B GIO DC V O TO

TRNG I HC NNG NGHIP H NI

PHM HNG QUN







NGHIN CU MT S C TNH SINH HC CA VI KHUN
STREPTOCOCCUS SPP. GY BNH XUT HUYT C R PHI
NUI TI MT S TNH MIN BC VIT NAM




LUN VN THC S







H NI 2013
B GIO DC V O TO
TRNG I HC NNG NGHIP H NI




PHM HNG QUN





NGHIN CU MT S C TNH SINH HC CA VI KHUN
STREPTOCOCCUS SPP. GY BNH XUT HUYT C R PHI
NUI TI MT S TNH MIN BC VIT NAM



Chuyn ngnh: CNG NGH SINH HC
M s: 06.42.02.01


NGI HNG DN KHOA HC:
TS. L VN KHOA
TS. HUNH TH M L





H NI - 2013


i
LI CAM OAN

Ti xin cam oan y l cng trnh nghin cu ca ring ti. Cc s liu, kt
qu c nu trong lun vn l trung thc v cha tng c cng b trong bt k
mt cng trnh no khc. ng thi tt c cc thng tin ti trch dn trong lun vn
u c ch r ngun gc.




Tc gi


Phm Hng Qun










ii
LI CM N !

hon thnh cc ni dung c bn trong lun vn tt nghip ny, ngoi s n
lc v c gng ca bn thn ti cn nhn c s gip khng nh ca nhiu t
chc, c quan v cc c nhn.
Li u tin, ti xin by t lng bit n chn thnh v su sc nht ti TS. L
Vn Khoa Cc Th Y, TS. Hunh Th M L - i hc Nng nghip H Ni l
nhng ngi nh hng v trc tip hng dn ti hon thnh lun vn.
Ti xin by t lng bit n chn thnh v su sc nht ti TS. Nguyn Hu
V, ThS. H Thu Thy cng cc anh ch Trung tm nghin cu Cng ty Hanvet
to mi iu kin tt nht ti thc hin ti.
Ti xin c by t lng knh trng, bit n chn thnh ti cc thy c gio
trong trng i hc Nng nghip H Ni, trong sut hai nm hc ti trng, ti
nhn c s dy d, du dt tn tnh ca cc thy c gio trong trng.
Nhn y, ti xin gi li cm n ti tt c bn b, cc bn ng nghip nhng
ngi gp chn thnh, gip v ng vin ti trong sut thi gian ti hon
thnh lun vn ny.
Cui cng, con xin cm n b m, cc anh ch em lun c v, ng vin
con trong nhng lc kh khn nht gip con c thm ngh lc c c ngy hm
nay.
Tc gi

Phm Hng Qun




iii
MC LC

Li cam oan i
Li cm n ! ii
Mc lc iii
Danh mc ch vit tt vi
Danh mc bng vii
Danh mc hnh viii
1 M U 1
1.1 t vn 1
1.2 Mc ch ca ti: 3
1.3 ngha khoa hc ca ti 3
1.4 ngha thc tin ca ti 3
2 TNG QUAN TI LIU 4
2.1 Mt s c im sinh hc ca c r phi 4
2.1.1 Ngun gc 4
2.1.2 Phn loi 4
2.1.3 c im mi trng sng v tp tnh dinh dng 5
2.2 Tnh hnh nui c r phi 6
2.2.1 Tnh hnh nui c r phi trn th gii 6
2.2.2 Tnh hnh nui c r phi ti Vit Nam 6
2.3 Tnh hnh nghin cu dch bnh c r phi 7
2.3.1 Tnh hnh nghin cu dch bnh c r phi trn th gii 7
2.3.2 Tnh hnh nghin cu dch bnh c r phi trong nc 10
2.4 Tnh hnh s dng thuc trong nui trng thy sn 15
3 NI DUNG, NGUYN LIU V PHNG PHP NGHIN CU 17
3.1 Thi gian v a im nghin cu 17
3.1.1 Thi gian nghin cu 17
3.1.2 a im nghin cu 17


iv
3.2 Ni dung nghin cu 17
3.3 Vt liu nghin cu 17
3.3.1 Dng c, thit b phc v nghin cu 17
3.3.2 Mi trng, ha cht phc v nghin cu 18
3.3.3 Vt liu nghin cu 18
3.4 Phng php nghin cu 19
3.4.1 Phng php thu mu c bnh 19
3.4.2 Phng php m c ly ni tng 19
3.4.3 Phng php phn lp vi khun Streptococcus spp. c trong mu bnh
phm 20
4.4.4 Phng php m mt vi khun 21
3.4.5 Phng php nh danh vi khun Streptoccocus spp. 21
3.4.6 Phng php xc nh c lc ca cc chng vi khun Streptococcus spp. 26
3.4.7 Phng php xc nh tnh khng nguyn 29
3.4.8 Phng php kim tra kh nng mn cm vi khng sinh ca vi khun 32
3.4.9 Phng php x l s liu 33
4 KT QU NGHIN CU V THO LUN 34
4.1 Kt qu phn lp vi khun Streptoccocus spp 34
4.1.1 Kt qu thu mu 34
4.1.2 Kt qu phn lp vi khun 35
4.2 Kt qu xc nh mt s c tnh sinh ha ca vi khun Streptoccocus
spp. phn lp c. 37
4.2.1 Kt qu xc nh mt s c tnh sinh hc 37
4.2.2 Kt qu nh danh vi khun 38
4.3 Kt qu xc nh c lc ca cc chng vi khun Streptococcus
agalactiae phn lp c 40
4.3.1 Kt qu gy bnh thc nghim ca vi khun Streptococcus agalactiae
c r phi 40
4.3.2 Kt qu tng cng c lc ca cc chng vi khun Streptococcus
agalactiae 44


v
4.4 Kt qu xc nh tnh khng nguyn ca vi khun Streptococcus
agalactiae phn lp c 46
4.4.4 Kt qu to khng nguyn cho tng chng vi khun: 46
4.4.2 Kt qu to khng th khng S.agalactiae trn c r phi 46
4.4.3 Kt qu phn ng ngng kt 47
4.5 Kt qu kim tra kh nng mn cm vi khng sinh ca vi khun
Streptococcus agalactiae phn lp c 49
5 KT LUN V NGH 51
5.1 Kt lun 51
5.2 ngh 51
TI LIU THAM KHO 52
PH LC 55
BI BO KHOA HC 73





vi
DANH MC CH VIT TT


Ctv: Cng tc vin
KS: Khng sinh
VK: Vi khun
















vii
DANH MC BNG

STT Tn bng Trang
2.1 Vi khun gy bnh trn c r phi nui ti khu vc ng Nam
(Lauke Labrie, 2007) 10
3.1 Thuc th v cch c kt qu cc phn ng sinh ha trong API 20
Strep 25
3.2 B tr th nghim xc nh c lc vi khun gy bnh xut huyt c
r phi 27
3.3 nh gi ng knh vng v khun chun 33
4.1 Kt qu thu mu c nghi b bnh xut huyt 35
4.2 Thnh phn loi vi khun phn lp c t mu c bnh 36
4.3 Kt qu phn lp vi khun Streptococcus spp. t cc c quan ca c
r phi 37
4.4 Kt qu gim nh v nh danh vi khun Streptococcus spp. 39
4.5 Kt qu gy bnh thc nghim ca cc chng vi khun Streptococcus
agalactiae thu ti Hi Dng 41
4.6 Kt qu gy bnh thc nghim ca cc chng vi khun Streptococcus
agalactiae thu ti H Ni 42
4.7 Kt qu gy bnh thc nghim ca cc chng vi khun Streptococcus
agalactiae thu ti Hi Phng 42
4.8 Kt qu gy bnh thc nghim ca cc chng vi khun Streptococcus
agalactiae thu ti Qung Ninh 43
4.9 Bng kt qu tng cng c lc cc chng vi khun S.agalactiae 45
4.10 Kt qu kim tra phn ng ngng kt vi khng th pha long 48
4.11 Kt qu th khng sinh ca 52 chng S.agalactiae vi 10 loi
thuc khng sinh thng dng 49




viii
DANH MC HNH

STT Tn hnh Trang
2.1 C r phi vn (Oreochromis niloticus) 4
2.2 C r phi (c iu hng) 5
2.3 Tc nhn gy bnh trn c r phi cc giai on nui 10
3.1 Cch m xoang bng c 20
3.2 Cch m no c 20
3.3 S nui cy, phn lp vi khun Streptococcus spp. 21
3.4 S b tr th nghim gy bnh cho c r phi 28
3.5 C r phi th nghim 30
4.1 Du hiu bnh l ca c lc thu mu. A: Mt c b li, c. B: Ni
tng c b xut huyt. C: C bi l , hot ng chm chp. D: Bng
c trng to v xut huyt 34
4.2 Hnh thi khun lc Streptococcus spp. khi nui cy trn mi trng
thch mu 38
4.3 Vi khun Streptococcus spp. 38
4.4 Hnh thi khun lc Streptococcus spp. khi nui cy trn mi trng
BHIA 38
4.5 Kt qu th kt API 20Strep nh danh Streptococcus agalactiae 38
4.6 C r phi c du hiu bnh l sau 24 gi gy nhim vi vi khun
Streptococcus agalactiae 44
4.7 Vi khun bt hot bng formalin trc ly tm (A); sau li tm (B); pha
vi nc mui sinh l (C) 46
4.8 Hnh nh thu huyt thanh c (A): Mu c; (B): Mu c sau khi gi
lnh v ly tm 47
4.9 Kt qu phn ng ngng kt nhanh trn phin knh 47
4.10 Hin tng ngng kt quan st bng knh hin vi (40X) 48
4.11 Kim tra kh nng mn cm vi khng sinh 50


1
1. M U

1.1. t vn
Nhng nm gn y, ngh nui trng thy sn (NTTS) khng ngng pht
trin v ngy cng chim v tr quan trng trong ngnh Thy sn ni ring v kinh t
t nc ni chung. Vi kim ngch xut khu nm 2010 t 4,94 t USD th y l
mt trong ba ngnh c ng gp ln nht cho tng kim ngnh xut khu ca Vit
Nam. Tng cc Thy sn (B Nng nghip v Pht trin Nng thn) cho bit, giai
on 2011 2015 ngnh Thy sn hng n s pht trin bn vng, l mt ngnh
xut khu hng ha ln, c kh nng cnh tranh cao v hi nhp vng chc vi th
gii. Mc tiu quan trng hng u l y mnh xut khu t kim ngch 6,5 t
USD vo nm 2015 v chim khong 37% trong khi nng lm ng nghip. V vy
vic qun l dch bnh trn cc i tng ch lc l yu t quan trng t c
mc tiu trn. Tuy nhin hin ti ngh NTTS ti Vit Nam ang gp phi nhng tr
ngi ln nh dch bnh BNP trn c tra c basa, dch bnh xut huyt trn c r phi,
bnh m trn c trm c, bnh m trng trn tm s, bnh virus trn c
chp. qun l cc dch bnh trn cc i tng quan trng, nhiu gii php
c t ra nh: la chn cc con ging sch bnh, qun l tt mi trng, dinh
dng, s dng thuc v ha cht, tuy nhin cha mang li hiu qu cao. V vy
vic pht trin v ng dng cc ch phm sinh hc, c bit l vacxin trong NTTS
c ngha cp thit trong vic qun l dch bnh t hiu qu cao hn.
Bn cnh s pht trin nhanh chng ca ngh nui ven bin v ngh nui bin
th ngh nui c nc ngt vn khng nh c vai tr ca mnh. Trong , i
tng c r phi vi nhng u im nh c t b sc vi bin i ca mi trng v
c kh nng khng c mt s bnh, thc n khng i hi cht lng qu cao, gi
thnh sn xut thp nn cc quc gia ang pht trin c bit ch trng n pht
trin nui loi c ny. Tuy nhin, khi pht trin nui c r phi vi mt cao v
nui thm canh th cng pht hin mt s bnh nh hng n nng sut v cht
lng thc phm. Qua nghin cu, ngi ta ch ra rng bnh c r phi ch yu


2
l do vi khun, virt, nm, v k sinh trng (Shoemaker, 2008). c bit l bnh do
do vi khun Streptococcus spp. (lin cu khun) gy ra l nguyn nhn gy nn thit
hi ln cho c r phi ni ring v c nc ngt ni chung, lm nh hng n hiu
qu kinh t ca ngnh nui trng thy sn. Theo thng k th lin cu khun gy
bnh trn c ch yu l hai loi Streptococcus iniae vStreptococcus agalactiae.
Hin nay, vic phng tr bnh trn c nc ngt nc ta vn ch yu da vo
vic s dng thuc khng sinh v ha cht. Hin ti ch c mt loi vc-xin bo v
c tra chng li vi khun Edwardsiella ictaluri. Vc-xin ALPHA J ECT Panga 1
c Cc Th y (B Nng nghip v Pht trin nng thn) va cng b cp php
tip th k t ngy 10/4/2013. Trong khi trn th gii c 36 loi vacxin phng
bnh do vi khun gy ra v hai loi vacxin phng bnh do virut. Vic phng tr bnh
ch yu ph thuc vo cc loi thuc khng sinh v ha cht gn y khin cho
vic xut khu thy sn ca Vit Nam gp rt nhiu kh khn do danh mc cc loi
thuc v ha cht cm s dng trong nui trng thy sn ngy cng tng. V d c
th l vic cm s dng chloramphenicol, flomequine v xanh malachite nh
hng ln cho ngh xut khu c Tra v c Ba Sa ca Vit Nam trong nm 2005 v
2006. M l th trng ln nht cho c da trn ca Vit Nam trc nm 2005 c
nhng chnh sch tng thu nhp khuu c tra v c Ba Sa vo nc ny. Bn cnh
chnh sch bo h ngh nui c da trn ni a ca chnh ph M th vic s dng
thuc thuc danh mc cm l mt trong nhng nguyn nhn dn n vic kh khn
tm th trng u ra cho cc sn phm ca hai i tng trn. V vy vic nghin
cu, pht trin cc phng php phng tr bnh c hiu qu nh s dng cc loi
tho dc, cht tch chit t tho dc v vacxin cho c nc ngt l rt cn thit
nhm m bo cho s pht trin bn vng ca ngh. S dng vacxin phng bnh
cho c gip gim t l cht, gim vic s dng cc loi khng sinh trong nui trng
thy sn v h gi thnh sn phm. Bn cnh vic s dng vacxin cng gp phn
vo vic to ra cc sn phm t tiu chun v sinh an ton thc phm. Hin nay
cha c bt k loi vacxin phng bnh Streptococcosis gy bnh trn c r phi
c nghin cu v ng dng vo sn xut ti Vit Nam. V vy, vic phn lp v
xc nh c tnh sinh hc ca Streptococcus spp. l cn thit, l c s khoa hc


3
gip cho vic nghin cu v sn xut vacxin phng bnh do vi khun Streptococcus
spp. c r phi nui ti Vit Nam.
Vi mc tiu nh vy, ti tin hnh thc hin ti: Nghin cu mt s c
tnh sinh hc ca vi khun Streptococcus spp. gy bnh xut huyt c r phi
nui ti mt s tnh min Bc Vit Nam nhm cung cp ngun ging vi khun
tin hnh nghin cu ch to kt v vacxin phc v cho chn on nhanh v phng,
tr bnh.
1.2. Mc ch ca ti:
Phn lp v xc nh c mt s c tnh sinh hc ca vi khun Streptococcus
spp. phc v cho nghin cu kit chn on v vacxin phng bnh xut huyt trn c
r phi ti mt s tnh min Bc.
1.3. ngha khoa hc ca ti
Phn lp v xc nh c mt s c tnh sinh hc ca vi khun Streptococcus
spp. gy bnh xut huyt c r phi ti mt s tnh min Bc.
Xc nh tnh khng nguyn ca chng vi khun la chn.
Xc nh kh nng mn cm, khng khng sinh ca vi khun la chn.
1.4. ngha thc tin ca ti
Nghin cu ny nhm chn ra nhng chng vi khun c khng nguyn p ng
min dch tt phc v nghin cu sn xut vacxin.
Xc nh kh nng mn cm, khng khng sinh ca vi khun la chn t
c c s khoa hc la chn khng sinh c tnh mn cm cao vi loi vi khun trn
iu tr bnh Streptococcosis.
Phc v cho chin lc phng tr bnh trn c r phi nhm tm ra phng php
sn xut vacxin hiu qu cao, chi ph s dng vacxin thp v d p dng iu kin
ca Vit Nam.
Gip ngi nui trng thy sn a ra bin php phng v chn thuc iu tr
theo ng nguyn tc s dng khng sinh trnh gy ra cc dng vi khun khng
thuc gy nhim mi trng v hn ch c tn d khng sinh trong c r phi,
m bo v sinh an ton thc phm cho ngi tiu dng.



4
2. TNG QUAN TI LIU

2.1. Mt s c im sinh hc ca c r phi
2.1.1. Ngun gc
C r phi c ngun gc t Chu Phi, c c nui u tin Kenya v sau
nui rng ri nhiu nc Chu Phi v trn th gii. C c nui nhiu nht l
nhng nc nhit i v cn nhit i. R phi en (Oreochromis mossambicus) l
loi c R phi u tin c nhp vo nc ta nm 1951. R phi vn (O. niloticus)
c nhp t i Loan nm 1973, sau c r phi c ci thin cht lng di
truyn (dng GIFT) c gii thiu vo Vit Nam t Thi Lan nm 1994.
2.1.2. Phn loi
C r phi thuc lp: Ostechthyes; Lp ph: Actynopterigii.
B: Perciformes; B ph: Perciidae.
H: Cichlidae
Ging: Tilapia, Sarotherodon, Oreochromis
Loi: Tilapia sp, Sarotherodon sp, Oreochromis sp
Hin nay c 2 loi chnh c ph bin ti Vit Nam l :
C r phi vn ( R phi i Loan, Oreochromis niloticus ) c nhp vo Vit
Nam nm 1973 t i Loan.

Hnh 2.1: C r phi vn
(Oreochromis niloticus)

C r phi (Oreochromis sp.), cn c gi l c iu hng, c mu hng
c nhp vo Vit Nam nm 1985 t Malaysia.


5

Hnh 2.2: C r phi (c iu hng)
2.1.3. c im mi trng sng v tp tnh dinh dng
R phi l loi c c ngun gc vng nhit i, nn kh nng thch nghi vi
nhit cao tt hn nhit thp. Nhit thch hp cho c sinh trng, pht trin
l 25 30
0
C. R phi l loi c c ngun gc nc ngt, nhng chng c kh nng
sng v pht trin trong mi trng nc l, mn c nng mui ti 35
o
/
oo
. Kh
nng thch ng vi mn ca mi loi u khc nhau. Loi O. niloticus c ngng
mui thp nht v loi c ngng mui cao nht l T. zillii, O. aureus (Philipart v
Ruwet, 1982). C r phi c th sng trong mi trng nc c hm lng oxy ha
tan thp ti 1mg/l nhng khng th ko di khi hm lng oxy di 0,7mg/l
(Balarin v Haller, 1982). Kh nng chu Amoniac ti 2,4mg/l. C r phi c kh
nng sng trong mi trng nc c bin pH rt rng 5 11, nhng thch hp
nht l 6,5 8,5. Theo Philipart v Ruwet (1982), R phi cht khong dao ng
ca pH =3,5 hay pH >12 sau 2 3 gi.
R phi l loi c n tp, khi cn nh c n sinh vt ph du thy sinh l ch yu,
20 ngy tui (17 18mm) c chuyn dn sang thc n nh c trng thnh. C
trng thnh n mn b hu c, to cc loi, u trng, cn trng, sinh vt y, ph
du sinh vt, thc vt thng ng loi mm, phn hu c Ngoi ra, trong ao nui
c th cho thm thc n b sung nh cm go, bt ng v cc ph phm nng
nghip khc. c bit c r phi c th s dng rt hiu qu thc n cng nghip v
thc n t ch bin (Balarin v Haller, 1982). y l mt c im gip cho vic
nui c r phi thm canh t nng sut cao. Vi nhng c im u vit c r
phi c phn b v ng nui kh rng ri trong cc vng min ca nc ta.


6
2.2. Tnh hnh nui c r phi
2.2.1. Tnh hnh nui c r phi trn th gii
Hin nay c r phi l i tng c nui ph bin nhiu nc trn th gii,
chim mt vi tr quan trng ch ng sau nhm c chp trong cc thy vc nc
ngt. Nh c nhng c tnh tt nh ph thc n a dng, t bnh tt, cht lng tht
thm ngon, u t chi ph hnh thnh ln sn phm thpv th m loi ny
c nui ph bin, din tch v sn lng cng nh cht lng sn phm khng
ngng tng ln, c bit trong nhng nm gn y. Trong tng lai, c R phi s l
sn phm thay th cho cc loi tht c trng ang ngy cng cn kit. i tng
chim u th v c nui ph bin l ging c R phi vn O. niloticus, vi tng
sn lng l 1,001,302 tn nm 2002, chim 84% ca tng sn lng c R phi
(FAO, 2004).
Ti Hi ngh ca INFOFISH TILAPIA 2010 v c R phi t chc ti Kuala
Lumper, Malaysia cui thng 10/2010, thng k tng sn lng c R phi ton cu
nm 2010 t 3,7 triu tn. Mc d c R phi c ngun gc khng phi t chu
nhng y li l khu vc sn xut c R phi quan trng nht th gii. Sn lng c
R phi chu trong thi gian qua c coi l tng nhanh nht th gii. Cc nc
chu i din c ngh nui c R phi pht trin mnh l Trung Quc,
Indonesia, Thi Lan, i Loan Tng sn ln c nm nc ny chim 94% tng
sn lng c R phi ca chu . Trong Trung Quc l quc gia sn xut c R
phi ln nht. Tnh n nm 2011, sn lng c R phi ca nc ny gi n nh
mc 1,1 1,2 triu tn v d kin vn tip tc tng vo cc nm ti.
2.2.2. Tnh hnh nui c r phi ti Vit Nam
Ngh nui c R phi nc ta c lch s hn 50 nm, khi u khi nhp ni c
R phi en (O. mosambicus) vo nc ta u nhng nm 1950. Nhng thp nin 50
v 60 ca th k trc, c R phi c nui ch yu hnh thc qung canh v
qung canh ci tin, nui chung c c v c ci. Phong tro nui c R phi c bit
pht trin t nhng nm u ca thp k 90 sau khi chng ta nhp li nhng dng
c R phi vn c cht lng tt, c bit l c chn ging dng Thi Lan v Israel.
C c nui nhiu a phng vi cc hnh thc khc nhau: nui n, nui ghp,


7
vi mc canh tc t qung canh, bn thm canh n thm canh.
Theo Cc Thng k nm 2005, din tch nui c R phi ca c nc ta l 22,340
ha chim 3% tng din tch nui trng thy sn, trong nui nc l, mn l
2,068 ha v nui nc ngt l 20,272 ha. Tng sn lng c R phi c tnh t
54,486,8 tn; chim 9,08% tng sn lng c nui. ng bng Sng Hng v ng
bng Sng Cu Long l hai vng nui ch yu, ln lt chim 17,6% v 58,4%
tng sn lng c R phi ca c nc. Sn lng c R phi trong c nc bao gm:
nui trong ao v trong m 37,931,8 tn; nui lng 10,182 tn. Mc tiu a ra n
nm 2015 sn lng c c nc t 200,000 tn/nm; trong ginh 40% cho xut
khu (Phm Anh Tun, 2006).
2.3. Tnh hnh nghin cu dch bnh c r phi
2.3.1. Tnh hnh nghin cu dch bnh c r phi trn th gii
C R phi l mt trong nhng i tng nui thy sn nc ngt ch yu trn
th gii v Vit Nam. Theo Gupta M.V v Acosta B.O. (2004) th gii c khong
70 loi c r phi khc nhau trong c 9 loi ang c nui trong cc h thng
khc nhau. Loi nui ch yu l Oreochromis niloticus vi sn lng nm 2007
t 2.12 triu tn (FAO., 2009). Ngh nui c r phi ngy cng m rng v pht
trin do c nhng u im nh nhanh ln, c kh nng nui vi mt cao, cht
lng tht ngon v sc chng chu tt vi cc iu kin mi trng khc nhau (El-
Sayed, A.M., 2006). Tuy nhin cng vi s pht trin ca cc hnh thc nui mi
vi mt cao nh nui cng nghip v nui thm canh th c r phi cng d b
nhim mt s tc nhn gy bnh nh vi rt, vi khun, k sinh trng v nm
(Shoemaker, 2008).
Ban u, c r phi c xem l c kh nng khng tt vi vi khun, k
sinh trng, nm v virus...so vi cc loi c khc trong cng mi trng nui. Tuy
nhin trong thi gian gn y, c R phi c tm thy l mn cm vi c vi
khun v k sinh trng. Cc tc nhn gy bnh ph bin cho c r phi bao gm
Streptococcus spp., Flavobacterium columnare, Aeromonas hydrophyla,
Edwarsiella tarda, Ichthyophitirius multifillis, Tricodhina sp., Gyrodactylus
niloticus (Klesius v ctv, 2008). iu quan trng cn lu rng nhim lin cu


8
khun tr thnh mt vn ln trong nui c r phi v gy thit hi kinh t nng
n. Streptococcus iniae v Streptococcus agalactiae l nhng loi vi khun chnh
nh hng n vic sn xut c r phi trn th gii (Evan v ctv, 2006).
Tc nhn gy bnh Streptococcosis l nhm vi khun thuc ging Streptococcus
spp. Vi khun Streptococcus spp. gy bnh c r phi ln u tin phn lp c
c r phi nui ti Nht Bn gm hai loi l Streptococcus shiloi v Streptococcus
difficile. Sau cc loi vi khun gy bnh trn c r phi c phn loi li nh
Streptococcus shiloi c lStreptococcus iniae, cn Streptococcus difficile c
xc nhn lStreptococcus agalactiae.
Vi khun gy bnh Streptococcus gy bnh trn c r phi bao gm hai loi
chnh l Streptococcus iniae v Streptococcus agalactiae (Bng 2.1). y l
nhm lin cu khun gram dng l tc nhn gy bnh chnh trn cc h thng nui
c r phi thm canh v gy thit hi ln cho ngh ny trn ton th gii (Perera
R.P., 1994). Vi khun c kh nng pht trin trn cc mi trng nui cy khc
nhau nh Tryptic Soy Agar, Brain Heart Infussion, Muller-Hinton v Blood Agar.
Khun lc ca vi khun c kch thc dao ng t 0,5 n 0,7mm sau 24 gi nui
cy. Vi khun c th to vng dung huyt trn mi trng thch mu (Nguyen v
Kanai, 1999). S. iniae thy phn esculin v tinh bt, khng thy phn gelatin v c
kh nng ln men glucose, maltose, mannitol, sucrose, khng ln men arabinose,
lactose, raffinose v xylose (Nguyen v Kanai, 1999).
Dch bnh c r phi nui Thi Lan c quan st thy trong lng nui
trn sng Mekong ti thnh ph Mukudahan, pha ng Bc Thi Lan vo thng 5
nm 2001. T l c b cht do dch bnh vo khong 40-60% sau hai tun b nhim
bnh. Du hiu in hnh ca c b bnh l chng bng, trong xoang bng cha
dch v hu mn b sng. Trong nm 2002 v 2003, ti thnh ph Lubuk Linggau,
min Nam Sumatra, Indonesia c r phi nui lng cng xut hin hin tng c
b cht vi du hiu bnh l hai mt c v i mu. Vi khun phn lp t b no
v cc c quan khc ca c r phi b nh hng t Thi Lan v Indonesia c
xc nh lStreptococcus agalactiae vStreptococcus iniae (Yuasa, 2005).
Nm 2005 ti mt s h cha ca Malaysia ghi nhn c hin tng c r


9
phi nui lng b cht, kt qu thu mu phn lp c vi khun t cc c quan.
c bit l mu thu mt, thn, no. Trong vi khun S. agalactiae chim 70%
tng s loi vi khun Streptococcus c xc nh, 30% cn li l Leuconostoc spp.
v S. constellatus. Du hiu in hnh quan st bao gm c bi li khng bnh
thng v b n. Hu nh tt c cc c r phi b bnh mt nh c gic mc hoc
ti mu, mt b li hoc xp (Yuasa, 2005).
Streptococcus agalactiae ngy cng c pht hin v khng nh l nguyn
nhn gy bnh cho c, c bit l c nc ngt (Plumb, 1999; Pretto-Giordano v
ctv, 2010a). Nhng nm gn y rt nhiu t dch bnh do nhim Streptococcus
agalactiae c ghi nhn nhiu trang tri nui c r phi c bit l c trang tri
chu (Musa v ctv, 2009; Suanyuk v ctv, 2005).
T thng 7 n thng 12 nm 2009 bnh Streptococcosis trn c r phi bng
pht ti bn tnh Guangdong, Guangxi, Hainan and Fujian ni chim ti 90% sn
lng nui i tng ny ti Trung Quc. Bnh Streptococcosis khng ch xy ra
ti ni c sn lng nui c r phi ln nht th gii (1.1 triu tn nm 2009). Ti
Thi Lan theo (Wongtavatchai & Maisak, 2008) t l Streptococcus agalactiae trn
Streptococcus iniae l 112/8 c r phi vn (Oreochromis nilotica), nghin cu v
dch t hc ca Intervet/Scheing Plough Animal Health cho kt qu Streptococcus
agalactiae chim 82% v Streptococcus iniae 18% trong tng s 500 mu phn lp
t 13 nc Chu v Chu M La Tinh trong 8 nm (Sheehan v ctv., 2009). Trn
c r phi (Oreochromis spp) cc kt qu nghin cu ca Hernandez v ctv.,
2009, Mian v ctv., 2009 v Zamri-saad v ctv., 2010 u kt lun tc nhn chnh
gy bnh Streptococcosis l Streptococcus agalactia.
Trong mi giai on nui khc nhau th c r phi thng nhim cc tc nhn
gy bnh khc nhau theo nh hnh 2.1.


10

Hnh 2.3: Tc nhn gy bnh trn c r phi cc giai on nui
(Intervet, 2006)
T cc kt qu nghin cu cho thy bnh do vi khun Streptococcus iniae v S.
agalactiae l rt ph bin v nh hng n c r phi nui ti khu vc ng nam .
Kt qu nghin cu c tng hp theo bng 2.1.
Bng 2.1: Vi khun gy bnh trn c r phi nui ti khu vc ng Nam
(Lauke Labrie, 2007)
Loi vi khun S mu nhim S im thu Quc gia
S. agalactiae 219 22 Indonesia, Singapore,
Malaysia, Philippin, Thi
Lan, Trung Quc v Vit
nam
S. iniae 75 14
Flavobacterium
colummnare
40 16

2.3.2. Tnh hnh nghin cu dch bnh c r phi trong nc
Nui trng thy sn l mt trong nhng ngnh kinh t quan trng ca Vit Nam
trong vic cung cp thc phm c gi tr cho th trng trong nc, xut khu thu
ngoi t v to cng n vic lm cho ngi dn. Theo thng k ca t chc nng


11
lng th gii sn lng thy sn ca Vit Nam nm 2007 t 4.525.750 tn ng
th 3 th gii sau Trung Quc v n . Theo cc thng k tng sn lng thy
sn ca Vit Nam nm 2009 t 4.847 triu tn trong nui trng thy sn t
2.569 triu tn (Cc Thng k., 2010). Nm 2009 tng kim ngch xut khu thy
sn t 4.2 t USD ch ng sau xut khu dt may v du th. Theo bo co ca
B Nng nghip v Pht trin Nng thn sn lng thy sn ca nc ta ng u
l c tra, basa (trn 1 triu tn), tip n l tm s (413 nghn tn) v c r phi ng
th 3 v sn lng.
Nm 2009, dch bnh gy cht hnh lot c r phi nui thng phm ti mt s
tnh Min Bc Vit Nam. y c coi l t dch bnh ln nht k t trc n
nay i vi ngh nui c r phi nc ta. Bc u, nguyn nhn gy cht c
xc nh chnh l bnh Streptococcosis do vi khun Gram (+), Streptococus spp.
gy ra.
Vit Nam rt nhiu loi c b nhim vi khun Streptococus spp. t nc ngt
nh c r phi, c trm c, c tra, basa, n cc loi c nc l nh c bp v cc
loi c nc mn nh c song, c chm, c gi v c hng m.
C b bnh thng c cc triu chng nh c bi l , mt nh hng, trng
bng, xut huyt, li mt, sng rut, cc c quan ni tng nh gan, thn, l lch bc
mu hoc xut huyt, sng to. c bit vi khun tn cng nim mc mt v no c
lm cho c bi khng nh hng v c du hiu tn thng thn kinh. Bnh
thng xy ra vo ma h c bit khi khi nhit nc cao. i vi ma ng v
ma xun, mt vi khun thng thp v khng ngng gy bnh. Tc nhn
gy bnh quan trng trn c r phi thng l vi khun, virus hoc protozoa... trong
ng ch nht l bnh do vi khun m c bit lStreptococcus agalactiae
gy cht hng lot c nui trong thi gia qua (ng Thanh H v ctv, 2010). Kt
qu nghin cu ca inh Th Thy, 2007 cc bnh nguy him thng gp c r
phi Oreochromis spp nui thm canh cho thy bnh Streptococosis thng xut
hin vo ma h c bit khi nhit nc cao, t l thit hi t 7 10% v c
giai on 1 4 thng tui. Ti An Giang v Vnh Long vi khun Streptococus spp.
c tn sut hin t 95 100% vo thng 1, thng 5, thng 9 v thng 11 (inh Th


12
Thy, 2007). T thng 4 n thng 9 nm 2009, hin tng c r phi b cht
xut hin tt c cc vng nui tp trung min Bc nh H Ni, Hi Dng, Hi
Phng, Qung Ninh, Bc Ninh, Bc Giang v H Giang. T l cht cao nht l
100% v trung bnh l 42,56%. y c coi l t dch bnh ln nht trn c r
phi nui ti min Bc Vit Nam. Kt qu nui cy phn lp tc nhn gy bnh trn
c r phi cho thy vi khun gram dng Streptococus spp. c xut hin trn mu
bnh (Cng ty Hanvet, 2009); Vin Nghin cu Nui trng Thy sn 1 thy trn
hu ht cc mu bnh (Khu, N.V v ctv., 2009).
Kt qu nghin cu tc nhn gy bnh Streptococcosis trn c r phi cho thy vi
khun Streptococus iniae v Streptococus agalactiae c th tn ti ngoi mi
trng quanh nm. Ngoi ra vi khun gy bnh c th phn lp c t ngun t,
cht hu c lng t, cht nhy ca c. Theo Bromage v ctv, 1999 vi khun gy
bnh Streptococus iniae c th do c b bnh qua khi t dch thi ra ngoi mi
trng. Do vi khun gy bnh Streptococcosis thch hp vi iu kin nhit cao
nn vo ma ng rt t khi phn lp c cc loi vi khun ny.
V theo ng Thanh H v ctv, (2010) dch bnh xy ra ln u vo ma h
nm 2009 cc tnh nh: H Ni, Hi Phng, Hi Dng, Qung Ninh, Bc Ninh,
H Giang; gy cht vi t l 90 100% c nui (c c ging v thng phm). Tc
gi ch ra rng tc nhn gy bnh trn c r phi min Bc Vit Nam l
Streptococus agalactiae v v kh nng pht trin ca vi khun 37
0
C v mn
37
o
/oo c xem l yu t nguy c ly nhim bnh cho ng vt c v v con ngi.
Cng theo ng Thanh H v ctv, (2010) vi khun Streptococus agalactiae c kh
nng sng st tt trong nc ao v bn y t 3 5 ngy hai mc nhit 25
0
C
v 30
0
C. T nhng mu c iu hng b bnh ph mt v xut huyt c thu t
nhng b nui c iu hng thm canh Tin Giang tin hnh kim tra vi khun
hc xc nh do vi khun Streptococus agalactiae gy ra (ng Th Hong Oanh v
ctv, 2012).
Triu chng v bnh tch ca c r phi khi nhim Streptoccocus spp.:
Vit Nam, dch bnh hng xy ra vi t l c cht rt cao vo cc thng cui ma
h v u ma thu. y l khong thi gian nhit nc cao nht trong nm. Ti


13
cc thi im khc trong nm c cht ri rc, ngoi tr nhng thng ma ng lc
nhit nc xung thp nht cc nc n i khng thy xut hin bnh. V
tui c thng c bnh, hu ht bo co cp bnh xy ra trong giai on nui
thng phm.
- Triu chng:
Hnh vi bt thng: C b bnh c triu chng chung kh in hnh trn nhiu
loi. Do vi khun gy bnh c hng tn cng vo h thng thn kinh trung ng
ca c nn c b bnh s c biu hin b hn m v mt phng hng, c bi l
hay mt nh hng gn mt nc. Nhng tn thng mt c th gp nh vim mt
hoc li mt, chy mu mt. Tuy nhin, khng phi con c no b bnh cng b
nhng tn thng v mt.
Cc p-xe: Nhng con c b nhim bnh do vi khun Streptococcus spp.
thng thy nhng vt p-xe c ng knh t 2-3mm v nhng vt lot ny nhanh
chng v ra to thnh nhng vt l lot xut huyt khng lnh. Nhng vt p-xe ln
hn c th gp thy vy ngc v phn ui ca c v nhng vt p-xe c cha
vt cht nh m bn trong.
Xut huyt da: Bnh do vi khun Streptococcus spp. l nguyn nhn gy xut
huyt bn ngoi da. Nhn chung cc im xut huyt thng c nhn thy
quanh ming c hoc cc gc vy. i khi cng c th quan st thy nhng vng
da hi xung quanh hu mn hoc l sinh dc ca c.
Dch c trng: S c mt ca dch cht lng trong bng ca c l du hiu ca
dch bnh thi k cp tnh. Bn ngoi c c biu hin b trng bng. Dch ny c
th c nhn thy chy ra t hu mn ca c.
- Bnh tch:
Cc du hiu bn trong bnh ny c nhiu im tng ng vi bnh nhim
trng mu ca c:
C b n: Nhn chung khng c s hin din ca thc n kh trong d dy hoc
rut ca nhng con c b bnh. Tuy nhin trong cc ao nui c thng phm khi c
b bnh giai on u bnh mi bng pht c vn c th n bng cch lc thc n.
Khi rut v d dy ca c trng rng thc n th s quan st thy ti mt rt to, l


14
c trng ca s vng mt hot ng tiu ha trong c th.
Nhim trng mu: Trong giai on cp tnh ca bnh vi khun nhanh chng i
n h thng mu v lan to n tt c cc c quan ni tng. Nhng du hiu lm
sng chnh lin quan n s nhim trng mu l s xut huyt, vim gan, thn, l
lch, tim, mt v ng rut. L lch thng m rng ra (trng v sng nh).
Vim mng bng: Khi c b nhim bnh nng c s dnh nhau ca cc c quan
ni tng vi mng trong khoang bng ca c. Hn na lc ny s hin din ca cc
t huyt (fibrinous) c th c quan st thy trong mng khoang bng ca c.
Ngoi ra khi c b nhim bnh nng, bnh cn kt hp vi nhng vi khun c
hi khc gy bnh cho c c sn trong mi trng nh vi khun Aeromonas spp.
nc ngt hay vi khun Vibrio spp. nc l.
- S phn b v lan truyn ca bnh
Dch bnh thng xy ra khi c nui tip xc vi s cng thng (stress) khi
nhit nc tng, lng oxy trong nc thp di mc cho php hoc c b
nui mt cao trong thi gian di.
V mt l thuyt th bnh ly nhim cho c mi la tui, kch c. Tuy
nhin c c kch thc ln (t 100g n c thng phm) d b mc bnh hn c.
Bnh giai on cp tnh vi nh im t vong trong khong t 2 3 tun khi
nhit nc cao. Tuy nhin bnh cng c th giai on mn tnh khi nhit
nc thp c th lm gim t l cht.
Bnh ly lan theo chiu ngang t c vi c (c kho n c b bnh, n tht ln
nhau, do vt thng trn da...) v cng c th ly truyn t mi trng n c.
- C ch ly nhim ca vi khun Streptoccocus spp.
Streptococcus spp. cho ra ngoi c t, ph hng cc kh quan trong c th c,
dn n lm cho c b ri lon chc nng nh khi vi khun tn cng vo h thng
thn kinh trung ng ca c lm c c biu hin b hn m v mt phng hng.
Do vy khi cha bnh khng ch dit mm bnh m ng thi phi dng thuc
gii cc c t hoc nng cao kh nng t gii c trong c th vt nui.
Streptococcus spp. trc tip ph hng h thng mu, gy nn hin tng c b
xut huyt ton thn, hin tng ny i vi vt nui rt quan trng, nh hng n
ton thn vt nui, kh quan v h thng mu b tn hi, lc bnh rt kh cha.


15
Hin nay cha c bt k loi vacxin phng bnh Streptococcosis gy bnh trn
c r phi c nghin cu v ng dng vo sn xut ti Vit Nam. V vy, vic
phn lp v xc nh c tnh sinh hc ca Streptococcus spp. l cn thit gip
cho vic nghin cu v sn xut vaccine phng bnh Streptococcosis trn c r phi
nui ti Vit Nam l rt cn thit.
2.4. Tnh hnh s dng thuc trong nui trng thy sn
Vn s dng thuc khng sinh ni ring v ha cht ni chung trong
nui trng thy sn cho n nay tng i ph bin. Song mt nghch l xy ra
l cha c thuc khng sinh dng ring cho ng vt thy sn m a phn
chng u dng ca ngi v gia sc (Inglis, V. 2000). Trc y ch c mt
s ha cht v thuc khng sinh c s dng nh vi bt, formalin, sulfate
ng, thuc tm, dipterex, rotanon v mt s thuc nh chloramphenicol,
furazolidon, tetracyclinc s dng trong nui trng thy sn. Ngy nay c
rt nhiu chng loi thuc, ha cht v ch phm sinh hc c s dng. c
hin tng nhn thuc trong cc tri sn xut tm ging nc ta (Phillips
Michael, 2000). Tuy nhin, thc t sn xut, kinh doanh v s dng cng nh
qun l cc sn phm nh th no l iu cn phi xem xt.
Ch xt ring Khnh Ha vi 65 tri sn xut ging thy sn s dng 44
loi khng sinh, mi tri trung bnh dng 5,8 loi. Trong s 44 loi thuc th c 5
loi l khng sinh cha bnh cho ngi (streptomycin, chloramphenicol, rifampicin,
furazonidon, erythromycin). Cc loi thuc, ha cht, ch phm sinh hc dng trong
nui trng thy sn ti khu vc nui Hi Phng v Qung Ninh c 10 loi thuc bao
gm: oxytetracycline, chloramphenicol, rifampicin, ampicilin, clocyte, ND
gentosine, panamin, penicillin, tetracyclin thng c dng phng v tr bnh
cho c trong sut qu trnh nui, ngoi ra cn c mt s loi thuc khng r nhn
mc v hng dn s dng ghi trn bao b do nhp lu t Trung Quc, hoc nhn
mc c vit bng ting Trung Quc (Mai Vn Ti, 2004).
Khi vn s dng thuc khng sinh tng ln theo cp s cng ng thi d
lng khng sinh trong cc sn phm thy sn xut khu tr nn bc xc. Hn th
na nng cao cht lng sn phm p ng nhu cu tiu dng ca ngi dn


16
cng nh yu cu ca cc nc nhp khu. Chnh ph cng nh B Nng nghip v
Pht trin nng thn c nhiu bin php kp thi ngn chn vic dng cc cht
cm. Ngy 17/03/2009 B Nng nghip v Pht trin nng thn ban hnh thng t
s 15/2009/TT-BNN v danh mc thuc, ha cht, khng sinh cm s dng, hn
ch s dng trong sn xut kinh doanh thy sn; v Thng t s 03/2012/TT-
BNNPTNT ngy 16 thng 01 nm 2012 v vic sa i, b sung Thng t s
15/2009/TT-BNN. (Ph lc 1)
Song song vi vn d lng thuc trong sn phm thy sn th vn khng
thuc ca vi khun cng c cp v quan tm. Mt trong s nhng nguyn
nhn gy ra hin tng vi khun khng li thuc khng sinh l do ngi dn s
dng thuc c tnh cht lm dng thuc, dng khng ng liu lng, dng mt
cch trn lan.

















17
3. NI DUNG, NGUYN LIU V
PHNG PHP NGHIN CU

3.1. Thi gian v a im nghin cu
3.1.1. Thi gian nghin cu
ti c thc hin t thng 04/2012 n thng 08/ 2013.
3.1.2. a im nghin cu
Mu bnh c thu ti 4 tnh min Bc : H Ni, Hi Dng, Hi
Phng, Qung Ninh.
Th nghim c thc hin ti : Cc Th y v Cng ty Hanvet - 88,
Trng Chinh, ng a, H Ni.
3.2. Ni dung nghin cu
Trong khun kh ca ti, chng ti thc hin cc ni dung nghin cu sau:
- Phn lp vi khun Streptoccocus spp. t c r phi b bnh xut huyt
- Xc nh mt s c tnh sinh hc ca vi khun Streptoccocus spp. phn
lp c.
- Xc nh c lc ca vi khun Streptoccocus spp. phn lp c.
- Xc nh tnh khng nguyn ca vi khun Streptoccocus spp. phn lp c.
- Kim tra kh nng mn cm vi khng sinh ca vi khun Streptoccocus
spp. phn lp c.
3.3. Vt liu nghin cu
3.3.1. Dng c, thit b phc v nghin cu
- Dng c:
Dng c thu mu: bng thm cn, ti PE tit trng, b gii phu (panh,
ko, dao)
Dng c dng phn tch v nghin cu vi khun : ng nghim, a petri,
u cn, ng eppendorf, ng fancol, que cy, n cn, dng c dng nhum
gram


18
Dng c dung ch cht gy p ng min dch v nh hiu gi khng
th: kim tim, lam knh
- Thit b:
+Ni hp kh trng +My vortex
+T m +My lc n nhit
+T lnh + My o OD
+B n nhit +My ly tm
+ My o pH +Knh hin vi quang hc
+T cy v trng + Cn in t
+My sc kh +Pipet t ng
+My khuy t
3.3.2. Mi trng, ha cht phc v nghin cu
- Mi trng:
Mi trng dng pha long vi khun: nc mui sinh l 0.85%.
Mi trng tng sinh vi khun: Brain heart infusion broth (BHIB, Merck)
Mi trng thch nui cy vi khun: Brain heart infusion agar (BHIA,
Merck)
BHI c b sung 5% mu hoc trn mi trng thch mu (Blood agar)
- Ha cht:
H
2
O
2,
NaOH, HCl, xylen, formalin, cn.
Cc ha cht dng trong lp khng sinh .
Kit API20 Strep (BioMerieux, Php).
Kit Strep-B-Latex (GBS) (an mch)
3.3.3. Vt liu nghin cu
Mu vi khun Streptoccocus spp. s dng trong ti c phn lp t mu c
r phi vi cc du hiu bnh l nh bi l , mt nh hng, trng bng, xut
huyt, li mt, sng rut, cc c quan ni tng nh gan, thn, l lch bc mu hoc
xut huyt, sng to, bi khng nh hng v c du hiu tn thng thn kinh thu
ti 4 tnh min Bc : H Ni, Hi Dng, Hi Phng, Qung Ninh.
C dng th nghim: c r phi c khi lng khong 60g, khe mnh, mu sc


19
c ti sng, y cc b phn v bi li bnh thng, c nui thun trong b
nui phng th nghim (khong 3 tun trc khi cm nhim) to s n nh hn
v iu kin sng, cc yu t mi trng, ngoi cnh tc ng n qu trnh sng
ca c so vi c sng bn ngoi t nhin.
3.4. Phng php nghin cu
Cc thao tc phi c thc hin trong iu kin v trng.
Lm thun vi khun: dng que cy ly vi khun cy ln mi trng BHIA. em
28
0
C - 30
0
C trong 18-24 gi kim tra kt qu.
Nui tng sinh vi khun: chn mt khun lc t a cy thun cho vo bnh tam
gic cha 25ml BHI t vo t nui lc n nhit 200 vng/pht sau 18 24 gi.
Kim tra kt qu.
Mi ch tiu kim tra sinh ha c lp li 3 ln.
3.4.1. Phng php thu mu c bnh
- Dng c: ti nilon v gng tay.
- Nguyn tc thu mu c bnh:
Phi kim tra ngay khi c c vt ln khi mt nc, cc biu hin bnh
tch bn ngoi. Ghi cc triu chng c bnh trc khi thu mu.
C quan s dng nui cy phn lp vi khun bao gm gan, thn, lch, no
v mt ca c r phi c du hiu bnh in hnh, c cn ang sng hoc mi cht.
Triu chng c trng ca c: du hiu bn ngoi (c c biu hin bt
thng, li mt hoc n mt, c cc p-xe, xut huyt da, dch c trng), du
hiu bn trong (vim mng bng, ti mt rt to, nhim trng mu).
Dng gng tay cho mu c bnh vo ti nilon.
Thu mu c bnh vn chuyn ngay v phng th nghim trong thi gian
ngn nht.
3.4.2. Phng php m c ly ni tng
* C c m bng ba ng ct:
ng th nht: bt u t trc hu mn, theo ng gia thnh bng cho n
phn u, dng trc np mang.
ng th hai: bt u t trc hu mn, chy ln pha trn, dc theo thnh


20
ngc n phn mang c.
ng th ba: ni hai ng th nht v hai li.

Hnh 3.1. Cch m xoang bng c
* M s no c: St trng mt ngoi ca vng da phn s no c bng cn
70. Sau ct bn ng ct, mi ng ct khong 0,5 1 cm sao cho l phn
no ra.

Hnh 3.2. Cch m no c
3.4.3. Phng php phn lp vi khun Streptococcus spp. c trong mu bnh
phm
Nui cy v phn lp vi khun Streptococcus spp. gy bnh trn c r phi bng
phng php nghin cu vi khun ca Frerich G.N. (1993), th cc c tnh sinh
hc bng test API20Strep.
Phn lp vi khun trn mi trng nui cy BHI c b sung 5% mu hoc trn
mi trng thch mu (Blood agar). Cc chng vi khun phn lp c tr -80C
trong mi trng Brain heart infusion broth (BHIB, Merck) c 25% glycerol.



21











Hnh 3.3. S nui cy, phn lp vi khun Streptococcus spp.
4.4.4. Phng php m mt vi khun
Phng php xc nh gin tip s lng t bo bng cch m s lng khun
lc mc trn mi trng thch.
Pha long dch huyn ph t bo theo c s 10 thnh cc nng : 10
-1
, 10
-
2
, 10
-3
,
10
-4
, 10
-5
, 10
-6
tin hnh cy mu cc pha long khc nhau vo cc a petri
(mi nng cy lp ba ln).
t cc a thch va cy vo t m nhit 30
0
C trong 24 gi.
Kt thc thi gian , ly c a thch ra, tin hnh m khun lc v tnh s
lng t bo trong 1 ml mu theo cng thc:
M
i
(CFU/ml) =A
i
x D
i
/V
Trong :A
i
: l s khun lc trung bnh/ a
D
i
: l pha long
V: l dung tch huyn ph t bo cho vo mi a (ml)
Mt t bo trung bnh M
I
trong mu ban u l trung bnh cng ca M
i
cc
nng pha long khc nhau.
3.4.5. Phng php nh danh vi khun Streptoccocus spp.
Hnh dng, kch thc ca vi khun c xc nh bng phng php nhum
Mu c bnh
Nui cy, phn lp
Phn loi vi khun
Thu mu bnh phm
Nhum Gram
Th phn ng
sinh ha


22
Gram. Tnh di ng ca vi khun c quan st bng cch nh mt git nc ct
ln lam, tri u ln lam mt t vi khun, y bng lamen v quan st bng knh
hin vi vt knh 40X. Cc c tnh sinh ha c xc nh da theo cm nang ca
Cowan v Steels (Barrow v Feltham, 1993) v s dng kt API 20 Strep
(BioMerieux, Php).
3.4.5.1. Xc nh cc ch tiu c bn
(Theo cm nang Cowan v Steel (Barrow v Feltham, 1993))
Quan st tnh di ng
Tin hnh th nghim trn mi trng thch bn lng, nhm xc nh kh nng
di ng ca vi khun trn mi trng thch bn lng (Brain heart infusion agar 0,6
%).
+Kt qu:
Vi khun di ng: s pht trin lan ra khi vt cy, lm nhe ng cy.
Vi khun khng di ng: s pht trin quanh ng cy.
Nhum Gram
T a thch cy vi khun c lm thun, chn nhng khun lc ri c
thi gian 24 - 72 gi tin hnh nhum gram.
+Quan st trn knh hin vi
Vi khun bt mu tm l vi khun G
+
.
Vi khun bt mu hng l vi khun G
-
.
Phn ng oxidase
Th nghim ny nhm xc nh s hin din ca enzyme oxidase.
+ c kt qu:
Nu sau 10 giy vt bi vi khun chuyn sang mu tm en: Phn ng oxidase
cho kt qu dng tnh.
Nu sau 60 giy mi chuyn mu: Phn ng oxidase cho kt qu m tnh.
Phn ng catalase
Nhm xc nh s c mt ca enzyme catalase ca mt s vi khun h hp
k kh ty .
+ c kt qu:


23
Kt qu c c ngay sau 1 2 giy.
Phn ng dng tnh: Xut hin bt kh.
Phn ng m tnh: Khng c bt kh.
Kh nng ln men v oxy ha ng glucose (Fermentation/oxidation:
O/F)
Dng kim tra kh nng s dng glucose ca vi khun trong iu kin c oxy
v khng c oxy.
- Kt qu:
Cch c kt qu phn ng O F test
ng khng ph du
Paraffin
ng ph du
paraffin
Kt qu
Xanh l cy Xanh l cy Khng phn ng vi glucose
Xanh l phn trn Xanh l cy Phn ng kim tnh
Vng Xanh l cy Phn ng oxy ha
Vng Vng Phn ng ln men

- nh danh bng b kit API 20 Strep (Biomrieux)
Kim tra cc phn ng sinh l v sinh ha ca Streptoccocus sp s dng b kt
API 20 Strep ca hng Biomrieux.
Cc ch tiu c bn (hnh dng, kh nng di ng, oxidase, catalase v phn ng
O/F) c thc hin trc khi s dng b kt API 20 Strep.
B kit gm 20 ging vi 9 ging nh gm cc phn ng: VP, HIP, ESC, PYRA,
GUR, GAL, PAL, LAP; v 11 ging ln gm cc phn ng: ADH, RIB, ARA,
MAN, SOR, LAC, TRE, INU, RAF, AMD, GLYG.
- Nguyn l:
Phng php ny cho php nh tn mt s loi lin cu v vi khun ng rut.
Kt API 20Strep gm cc ng nghim nh (microtube) trong c cha cc cht nn
kh nc. Trong qu trnh , hot ng ca vi khun s lm chuyn mu hoc
lm c mi trng. c kt qu sau 4gi v 24gi bng cch quan st cc phn
ng chuyn mu khi tip xc vi nhng thuc th, i chiu vi bng kt qu chun


24
nh danh vi khun bng phn mm: https://apiweb.biomerieux.com
- Cc bc tin hnh:
Chun b khay : Cho 5 ml nc mui sinh l vo trong khay gi m trong
sut qu trnh .
To huyn ph: Ly mt khun lc c phn lp vo 0,3 ml nc mui sinh
l to huyn ph.
Ht 100 l huyn ph cho vo mt na s ging t VP LAP. Ring ging
ADH cho y ming ging.
Cho 0,5 ml huyn ph vo ng API GP medium, sau cho vo mi ging t
ging RIB ti ging GLYG.
Cho paraffin v trng vo cc ging t ging ADH n ging GLYG.
y np khay li, em b test nh danh t m 30
o
C.
c kt qu v thm ha cht:
Sau 4 gi em khay ra khi t v nh ha cht vo cc ging:
Ging VP: nh vo mt git VP1, VP2.
Ging HIP: nh 2 git NIN.
Ging PYRA, GAL, GUR, GAL, PAL v LAP: nh mt git ZYMA v
mt git ZYMB.
10 pht sau c kt qu ln 1. em b kit thm 24 gi na sau c kt qu
ln 2.


25
Bng 3.1. Thuc th v cch c kt qu cc phn ng sinh ha
trong API 20 Strep
Tests
Kt qu
m tnh (-) Dng tnh (+)
VP
VP1 + VP2 / i 10 pht
Khng mu hng
HIP
NIN / i 10 pht
Khng mu / xanh nht / xm nht Xanh sm / tm
ESC
4 gi 24 gi 4 gi 24 gi
Khng mu / vng
nht
Khng mu / vng nht /
xm nht
en /
xm
en
PYRA
ZYM A + ZYM B / i 10 pht (PYRA n LAP)
Khng mu / cam rt nht Cam
GAL Khng mu Tm
GUR Khng mu Xanh
GAL Khng mu / tm rt nht Tm
PAL Khng mu / tm rt nht Tm
LAP Khng mu Cam
ADH Vng
RIB
4 gi 24 gi 4 gi 24 gi
Cam /
Cam /
vng
Vng
ARA Cam /
Cam /
vng
Vng
MAN Cam /
Cam /
vng
Vng
SOR Cam /
Cam /
vng
Vng
LAC Cam /
Cam /
vng
Vng
TRE Cam /
Cam /
vng
Vng
INU Cam /
Cam /
vng
Vng
RAF Cam /
Cam /
vng
Vng
AMD Cam /
Cam /
vng
Vng
GLYG Cam / Vng



26
Phn ng Hemolysis
Nhm xem kh nng tiu mu ca vi khun trn mi trng thch mu.
+ Cch tin hnh:
S dng que cy v trng ly 1 khun lc v cy ria ln mi trng thch mu,
thc hin cc thao tc trong t cy vi sinh.
+ Kt qu:
hemolysis xut hin vng dung huyt mu xanh.
hemolysis xut hin vng dung huyt trong sut xung quanh khun lc.
hemolysis khng c vng dung huyt.
- Xc nh kiu huyt thanh
Kiu huyt thanh c xc nh bng phng php ngng kt min dch s
dng kit Strep-B-Latex (GBS) (an mch). Hai git dung dch latex (khong 10
l/git) c nh ln hai lam. Dng que cy tit trng ly khong t 3-5 khun lc
cho vo 3ml nc mui sinh l, lc u ri nh mt git dung dch vi khun ln mt
lam. Mt git nc mui sinh l c nh ln lam cn li lm i chng m.
Dng tm tit trng trn u 2 dung dch. Phn ng dng tnh s c ngng kt
xut hin trong 5 10 giy gip xc nh Streptococus spp. c kiu huyt thanh Ib
(serotype Ib) hay kiu sinh hc 2 (biotype 2).
3.4.6. Phng php xc nh c lc ca cc chng vi khun Streptococcus spp.
Th c lc ca vi khun bng phng php gy nhim vi khun Streptococcus
spp.. Th nghim c b tr trong phng th nghim t c iu khin c cht
lng nc v nhit , vi khun c tim vo xoang bng ca c . C s dng
lm th nghim c khi lng khong 60 gram/con, gm 4 l (3 l th nghim v 1
l i chng), mi l 20 con. Gy nhim vi khun vi cc nng t 10
6
10
10

cfu/c th. L i chng s dng nc mui sinh l 0.85% (bng 3.2). Tin hnh
theo di, quan st, thu thp s liu v tin hnh phn lp li vi khun t cc c th
c c du hiu bnh in hnh (phn lp t t chc gan, thn, no, mt). Thi gian
theo di mt ln th nghim ko di 4 tun.



27
Bng 3.2. B tr th nghim xc nh c lc vi khun gy bnh xut huyt
c r phi
Nng vi khun gy bnh Chng vi khun xc nh c lc
L i chng 20 c/b x 3 b
10
6
cfu/ml 20 c/b x 3 b
10
7
cfu/ml 20 c/b x 3 b
10
8
cfu/ml 20 c/b x 3 b
10
9
cfu/ml 20 c/b x 3 b
10
10
cfu/ml 20 c/b x 3 b
Tng 360 con x 52 chng =18720 con c

Cc bc tin hnh th nghim c lc ca vi khun:
- Nui cy tng sinh cc dng vi khun chn c t cc vng khc nhau
trn mi trng (BHI) lng 30
0
C trong vng 24 gi theo phng php ca
Hernandez v ctv., 2009.
- nh lng vi khun bng phng php m khun lc.
- Gy bnh nhn to bng phng php tim xoang bng sau khi c c
gy m bng MS 222 vi nng 50-75ppm.
- B tr cc l th nghim trong phng th nghim t. Mi nng th
nghim lp li 3 ln.
- Theo di th nghim, ghi chp s lng c cht v kt thc th nghim sau
khi c ngng cht 3 ngy lin tc. Thi gian th nghim ko di trong 4 tun.
- Tin hnh phn lp li vi khun t mu c bnh in hnh sau khi gy
bnh nhn to trn mi trng BHIA, kim tra hnh thi vi khun, th phn ng
sinh ha v test API20 Strep khng nh c b bnh do vi khun cm nhim gy
ra (Hnh 3.4)


28
-















Hnh 3.4. S b tr th nghim gy bnh cho c r phi
- Tng cng c lc ca cc chng vi khun phn lp
Cc chng vi khun sau khi c xc nh c c lc, m bo n nh v
c tnh cng nh tng cng c lc ca vi khun. Chng ti tin hnh tip i
lin tc qua c r phi (vt ch chnh ca vi khun) sau ti phn lp li chng vi
khun v gi ging phc v nghin cu sn xut kt chn on v vacxin.
Cc bc tin hnh:
- Nui cy tng sinh cc chng vi khun trn mi trng BHIB.
- Gy nhim thc nghim vi liu gy cht
- Phn lp li vi khun trn c b bnh.
Nh vy, sau bc ny chng ta tuyn chn c cc chng Streptococcus
spp. c c lc cao, n nh v c tnh v c m trnh t gen tng ng lm b
ging chun.
Cc chng vi khun thun phn lp c
Gy nhimtrn c khe
Nui cy tng sinh trn mi trng BHI
La chn c th c du hiu bnh in hnh
phn lp li vi khun
Phn loi vi khun bng phn ng sinh ha
v test API 20Strep
Xc nh hnh thi vi khun
Khng nh c b bnh do vi khun
cmnhimgy ra


29
3.4.7. Phng php xc nh tnh khng nguyn
Nghin cu ny nhm chn ra nhng chng vi khun c khng nguyn p ng
min dch tt phc v nghin cu sn xut vacxin.
Ch to khng nguyn ring bit cho tng chng vi khun tim min dch
cho c r phi vi liu 0,1ml/con vo xoang bng, mi chng cho 10 con (mi chng
lp 3 ln). Sau 21 ngy tin hnh ly mu v xc nh hiu gi khng th ca huyt
thanh.
- Ch to khng nguyn cho tng chng vi khun:
Sau 24gi vi khun tng sinh mnh, sau bt hot vi khun bng Formalin vi
liu lng c tnh bng cng thc:
V
Formalin
=0,5% x V
dd

Trong : V
Formalin
: Th tch Formalin
V
dd
: Th tch dung dch ca mi trng canh BHI
Bo qun trong iu kin 4
0
C khong 24 48 gi
Sau khi bt hot vi khun bng Formalin, ta tin hnh thu nhn vi khun bng
cch li tm mi trng nui tng sinh vi khun ly phn c c nm di y
ng ly tm, c mu trng.
Thc hin ly tm 5000 vng/pht trong vng 15 pht.
Ra phn c c bng nc mui sinh l 3 ln. Sau mi ln ra ly tm li ra
sch ht formalin.
Sau khi ra ta em pha vi nc mui sinh l t nng 10
9
cfu/ml. Bo qun
nhit 4 10
0
C.
- To khng th bng cch tim vo c th c r phi:
La chn c r phi khe mnh, khng b nhim bnh, khng b d hnh. C c
khi lng 60 70g.


30

Hnh 3.5. C r phi th nghim
Tim hn hp dung dch huyn ph vi khun nc mui sinh l vo xoang
bng ca c vi liu lng 0,1ml/ln.
Quan st tnh trng sc khe ca c trong sut qu trnh to khng th.

- Kim tra khng th:
Kim tra xem trong huyt thanh c c khng th khng lin cu khun hay
khng. S dng vi khun Streptococcus spp. c phn lp t c bnh qua cc ln
thu mu v c gy bnh thc nghim.
Phng php kim tra: ly mu c cho vo ng nghim t nghing v yn t
1 2gi v gi lnh khong 1gi, sau em ly tm vi tc 2000 vng/pht
trong vng 15 pht.
Sau ht ly huyt thanh l phn trong nm trn c mu vng nht ta thc
hin phn ng ngng kt vi vi khun sng v vi khun c bt hot bng
formalin kim tra xem c khng th khng lin cu khun hay khng.
- Thc hin phn ng ngng kt trn phin knh:
Nh vi git huyt thanh vi khun sng, huyt thanh vi khun bt hot trn
phin knh quan st trong vng 2 pht. i vi vi khun sng, dng que cy vng
ly mt t vi khun thun ang nui trn a thch ha vo vi git nc mui sinh
l c sn trn lame v cng ha vi vi git huyt thanh quan st trong vng 2
pht. Sau thu nhn kt qu.


31

kim tra xem hot lc ca khng th c mnh hay khng ta pha long huyt
thanh ra lm 2, 4, 8 ln sau kim tra nh trn.
Nu trn knh c nhiu ngng kt mu trng c li ti th ta thu c kt qu c
khng th khng vi khun Streptococcus spp.
- Thc hin phn ng trn microplate 96 ging

a microplate 96 ging (y hnh ch U)
Phng php thc hin trn a microplate 96 ging theo cc bc nh sau:
Thm 25l nc mui sinh l vo tt c cc ging tr ct th nht.
Cho 25l huyt thanh vo cc ging ct 1 v ct 2.
Dng pipette trn hn hp trong ging th 2. Nh vy, ta s thu c
hn hp huyt thanh pha long 2 ln.
Chuyn 25l ca ging th 2 sang ging th 3 ri trn hn hp cho u.
Tip t nh th cho n ging th 12. Cui cng, ta c mt dy pha long bc 2.
Thm 25l huyn ph vi khun vo mi ging ri bc a li bng mt
ming phim. Lc nh a trn mt phng bn hn hp ha trn li vi nhau.
Bo qun nhit phng trong vng 24gi


32
Ghi nhn kt qu nhng ging cui cng c xut hin ngng kt, ta kt
lun c ln pha long no th cho phn ng ngng kt.

3.4.8. Phng php kim tra kh nng mn cm vi khng sinh ca vi khun
Kh nng mn cm khng sinh ca cc chng vi khun Streptococcus spp. c
kim tra bng phng php khuch tn trn a thch (Theo phng php ca Kirby
Bauer) v nh gi kt qu theo Hi ng quc gia Hoa k v cc tiu chun lm
sng phng th nghim (National Committee of Clinical Laboratory Standards -
NCCLS, 1999).
Vi khun sau khi c gim nh th tin hnh lm kim tra kh nng mn cm
vi khng sinh.
Dng que cy tit trng ly khun lc trn a vi khun cho vo ng nghim
cha 10ml nc mui sinh l (0,85%) tit trng. Trn u v tin hnh xc nh
mt vi khun bng my so mu quang ph bc sng 610 nm v iu chnh
xc nh mt s vi khun t 10
8
cfu/ml (OD =0,1 0,02).
Sau khi xc nh mt s vi khun th tin hnh lng dung dch vi khun ln mi
trng thch.
Dng tm bng tit trng nhng vo dung dch vi khun, lng u trn mi
trng thch BHIA. Sau yn khong mt pht ri dng pank tit trng ly a
giy tm thuc khng sinh t vo a petri sao cho khong cch gia hai tm ca
a thuc khng sinh khong 24mm v khong cch gia tm a khng sinh vi
mp a petri 10-15mm. Mi a petri (ng knh 100mm) mi trng t ti a 6


33
a khng sinh.
Sau khi hon tt vic dn a thuc khng sinh, t a petri vo t m iu
kin 30
0
C. Sau 24 gi tin hnh c kt qu.
Ghi ch:
- Phi lc u vi khun v c lp li 3 ln.
- Khng s dng cc khng sinh c trong danh mc ha cht, khng sinh cm
s dng trong sn xut, kinh doanh Thy sn (Ph lc)
c kt qu: o ng knh vng v trng (mm) da vo chun ng knh
vng v trng ca nh sn xut xc nh loi khng sinh nhy, trung bnh nhy
v khng. Kt qu ng knh vng v trng ca 2 trong 3 ln lp li sai khc
khng ng k th ghi nhn kt qu ca 2 ln lp li hoc kt qu trung bnh ca
3 ln lp .
Bng 3.3. nh gi ng knh vng v khun chun
Loi KS
Lng KS
(g)
R ()
(mm)
I
(mm)
H ()
(mm)
Amoxicillin (Ax) 10 13 14 - 16 17
Ampicillin (Am) 10 13 14 - 16 17
Enrofroxacin (En) 30 16 17 19 20
Erythromycine (Er) 15 13 14 22 23
Kanamycine (Kn) 30 13 14 17 18
Streptomycine (Sm) 10 11 12 14 15
Rifampin (Rf) 5 16 17 - 19 20
Doxycilline (Dx) 30 10 11 13 14
Tetracycline (Te) 30 11 12 14 15
Sulfamethoxazol/Trimethoxazol(Bt) 1,25/23,75 10 10 15 16
(Ngun: Oxoid t NCCLS (1990) M
2
A
4
(Oxiod, 1982))
3.4.9. Phng php x l s liu
S liu c x l bng chng trnh Excel 2003; so snh s sai khc gia cc
yu t bng php th
2
vi phn mm Minitab 14.0 v php th Fisher Exact Test
(phn mm SAS 9.1).


34
4. KT QU NGHIN CU V THO LUN

4.1. Kt qu phn lp vi khun Streptoccocus spp
4.1.1. Kt qu thu mu
Tin hnh thu mu ti 4 tnh =60 mu c r phi c biu hin bnh xut huyt.
Mu bnh phm thu t c r phi c biu hin bnh nh: c bnh bi l , hot
ng chm chp, km linh hot, bi li mt phng hng, mt li v c, trn thn
c nhng m xut huyt vy ngc v vy bng, mang ti nht, bng trng to,
xoang bng c cha dch mu vng, ni tng b xut huyt, mm nhn (Hnh 4.1).




Hnh 4.1. Du hiu bnh l ca c lc thu mu. A: Mt c b li, c. B: Ni
tng c b xut huyt. C: C bi l , hot ng chm chp. D: Bng c
trng to v xut huyt

A B
C D


35
Bng 4.1. Kt qu thu mu c nghi b bnh xut huyt
Giai on
Kch thc
(cm)
Trng lng c
(gam)
Hnh thc
nui
S lng c
thu c (con)
C ging 5 10 30 50
Lng 10
Ao 15
C thng
phm
10 n xut
bn
50
Ao 18
Lng 17
Tng s
60

Song song vi thu mu c, chng ti cng tin hnh o cc yu t mi
trng ao, lng nui xut hin bnh: nhit dao ng t 18 27
0
C, pH dao ng
t 7,5 9; hm lng oxy ha tan dao ng t 5 10mg/l. Cc thng s mi trng
trn l thch hp cho s tn ti v pht trin ca c r phi.
Theo kt qu thu mu, chng ti nhn thy nhng mu bnh thng thu c
nhng ao nui th theo hnh thc nui thm canh cao, mt dy, nc ao b
nhim nng, cc yu t mi trng khng thch hp cho i sng ca c (DO thp,
hm lng NH
3
, amoniac cao). y c th l nhng yu t khin cho sc
khng ca c gim, nguy c mc bnh tng ln.
4.1.2. Kt qu phn lp vi khun
Trc khi tin hnh phn lp, gim nh vi khun gy bnh, chng ti kim
tra loi b nhng mu c thu c b bnh ngoi da do ngoi k sinh trng hay
nm. Kt qu cho thy 100% cc mu thu c u sch bnh vi cc tc nhn l
k sinh trng v nm.
Tin hnh gii phu, m khm thu mu kim tra vi khun trong cc c quan
ni tng gm: gan, thn, no, mt bng cch dng que cy v trng ly vi khun t
nhng c quan trn ria cy trn mi trng thch a BHIA, tm khun lc.
Kt qu phn lp vi khun ca 60 mu c cho kt qu nh trnh by bng 4.2:



36
Bng 4.2. Thnh phn loi vi khun phn lp c t mu c bnh

a im S
mu
Aeromonas
spp.
Pseudomonas
spp.
Staphylococcus
spp.
Flavobacterium
spp.
Streptococcus
spp.
Mu
(+)
T l
(%)
Mu
(+)
T l
(%)
Mu
(+)
T l
(%)
Mu
(+)
T l
(%)
Mu
(+)
T l
(%)
H Ni 15 8 53,33 0 0,00 0 0,00 2 13,33 13 87,27
Hi Dng 15 2 13,33 1 6,67 0 0,00 1 6,67 14 93,33
Hi Phng 15 3 20,00 1 6,67 1 6,67 0 0,00 13 86,67
Qung Ninh 15 3 20,00 1 6,67 1 6,67 2 13,33 12 80,00
Tng 60 16 26,67 3 5,00 2 3,33 5 8,33 52 86,67
Ghi ch: (+): s mu nhim
Khi tin hnh cy ria tm vi khun t cc c quan ch l: gan, thn, no, mt
ca c biu hin bnh l trn mi trng nui cy c bn chng ti pht hin thy
khun lc mc ln kh thun (ch yu l mt loi khun lc/a mi trng, mt s
t a thch c 2 3 loi khun lc).
Kt qu kim tra, trong tng s 60 mu c r phi b bnh chng ti thy xut
hin 5 loi vi khun l: Aeromonas spp., Pseudomonas spp., Staphylococcus spp.,
Flavobacterium spp. v Streptococcus spp.. Trong s mu tm thy
Streptococcus spp. l cao nht, c 52/60 chim t l 86,67%; tip n s mu xut
hin Aeromonas spp. c 16/60 chim t l 26,67%; s mu xut hin Pseudomonas
spp. l 5,00%; Staphylococcus spp. l 3,33%; sau cng l s mu dng tnh vi
Flavobacterium spp. l 8,33%; S sai khc ny c ngha thng k vi p<0,01.
Ti thi im thu mu th t l cht trung bnh pht hin c vi khun
Streptococcus spp. thp nht l Qung Ninh (80,00%), tip n l mu Hi
Phng v H Ni (86,67%), thp hn so vi cc mu thu c Hi Dng
(93,33%); tuy nhin, s sai khc ny khng c ngha thng k (p>0,05).
Kt qu nghin cu ca chng ti c s sai khc vi nghin cu ca mt s tc
gi nh: Nguyn Vit Khu v cs (2009) thng bo c 74/86 mu dng tnh vi vi
khun Streptococcus spp. chim t l 86,05%; Liu v ctv, (2012) cng ch ra t l
dng tnh vi vi khun ny l 90%; S sai khc ny c th do ngun mu thu
c t cc a phng khc nhau l khc nhau. Cc kt qu trn cho thy vi khun
Streptococcus spp. xut hin nhiu ti cc a phng v ngy cng gy thit hi


37
cho ngnh nui trng thy sn.
Nhm so snh t l phn lp c Streptococcus spp. t cc c quan khc nhau
ca c bnh, chng ti c kt qu trnh by bng 4.3.
Bng 4.3. Kt qu phn lp vi khun Streptococcus spp.
t cc c quan ca c r phi
STT
C quan
phn lp
S mu kim tra
S mu nhim
(+)
T l
(%)
1 Gan 52 50 96,15
2 Thn 52 52 100
3 No 52 52 100
4 Mt 52 49 94,23

4.2. Kt qu xc nh mt s c tnh sinh ha ca vi khun Streptoccocus spp.
phn lp c.
4.2.1. Kt qu xc nh mt s c tnh sinh hc
Chng ti tin hnh gim nh c tnh sinh hc ca vi khun phn lp c.
Kt qu cho thy: trn mi trng thch mu, sau 24 gi nui cy, trn a thch
mc ln khun lc mu trng sa, trn, ra u, tm hi m, khun lc to vng
dung huyt beta hoc gamma nh, trong sut, ra khng r (hnh 4.2). Lm tiu bn
nhum gram xem hnh thi vi khun, quan st di knh hin vi vt knh du ghi
nhn c: vi khun bt mu tm, gram dng, dng hnh cu, c th ng ring l,
thnh tng cp, v thng xp vi nhau thnh chui di (hnh 4.3).
Trn mi trng BHIA (Brain Heart Infusion Agar), nui nhit 28 - 30C
trong 24 gi, chng ti xc nh c a s cc khun lc mc trn a thch BHIA
u c hnh trn, ra u, bng, li thp, tm hi m, ng knh t 0,5 0,7 mm
(hnh 4.4.).


38
Hnh 4.2 Hnh thi khun lc
Streptococcus spp. khi nui cy trn
mi trng thch mu
Hnh 4.3. Vi khun Streptococcus spp.


Hnh 4.4. Hnh thi khun lc
Streptococcus spp. khi nui cy trn
mi trng BHIA

4.2.2. Kt qu nh danh vi khun
Nhm mc ch nh danh vi khun Streptococcus spp. phn lp c, chng ti
s dng b kt API 20 Strep ca hng Biomrieux (hnh 4.5)

Hnh 4.5. Kt qu th kt API 20Strep nh danh Streptococcus agalactiae
Kt qu gim nh v nh danh vi khun bng kit API 20 Strep c trnh by
bng 4.4.


39
Bng 4.4. Kt qu gim nh v nh danh vi khun Streptococcus spp.
TT Ch tiu
Kt qu kim tra (n = 52)
c tnh
S chng
(+)
T l (+)
(%)
1 Nhum Gram Gram (+) 52 100
2 Hnh dng Cu khun 52 100
3 Di ng - 52 100
4 Sinh catalaza - 52 100
5 Sinh oxidaza - 52 100
6 Phn ng ln men ym kh - 52 100
7 Phn ng ln men hiu kh - 52 100
8 Mc trn mi trng mu + 52 100
9 Gy tan huyt
Dng 4 7,69
Dng 48 92,31
10 Phn ng Voges-Proskauer + 52 100
11 Hippurate hydrolysis + 52 100
12 Bile-esculin tolerance - 52 100
13 Pyrrolidonyl arylamidase - 52 100
14 Sinh -galactosidase - 52 100
15 Sinh -glucuronidase - 52 100
16 Sinh -galactosidase - 52 100
17 Alkaline phosphatase + 52 100
18 Leucine AminoPeptidase + 52 100
19 Arginine Dihydrolase + 52 100
20 S dng ng
Ribose - 52 100
Arabinose - 52 100
Manitol - 52 100
Sorbitol - 52 100
Lactose - 52 100
Trehalose + 52 100
Inulin - 52 100
Raffinose - 52 100
Amidon - 52 100
Glycogen - 52 100
21 Kiu huyt thanh Ib 52 100
(+): dng tnh; (-): m tnh


40
Da trn cc ch tiu sinh ha v cn c vo m s nh danh ca kit API 20
Strep, tt c 52 chng vi khun phn lp c nh danh l Streptococcus
agalactiae. Kt qu ny ph hp vi mt s ti liu trc m t v vi khun
Streptococcus agalactiae Buller (2004); Salvador v cs (2005). ng Thanh H v
cs, (2010); ng Th Hong Oanh v Nguyn Thanh Phng (2012) cng c kt
qu tng t khi kt lun Streptococcus agalactiae l tc nhn gy bnh thu c
trn c r phi b bnh xut huyt do vi khun Streptococcus spp. gy ra.
T kt qu gim nh vi khun hc trn, chng ti khng nh c vai tr
quan trng ca Streptococcus spp. ( c nh danh loi l Streptococcus
agalactiae) trong qu trnh gy bnh cho c ti cc tnh thuc a bn nghin cu.
Kt qu ny rt c ngha, phc v cho cc nghin cu tip theo vi mc ch
phng v tr bnh; c bit l vic la chn chng vi khun sn xut vacxin
phng bnh.
4.3. Kt qu xc nh c lc ca cc chng vi khun Streptococcus agalactiae
phn lp c
4.3.1. Kt qu gy bnh thc nghim ca vi khun Streptococcus agalactiae c
r phi
Qua kt qu phn lp v nh danh vi khun cho thy c r phi b bnh b nhim
vi khun Streptococcus agalactiae, tuy nhin kim tra xem vi khun ny c phi
l tc nhn gy bnh cho c r phi hay khng chng ti tin hnh cm nhim gy
bnh bng 52 chng vi khun phn lp c cho c r phi trong phng th
nghim.
C c a vo gy bnh hon ton khe mnh, c nui thun ha 2 ngy
trc khi tin hnh cm nhim nhn to. C c nui trong iu kin cc yu t
thy l, thy ha thch hp (Ph lc 4). C gy cm nhim nhn to c tim canh
khun vo xoang bng, liu tim 0,1ml/c th (nng vi khun: 10
6
10
10
cfu/ml
canh thang hay pha long t 10
0
10
-5
). C l i chng tim 0,1ml nc
mui sinh l 0,85%/con.
Kt qu tin hnh cm nhim gy bnh thc nghim bng chng vi khun
phn lp c cho c r phi nhng nng khc nhau trong phng th nghim c


41
kt qu c th nh sau: kt qu sau 24 gi gy nhim c r phi c biu hin b
bnh vi du hiu bnh l in hnh l xut huyt, b li mt, c b nhim bnh cht
sau 36 gi xut hin du hiu bnh l. Chng ti phn lp li c vi khun
Streptococcus agalactiae t cc t chc gan, thn, no, mt ca c b bnh sau khi
cm nhim vi nhng du hiu bnh l in hnh. tt c cc liu th nghim u
c c cht ngoi tr l i chng v chng H2 liu 10
6
cfu/ml khng gy cht c
th nghim. Kt qu gy bnh thc nghim c th hin r qua cc bng 4.5 4.8:
Bng 4.5. Kt qu gy bnh thc nghim ca cc chng vi khun Streptococcus
agalactiae thu ti Hi Dng
Chng vi
khun
S c th
nghim
(con)
T l cht trn 50% c th nghim cc nng
(cfu/ml)
10
10
10
9
10
8
10
7
10
6

T1 20 - - - - -
T2 20 + + - - -
T3 20 + + + + -
T4 20 - - - - -
T5 20 + + + + -
T6 20 - - - - -
T7 20 - - - - -
T8 20 - - - - -
T9 20 + + - - -
T10 20 + + + + -
T11 20 + + - - -
T12 20 + + - - -
T13 20 + + - - -
T14 20 + + + + +
i chng 20 - - - - -





42
Bng 4.6. Kt qu gy bnh thc nghim ca cc chng vi khun
Streptococcus agalactiae thu ti H Ni
Chng vi
khun
S c th
nghim
(con)
T l cht trn 50% c th nghim cc nng
(cfu/ml)
10
10
10
9
10
8
10
7
10
6

C1 20 + + - - -
C2 20 + + + + +
C3 20 + + + - -
C4 20 + - - - -
C5 20 + + - - -
C6 20 + + - - -
C7 20 + + + + -
C8 20 + + - - -
C9 20 - - - - -
C10 20 - - - - -
C11 20 - - - - -
C12 20 + + - - -
C13 20 - - - - -
i chng 20 - - - - -

Bng 4.7. Kt qu gy bnh thc nghim ca cc chng vi khun
Streptococcus agalactiae thu ti Hi Phng
Chng vi
khun
S c th
nghim
(con)
T l cht trn 50% c th nghim cc nng
(cfu/ml)
10
10
10
9
10
8
10
7
10
6

H1 20 - - - - -
H2 20 - - - - -
H3 20 + + + - -
H4 20 + + - - -
H5 20 + + + + -
H6 20 + - - - -
H7 20 + + + - -
H8 20 - - - - -
H9 20 - - - - -
H10 20 - - - - -
H11 20 - - - - -
H12 20 - - - - -
H13 20 + + - - -
i
chng
20 - - - - -



43
Bng 4.8. Kt qu gy bnh thc nghim ca cc chng vi khun Streptococcus
agalactiae thu ti Qung Ninh
Chng vi
khun
S c th
nghim
(con)
T l cht trn 50% c th nghim cc nng
(cfu/ml)
10
10
10
9
10
8
10
7
10
6

V1 20 + + - - -
V2 20 + + - - -
V3 20 + + + - -
V4 20 + + + - -
V5 20 - - - - -
V6 20 - - - - -
V7 20 - - - - -
V8 20 + + - - -
V9 20 - - - - -
V10 20 - - - - -
V11 20 + + - - -
V12 20 - - - - -
i chng 20 - - - - -

T l gy cht c th nghim ca vi khun Streptococcus agalactiae nng
10
6
cfu/ml l thp nht trung bnh di 50%,. nng 10
10
cfu/ml hu ht gy
cht c th nghim vi t l rt cao nh T3 (100%). nng 10
9
cfu/ml t l gy
cht c th nghim trung bnh t 50%. Cc l i chng khng c c cht.
Nhng c gn cht hoc cht sau khi gy bnh thc nghim u c gii phu
kim tra, quan st s bin i bnh l ca cc c quan ni tng bn trong c th.
Sau li tin hnh ti phn lp vi khun t gan, thn, mt v no c trn mi
trng BHIA sau 24gi nhit 30
0
C. Khun lc cc a BHIA c mu sc v
hnh dng khun lc ging vi khun lc ca vi khun phn lp t mu c r phi lc
thu mu. Vi khun ti phn lp t nhng c bnh trong khong 24-48 gi sau khi
gy nhim c xc nh l c cc ch tiu hnh thi, sinh ha ging nh chng vi


44
khun cm nhim S.agalactiae. Qua kt qu cm nhim cho thy vi khun
S.agalactiae l tc nhn gy bnh cho c r phi nui thng phm.

Hnh 4.6. C r phi c du hiu bnh l
sau 24 gi gy nhim vi vi khun Streptococcus agalactiae
4.3.2. Kt qu tng cng c lc ca cc chng vi khun Streptococcus
agalactiae
Mc ch nhm tuyn chn c cc chng Streptococcus agalactiae c c lc
cao, n nh v c tnh lm b ging chun.
T kt qu cm nhim vi khun, chng ti chn nng vi khun gy c lc
cho c th nghim l 10
9
cfu/ml vi liu tim 0,1ml/1 c th nghim gy cht 50% c
th nghim . Chng ti chn c 29 chng vi khun v tin hnh tip i qua c r
phi (vt ch chnh ca vi khun); dng th nghim sau 3 ngy lin tip c th nghim
khng cht sau ti phn lp li chng vi khun v gi ging phc v nghin
cu sn xut kt chn on v vacxin. Kt qu c trnh by bng 4.9.


45
Bng 4.9. Bng kt qu tng cng c lc cc chng vi khun S.agalactiae
K hiu
chng
S c
tim
(con)
S lng c cht sau cc ngy tim (con)
Tng
s c
cht
(con)
T l
cht tch
ly (%)
1 2 3 4 5 6 7
T2 20 0 9 4 4 0 0 0 17 85,00
T3 20 0 14 6 0 0 0 0 30 100
T5 20 0 15 3 2 0 0 0 30 100
T9 20 0 12 4 3 0 0 0 19 95,00
T10 20 0 12 3 3 0 0 0 18 90,00
T11 20 0 10 3 3 0 0 1 17 85,00
T12 20 0 8 5 2 1 0 0 16 80,00
T13 20 0 9 6 2 1 1 0 19 95,00
T14 20 0 7 3 3 1 1 0 15 75,00
C1 20 0 12 4 1 1 0 0 18 90,00
C2 20 0 16 3 1 0 0 0 20 100
C3 20 0 10 3 3 2 2 0 20 100
C4 20 0 5 4 4 1 2 0 16 80,00
C5 20 0 1 4 5 2 2 1 15 75,00
C6 20 0 3 3 2 5 1 2 16 80,00
C7 20 0 8 5 3 2 0 1 19 95,00
C8 20 0 7 4 2 2 1 1 16 80,00
C12 20 0 7 8 2 2 0 0 19 95,00
H3 20 0 11 5 3 0 1 0 20 100
H4 20 0 5 5 3 2 1 1 17 85,00
H5 20 0 9 3 3 3 0 0 18 90,00
H7 20 0 12 1 3 3 1 0 20 100
H13 20 0 4 4 3 3 1 1 16 80,00
V1 20 0 1 9 3 3 1 1 18 90,00
V2 20 0 10 7 3 0 0 0 20 100
V3 20 0 5 3 4 3 1 0 16 80,00
V4 20 0 8 5 2 1 0 0 16 80,00
V8 20 0 2 9 3 1 1 1 17 85,00
V11 20 0 6 6 2 2 1 0 17 85,00
T bng 4.8, sau khi tin hnh cng cng c qua c r phi (vt ch chnh ca
vi khun), chng ti xc nh c 7 chng c c lc cao v n nh v c tnh:
C2 (100%), C3 (100%), T3 (100%), T5 (100%), V2 (100%), H3 (100%), H7
(100%). T kt qu ny chng ti tin hnh cc th nghim tip theo.


46
4.4. Kt qu xc nh tnh khng nguyn ca vi khun Streptococcus agalactiae
phn lp c
4.4.4. Kt qu to khng nguyn cho tng chng vi khun:
Sau khi bt hot vi khun bng formalin 0,5%, chng ti tin hnh ly tm v ra
vi khun (Hnh 4.7).
Pha vi khun vi nc mui sinh l vi th tch bng vi th tch trc khi bt
hot vi khun ta c hn hp dung dch v nc mui sinh l. Bo qun 4
0
C.




Hnh 4.7. Vi khun bt hot bng formalin trc ly tm (A); sau li tm (B);
pha vi nc mui sinh l (C)
4.4.2. Kt qu to khng th khng S. agalactiae trn c r phi
Sau khi tim hn hp dung dch huyn ph vi khun nc mui sinh l vo xoang
bng ca c vi liu lng 0,1ml/c, c r phi khng c biu hin g khc thng.
n ngy th 14 ly mu c. Sau khi ly mu c t nghing ng eppendorf
tng din tch mt thong v gi yn trong iu kin lnh t 1-2gi cho n khi mu
hon ton ng c li.
em ly tm v ht ly phn huyt thanh c mu vng nht, trong nm phn
A
B
C


47
trn ca ng eppendorf (Hnh 4.8).

Hnh 4.8. Hnh nh thu huyt thanh c
(A): Mu c; (B): Mu c sau khi gi lnh v ly tm
4.4.3. Kt qu phn ng ngng kt
Vi mc ch ca ti l la chn nhng chng vi khun sn xut vacxin
do cn thit phi la chn nhng chng vi khun c sinh p ng min dch v
c c lc cao. Phn ng ngng kt da trn nguyn tc ca s lin kt gia khng
nguyn v khng th c th nhn thy c dng kt khi (Hnh 4.9 v 4.10).
Phn ng dng tnh: khng nguyn b ngng kt thnh tng m lm tm trn
phin knh, mt thung nhn thy c.
Phn ng m tnh: Khng c hin tng ngng kt, khng nguyn ha u trong
hn dch ging nh bn i chng.

Hnh 4.9. Kt qu phn ng ngng kt nhanh trn phin knh
A: m tnh; B: Dng tnh vi vi khun sng;
C: Dng tnh vi vi khun bt hot.
A B C
A B


48

Hnh 4.10. Hin tng ngng kt quan st bng knh hin vi (40X)
Qua th nghim kim tra phn ng ngng kt min dch, chng ti chn
c 7/7 chng vi khun c lc cao c phn ng ngng kt. Kt qu trnh by
bng 4.10.
Bng 4.10. Kt qu kim tra phn ng ngng kt vi khng th pha long
Ngy
thu
mu
Chng
vi
khun
S ln pha long khng th
Vi khun sng Vi khun bt hot
0 1/2 1/4 1/8 0 1/2 1/4 1/8
14
ngy
C2 + + + - + + + -
C3 + + + - + + + +
T3 + + + + + + + +
T5 + + - - + + - -
H3 + + - - + + - -
H7 + + + - + + + +
V2 + + + - + + + -
(-): Kt qu m tnh, phn ng ngng kt khng xy ra.
(+): Kt qu dng tnh, c xy ra phn ng ngng kt.
3/7 mu huyt thanh kim tra c khng th khng lin cu khun S.agalactiae
v cho phn ng ngng kt vi huyn ph t bo pha long 8 ln.
T bng kt qu trn, chng ti chn c 7 chng: C2, C3, T3, T5, H1, H7, V2
lm b ging chun tin hnh nghin cu ch to kt v vacxin phc v cho
chn on nhanh v phng, tr bnh.


49
4.5. Kt qu kim tra kh nng mn cm vi khng sinh ca vi khun
Streptococcus agalactiae phn lp c
Mi lo ngi hin nay i vi y h ni chung v ngnh th y thy sn ni ring l
tnh trng khng thuc ca vi khun. Mi lo ngi ny cn ln hn gp bi khi vi
khun khng ch n khng vi mt loi khng sinh no m cng mt lc vi
nhiu loi khng sinh. Ngy nay vic s dng khng sinh trong phng v tr bnh
hay b sung trong thc n chn nui l rt ty tin, khng ng nguyn tc v dn
n hin tng khng thuc trn lan. Nhng chng vi khun khng nhiu loi khng
sinh khng ch lan truyn trong mi trng nui thy sn m rt d dng lan truyn
trong t nhin gy hu qu xu, gy nhim mi trng, nh hng n hiu qu
iu tr v sc khe con ngi cng nh vt nui.
c c s la chn loi khng sinh thch hp s dng iu tr bnh xut
huyt c r phi do vi khun Streptococcus agalactiae (S. agalactiae) gy ra cc
tnh thuc a bn nghin cu, chng ti tin hnh kim tra tnh mn cm ca vi
khun vi 10 loi khng sinh v ang c s dng ph bin trong nui trng
thy sn (hnh 4.11). Kt qu c trnh by bng 4.11.
Bng 4.11. Kt qu th khng sinh ca 52 chng S.agalactiae vi 10 loi
thuc khng sinh thng dng
STT Tn thuc khng sinh
Khng thuc
Mn cm
trung bnh
Mn cm
cao
S
chng
T l
(%)
S
chng
T l
(%)
S
chng
T l
(%)
1 Ampicillin 49 94,23 3 5,77 0 0,00
2 Amoxicillin 52 100 0 0,00 0 0,00
3 Enrofroxacin 0 0,00 0 0,00 52 100
4 Erythromycine 0 0,00 46 88,46 6 11,54
5 Rifampin 52 100 0 0,00 0 0,00
6 Streptomicine 22 42,31 30 57,69 0 0,00
7 Kanamycine 9 17,31 43 82,69 0 0,00
8 Doxycyline 0 0,00 0 0,00 52 100
9 Tetracycline 0 0,00 52 100 0 0,00
10 Sulfamethoxazol/Trimethoxazol 51 98,08 1 1,92 0 0,00



50
Qua bng 4.11 cho thy vi khun S.agalactiae phn lp c mn cm vi hai
loi khng sinh l Enrofroxacine v Doxycyline. V vy, c th la chn nhng
thuc c thnh phn hai loi khng sinh trn iu tr trong thc t ti a bn
nghin cu. Tuy nhin do hin nay khng sinh Enrofroxacine thuc trong nhm
thuc cm s dng trong nui trng thy sn nn khng nn s dng iu tr
trong thc t.


Hnh 4.11. Kim tra kh nng mn cm vi khng sinh






51
5. KT LUN V NGH

5.1. Kt lun
T cc kt qu nh trnh by trn, chng ti rt ra mt s kt lun sau:
- T l phn lp c vi khun Streptococcus spp. t cc mu c b bnh
xut huyt l 86,67%.
- Vi khun Streptococcus spp. phn lp c mang y cc c tnh sinh
hc nh ti liu kinh in m t. Ton b 52 chng Streptococcus spp. u c
gim nh v l S.agalactiae.
- Chng ti chn c 7 chng vi khun Streptococcus agalactiae: C2, C3,
T3, T5, H3, H7, V2 c c lc cao, n nh v c tnh v c sinh p ng min dch.
- Cc chng vi khun rt mn cm vi hai loi khng sinh l Enzofroxacine
v Doxycyline.
Gi ging phc v nghin cu sn xut kit chn on v vaccine.
5.2. ngh
Tip tc nghin cu v tc nhn gy bnh trn c r phi v xy dng quy
trnh phng tr bnh cho c r phi
T 7 chng vi khun thu c, tip tc nghin cu kit chn on v vacxin
phng bnh do vi khun Streptococcus spp. gy bnh xut huyt trn c r phi ti
mt s tnh min Bc.








52
TI LIU THAM KHO

Ti liu ting Vit
1. ng Thanh H, Nguyn Vit Khu, Nguyn Th Hnh, (2010). Mt s c im ca
Streptococcus agalactiae, tc nhn gy bnh Streptococcosis trn c r phi min Bc Vit
Nam. Trung tm nghin cu quan trc cnh bo mi trng v phng nga dch bnh thy
sn min Bc Vin Nghin cu Nui trng Thy Sn I.
2. Nguyn B Hin, Trn Th Lan Hng. Gio trnh min dch hc th y. Nh xut bn
Nng nghip.
3. Nguyn Khang (2005). Khng sinh hc ng dng, nh xut bn y hc, H Ni.
4. Nguyn Vit Khu, Trng Th M Hnh, ng Thanh H, Nguyn Th H, Phm
Thnh , Bi Ngc Thanh, Nguyn Th Nguyn, Nguyn Hi Xun, Phm Thi
Giang v Nguyn Th Thu H, (2009). Xc nh nguyn nhn gy cht hng lot c r
phi nui thng phm ti mt s tnh min Bc. Bo co khoa hc Vin Nghin cu
Nui trng Thy sn 1.
5. ng Th Hong Oanh v Nguyn Thanh Phng, 2012. Phn lp v xc nh c im
ca vi khun Streptococus agalactiae t c iu hng (Oreochromis sp) bnh m mt
v xut huyt. Tp ch khoa hc 2012, trng i hc Cn Th, 22c 203-212.
6. Mai Vn Ti (2004), iu tra nh gi hin trng cc loi thuc, ha cht v ch phm
sinh hc dng trong nui trng thy sn nhm xut cc gii php qun l. Tuyn tp
bo co khoa hc Vin Nghin cu nui trng thy sn I.
7. Trn Th Minh Tm, (2004). Nghin cu bnh nguy him thng gp trn c r phi
(Oreochromis spp) nui thm canh. Vin Nghin cu Nui trng Thy Sn II.
8. Bi Quang T, (2006). Bnh hc Thy sn. Vin Nghin cu Nui trng Thy sn I.
9. Phm Anh Tun (2006), Bo co qui hoch pht trin c r phi giai on 2006-2015.
10. inh Th Thy, (2007). Nghin cu cc bnh nguy him thng gp c r phi nui
thm canh. Thng tin KHCN & Kinh t Thy sn 12.
Ti liu ting Anh
1. Balarin, J .D and R.D. Haller, 1982, The intensive culture of tilapia in tanks, receways
and cages. In: Recent advances in aquaculture (eds. J .F. Muir and R.J . Roberts),
pp.266-355. Westview, Boulder.
2. Bromage E. S., Thomas A. and Owens L. (1999) Streptococcus iniae, a bacterial
infection in barramundi Lates calcarifer. Diseases of Aquatic Organisms, 36: 177-
181.
3. Buller, N.B., 2004. Bacteria from fish and other aquatic animals: a pratice identification
manual, 361 pp.
4. El-Sayed, Abdel - Fattah M., (2006). Tilapia culture. CABI Publishing. ISBN-13: 978-
0-85199-014-9.
5. Evans, J ., Klesius, P.H. and Shoemmaker, C.A. 2006. Sreptococcus in warm-water fish.


53
Aquaculture Health International. 10-14
6. FAO (2004), State of World Fisheries and Aquaculture 2004, FAO, Rome, Italy.
7. Frerichs, G.N & Millar (1993). Manual for the isolation and identification of fish
bacterial pathogens. Pisces Press. Stirling, pp. 58
8. Gupta M.V v Acosta B.O. (2004). Review of global tilapia farming practices.
Aquaculture Asia IX, 7 - 12.
9. Hernandez, E., J . Figueroa and C. Iregui, (2009). Streptococcosis on a red tilapia,
Oreochromis sp., farm: A case study. J . Fish Dis., 32: 247-252.
10. Inglis, V. (2000), Antibacterial Chemotherapy in Aquaculture, Review of Practice,
Associated Risks and Need for Action, In: Use of Chemicals in Aquaculture in Asia,
Arthur J . R; Lavilla-Pitogo C. R. and Subasinghe R. P., 2000, pp. 7-22
11. Intervet, (2006). Diseases of Tilapia An Introduction
12. Klesius P.H, Shoemaker CA, Evans J .J . Efficacy of a single and combined
Streptococcus iniae isolates vaccine administered by intraperitoneal and intramuscular
routes in tilapia (Oreochromis niloticus). Aquaculture 2000; 188 (3-4):327-246.
13. Lauke Labrie, J .N., Cedric Komar and Brian Sheehan, 2007. Bacterial Diseases of
Finfish in the South East Asian Region. Intervet.
14. Liu Liping, Zhang Zongfeng, Zhang Wembo, Francis Murray, David Little. 2012 Tilapia
aquaculture in China: Low market prices, other issues challenge as sector seeks
sustainability. Global Aquaculture Advocate, Vo 15. Issue 2, March/ April 2012, pp.20-21
15. Mian, G.F., D.T. Godoy, C.A.G. Lea, Y.T. Yuhara, G.M. Costa and H.C.P. Figueiredo,
2009. Aspects of the natural history and virulence of S. agalactiae infection in Nile
tilapia. Vet. Microbiol., 136: 180-183.
16. Nguyen, H.T., Kanai, K 1999 Selective agars for the isolation of Streptococcus iniae
from J apanese flounder. Paralichthys olivaceus, and its cultural environment.J .Appl.
Microbiol. 86, 769-776.
17. Perera R.P., J .S.K., Collins M.D. and Lewis D.H, (1994). Streptococcus iniae
Associated with Mortality of Tilapia nilotica x T. aurea Hybrids. J ournal of Aquatic
Animal Health, 10: 294 299.
18. Philipart.J .C.L. Ruwet, (1982), Ecolapia, logy and Distrisbution of Tilapia, In: R.S.V.
Pullin and R.H. Lowe-Mc Connell (Eds), Biology and Culture of Tilapia, ICLAM
conference Proceedings 7,432. ICLARM, Mamila, Philippines, pp 15-59.
19. Phillips Michael (2000), The use of Chemicals in Carp and Shrimp Aquaculture in
Bangladesh, Cambodia, Lao PDR, Nepal, Pakistan, Sri Lanka and Viet Nam, Use of
chemicals in Aquaculture in Asia, pp. 75-85
20. Plumb, J .A., 1999. Health Maintenance and Principal Microbial Diseases of Cultured
Fishes. Iowa State University Press, Ames.
21. Pretto-Giordano, LG., E.E. Muller, J .C de Frritas and V.G. da Silva, 2010a. Evaluation
on the Pathogenesis of Streptococcus agalactiae in Nile Tilapia (Oreochromis
nilonicus). Brazilian Arch. Biol. Technol., 53: 87-92.


54
22. Salvador, R., E.E. Muller, J .C. Freitas, J .H. Leonhadt, L.G. Pretto-Giordano and J .A.
Dias, (2005). Isolation and characterization of Streptococcus spp. Group B in Nile
tilapias (Oreochromis niloticus) reared in hapas nets and earth nurseries in the northern
region of Prana State, Brazil. Ciencia Rural, 35: 1374-1378.
23. Sheehan, (2009). Streptococcosis in Tilapia: A more complex problem.
(http://www.thefishsite.com/articles/812/)
24. Shoemaker, C.A., Xu, D., Klesius, P.H., Evans, J .J , (2008). Concurrent infections
(parasitism and bacterial diesease) in tilapia, The 8
th
International Symposium on
Tilapia in Aquaculture, Cairo, Egypt pp. 1365-1375.
25. Yuasa, Kamaishi, Hatai, Bahnnan and Borisuthpeth, (2005). Two cases of streptococcal
infections of cultured tilapia in Asia. In: Sixth Symposium on Diseases in Asian
Aquaculture (ed Bondad-Reantaso MG, Mohan, C.V., Crumlish, M. and Subasinghe,
R.P.) Fish Health Section, Asian Fisheries Society, Colombo Sri Lanka, pp. 259-268.
26. Zamri-Saad M, Amal MN, Siti-Zahrah A.. Pathological changes in red tilapias
(Oreochromis spp.) naturally infected by Streptococcus agalactiae. J ournal of
comparative pathology,2010Aug-Oct;143(2-3):227-9.doi: 10.1016/j.jcpa. 2010.01.020.
Epub 2010 Mar 23.
27. Wongtavatchai & Maisak, (2008). Pathobiological Characteristic of Streptococcosis in
Farmed Tilapia, Oreochromis nilotica, in Thailand. Proceedings of 5th world fisheries
congress.
Web tham kho
https://apiweb.biomerieux.com



55
PH LC

Ph lc 1.
DANH MC HO CHT, KHNG SINH CM S DNG TRONG SN
XUT, KINH DOANH THY SN
( Ban hnh km theo Thng t s 15/2009/TT-BNN ngy 17 thng 3 nm
2009
ca B trng B Nng nghip v Pht trin nng thn)
TT Tn ho cht, khng sinh i tng p dng
1 Aristolochia spp v cc ch phm t chng
Thc n, thuc th y, ho
cht, cht x l mi
trng, cht ty ra kh
trng, cht bo qun,
kem bi da tay trong tt
c cc khu sn xut
ging, nui trng ng
thc vt di nc v
lng c, dch v ngh
c v bo qun, ch bin.
2 Chloramphenicol
3 Chloroform
4 Chlorpromazine
5 Colchicine
6 Dapsone
7 Dimetridazole
8 Metronidazole
9 Nitrofuran (bao gm c Furazolidone)
10 Ronidazole
11 Green Malachite (Xanh Malachite)
12 Ipronidazole
13 Cc Nitroimidazole khc
14 Clenbuterol
15 Diethylstilbestrol (DES)
16 Glycopeptides
17 Trichlorfon (Dipterex)
18 Gentian Violet (Crystal violet)
19 Nhm Fluoroquinolones (cm s dng trong sn
xut, kinh doanh thu sn xut khu vo th trng
M v Bc M)


56

Ph lc 2
DANH MC HO CHT, KHNG SINH HN CH S DNG
TRONG SN XUT KINH DOANH THY SN
(Ban hnh km theo Thng t s 15/2009/TT-BNN ngy 17 thng 3 nm 2009
ca B trng B Nng nghip v Pht trin nng thn)

TT Tn ho cht, khng sinh D lng ti a
(MRL)(ppb)
1 Amoxicillin 50
2 Ampicillin 50
3 Benzylpenicillin 50
4 Cloxacillin 300
5 Dicloxacillin 300
6 Oxacillin 300
7 Oxolinic Acid 100
8 Colistin 150
9 Cypermethrim 50
10 Deltamethrin 10
11 Diflubenzuron 1000
12 Teflubenzuron 500
13 Emamectin 100
14 Erythromycine 200
15 Tilmicosin 50
16 Tylosin 100
17 Florfenicol 1000
18 Lincomycine 100
19 Neomycine 500
20 Paromomycin 500
21 Spectinomycin 300
22 Chlortetracycline 100
23 Oxytetracycline 100
24 Tetracycline 100
25 Sulfonamide (cc loi) 100
26 Trimethoprim 50
27 Ormetoprim 50
28 Tricainemethanesulfonate 15-330
29 Danofloxacin 100
30 Difloxacin 300
31 Enrofloxacin +Ciprofloxacin 100
32 Sarafloxacin 30
33 Flumequine 600


57

Ph lc 3: c im hnh thi, sinh l, sinh ho ca vi khun phn lp t
c iu hng bnh
Ch tiu
Chng vi khun
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
Nhum Gram + + + + + + + + + + + +
Hnh dng
Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu
Di ng - - - - - - - - - - - -
Sinh catalaza - - - - - - - - - - - -
Sinh oxidaza
- - - - - - - - - - - -
Phn ng ln men ym kh
- - - - - - - - - - - -
Phn ng ln men hiu kh
- - - - - - - - - - - -
Mc trn mi trng mu + + + + + + + + + + + +
Gy tan huyt
Phn ng Voges-Proskauer + + + + + + + + + + + +
Hippurate hydrolysis + + + + + + + + + + + +
Bile-esculin tolerance
- - - - - - - - - - - -
Pyrrolidonyl arylamidase
- - - - - - - - - - - -
Sinh -galactosidase
- - - - - - - - - - - -
Sinh -glucuronidase
- - - - - - - - - - - -
Sinh -galactosidase
- - - - - - - - - - - -
Alkaline phosphatase - - - - - - - - - - - -
Leucine AminoPeptidase + + + + + + + + + + + +
Arginine Dihydrolase + + + + + + + + + + + +
S dng ng
Ribose + + + + + + + + + + + +
Arabinose
- - - - - - - - - - - -
Manitol
- - - - - - - - - - - -
Sorbitol - - - - - - - - - - - -
Lactose - - - - - - - - - - - -
Trehalose + + + + + + + + + + + +
Inulin
- - - - - - - - - - - -
Raffinose
- - - - - - - - - - - -
Amidon
- - - - - - - - - - - -
Glycogen
- - - - - - - - - - - +
Kiu huyt thanh Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib





58

Ch tiu
Chng vi khun
T13 T14 C2 C2 C3 C4 C5 C6 C7 C8 C9 C10
Nhum Gram + + + + + + + + + + + +
Hnh dng
Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu
Di ng
- - - - - - - - - - - -
Sinh catalaza
- - - - - - - - - - - -
Sinh oxidaza
- - - - - - - - - - - -
Phn ng ln men ym kh
- - - - - - - - - - - -
Phn ng ln men hiu kh - - - - - - - - - - - -
Mc trn mi trng mu + + + + + + + + + + + +
Gy tan huyt
Phn ng Voges-Proskauer + + + + + + + + + + + +
Hippurate hydrolysis + + + + + + + + + + + +
Bile-esculin tolerance
- - - - - - - - - - - -
Pyrrolidonyl arylamidase
- - - - - - - - - - - -
Sinh -galactosidase
- - - - - - - - - - - -
Sinh -glucuronidase - - - - - - - - - - - -
Sinh -galactosidase - - - - - - - - - - - -
Alkaline phosphatase
- - - - - - - - - - - -
Leucine AminoPeptidase + + + + + + + + + + + +
Arginine Dihydrolase + + + + + + + + + + + +
S dng ng
Ribose + + + + + + + + + + + +
Arabinose
- - - - - - - - - - - -
Manitol - - - - - - - - - - - -
Sorbitol - - - - - - - - - - - -
Lactose
- - - - - - - - - - - -
Trehalose + + + + + + + + + + + +
Inulin
- - - - - - - - - - - -
Raffinose
- - - - - - - - - - - -
Amidon
- - - - - - - - - - - -
Glycogen
- - - - - - - - - - - +
Kiu huyt thanh Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib







59

Ch tiu
Chng vi khun
C11 C12 C13 H1 H2 H3 H4 H5 H6 H7 H8 H9
Nhum Gram + + + + + + + + + + + +
Hnh dng
Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu
Di ng
- - - - - - - - - - - -
Sinh catalaza
- - - - - - - - - - - -
Sinh oxidaza
- - - - - - - - - - - -
Phn ng ln men ym kh
- - - - - - - - - - - -
Phn ng ln men hiu kh - - - - - - - - - - - -
Mc trn mi trng mu + + + + + + + + + + + +
Gy tan huyt
Phn ng Voges-Proskauer + + + + + + + + + + + +
Hippurate hydrolysis + + + + + + + + + + + +
Bile-esculin tolerance
- - - - - - - - - - - -
Pyrrolidonyl arylamidase
- - - - - - - - - - - -
Sinh -galactosidase
- - - - - - - - - - - -
Sinh -glucuronidase - - - - - - - - - - - -
Sinh -galactosidase - - - - - - - - - - - -
Alkaline phosphatase
- - - - - - - - - - - -
Leucine AminoPeptidase + + + + + + + + + + + +
Arginine Dihydrolase + + + + + + + + + + + +
S dng ng
Ribose + + + + + + + + + + + +
Arabinose
- - - - - - - - - - - -
Manitol - - - - - - - - - - - -
Sorbitol - - - - - - - - - - - -
Lactose
- - - - - - - - - - - -
Trehalose + + + + + + + + + + + +
Inulin
- - - - - - - - - - - -
Raffinose
- - - - - - - - - - - -
Amidon
- - - - - - - - - - - -
Glycogen
- - - - - - - - - - - +
Kiu huyt thanh Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib



60

Ch tiu
Chng vi khun
H10 H11 H12 H13 V1 V2 V3 V4 V5 V6 V7 V8
Nhum Gram + + + + + + + + + + + +
Hnh dng
Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu
Di ng
- - - - - - - - - - - -
Sinh catalaza
- - - - - - - - - - - -
Sinh oxidaza
- - - - - - - - - - - -
Phn ng ln men ym kh
- - - - - - - - - - - -
Phn ng ln men hiu kh - - - - - - - - - - - -
Mc trn mi trng mu + + + + + + + + + + + +
Gy tan huyt
Phn ng Voges-Proskauer + + + + + + + + + + + +
Hippurate hydrolysis + + + + + + + + + + + +
Bile-esculin tolerance
- - - - - - - - - - - -
Pyrrolidonyl arylamidase
- - - - - - - - - - - -
Sinh -galactosidase
- - - - - - - - - - - -
Sinh -glucuronidase - - - - - - - - - - - -
Sinh -galactosidase - - - - - - - - - - - -
Alkaline phosphatase
- - - - - - - - - - - -
Leucine AminoPeptidase + + + + + + + + + + + +
Arginine Dihydrolase + + + + + + + + + + + +
S dng ng
Ribose + + + + + + + + + + + +
Arabinose
- - - - - - - - - - - -
Manitol - - - - - - - - - - - -
Sorbitol - - - - - - - - - - - -
Lactose
- - - - - - - - - - - -
Trehalose + + + + + + + + + + + +
Inulin
- - - - - - - - - - - -
Raffinose
- - - - - - - - - - - -
Amidon
- - - - - - - - - - - -
Glycogen
- - - - - - - - - - - +
Kiu huyt thanh Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib



61

Ch tiu
Chng vi khun
V9 V10 V11 V12
Buller
(2004)

Nhum Gram + + + + +
Hnh dng
Cu Cu Cu Cu Cu
Di ng - - - - -
Sinh catalaza - - - - -
Sinh oxidaza - - - - -
Phn ng ln men ym kh - - - - -
Phn ng ln men hiu kh - - - - -
Mc trn mi trng mu + + + + +
Gy tan huyt
Phn ng Voges-Proskauer + + + + +
Hippurate hydrolysis + + + + +
Bile-esculin tolerance
- - - - -
Pyrrolidonyl arylamidase
- - - - -
Sinh -galactosidase - - - - -
Sinh -glucuronidase - - - - -/+
Sinh -galactosidase
- - - - -
Alkaline phosphatase - - - - -
Leucine AminoPeptidase + + + + +
Arginine Dihydrolase + + + + -/+
S dng ng
Ribose + + + + +s
Arabinose - - - - -
Manitol - - - - -
Sorbitol
- - - - -
Lactose
- - - - -
Trehalose + + + + -
Inulin
- - - - -
Raffinose
- - - - -
Amidon
- - - - -
Glycogen - - - - +
Kiu huyt thanh Ib Ib Ib Ib

Ghi ch: (+) dng tnh; (-) m tnh; (+s) phn ng chm





62
Ph lc 4. Kt qu gy bnh thc nghim ca cc chng vi khun
Streptococcus agalactiae thu ti Hi Dng
M lu
Mt VK tim
(cfu/ml)
S lng c tim
(con)
T l cht (%)
T1
10
10
20 40,00
10
9
20 40,00
10
8
20 30,00
10
7
20 20,00
10
6
20 20,00
T2
10
10
20 60,00
10
9
20 50,00
10
8
20 40,00
10
7
20 40,00
10
6
20 30,00
T3
10
10
20 100,00
10
9
20 80,00
10
8
20 70,00
10
7
20 60,00
10
6
20 40,00
T4
10
10
20 40,00
10
9
20 40,00
10
8
20 30,00
10
7
20 30,00
10
6
20 20,00
T5
10
10
20 80,00
10
9
20 60,00
10
8
20 50,00
10
7
20 50,00
10
6
20 20,00
T6
10
10
20 40,00
10
9
20 30,00
10
8
20 20,00
10
7
20 10,00
10
6
20 10,00
T7
10
10
20 10,00
10
9
20 10,00
10
8
20 0
10
7
20 0


63
10
6
20 0
T8
10
10
20 40,00
10
9
20 40,00
10
8
20 30,00
10
7
20 10,00
10
6
20 10,00
T9
10
10
20 60,00
10
9
20 50,00
10
8
20 30,00
10
7
20 30,00
10
6
20 20,00
T10
10
10
20 70,00
10
9
20 70,00
10
8
20 70,00
10
7
20 60,00
10
6
20 40,00
T11
10
10
20 70,00
10
9
20 60,00
10
8
20 40,00
10
7
20 40,00
10
6
20 30,00
T12
10
10
20 50,00
10
9
20 50,00
10
8
20 30,00
10
7
20 10,00
10
6
20 0,00
T13
10
10
20 60,00
10
9
20 50,00
10
8
20 40,00
10
7
20 40,00
10
6
20 20,00
T14
10
10
20 70,00
10
9
20 60,00
10
8
20 50,00
10
7
20 50,00
10
6
20 30,00
i chng 0,85% 20 0



64
Ph lc 5. Kt qu gy bnh thc nghim ca cc chng vi khun
Streptococcus agalactiae thu ti H Ni
M lu
Mt VK tim
(cfu/ml)
S lng c tim
(con)
T l cht (%)
C1
10
10
20 60,00
10
9
20 50,00
10
8
20 40,00
10
7
20 40,00
10
6
20 20,00
C2
10
10
20 90,00
10
9
20 80,00
10
8
20 60,00
10
7
20 60,00
10
6
20 50,00
C3
10
10
20 90,00
10
9
20 70,00
10
8
20 60,00
10
7
20 40,00
10
6
20 40,00
C4
10
10
20 50,00
10
9
20 40,00
10
8
20 30,00
10
7
20 10,00
10
6
20 10,00
C5
10
10
20 60,00
10
9
20 60,00
10
8
20 40,00
10
7
20 30,00
10
6
20 20,00
C6
10
10
20 50,00
10
9
20 50,00
10
8
20 40,00
10
7
20 10,00
10
6
20 10,00
C7
10
10
20 70,00
10
9
20 70,00
10
8
20 60.00


65
10
7
20 50,00
10
6
20 40,00
C8
10
10
20 60,00
10
9
20 50,00
10
8
20 30,00
10
7
20 10,00
10
6
20 10,00
C9
10
10
20 20,00
10
9
20 20,00
10
8
20 10,00
10
7
20 0
10
6
20 0
C10
10
10
20 40,00
10
9
20 40,00
10
8
20 20,00
10
7
20 10,00
10
6
20 10,00
C11
10
10
20 30,00
10
9
20 20,00
10
8
20 0
10
7
20 0
10
6
20 0
C12
10
10
20 50,00
10
9
20 50,00
10
8
20 30,00
10
7
20 10,00
10
6
20 0
C13
10
10
20 10,00
10
9
20 10,00
10
8
20 0
10
7
20 0
10
6
20 0
i chng 0,85% 20 0





66
Ph lc 6. Kt qu gy bnh thc nghim ca cc chng vi khun
Streptococcus agalactiae thu ti Hi Phng
M lu
Mt VK tim
(cfu/ml)
S lng c tim
(con)
T l cht (%)
H1
10
10
20 40,00
10
9
20 30,00
10
8
20 20,00
10
7
20 10,00
10
6
20 10,00
H2
10
10
20 0
10
9
20 0
10
8
20 0
10
7
20 0
10
6
20 0
H3
10
10
20 70,00
10
9
20 60,00
10
8
20 60,00
10
7
20 40,00
10
6
20 40,00
H4
10
10
20 60,00
10
9
20 50,00
10
8
20 40,00
10
7
20 20,00
10
6
20 20,00
H5
10
10
20 70,00
10
9
20 60,00
10
8
20 50,00
10
7
20 50,00
10
6
20 20,00
H6
10
10
20 50,00
10
9
20 40,00
10
8
20 20,00
10
7
20 20,00
10
6
20 0
H7
10
10
20 70,00
10
9
20 70,00
10
8
20 50,00


67
10
7
20 30,00
10
6
20 20,00
H8
10
10
20 40,00
10
9
20 20,00
10
8
20 10,00
10
7
20 10,00
10
6
20 0
H9
10
10
20 40,00
10
9
20 30,00
10
8
20 10,00
10
7
20 0
10
6
20 0
H10
10
10
20 10,00
10
9
20 10,00
10
8
20 0
10
7
20 0
10
6
20 0
H11
10
10
20 40,00
10
9
20 40,00
10
8
20 30,00
10
7
20 20,00
10
6
20 20,00
H12
10
10
20 30,00
10
9
20 20,00
10
8
20 20,00
10
7
20 10,00
10
6
20 0
H13
10
10
20 60,00
10
9
20 50,00
10
8
20 40,00
10
7
20 40,00
10
6
20 20,00
i chng 0,85% 20 0




68
Ph lc 7. Kt qu gy bnh thc nghim ca cc chng vi khun
Streptococcus agalactiae thu ti Qung Ninh
M lu
Mt VK tim
(cfu/ml)
S lng c tim
(con)
T l cht (%)
V1
10
10
20 60,00
10
9
20 50,00
10
8
20 40,00
10
7
20 20,00
10
6
20 20,00
V2
10
10
20 70,00
10
9
20 60,00
10
8
20 40,00
10
7
20 30,00
10
6
20 20,00
V3
10
10
20 60,00
10
9
20 50,00
10
8
20 50,00
10
7
20 30,00
10
6
20 10,00
V4
10
10
20 70,00
10
9
20 60,00
10
8
20 50,00
10
7
20 30,00
10
6
20 20,00
V5
10
10
20 40,00
10
9
20 40,00
10
8
20 30,00
10
7
20 10,00
10
6
20 10,00
V6
10
10
20 40,00
10
9
20 30,00
10
8
20 20,00
10
7
20 10,00
10
6
20 10,00
V7
10
10
20 10,00
10
9
20 10,00
10
8
20 0


69
10
7
20 0
10
6
20 0
V8
10
10
20 50,00
10
9
20 50,00
10
8
20 40,00
10
7
20 20,00
10
6
20 10,00
V9
10
10
20 40,00
10
9
20 30,00
10
8
20 10,00
10
7
20 0
10
6
20 0
V10
10
10
20 20,00
10
9
20 20,00
10
8
20 10,00
10
7
20 0
10
6
20 0
V11
10
10
20 50,00
10
9
20 50,00
10
8
20 30,00
10
7
20 20,00
10
6
20 20,00
V12
10
10
20 10,00
10
9
20 10,00
10
8
20 0
10
7
20 0
10
6
20 0
i chng 0,85% 20 0

Ph lc 8: iu kin mi trng trong qu trnh gy bnh thc nghim
STT Cc yu t mi trng bin ng
1 Nhit 26 33
0
C
2 O
2
4 6 (mg/l)
3 pH 7 8
4 NH
3
/NH
4
+
0,007 0,009 (mg/l)
5 Kim 102 120 (mg CaCO
3
/l)


70
Ph lc 9: ng knh vng v khun ca 52 chng vi khun Streptococcus
agalactiae vi 10 loi thuc khng sinh thng dng
Ax Am En Er Kn Sm Rf Dx Te Bt
T1 13,0 11,1 29,0 21,0 15,0 14,4 12,7 21,4 15,8 15,4
T2 12,3 12,0 29,7 21,5 15,1 17,0 14,3 24,3 17,2 13,3
T3 13,7 11,4 29,6 20,8 16,3 16,3 16,0 27,6 15,8 16,2
T4 12,6 11,6 29,8 23,1 14,2 16,4 16,4 21,0 16,4 14,3
T5 13,5 11,6 27,3 22,0 15,5 16,1 13,0 25,8 15,1 13,7
T6 14,0 13,1 25,0 20,7 14,0 15,5 15,6 25,3 17,4 14,1
T7 11,8 12,0 29,5 21,4 14,7 15,9 15,7 24,1 15,6 14,0
T8 12,3 11,3 29,3 21,6 14,9 14,7 15,0 22,0 15,9 15,4
T9 12,3 11,4 24,9 23,4 15,1 16,0 14,9 26,7 17,0 14,9
T10 12,5 11,3 25,5 22,0 16,0 16,0 14,2 27,3 16,5 15,2
T11 13,0 11,9 26,0 22,4 13,9 15,2 14,9 27,0 16,1 15,0
T12 14,2 11,8 26,0 20,9 14,5 15,8 14,3 27,4 16,0 15,6
T13 13,5 11,8 28,4 20,0 14,3 15,3 12,9 27,1 17,9 13,1
T14 12,5 13,0 24,3 22,1 15,0 14,6 12,9 26,8 15,9 14,0
C1 12,4 12,3 27,8 20,1 15,8 14,9 13,0 26,8 15,5 14,9
C2 12,9 12,1 26,9 20,5 15,4 16,0 15,7 25,0 16,2 14,3
C3 13,5 12,3 28,0 20,5 15,0 17,2 16,2 24,3 15,4 14,8
C4 13,3 11,8 27,6 21,0 14,3 13,6 15,6 23,1 15,6 13,5
C5 13,3 11,7 25,3 22,2 14,0 14,5 15,0 21,0 16,3 13,5
C6 13,0 11,5 27,3 22,8 16,0 14,0 15,3 21,9 16,5 14,0
C7 13,1 11,9 27,0 21,3 16,4 13,7 15,8 25,3 16,5 14,7
C8 12,9 11,9 26,7 21,3 16,4 13,0 15,0 24,3 17,1 14,3
C9 12,9 11,7 28,4 22,1 15,1 16,1 14,5 24,9 17,4 14,4
C10 13,0 11,6 25,8 22,5 15,2 17,3 15,5 27,1 17,5 14,8
C11 13,0 11,4 26,0 22,8 15,2 13,9 16,0 21,6 16,0 14,8
C12 13,2 11,1 27,8 22,4 14,9 14,5 14,3 22,9 16,4 14,9


71
C13 13,2 11,5 26,0 22,4 14,8 14,0 16,4 24,4 16,2 15,3
H1 13,6 11,6 25,2 20,3 14,0 13,6 15,3 24,0 17,0 15,1
H2 13,5 11,6 29,1 20,8 13,4 14,8 14,2 23,2 15,4 13,7
H3 13,0 12,0 27,1 23,0 15,0 17,0 15,9 23,9 15,9 14,3
H4 13,5 12,0 28,4 21,0 15,7 16,5 15,9 21,8 15,7 14,2
H5 13,0 12,3 27,3 22,9 14,6 16,1 16,3 21,7 15,9 13,7
H6 12,7 11,3 26,8 21,3 14,1 13,9 16,6 25,2 16,4 14,4
H7 12,8 11,5 25,6 21,3 16,2 15,5 16,6 26,3 16,0 15,5
H8 13,0 11,7 28,5 20,1 16,0 16,1 17,0 26,0 17,2 15,0
H9 13,4 11,5 26,5 20,4 14,2 17,1 13,4 25,1 15,3 15,0
H10 13,3 11,5 28,0 21,0 15,0 14,0 15,2 24,3 15,8 14,4
H11 14,1 12,0 27,9 22,7 13,9 13,2 15,8 25,0 15,7 14,5
H12 12,4 11,9 26,8 23,1 13,9 14,7 15,0 21,9 16,4 14,9
H13 12,4 11,9 26,8 23,0 14,1 13,0 13,2 26,2 15,7 14,2
H14 12,8 11,2 27,7 21,9 14,2 15,1 15,8 22,0 16,0 14,8
V1 13,0 11,6 25,9 22,0 14,2 16,1 16,3 21,1 16,8 14,1
V2 13,5 11,7 26,3 20,2 15,3 15,4 12,9 24,5 16,3 13,8
V3 13,0 11,8 25,9 20,5 15,1 14,0 14,4 22,3 17,1 15,2
V4 12,5 12,0 26,7 20,9 13,7 16,2 15,1 25,9 17,0 15,0
V5 12,7 12,3 26,7 20,1 15,8 15,7 16,4 21,7 15,0 15,4
V6 12,9 12,1 28,1 23,0 15,0 13,5 15,3 24,8 16,3 14,8
V7 13,1 12,0 28,5 21,0 16,0 14,3 15,7 24,5 16,5 14,3
V8 13,0 11,7 28,9 21,8 13,5 17,0 13,7 24,5 16,9 14,2
V9 12,5 11,9 27,2 22,1 14,0 16,1 14,3 21,6 15,5 14,0
V10 12,7 11,8 26,0 20,9 14,9 15,2 14,0 21,1 15,8 15,2
V11 13,1 11,3 26,0 20,4 15,5 15,9 15,4 25,0 15,6 14,6



72
Ph lc 10: Hnh nh 4 loi vi khun khc phn lp c

Aeromonas spp. Staphylococcus spp.

Flavobacterium spp. Pseudomonas spp.










73
Ph lc 11: Mt s hnh nh trong qu trnh thc hin ti
a im thu mu c

Ao nui

Lng nui

Tin hnh th nghim

B th nghim Gy bnh thc nghim Ly mu c