BAO TON NGOON GEN COA ENTEROTOXIGENIC EMU CHIJNG PD17 VA CHONG TM21 DONG TRONG THO Y
Phgm Hong Ngdn, Vd Thi Thu Trd, Cam Thi Thu Hd, Lgi Thi Lan Huffng Khoa thu y - Dgi hoc Nong nghiep Hd Npi
TOM TAT
Muc dich cua nghien cihi nay la khao sat, danh gia cac dac linh sinh h6a ctia enterotoxigenic E.coli chiing PD17 (ETEC-PD17) ya chting TM21 (ETEC-TM21), d6ng thai xac dinh difiu kien d6 luu giu. bao ton cac chiing vi khuan nay. Kgt qua nghien ciru cho th4y cac chung ETEC-PD17 va ETEC-TM21 bieu hien nhirng dac diem dac tnmg cua vi khuan E.coli. Sau 20 doi cky truyen, cac chitng nay vin co cac dac linh sinh hoa va linh chat nuoi cay on dinh so vai gi6ng g6c lim giir, d6ng thai luon giG duac kha nang san sinh doc t6 duong rugt enterotoxin. Da xac dinh dugc cac dac tinh sinh vat, hoa hgc, dac diem nuoi cay va tinh on dinh qua cac doi cay truyen cua cac chiing ETEC- PD17 va ETEC-TM21 khi bao quan a nhiet do va thai gian khac nhau chiing da cho k^t qua phat trien binh thuong. Trong do, phuang phap bao quan dong kho la phuang phap l6i im de bao quan cac chung vi khuan.
Tirkhoa- Bao ton quy gen. Enterotoxigenic E.coli, Dac linh smh hoa.
Conservation of gene pool of enterotoxigenic E.co//strain PD17 and strain TM21 for veterinary use
Pham Hong Ngan, Vu Ttii Thu Tra, Cam Thi Thu Ha, Lai Thi Lan Huong
SUMMARY
The objectives of this study were survey and evaluation of the biochemical characteristics of the enterotoxigenic E.coli strain PD17 (ETEC-PD17) and strain TM21 (ETEC-TM21) as well as study on the conditions tor preservation, conservation of these microorganisms The studied result indicated that both strains ETEC-PD17 and ETEC-TM21 showed all the typically biochemical characteristics of E. coli After 20 subculture lifes, these strains showed the biochemical characteristics and stability properties of culture compared with the conservative origin breed. Besides, all the tested strains always kept the ability to produce enterotoxin in the intestine. Biologicaf, chemical, stability characteristics of the strains ETEC-PD17 and ETEC- TM21 were determined. These strains were kept at the different conservative temperatures and durations showed the normal performances. Of which, freeze-dned conservation is an optimized method for microorganism conservation.
Key words: Conservation of gene pool, Enterotoxigenic E.coli, Biochemical characteristic,
I. DAT VAN D E khoa hgc dang lim kiem cac san pham thay the
^, , . , , , . ; , nham nang cao hieu qua trong viec ph6ng benh Ngay nay, chan nuoi ngay mol phat tnen tren ,. . * - i , u ' u - - i , = ^ ' - , . - *j , , • " • ' • ' ^; ... . VOI muc dich han che o nhiem moi truang do khap cac tinh thanh trong ca nuac de dap ung ^. - ' • i. - u-'. - c A i - u- - u
, "^ . ^ , . Ihuoc \ a hoa chat gay ra. Sudung lieu phap smh nhu cau dinh ducmg ngay cang cao cua \a hoi. , • .L- .u - •• -•. • u '
& ^ J ° • hoc va than thien vai moi truang hay viec cung Chinh vi the khong the tranh khoi co cac rui ro ; . u ••* • i • A „ r '• U-
° - • cap cac M sinh val CO lgi duac xem la giai phap do dich benh trong moi trirong nuoi gay ton that
va thi?t hai cho nguoi chSn nuoi. Ben canh cac lo^i thuoc khang sinh dimg de tri b?nh, cac nha
can huang ten gitip ngan chan nguy co dich benh xay ra va lir d6 giam thieu thiet hai cho
ngucri chan nuoi. Cac vi sinh vat co lgi co kha nang cai Ihien moi tmong, uc che sir phat trien cua vi khuan gay benh nham giam dgc luc va kha nang phat trien ciia chung de co the san xuat vacxin Hien nay mgt so chimg giong vi sinh vat CO cac uu diem thoa man yeu cau tren dang duac tap trung nghien ciiu, trong do co Bacillus subtilis, Geobacillus stearothermophilus va mgt so chiing enterotoxigenic E.coli co tiem nang khai thac va phat trien nguon gen.
Bao ton nguon gen vi sinh val da va dang dugc nhieu quoc gia tren the gioi quan tam, trong do CO Viet Nam do chiing co the phuc vu cho cong lac nghien ciru, ung dung trong sinh hgc va cong nghe sinh hgc de tao ra cac san pham da dang phuc vu dai song. Bao ton giong vi khuan E coli noi rieng va bao ton giong vi khuan, vi sinh vat n6i chung la viec Ihuc hien cac kT thual can thiet de giii cho vi sinh vat co ti le song sol cao, cac dac linh di truyen khong bi bien doi va khong bi lap nhiem bai cac sinh vat la.
Nghien cim nay dugc tien hanh tai Truang Dai hgc Nong nghiep Ha Noi nham muc dich bao ton nguon gen vi sinh vat phuc vu nghien Cliu khoa hoc thugc ITnh vuc an loan ve sinh thuc pham. phong trj benh cho gia siic, gia cam va phuc vu dao tao, nghien ciiu khoa hgc.
II. VAT LIEU VA PHlTONG PHAP
2.1. Vat lieuVi khuan enterotoxigenic E.coli cac chiing PD17 (ETEC-PD17) va TM21 CETEC-TM21) la nhiing vi khuan dugc phan lap tir be tieu chay a Viet Nam, la ket qua nghien ciiu cua de tai cap Bg dugc luu trir tai phong thi nghiem Bo mon thii y cong dong, Khoa thii y, DHNN Ha Ngi.
Moi truong va hoa chat dung de xac dmh dac tinh sinh hgc, bao ton va luu giii ngudn gen ciia ETEC-PD17 va ETEC-TM21: MacC, EMB, BGA, TSA, TSI, Glucoza, Lactoza, Saccharoza... do hang Oxoid cung cip. Thiet bi thong dung trong ph6ng thi nghiem cua Khoa
Thu y - Truong Dai hgc Nong nghiep Ha Ngi:
Tu am, noi hap cao ap, tii say, tii lanh am, can dien tir, may khuay tir gia nhiel..
2.2. Phirong phap nghifin cihi
2.2.1. Phuffngphdp xdc dinh dgc tinh sinh hoa cua vi khuan
Xac dinh dac tinh sinh vat hoa hgc ciia vi khuan E.coli theo phuang phap mo ta boi Quinn va cgng sir (1994) tom tat nhu sau: Cay 2 chimg VI khuin ETEC-PD17 va ETEC-TM21 vao moi truang TSA, nuoi trong tii am 37''C. Sau 24 gio quan sat khuan lac va nhupm Gram quan sal dac diem hinh thai, thu cac phan img sinh hoa. Tir cac khuan lac tiep tuc nuoi cay tren cac moi tru6Tig thach MacConkey, EMB, TSI, BGA nuoi trong tu am 37''C sau 24 gi6 quan sat hinh thai, mau sac khuan lac.
2.2.2. Phuffng phdp kiim tra tinh dn dinh cda ETEC-PD17 vd ETEC-TM21 qua cdc d&i dy chuyin
ETEC-PD17 va ETEC-TM21 sau khi da kiem tra cac dac tinh sinh hgc dugc cay truyen qua 20 doi tren moi tmong TSA va moi truong Nutrient broth, sau do tien hanh kiem tra tinh on dinh ve cac dac linh sinh hgc va kha nang san sinh khang nguyen pili va dgc to enterotoxin a doi thii 10va20.
2.2.3. Phuffng phdp bdo tdn vd luu giir nguon gen cua ETEC-PDI7 vd ETEC-TM2I
Vi khuin ETEC-PD17 va ETEC-TM21 duac bao ton theo 2 phuong phap:
- Su dung moi tmong TSB + Glycerin theo ty le 1:1 sau do bao quan a nhiet do 4 - 6''C va -20"C. Sau 3 thang, 6 thang liSn hanh kiem tra lai so lugng vi khuan.
- Dong kho, bao quan 6 4 - 6''C. Sau 6 thang kiem tra lai so lugng vi khuan.
Dia diem nghien cim: Ph6ng thi nghiem trgng diem cong nghe sinh hgc thii y, Khoa ihii y va Phong thi nghiem Bg mon thu y cgng dong, Khoa thii y, DHNN HN.
2.2.4. Phuffng phdp xdc dinh khdng nguyen pili cua cdc chung E.coli
Xac dinh khang nguyen pili theo phuong phap dong ngung ket (coagglutination test) mo ta boi Murray (19S7), tom tit nhu sau: Dung phien kinh trong, sach, sau khi dS ngam c6n ethanol 90", de kho, siy a nhiet do 120"C trong 30 phut. Chia phien kinh lam 2 phin: Nho len moi phan da chia mgt gipt nuoc sinh ly 0,85%.
Diing que cay trich liy 1 - 2 khuan lac E coli p-en moi tmong Minca agar, trgn deu voi niroc sinh ly roi nho khang the khang pili F5 hoac F41, F6, F5 + F4 len gigt khang nguyen da chuan bj, phia doi dien nho mot giol nuac sinh iy 0,85%.
Thi nghiem d6i chung dugc tien hanh voi khang nguyen chuan va khang the chuan.
Phan ling dugc coi la dutmg tinh khi nhirng gigt khang nguyen va khang the chuan hinh thanh nhirng dam ngung ket mau trang nhat va
phan irag am tinh khi cac huyen dich due deu, khong CO ngung ket.
III. KET QUA VA THAO LUAN
3.1. Ket qua kiem tra mot so dac dilm hinh thai, dac tinh nuoi cay va dac tinh sinh h6a cua vi khuan enteroxigenic E.coli ehung PD17vaTM2lTir 6ng gi6ng vi khuin ETEC-PD17 va ETEC- TM21 luu giir lai Phong thi nghiem Bg mon thii y cgng dong, cac dac tinh sinh hgc ciia vi khuan ETEC-PD 17 va ETEC-TM21 dugc ti^n hanh kiem tra tren mot so moi tmong chon loc. Ket qua kiem tra cho thay ca 2 chung bieu hien nhirng dac diem dac tnmg cua vi khuan E coli (Bang 1).
Cac chiing nay phat trien lot Iren cac moi tmong chgn lgc, the hien cac dac linh dien hinh ciia enterotoxigenic E.coli nhu Guinee va cgng su (1977) da mo ta (Pham H6ng Ngan, 2010).
Bang 1 . Ket qua kiem tra mpt so dac tinh nuoi cay, sinh hoa cua ETEC-PD17 va ETEC-TM21
M6i triFffng kiem tra MacC BGA EMB TSA TSI Indole MR VP Simmon's oltrate Dldpng Ureaza Glucoza Lactoza Saccharoza
N
30 30 3D 30 30 30 30 30 30 30 30 30 30 30
Dac tinh nuoi cay, sinti ti6a Khuan lac tron, ria gon, mat hoi vong (dang S), mau hong canh sen
Khuan igc dang S, m^u vang chanh tren nen mot tru'ong mau vang nhat
Khuan lac dang S, mau den, anh kim Khuan lac dang S, xam nhat
Ca phSn day moi trirong va mat nghieng deu chuyen mau vang, khong smh H,S, san sinh acid, len men smh hoi
+ +
+
+ + +
n
30 30 30 30 30 30 30 30 30 19 30 30 30 30
Ty ie (%) too
100 100 100 100 too 100 100 100 63 3 100 100 100 100 Ghi chu:
N: Sd ong giong kiem tra (Bao gom 15 ong ETEC-PD 17 vd 15 ong ETEC-T\I2I, hao quan (rnliicl dQ - 20"C trong mot truang TSB + Glycerin)
n: so mdu duang tinh (+)
39
3.2. Ket qua kiem tra tinh on dinh dac tinh sinh hoa cua ETEC-PD17 va ETEC-TM21 qua cac doi cay truyen
Cay tmyen vi khuan nhieu doi tren cac moi tmong nhan tao c6 the lam mat di mgt so dac tinh sinh hoa ciia chung. Do vay viec kiem tra tinh on dinh cac dac tinh sinh hoa ciia vi khuan la can ihiet trong cong tac bao ton cac giong vi khuin. ETEC-PDI7 va ETEC-TM21 sau khi da kiem tra cac dac tinh sinh hoa, duac cay tmyen qua 20 ddi tren moi tm6Tig TSA song hanh cung moi tmong Nutrient broth, cac dac linh sinh h6a dugc kiem tra lai a doi thir 10 va 20. Ket qua sau 20 dai cay tmyen, dac tinh sinh vat hoa hgc ciia ETEC-PD17 on dinh so vai chiing goc.
Tren moi tmong TSA a dai thii 20 xuat hien mgt so khuan lac dang R, nhay (Gyles va cgng sir, 1993, Pham H6ng Ngan, 2010).
Ket qua kiem tra cac dac tinh sinh hoa ciia ETEC-TM21 cho kel qua tuong tu nhu ciia ETEC-PD 17. Sau 20 doi cay tmyen, cac dac linh sinh vat, hoa hgc ciia ETEC-TM21 on dinh so vai giong goc bao quan. Tren moi tmong TSA 6 lan nuoi cay thir 20 xual hien mpl so khuan lac dang R, nhay, duang kinh >3mm.
Dieu nay cung da dugc mo ta trong mgt so tai lieu nghien ciiu ve ETEC nhu Gyles va cong su, 1993, Acres, !985, Pham H6ng Ngan, 2010).
3.3. Ket qua kiem tra tinh on djnh kha nang san sinh khang nguyen pili va d^c t6 entero- toxin ciia ETEC-PD17 va ETEC-TM21
ETEC-PD 17 va ETEC-TM21 co kha nang san sinh khang nguyen pili va doc t6 duong mot
enterotoxin, ia nhung yeu to dgc luc quan Irgnj gay tieu chay 6 be. Vi vay, kiem tra cac yeu tc doc luc sau nhieu dm cay tmyen la can thiet di khang djnh kha nang bao ton cac yeu to dgc lire ciia vi khuan ETEC (Quinn va cgng su, 1994).
c i y tmyan ETEC-PD 17 va ETEC-TM21 qua 20 doi tren moi tm6ng TSA, kiem tra kha nang san sinh khang nguyen pili va dgc to enterotoxin 6 d6fi thu 10 va dai thii 20. Ket qua cho thay 6 dai thif 10, ETEC-PD 17 va ETEC-TM21 v§n gia dugc dac tinh san sinh hai yeu to dgc lire co ban la khang nguyen pili va enterotoxin, 100%
cac ong giong cay tmyen deu cho ket qua duong tinh. Sau 20 dai ciy huyen, 13/15 (86,67%) si mau ong giong ETEC-PD 17 khong con bao ton dugc kha nang san sinh khang nguyen pili F5;
10/15 (66,67%) s6 mau ong giong ETEC-TM21 khong c6n bao ton dugc kha nang san sinh khang nguyen pili F4I, mac dii vay kha nang san sinh dgc to enterotoxin van dugc luu giir (Bang 2 va 3). Nhu vay, tren mgt so moi tmcmg nhan lao nhu TSA sau 20 doi nuoi cay, vi khuan ETEC CO the mat kha nang san sinh khang nguyen pili, Kel qua tren phii hgp voi nghien cim ciia Duchet-Suchaux (1992) va nghien ciiu cua Gyle va Thoen (1993). Khi nghien cuu khang nguyen pili F5 va F41, Duchet-Suchaux cho biel mot so moi lru6ng nhan tao phu hgp cho vi khuan enterotoxigenic E. coli. Chi co mgt so moi Imimg nhat djnh ETEC moi san sinh khang nguyen pili nhu moi tmong Minca, moi tmong TSA. Mac du vay, sau nhieu lan cay iruyen (tir dai thir 20 tra di) ETEC co the mat kha nang san sinh khang nguyen pili.
Bang 2. Ket qua kiem tra tinh on djnh kha nang san sinh khang nguyen pili va doc to enterotoxin cua ETEC-PD17
Chi tieu i<iem tra
Khang nguyen F5 Enterotoxin
So miu
15 15
Do'i thLP 10 So miu (*)
15 15
Tyle(%) 100 100
Do'i thij' 20 So miu (+)
13 15
TyiS(%) 86,67
100
Bang 3. Ket qua kiem tra tinh on djnh kha nang san sinh khang nguyen pili va doc to enterotoxin cua ETEC-TM21
Chi tieu i<ieni tra Khang nguyen F41 Enterotoxin
So m i u 15 15
Dd'i thi> 10 So miu (*)
15 15
Tyle(%) 100 100
Dcfi thl> 20 So miu (+)
10 15
Tyle(%j 66,67 100
3.4. Ket qua bao tfln va lun giu- ngu6n gen cua ETEC-PD17 va ETEC-TM21 Bang 4. Ket qua bao quan ETEC-PD17 ff nhiet do va tho'i gian khac nhau Moi tru'O'ng
bao quan
TSB + Glycerin Dong kho (Sua lach ba)
N
15 15 15
Nhiet dp bao quan
rc)
4 - 6 -20 4 - b
Tho'i gian bao quan 3 thang
N1 15(100%) 15(100%)
K
CFU/ml 5,2x10'-3,4x10^
2,5 X 10S-6,4x10«
K
6 thang N2 12 (80%) 15(100%) 15(100%)
CFU/ml 0- 1,5x108 5,2 X l O ' - 6 , 4 xlO^
4,2x10'-6,6x10f' Ghi chit: N - so ong giong kiem tra. K - khong kiim tra; Nl, N2 - so dng giong phdt trien binh thirdng.
Bang 5. Ket qua bao quan ETEC-TM21 cr nhiet do va tho'i gian khac nhau Moi tru'O'ng
bdo qudn
TSB + Glycerin Dong kho (Sifa tach bo)
N
15 15 15
Ntiiet do bao quan CC) 4 - 6 -20 4 - 6
Thd'i gian bao quan 3 thang
N1 15(100%) 15(100%)
K
CFU/ml 4,8 XlO'-5,4x10"
1,8 X 109-6,3x10^
K
6 thang N2 12 (80%) 15(100%) 15(100%)
CFU/ml 0-1.5x10' 5,2x10'-6.3x10»
4,2 X l O ' - 6 , 6 XlO' Ghi chu: N - s6 ong gidng kiem tra, K - khong kiem ira: Nl, N2 - sd ong gidng phdl trien binh Ihudng
Kel qua bao quan ETEC-PD 17 va ETEC- TM21 a nhiel do va thai gian khac nhau (Bang 4 va 5) cho Ihay: C nhiet do bao quan giong nhau nhung moi trucmg bao quan va thai gian bao quan khac nhau se cho ket qua ve ty le so lugng vi khuan sau bao quan phat trien binh Ihucmg
\i khac nhau. 6 nhiet dp bao quan la 4 - 6"C, voi moi truong bao quan la TSB + Glycerin, sau thai gian bao quan la 3 thang, 100% so ong phat
tnen binh thuang, nhung sau then gian bao quan 6 thang, so ong phat trien binh thirong la 12/15 (chiem 80%). 0 nhiet do bao quan la -20"C, phuang phap bao quan bang TSB + Glycerin s6 cho ket qua toi uu hem so vai bao quan a nhiet do 4 - 6"C. Doi vai phucmg phap dong kho, sau thai gian bao quan 6 thang cho ket qua 100%
cac ong giong phat tnen binh ihucmg
IV. K E T L U A N
Cac giong vi khuan enteroxigenic E.coli PD17 {ETEC - PD17) va enteroxigenic Ecoli TM21 (ETEC - TM21) liru giir tai phong thi nghiem Bo mon thu y cgng dong mang day dii nhiing dac diem dac tnmg ciia vi khuan E.coh nhu tai heu kinh dien da mo ta.
Chung vi khuSn ETEC - PD17 va ETEC - TM21 da dugc kiem tra cac dac tinh sinh val hoa hgc va dac diem nuoi cay, sau 20 dai cay truyen c6 cac dac tinh sinh hoa va tinh chat nuoi cay on dinh so voi giong goc bao quan.
Cac chiing vi khuan E.coli mang khang nguyen pili F5, F41 da dugc kiem tra kha nang sinh doc to duong rugt Enterotoxin
Sau 20 doi cay Iruyen, du khong duy tri dugc kha nang sinh khang nguyen piH, ty le 13/15 (chiem 88,67%) doi voi viec san sinh khang nguyen pili F5 cua ETEC - PD 17, 10/15 (chiSm 66/67%) doi vol viec san sinh khang nguyen pili F41 ciia ETEC - TM21, nhtrng cac chung vi khuan luon giu dugc kha nang san sinh doc to duang rugt enterotoxin, yen to doc luc quan trgng ciia vi khuan E coli.
Cac chiing vi khuin ETEC - PD 17 va ETEC - TM21 da xac dinh cac dac linh sinh val, hoa hgc, dac diem nuoi cay va tinh on djnh qua cac dai cay truy6n dugc bao quan a nhiel do va thai gian khac nhau cho ket qua phat trien binh thuang.
Trong do phuong phap bao quan dong kho la phuong phap toi uu, cho ket qua 15/15 so 6ng phat Inen binh thuang sau bao quan (dal 100%).
T A I L I E U T H A M K H A O 1. Acres S. D. (1985), "Enterotoxigenic
Escherichia coli in newborn calves - A review", J Dairy. Sci., (68), pp. 229-256.
2. Duchet-Suchaux, M. (1992), "Charactenstic of enterotoxigenic Eschenchia coli bearing F5, F41 antigens". Infect Immun., pp. 4468- 4474.
3 Guinee, P. A. M., J. Veldkamp and W. H.
Jansen (1977), "Improved Minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli isolated from calves and correlation with enterotoxigenicity". Infect. Immun., (13), pp. 1369-1377.
4. Gyles, C A, C. O Thoen (1993), ''''Escherichia coli, Pathogenesis of bacterial infection in animals", Iowa state. University Press, United slate of America, pp. 164-187, 5. Pham Hong Ngan (2010). Nghien cuu mgt so
dac tinh gay benh ciia vi khuan Escherichia coli. Salmonella gay tieu chay a be giong siJa nuoi tai ngoai thanh Ha Ngi va bien phap phong tri. Luan an tien sT nong nghiep, Truang Dai hgc Nong nghiep Ha Ngi.
6. Quinn, P. J., M. E. Carter, B. Markey and R.
G. Carter (1994), "Bacterial cell counting technique". Clinical Veterinary Microbiology, Wolfe Publishing, Mosby-Year Book Europe Limited, England, pp. 61-66.
7. Quinn, P. J,. M. E. Carter, B. Markey, G.
R. Carter (1994), "Enterobacleriaceae", Clinical Vetennary Microbiology, Wolfe Publishing, Mosby - Year Book Europe Limited, England, pp, 209-236.
Nhan ngay: 19.12.2013 Phan bien ngay: 5.3.2014