilin, ampieillin va amociUin), nhung khang voi oxytetracycline. Day la bao cao dau tien ve bfenh do S. agalactiae nhom III tren ech nuoi o Viet Nam.
Tii khda: ^h Thai Lan, Rana tigenna, Streptococus agalactiae, kieu huydt thanh lb, kieu huydt thanh III
LIVKTBAU
Nud'c t a da du n h a p , t h u a n d u o n g va n b a n g i d n g t h i n h e o n g g i d n g e c h T b l i Lan {Rana tigerina). D a y 11 l o l i e c h cd tdc dd tang t r u d n g n h a n h , ty le s d n g eao v l thich h p p e h o q u y m d nudi c o n g nghidp so vdi g i d n g e c h d d n g Viet N a m (R.
mgulosa). Tir n a m 2005, ech T b l i Lan d u p e d l n h g i l 11 ddi t u p n g nuoi m a n g lai loi n h u a n cao e h o n o n g d a n [3, 4 ] . Hien nay, Viet N a m x e p thii hai t r e n thd gidi ve nuoi e c h d e san x u a t t h u e p h a m vdi s i n l u p n g u d c tinh t r e n 20.000 tan e c h thit e u n g cap e h o thi t r u d n g t h e gidi m 6 i n a m (Bao T h a n h nien online, sd n g l y 2 3 / 7 / 2 0 1 8 ) . N g h e nuoi dch tai Thiia T h i e n - H u e b i t d i u tii n a m 2009 vdi cac m o b i n h nudi t h u n g h i e m c u a T r u n g t l m K h u y e n N o n g Lam N g u tai h u y e n Phii Lpc, d e n nay m o h i n h nuoi e c h T h l i Lan d a d u p e n h a n r o n g t r e n dia b i n tinh Thiia T h i e n - Hue. M p t t r o n g n h u n g t h a c h tbiic ldn c h o viec p h I t trien b o a t d p n g nuoi e c h 11 dich b e n h . D i e h b e n h g a y r a t r e n e c h da U m giam d i n g k e s i n l u p n g e c h nuoi t r o n g nbiing n a m g i n day. T r o n g sd cac t i e n h a n gay b e n h t b u d n g g a p tren e c h T b l i Lan (R. tigerind), Aeromonas hydropbila va Streptococcus
Khoa Thiiy san. Trucmg Dai hoc Nong Lam, Dai hoc Hue Email. [email protected]
n h o m B 11 n h u n g t i c n h a n g a y b e n h g a y thiet hai n a n g n e n h a t T y le m a c b e n h d o vi k h u a n A hydropbila g a y r a t r e n e e h e h i e m t y Id tii 64,5-72,9%
[4]. T r o n g k h i d o , vi k h u a n 5. agalactiae n h o m B cd t h e gay r a ty le e h e t len d e n 80% d i n e c h bullfrog {R
nudi tai Brazil [6]. Vi k h u a n S.
I t i e n h l n g a y b e n h p h d bien c h o d p n g vat t h u y san, l u o n g e u t r e n t h e gidi va Viet N a m [1, 2, 5, 191. T u y n h i d n , c h o d d n n a y e h u a cd n g h i d n ciiu n a o vd b e n h do vi k h u a n S. agalactiae gay r a t r e n ech nudi tai Viet N a m ndi c h u n g va Thira T h i e n - H u e ndi rieng. D o dd viee n g h i e n ciiu b e n h d o vi k h u a n S.
agalactiae trdn ech nudi tai Thira T b i e n - H u e n h a m g d p p h a n g i a m tbieu t h i e t hai d o vi k b u a n n l y g a y ra 11 cin thiet.
2. PHUONG PHAP NGHIEN CUU 2.1. P h u a n g p h I p t h u m l u
M l u e e h b e n h (20 m l u ) d u p e tbu tii 4 h o nuoi e e h tai 2 h u y e n H u o n g T r a (2 hp) va h u y e n P h u Ldc (2 h d ) , tinh Thiia T h i e n - H u e t r o n g t h a n g 5 va t h i n g 6 n a m 2019. M d i h p thu 5 c o n e e h voi dau hidu b d n h ly didn h i n h 11 da tdi s I m , m i t bi ldi due,
2.2. P h u o n g p h a p p h i n lap v l dinh d a n h vi k h u a n
M l u vi k h u a n d u p e thu tai hien truoi e a c h d u n g vat n h p n d a m vao cot s d n g de b a n g
flmh NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG '020
KHOA HOC CONG NGHE gch, sau do dimg dao mo da dupe tiet trung bing cdn
90", md phin bung vl hdp sp eua dch, diing que cay nhua tidt trimg dim thing vao khdi nao roi eay ldn mdi trucmg Tryptic Soy Agar (TSA. Hunedia, An Dp) da duoc chuan bi sin. Cic cBa thgch sau do dupe li o- nhiet dp 28''C trong vdng 2 4 - 4 8 gid-.
Quan sit su phIt tri^n eiia khuan lac tren dia thach, neu khuIn lac khdng thuIn (2 hoac nhidu loai khuan lac cimg phIt trien tren cimg 1 dia TSA) thi khuan lac chidm uu the se dupe cay thuIn lln thii hai. Tat cl cle khuan lac phin lap se dupe dinh danh bing bp kit API 20 Strep (analytical profile index) (Bio-Merieux, Phap) va bp kit ngimg ket khang nguydn Lancefield Strep-B-Latex (Prolex, Prolab Diagnostics, Richmond Hill, Ontario, Canada) theo quy trinh tien hanh ciia nha san xuat.
2.3. Hiuong phIp xlc ftinb d ^ di^m a n h hoa cle chiing vi khuIn
Xlc dinh dac didm sinh hoa cac chiing vi khuan da phan lip dupe tidn hinh theo phuong phIp eua Austin vl Austin (2012) vl Gagnon va cdng su (1959) [9,141.
Cic phan ling co ban duoc tidn hanh gom:
nhudm Gram, phan iing oxidase, catalase, bde escuhn, phin ting oxi hoa/ len men dudng ( 0 / F glucose), kha nang di dpng vl kha nang huydt trdn mdi truong thach cd chiia 5% mlu ngua. Ngoli ra, cae phin iing smh hoi khic bao gdm: kha nang phlt trien tren mdi tnrong TSB + 6,5% NaCI, phan ling Lysm decarboxylase, kha nang lim ket tua huyet thanh tho dong kho cung dupe tien hinh de nghidn Cliu day dii cle dac diem sinh hoi ciia cic chung vi khuan pbln lap diroc. Chiing vi khuan 5. agalactiae NCIMB 701348 duoc su dung de lam ddi chiing dirong.
2.4. Phuong phIp xlc dinh kilu huy^t thanh Kieu huyet thanh cua vi khuan dupe xac dinh bing phuong phIp ngung ket midn dich [ 15]. Nhd 10 nL huyen phii vi khuan da dupe nudi sau 24 gia b nhidt dp 28''C len tlm giay thu, sau do nhd 10 \i\
dung dich latex (thudc thii la, lb va II den DC; bp Strep-B-latex, Statens Serum Instiit, Copenhagen, Dan Mach) len huyen phii vi khuan. Diing que nhua tiet triing trdn ddu hai dung dich. Phan ling duong tinh se cho kdt qua ngung ket xuat hien tdi da trong 30 gily. Ndu chung vi khuan nIo cho phan iing ngung ket manh vdi 2 loai khing nguyen thi kdt luan
11 khdng xac dinh dupe kidu khing nguyen ciia chting vi khuan do.
2.5. Phuong phap xac dinh kha nang gdy bfnh ciia elc chiing vi khuan phin llip
25.1. Ecb thi nghiim
Ech Thli lan {R tigerina) co khdi Iupng 25-30 g/eon, khdng di tat, mau sie tiroi sing, boat dpng nhanh nhen duge mua tir Trung tam gidng thuy san TTiua Thien - Hue. Ech dupe nuoi thuIn 14 ngly trude khi thi nghiem trong be nhua ed the tieh 0,5 m^
vdd miic nuoc khoang 0,2 cm (ngap 1/3-1/2 than eeh). Thuc an Green feed (Viet Nam) vdi ham lupng dam 25% dupe cung cap hang ngly vdd Heu luong 5%
khdi lupng than.
Trude khi tien hanh thi nghiem elm nhidm, dch dupe Idem tra khong nhidm vi khuan bang each thu mlu VI khuan bi nao cua 5 el the nglu nhidn. Diing que cay nhua tiet tning dam thing vIo khdi nao dch roi cay ldn ffia thach cd chiia moi tmdng TSA da dupe ehuan bi sin, ii dia thach d' nhidt dp 28''C trong 24 gio.
2.5.2. Pbuong phap cdng cuong dpc lire Cac chung vi khuan S. agalactiae ^han lap tii ech dupe nudi cay thuIn trdn mdi trudng TSA. Lay 1 khuan lac eho vao nuoi tang sinh trong 5 mL mdi trudng Tryptic Soy Broth fTSB, Himedia, An Dp) trong 24 gid- d- nhiet dp 28''C. Sau do, ly tam dung dich huyen phu d- tdc dd 4.000 vong/phiit bing miy ly tlm (Lab Centrifuge, Diic), loai bd phan dich ndi, cho them ImL nude mudi smh ly (0,86% NaCI) rdi trpn deu trdn may Vortex. Lay 0,1 mL dung dich vi khuan trdn tiem eho eeh. Sau 1-2 ngly, ndu eeh chet thi tien hanh thu mlu vi khuan bing cleh lay que cay vd triing dua vIo tlian vl nao ech, ria que cay ldn dia thach TSA, u 24 gio a nhiet dp 28''C. Vi khuan thu dupe sau khi dinh danh lai bing Lancefield kit 11 S.
agalactiae, luu giu huyen phu vi khuan trong dung dich glycerol 15% vl bao quan d' -20''C bing tii lanh sau de dimg cho cae thi nghidm elm nhidm. Chpn mdi loai khing nguyen mpt chung gay chet ech sau 24 gid- de tidn hlnb thi nghidm xie dinh beu gay chet LDeo.
2.5.3. Phuong pbap xac dinb LDgo
* Chuan hi vi kbuan
Chiing VI khuan 5. agalactiae sau khi cdng cudng ddc luc dupe nuoi cay trdn moi truong thach
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1/2020 59
KHOA HOC CONG NGHE TSA a nhiet dp 2 8 ^ trong 24 gia. Sau do, lay 1
khuan lac rdi tren dia thach nuoi cay tang smh trong 10 mL moi trudng Tryptone Soya Broth (TSB, HiMedia, An Dp) ti-ong miy u lac Shaking mcubator (LM-4200, Ymder, Tmng Qudc) a nhiet dd 28"C, toe d6 100 vong/phiit trong 24 gid'. Sau dd ly tam vi khuan 4000 vdng/pbut tirong 20 phut, nia vi khuan 2 lan bang dung dich nude mudi sinh ly 0,86% NaCI vl xac dinh mat dp vi khuan bang miy so mlu quang phd (Specti-ophotometer model 4111 RS, Zuzi, Tly Ban Nha), a bude sdng 620 nm kdt hop vdd phuang phIp dem sd lupng khuan lac tren moi ti-udng TSA (cfu/mL). Diing nuac mudi smh ly pha loang huydn phil vi khuIn den gil ti-i OD62o= 1 (tuong duang 10"
chi/mL, sd heu khong cong bd). Sau dd pha loang mat dd vi khuan bi 10^-10^ cfu/mL de dimg eho thi nghiem elm nhidm
*Bd tn thi nghiim
Thi nghiem gay benh thuc nghidm de xlc dinh lieu gay chet 60% (LDa,- Letiial dose 60) dupe tien hinh d- phdng thi nghiem udt, bd mdn Benh thuy san, Khoa Tliuy san, Truong Dai hpc Nong Lam, Dai hpc Hue.
Tir ket qui ciia thi nghiem cong eudng dfic luc, 2 chimg VI khuan S. agalactiae TTHRTOOl (kieu huyet thanh lb) vl TTHPL008 (kidu huyet thanh IID dupe chon dd xac dinh beu gay chdt LDgo- Thi nghiem xlc dinh gia tn LDgo dupe bd ti-i trdn 7 nghiem thiic bao gom: 6 nghiem thiic thi nghiem va 1 nghiem thiic ddi chiing. Moi nghiem thiie gdm 10 eon eeh dupe nuoi trong be nhua (V = 50L). Eeh dupe elm nhidm bing phuong phIp tiem vIo ea diii. Trong 6 ngbiem tbiic thi nghidm: dch d' mdi nghidm thiic duoe tiem 0,1 mL v6i mpt trong slu mat dp vi khuan 5. agalactiae tii 1 x 10^ den 1 x 10^ cfu/mL. O nghiem thiie ddi ehung, ech dupe tidm 0,1 mL nude mudi sinh ly (0,86%
NaCI) vd trung.
Ech sau khi tidm duoc nudi trong be nhua SOL vdi mice nude khoang 0,2 em (ngap 1/3-1/2 thin ech).
Thiic an Green feed (Viet Nam) vdi him lupng dam 25% duae cung cap hang ngly vdi bdu lupng 5% khdi luong than. Ty Id chet dupe theo ddi trong 14 ngly.
Gil tri LDfi(i dupe xae dinh theo phuong phap cua Reed-Mueneh (1938). Dua vIo sd luong dch ehet d- cic nghidm thuc de tinh LDg,, theo cdng thiie sau;
LDfin^lO^-"
Trong do: a la sd luy thira m l tai do vi khuan gay chet tiiap nhat (ti-dn 60%).
x dupe tinh dua vao eong tinic: x = (Pa - 60)/(Pa - P u ) .
Vdi: Pa la ty le ehdt can trdn va Pu la ty le chdt can dudi cua lieu gSy chet 60%.
2.6. Phuong phIp xlc dinh dO min cam khing sinh
Dp min elm kbIng sinh eiia 20 chung vi khuan phan lap dirpc tien hinh theo pbuong phap khuech tan khing smh trdn dia thach ciia Bauer va cpng su (1950) [10] vol 6 loai khang sinh tiiudc 4 nhom:
ofloxaem thupc nhdm fluroqumolon, oxytetracyclme thupc nhdm tetracycline, oxacilbn, ampieiUin, amoedhn thupc nhdm p-lactam va streptomycin thudc nhdm aminoglyeosid.
Lay 2-3 khuan lac ridng le tren dia thach chiia S.
agalactiae phan lap duoc, hda vIo 4 mL nude mudi sinh ly va trpn ddu bing Vortex. Lay 1 mL huydn phii vi khuan vl tidn blnh do mat dp quang hpc (optical density- OD) bing may so mlu quang pho (4111 RS, Zuzi, Tay Ban Nha) a buoc sdng 620 nm; sau do diing nude mudi sinh ly pha loang den gil tn 00^20 = 1 (tuong duong 10* chi/mL, sd beu khong cong bd).
Pha bang mgt dp vi khuan ve 10^ chi/mL Diing micropipette lay O.lmL huyen phii vi khuan cly din deu ldn dia mdi tmdng Mueller Hinton (Himedia, An Dp) vl de d nhiet dp phdng ti-ong 30 pbiit. Sau dd diing kep kbu tning gIp dia giay ed tam khing sinh dat vao dia thach da cay din vi khuan ti-udc do, u a nhiet do 28°C ti-ong 48 gid'. Do duong kinh vong v6 khuan vl dlnh gil khi nang nhay elm khing smh cua vi khuan dua tren dua tren dudng kinh vong vo khuan theo tieu chuan cua Cbnical and Laboratory Standards Institiite (CLSI) [12].
3. KET QUA NGHEU CUU
3.1. DIu hi^u b$nh ly ciia 6ch bi b^nh do vi khuIn S. agahtiaegkyra
Tir 20 mlu ech hi benh thu dupe tii 4 hp nuoi ech tiiudc 2 huyen Huong Tra vl Phii Loc deu cho tiiay elc diu hieu dac trung: eeh boat dpng cham chap, kem Iinh hoat, bd an. Da ech tdi sim, mit cd mlu h-Ing due (mot hoac cl hai mit). Bung trudng to vl mdm nhiin (Hinh LA), Noi quan xuat huyet, gan sung to (Hinh IB).
60 N O N G NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1/2020
KHOA HQC CdNG NGHE
agalactiae. Ket qua phan img ngung ket mien dich cho thay 7/20 ehiing vi khuan S. agalactiae (ehiem 35%) cho duong tinh nhdm III vl 13/20 chiing vi khuan S. agalactiae (ehiem 65%) cho phan iing ngung ket vdi nhdm lb (Bang 1).
Hinh 1. D ^ hidu bdn ngoli ciia ^ch bi nhi^m 5. a^actiae vdi mit c6 mlu trIng due (mui tdn), da t6i Sim (A) vl npi quan xuit huy^t, gan sung to (B) 3.2. K^t qua phan Ifip, dinh danh v l d i e di^m sinh hoi cic chung S. agalatiae
Ket qua tii Lancefield kit cho thay tat ea cic mlu vi khuan phin lap dupe tir nao ech benh d^u duong tinh vdi nhdm B vl dupe xlc dinh 11 vi khuan S.
Bing 1 • Sd chiing vi k h u ^ phin lap dupe tfr than va nao cua ^ch bj b ^ tai dja di^m thu mlu Hmh 2. KhuIn Igc tr&i moi trudng TSA (Pi) va g$iy
tan huy^t trdn mdi trudrng thach mlu (B)
Dia diem thu mlu
Huyen Huong Tra
Huyen Phii Loc
H d i
Ho 2
H g 3
Hp4
Chung TTHHTOOl
•nHHT002 TTHHT003 TmHT004 TTHHTOOS ITHHTOOe TTHHTOO?
TTHHTOOS TTHH-TOOg TTHHTOIO TTHPLOOl TrHPL002 TTHPL003 TTHPL004 TTHPL005 TTHPLOOe TTHPL007 TTHPLOOS TTHPLOOg TTHPLOIO
Nhom Lancefield B B B B B B B B B B B B B B B B B B B B
Kieu huyet thanh lb lb lb lb lb lb lb lb III III lb III III lb lb lb lb III III III
K6't qua dinh danh 5. agalac^tiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae Ket qua xlc dinh dae diem hinh thli, sinh hoi
ciia cic chiing vi khuan phin lap tii ech dupe trinh biy a bang 1. Tat ca 20/20 ehung vi khuan phan lap duoe khi phlt tridn trdn mdi tnrdng TSA tao khuan lae nho c6 mlu kem (Hinh 2A). Dac diem sinh hoi cho thay elc ehung nly ddu 11 vi khuan Gram duong, hinh cau, khong di ddng, phan iing Im tinh vdi oxidase, catalase va bile esculin. Mae dii khae nhau vd kieu huyet thanh, nhung dac diem sinh hoa eua
cic chung khi ddng nhat (Bing 2). Tat ea eac chiing deu khong phlt trien tren moi trudng TSB + 6,5%
NaCI. Cd 7/7 chung kieu huyet thanh III cd khi nang gay tan huydt holn toln (p haemolytic) (Hinh 2B), trong khi dd cac chiing kieu huyet thanh lb khong gay tan huyet (y haemolytic) trdn mdi trudng thach mlu. Tat cl cle chung deu phin iing duong tinh vdi huyet thanh thd ddng khd. Da sd elc chiing khdng ed khi nang sir dung elc loai dudng (Bang 2).
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020
KHOA HOC CONG NGHE
___„„
Bang 2. Dac di^m sinh hda ciia cac chung vi khuIn S. aga&c&aephSai l§p dugc tir 6ch, chmg S. agalacUae
NCIMB 701348 dupe sii dune dk Um k^t nnii wi sAnh __—, Chi tieu
Nhudm Gram Hinhtiili Didong Kha nang tan huyet Oxidase Catalase Bile Esculine Huydt tiiang thd dong kho LDC
Voges-Proskauer Hypurate hydrolvsis Pyrohdonylarylamidase Ribose
Arabinose Mamtol Sorbitol Lactose Trehalose Inulin Rafinose Glycogen Amygdalm TSB 6,5% NaCI
Streptococcus agalactiae NCIMB 701348
(-H)
Hinh clu Khong
p
(-) C-)
(-)
(+) (+) {+) (+)
M
(-) (-) (-)
(+)
(-)
W
(-) (-) (-) (-) (-)
Ty le (%) chung vi khuan phan lap Duong tinh
100
Am tinh 0 _ _ - Hinh cau
Khong — 65% (tanhuvetp)
100 90 80 100 10 100
100 10 10 20
35%(tanh!2etl) 100 100 100 0 10 20 90 100 100 100 100 100 90 90 80 100 (•r):phan dng duong tinh; (-) pban iing am tinh
Dac didm sinh hda eiia cic chiing vi khuan S.
agalactiae TTHHTOOl (kidu huydt tiianh lb) va TTHPL008 (kieu huyet thanh III) gidng vra dae diem sinh hda eiia cic chiing vi khuan S. agalatiae ti-en dpng vat thiiy sin va ngudd [1, 5, 7, 11, 19]. Ngbien Cliu eiia Delannoy va cdng su (2010) da ehi ra ring S.
agalactiae kieu huyet thanh lb cam nhidm tren ddng vlt thuy san tren the gidi cdn S. agalacQ'aekiku huyet thanh III ehi phat hien tren mpt sd dpng vat thuy san d" viing Dong Nam A [13], va diy II nghien eiiu dau tien xlc dinh 5. agalactiae kieu huydt thanb III da xuat bien trdn ech hi benh due mit d'Viet Nam.
3.3. Kha ning gly bdnh ciia cle chung vi khuan phin lip dugfc
Tii ket qui thi nghiem cam nhidm d' hinh 3 va hinh 4 cho thay hai chung S. agalacti'aeTniRTdOl (kieu huyet tiianh lb) vl S. agalactiae TFHPLOOS (kieu huyet thanh III) d" nhiing ndng dp khac nhau gay ra ti le chet khac nhau 6' ech thi nghidm. O tat ca eae mat do vi khuan elm nhidm deu gly ehdt trdn
ech ngoai tni nghiem thiic ddi chiing chi tiem nude mudi sinh ly 0,86% (Hmh 3 vl hmh 4).
Hmh 3. Ty Id ch^t cua dch sau khi cam nhidm chung 5. ^a/ac&ae TTHHTOOl (ki^u h u y ^ thanh lb)
Hmh 4. Ty 1^ ch^t ciia ^ h sau khi cam nh chung 5. agalactiaeTniPLim (ki^u h u y ^ tha. ni)
62 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANc
KHOA HOC CONG NGHE Tir k^t qui thi nghiem xlc dinh dupe LDgo cua
hai chung S agalactiae TTHHTOOl (lb) va 5.
agalactiae TIHPL008 (III) lln lupt 11: 2 x 10^ cfu/mL vl 6,3 X 10^ cfu/niL cho thay kha nang gly bdnh cua ehung vi khuan S. agalactiae ITHPLOOS manh han chimg S. agalactiae TTHHTOOl. Ket qui nly cOng phil hpp vdi nghidn ciiu cua Ip vl cpng su (2006) cho rang S. agalactiae kieu huyet thanh III ed doe luc cao hon eac kidu huydt ihanh la hay lb [16], Day la nghidn ciiu gay benh thuc nghiem dau tien tren ech ciia chung 5. agalactiae. Chua co nghidn ciiu nao xac dmh gia tri LDgg cua vi khuan S. agalactiae trdn dch tai Vidt Nam, tiiy nhien tii ket qui ciia elc nghidn ciru khle khi tien hinh gay benh thuc nghiem S.
agalactiae trtn ca r6 phi (Oreoehromis niloticus) ddu ghi nhan duoc gil tri ID50 dao ddng tir 3,6 x 10^ - 1,7 X 10^ cfu/mL [1,18,19]. Ech chet d" thi nghiem cam nhidm ed dau hidu benh Iy gidng nhau la da tdi sam
cam nhi&n dup-c giai phlu vl tai phan lap vi khuan a nao trdn moi tiudng TSA sau 24 gid- u 6- nhiet dd 28''C deu cho khuan lac cd mau sae va hinh dang tiiang tii nhau 11 mau kem, hmh trbn, Idi, kich thuoc 1 mm, gidng vdi khuIn lac ciia vi khuan phan lap duoc tii mlu ech bdnh thu dupe tir hd nuoi.
Vi khuan tii phan lap duoe bi nhimg eeh bdnh sau khi elm nhidm eo cac dae didm hinh thai sinh Iy, smh hda v l kieu huydt thanh lb vl III gidng nhu 5. agalactiae TTHHTOOl (kidu huyet tiianh lb) v l TTHPL008 (kieu huydt thanh III). Didu nay chiing td hai chung vi khuan nly cd kha nang gay bdnh thuc nghidm trdn eeh khoe.
3.4. K^t qua thtr nghiran d6 min cam khang sinh Ket qua thu kha nang min cam khing sinh ciia 20 chung vi khuan phan lap dupe trinh biy a bang 3.
vl mat hi due. Nhiing dch gin chdt sau khi gly
Bang 3. Kha ndng min cam khing sinh cua cic chung vi khuan S. agalactiae phSn 14^ trdn ^ h tai Thira Thidn - Hud
Chung vi khuan TTHHTOOl TTHHT002 TrHHT003 TrHHT004 TTHHTOOb TraHT006 TTHHT007 TTHHT008 TrHHT009 TTHHTOIO TTHPLOOl TraPL002 TrHPL003 TrHPL004 TTHPL005 TTHPL006 TraPL007 TTHPLOOS TTHPL009
rraPLoio
Nhdm khang thd
lb lb lb lb lb lb lb lb III III lb III III lb lb lb lb III III III
Khing sinh Nhom p-lactam
Oxacillin
-H + -1- +
+ + +
+ +
+ + + +
Ampieillin
-H + + +
+ + +
+ -H +
+ +
Amocillin -t- + + + + + +
+ -h + + -I- + + + +
Nhdm fluro- quinolon Ofloxacin
+ + + + + + + + + + + -H + + + + + -H + +
Nhdm amino- glycoside Streptomycin
+
-H
+
+ + +
Nhdm Tetracycline Oxytetracycline
(•f): nhay vol khang sinb; (-); khang vdi khang sinb tbirnghlim
Tat ca cic chung vi khuan S. agalactiae (20/20 teti-acyline. 100% cac chung nhay cam vdi khing sinh ehung) thuoc hai kidu huyet thanh lb vl III deu ofloxacin thudc nhdm fluroquinolon, day la nhdm khang vdd khang sinh oxytetracyclme thudc nhdm khang smh cd pho khing khuan rpng dac bidt ddi vdi
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020
KHOA HOC CONG NGHfi
vi k h u a n 5. agalactiae [8]. Da sd c a c c h i i n g d e u n h a y e l m vdi 3 loai k h a n g s m h t h u p c n h o m p-lactam la n h d m k h a n g sinh ed t i e d u n g tdt vdi vi k h u a n G r a m d u o n g vi tac d o n g v I o ldp vd peptidoglycan eiia m a n g td b i o , tuy nhidn amoeiUin c h o kdt q u a tdt n h a t vdi 1 6 / 2 0 e h i i n g (chiem 80%) n h a y c a m vdi k h i n g sinh n l y . Oxaeihn ed k h i n a n g diet k h u a n tdt, 1 3 / 2 0 c h u n g n h a y vdi k b I n g sinh n a y n b u n g d a y la loai k h a n g sinh ehi diing c h o d u d n g tiem, t r o n g k h i d o ampieilbn v l amocillin cd t h e h a p t b u q u a t h i n h r u o t n e n cd thd diing b i n g c l e h trpn vao thiie an. Chi ed 6 / 2 0 chiing n h a y c a m vdd streptomycin, dae biet k h o n g cd chiing vi khuan nao thupc n h d m h u y e t t h a n h i n n h a y cam vdi streptomycin. Ket q u i nay tuong d d n g vdi nghien ciiu ciia Musa v l e d n g s u (2009) khi eho r i n g S. agalactiae cd k h i n a n g I d l i n g lai streptomycin [17], d o dd khuyen c l o Idiong ndn dimg k b I n g sinh n l y trong didu b i bdnh ech.
4.KETLUANVAKIBVNGHI
4.1. K ^ t l u ^
N g h i d n ciiu da p h a n lap dugc 20 e h u n g vi k h u a n S. agalactiae thupe hai n h d m k h a n g t h e lb (13 ehiing) v a III (7 chiing) tren dch bi b d n h ldi va d u e m i t nudi tai T h u a Thidn - H u e . Day 11 n g h i e n ciiu d i u tien p b l n lap d u p e S. agalactiaekxkn huydt t h a n h III t r e n e e h d" Viet N a m .
Ket q u a gay b d n h t h u c n g h i e m cho g i l tri LDgo ciia c h u n g S. agalactiae TTHHTOOl (kieu h u y e t t h a n h lb) va S agalactiae TTHPLOOS (kieu h u y e t t h a n h III) l l n lupt 11: 2 x lO^efu/mL v l 6,3 x
Wcin/mL.
C i c c h i i n g VI k h u a n p b l n lap d u a e deu k h i n g vdd oxytetraeyebne. T r o n g k h i dd, ofloxaem v l 3 loai k h i n g sinh oxacibn, ampieillin va amocillin thupe n h d m p-laetam cd tac d u n g tdt vdi cac chiing vi k h u a n n l y .
4.2. Ki^n n g h i
D i y 11 n g h i e n ciiu b u d c d i u v e b d n h do vi k h u a n S. agalactiae trdn e c h nudi, c i n n g h i e n euu sau h o n vd d a c diem b e n h h o c , miia vu x u a t hien b e n h va cac bidn p h I p p h d n g t n i dich b e n h do tac n h a n nay g a y r a d e b a n chd viec d u n g k b I n g sinh h-ong q u a tiinh nuoi.
LOI CAM DIU
Nghiin Cliu duoc tai trg bdi du an: Of Fish and Men - Public Health Risks Posed by Group B Sti'eptococcus in Aquaculture in Soutlieast Asia do td
chdc Academy of Medical Sciences Newton intemational Fellowships (Vuong qudc Anbf
TAI LIEU THAM KHAO
1. D a n g T h i H o i n g O a n h va Nguydn Tiianh
64
P h u a n g (2012). P h a n lap v l x l c dinh d a c diem cua vi k h u a n Streptococus agalactiae b i c l dieu h d n g (Oreocbmmis sp.) b d n h mii m i t v l x u i t huydt. T a p chi K h o a h p c 2012, T r u o n g Dai h p c C i n T h a , (22) 203-212.
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ISOLATION AND CHARACTERIZATION OF Sti-eptococus agalacdaeFROM THAILAND FROG {Rana tigerina) CULTURED IN THUA THIEN - HUE PROVINCE, VIETNAM
Nguyen Ngoc Phuoc^, Nguyen Thi Hue Linh^, Nguyen Thi Xuan Hong^
^Faculty of Fishery, University of Agriculture and Forestiy, Hue University Email [email protected]. vn Summary
From 20 diseased frog (Rana tigenna) that culture at Huong Tra and Pbu Loc district, Thua Thien - Hue provmce, 20 small cocci, gram possitive bactena isolates were recovered. Bactena isolates were non-motile, oxidase, catiase and bile-esculin negative and they were identified as Streptococus agalactiae serotype lb (13 isolates) and S. agalactiae serotype III (7 isolates) using a Lancefield test kit, API 20 strep system and slide agUuhnation method. Challenge experiments usmg injection method showed that they can cause the observed disease signs with the ID^^ value of 2 x lO^cfu/mL (5. a^a/ac&aeTTHHTOOl, serotype lb) and 6.3 X 10' cfii/mL (5. a^^^cflaeTTHPLOOS, serotype III). The results of anUbiotic resistant tests showed tiiat most of isolates were sensitive to ofloxacin and 3 antibiotics that belong to p-Iactam group (oxacibn, ampieillin va amocillin), however all isolates resistant to oxytetiacyclme This is tbe first report of S.
agalactiae serotype III outbreak in Thailand frog in Vietnam.
Keywords: Thailandd-og, Rana tigerina, Streptococus agalactiae, serotype lb, serotype III Ngudi phan bidn: TS. Ha K;^
Ngly nhln bii: 23/8/2019 Ngly thong qua phan bidn: 24/9/2019 Ngly duydt dang: 01/10/2019
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