Tap chi so 32, thang 10-2018. trUdng Dai hgc Tay Nguyen ISSN 1859-4611
H,N, TAI C A C C H O DAU M O I HUYEN DU'C T R O N G TINH L A M D O N G Dang Manh Hung', Hoang Thi Anh Phttffng\ Dinh Nam Lam^
Ngay nhan bai: 01/9/2018; Ngay phan bien thdng qua: 15/9/2018; Ngay duyet dang: 30/9/2018 TOM TAT
Nghien ciiu duge thuc hien nham khao sat sir luu hanh ciia virus ctim A types H^N,, HjN^ va H,N^j tren gia cam tai eho dau mdi huyen Diic Trgng, tinh Lam Ddng. Nghien ciiu tien hanh mgt nam va chia thanh 4 dgt liy mlu chinh, la thang 4 (dot 1), thang 5 (dot 2), diang 10 (dgt 3) va thang 11 (dgt 4). Tong 72 man swab tir hau hgng eua ga, vit va mdi trudng duge thu thap va xet nghiem virus ciira A vdi cac subtype Hj, N,, N^, H, va N^ bang phuang phap Real-time PCR. Ket qua cho thay ty le duong tinh vdi vims cum A cao 72,22% (52/72). Ty le duong tinh virus ciim A tren ga, vit va mdi trudng tuong lan lugt la 87,5% (21/24), 66,67% (16/24) va 62,5% (15/24). Ty Ie nhiem virus cum A d hai miia dSu cao, miia khd 77,78% (28/36) va miia mua la 66,67% (24/36). Khoang 7% (5/72) ducmg tinh vdi virus cum A type HjN^. Chua phat hien vi riit cum A type H^N, trong cae mau tu gia cam sdng ban tai chg va mau mdi trudng trong khoang thdi gian nghien cuu.
Tir khoa: virus. ciimA. H^^. H^^ H^^ PCR.
1. MO DAU
Trong nhiing nam gan day, nganh chan nudi ndi chung va chan nuoi gia cam ndi rieng dang ngay cang phat trien. Su phat trien dd da mang lai hieu qua kinh te tao nhieu ea hdi viec lam va nang cao ddi sdng vat chit va tinh thin cho ngudi dan, gdp phan xda ddi giam ngheo, tao co hgi vuon len lam giau cho nhieu gia dinh va gdp phan vao nen kinh te dat nude. Tuy nhien, ben canh su phat trien dd ciing cd rat nhieu thach thiie nhu su gia tang va diln bien phirc tap eua dich benh. Nhieu dich benh xay ra budc phai tieu huy toan dan gay thiet hai ldn cho ngudi chan nudi. Trong dd phai ke den benh eiim gia cim do vmis ciim types A/H^N,, A/
HjN^ va A/HjN,. Day la mot benh truyen nhilm nguy hiem, lay lan nhanh va ty le chet cao. La mgt trong nhimg trung tam giao dich chinh ve mua ban gia cam sdng, cac chg dau mdi huyen Diic Trgng eung dang tiem an nhiing nguy co din den su lay lan eiim gia cim. Nhara gdp phan vao viec nghien euu eae chung virus cum hien dang luu hanh, tao CO sd dl xay dung chuong trinh san xuat vaccine phu hgp vdi chimg virus luu hanh va xay dung ke hoach phdng chong phii hgp vdi dia phuong, ehiing tdi tien hanh thirc hien de tai: "Khao sat su luu hanh virus cum gia cam types A/H^N,, A/H;N^
va A/H,N, tai cac chg dau mdi huyen Diic Trpng tinh Lam Ddng".
2. NOI DUNG VA PHU'ONG PHAP NGHIEN CUti
2.1. Vdt lieu nghien ciru
Ddi tugng nghien cim: Cac chiing virus ciim A/
H5N,;A/H5N^vaA/H,N,
Thdi gian nghien cim: De tai thuc hien tir thang 01 den thang 12/2017. Tdng sd mlu lay trong thdi gian nghien cuu la 72 mau swab hau hgng ga, vit, mdi tnrdng, Thdi gian lay mSu duge ehia thanh 4 dgt, gdm thang 4 (dgt 1), thang 5 (dcrt 2), thang 10 (dgt 3) va thang 11/2017 (dgt 4).
Dia diem nghien ciiu: Man nghien cuu duge thu thap tai eac chg dau radi cua huyen Dire Trong.
Xac dmh chung virus duoc thuc hien tai Phdng Virus - Tram chan doan xet nghiem benh dgng vat - Chi cue Thu y viing V.
Nguyen lieu-
- Dung dich dem phdt phat (PBS); cae primers, probe do Trung tara chan doan Thii y Trung uong eung cap; ddi chiing duang tinh (+), doi chimg am tinh (-) eua chung cum H^N,,^; H,N,(Cuc Thii y, 2007).
- Thiet bi va dung cu trong phdng thi nghiem:
til lanh am -20°C, -SO^C; tii lanh thudng 2 - S^C;
budng an toan sinh hpc; he thong chiet xuit RNA bing may ly tam (may ly tam lanh dng Eppendofd 1,5 ml tdc do cao > 14000 rpm; may ly tam dng 15 rai - 50 rai; may ly tam nhd (Spin down); may trgn dng nghiem (vortex mixe); ong chay PCR 0,2 ml, dia chay PCR 96 gieng, trip chay PCR; bd kit chiet tach RNeasy Mini kit - Cat Qiagen. No. 74104 - 70106 do hang Qiagen eiia Diic san xuat.
/ ThS.. Chi cue Thu y vung V;
2 ThS, Khoa Chan nuoi thii y, Trudng Dai hgc Tdy Nguyen;
3 TS.. Khoa Chdn nuoi thuy, Trudng Dgi hgc Tdy Nguyen;
Tdc gid lien he: Dang Mgnh Himg, DT: 01674779779; Email- manhhungtyvl
Tap chi so 32, thang 10-2018, trUdng Dai hgc Tay Nguyen ISSN 1859-4611 2.2. Npi dung nghiin ciru
Xac dinh ty le chiing virus tai bdn thai diem trong nam, gom thang 4, 5, 10 va thang 11.
Xac dinh ty le nhiem virus ciira cac ddi tugng, gdm ga, vit va moi trudng.
Xac dinh ty le nhilm virus ciim theo miia vu.
2.3. Phuffng phdp nghien ciiu
Phuang phap lay miu Swab: Dung tam bdng vd triing ngoay sau viing hau, khi quan ciia vit, ngan, tam bdng sau khi ngoay cho vao dng chiia dung dich bao quan, day nap kin. Phuong phdp tien hdnh phdn img Real Time PCR:
Sau khi thu mau, tien hanh kiem tra mau va de lua chon mau dat yeu cau xet nghiem; tien hanh xet nghiem theo quy trinh cua phucmg phap RT
- PCR (QIAGEN, 2017). Hda chit duoc sir dung trong phan img Real Time RT - PCR la Taqman probe.
Trinh tu cac phan irng Realtime RT-PCR dg xac dinh cac gen gdm 4 budc: 1) Tach ARN vdi bd kit Qiagen (Hilden. Germany) theo hudng dan ciia nha san xuat; 2) ARN duge khucch dai vdi Invitrogen Superscript 3 qRT-PCR kit nham xac dinh su cd mat eiia gen M (matrix gene); 3) Cac mlu duong tinh vdi gen M duge tiep tuc xac dinh su cd mat cua gen H^ va H, bdi Invitrogen Superscript 3 qRT-PCR kit; 4) Cac mlu ducmg tinh vdi gen H^ duge xae dinh sy cd mat ciia gen N,; N^, cac mau ducmg tinh vdi H, duoc tilp tuc xac dinh su cd mat cita gen N^ bdi Invitrogen Superscript 3 qRT-PCR kit.
Primer va probe diing xac dinh nhiem virus ciim typeA/HgN|, A/H^N^ va A/H,N, PPP Name
(Source)
Primer/
Probe Sequence ( 5 ' - 3 ' )
5' FAM None None HEX TET None None FAM None None FAM None None FAM None None FAM None None
3' BHQl None None BHQl BHQl None None BHQl None None BHQl None None BHQl None None BHQl None None M-4
(CDC)
H5-3S (AAHL+
Probe HA 2-3)
NI-2 (China)
H7-6 (CODA)
N9-1 (CNIC)
Probe TGCAOTCCTCGCTCACTGGGCACG Forward CATGGARTGGCTAAAGACAAGACC Reverse AGGGCATTTTGGACAAAKCGTCTA Probe TCAACAGTOGCGAGTTCCCTAGCA Probe TCAACAGTTGCGAGTTCTCTAGCA Forward ACG TAT GAC TAC CCG CAG TAT TCA Reverse AGA CCA GCT ACC ATG ATT GC Probe TGOTCTTGGCCAGACGGTGC Forward TGGACTAOTGGGAGCAGCAT Reverse TGTCAATGGTTAAGGGCAACTC Probe CCCCACCAATGGGAACTG Forward TCTAGGAATGCAAACCCTTTTACC Reverse CCAATAACAGGAGGGAGCCCAGACCC Probe TGOTTTAGCTTCGGGGCATCATG Forward GYAGYGGYTACAAAGATGTG Reverse GAAGACAAGGCCCATTGCAA
Probe AGACAATCCCCGACCGAATGAATGACCC Forward TAGCAATGACACACACTAGTCAAT Reverse ATTACCTGGATAAGGGTCATTACACT
2.4. PhiroTigphdp xii iy so lieu
Phan meni Excel diing xu ly th6ng ke va quan Iy so lieu. Phan mem Minitab 17 diing so sanh su khac biet ctia mau nhifim virus cum giira 4 dot, giiia mlu nhilm tir ga, vit, moi trucmg, va giira miia mua va miia kho bang phep thir Turkey vai dp tin cay 95%.
3. KET QUA VA THAO LUAN
S.l. Sir Imi hdnh chiing virus cum tai cdc tlitfi diem lihdc nhau trong ndm
Chijng toi tien hanh khao sat su Itm hanh eiia caechiingviruseLimAtaibon thai diem khae nhau trong nam, ket qua duac ghi nhan a bang sau:
Tap chi so 32, thang IQ-2Q18, trtt6ng Dai hoc Tay Nguyen ISSN 1859-4611 Bang I, Ty le chdng virus phat hien tai bon thM diem dfft 1 (thang 4), dot 2 (thang 5), dot 3
(thang 10) va dot 4 (thang 11)
Dot 1 (n=18) Dfft 2 (n=18) Dfft 3 (n=18) Dot4(n=18) Mau
nhiem Tyle(%) Mau
nhiem Ty le (%) Mau
nhiem Ty le (%) Mau
T y l e ( % ) Ciim A
H, N, N, H, N,
15 0 0 0 0 0
83,33 0,00' 0,00 0,00' 0,00 0,00 .
13 0 0 0 0 0
72,22 0,00'' 0,00 0,00' 0,00 0,00
II 0 0 0 0 0
61,11 0,00' 0,00 0,00' 0,00 0,00
13 7 0 5 0 . 0
72,22 38,89' 0,00 27,78"
0,00 0,00 Ghi chu. cdc chir cdi khde nhau trong mot hdng
Ket qua phan tich bang 1 cho thay su luu hanh chiing virus cum A trong 4 dgt khdng cd su khac biet. Ket qua nghien cuu ghi nhan rang ty Ie luu hanh virus ciim A tuang ddi cao trong ni 61,11 % - 83,33%/ nam. Nghien ciiu ciia cac tac gia Nguyen Tiln Dung va cs (2005), Cin Xuan Minh (2012), cting cho thay ty le luu hanh ddi vdi eiim gia cam type A cao hon 60% qua cac nam. Chimg tdi nhan thay 3 dot dau lay mau xet nghiem khdng phat hien mau nao duong tinh vdi subtype H^ va H,.
CO y nghia thong ke (P<0.05).
Tuy nhien, trong dgt 4 chiing tdi phat hien duge 7/18 (38,89%) miu ducmg tinh vdi subtype H^
trong dd 5 (27,78%) mlu duong tinh vdi subtype Nj va khdng phat hien mau duong vai subtype N^
(P<0.05).
3.2. Su luu hdnh virus cum trin ga, vit va moi tru&ng
Mau thu thap trong khao sat chiing virus cum A tai cac chg dau mdi huyen Dire Trgng, gdm ga, vjt va mdi trudng. Ket qua duge trinh bay d bang 2:
Bang 2. Ty Ic lini hanh virus cum tir cac mau thu thap tren ga, vit va moi truoTig
CiimA H, N, N.
H, N,
Mau nt 21
5 0 3 0 0
Ga(n^
liem
=24) Ty le (%)
87,50 20,83' 0,00 12,50"
0,00 0,00
Vit (n Mau nhiem
16 2 0 2 0 0
=24) T y l e ( % )
66,67 8,33'' 0,00 8,33"
0,00 0,00
Moi truffng (n=24) Mau nhiem
15 0 0 0 0 0
T y l e ( % ) 62,50 0,00' 0,00 0,00' 0,00 0,00 Ghi chu. cdc chu cdi khac nhau trong mgt hdng cd y nghta thong ke (P< 0.05)
Ket qua cho thiy khdng cd su khac biet ciia mlu nhilm virus cum A va subtype N^ tir ga, vit va mdi tmdng. Tuy nhien ket qua ghi nhan duge ty le nhiem virus ciim A tuong ddi cao tir 62,5% - 87,5% tren ba Ioai mau xet nghiem tu ga (21/24), vjt (16/24) va mdi trudng (15/24) (bang 2). Ty le nhiem ciira A subtype H^N^ da duge phat hien trong dgt 4, tren ga la 20,83% va 12,5%, tren vit la 8,33% (H,va N^_). Virus cum A subtype H,N, khdng xuat hien d tat ca cac mlu xet nghiem.
3.3. Suluu hdnh virus ciim theo miia vu Khao sat su luu hanh cum A tren mau nghien Cliu duge tien hanh theo hai miia, miia khd va miia mua. Ket qua duge trinh bay d bang 3:
Bang 3. Ty le liru hanh virus cum theo mua vu, mija kho (thang 4-5) va miia mua (thang 10-11)
Cum A H, N, N, H, N.
Miia kho (n=36) Mau nhiem
28 0 0 0 0 0
Tyle 77,78
(%)
0,00' 0,00 0,00' 0,00 0,00
MiJa mira Mau nhiem
24 7 0 5 0 0
I (n=36) TylS 66,67
(%)
19,44' 0,00 13,89"
0,00 0,00 Ghi chii: cdc chir cdi khde nhau trong mgl hdng cdy nghia thdng ke (P<0.05).
Tap chi so 32, thang 10-2018, tril6ng Dai hpc Tay Nguyen ISSN 1859-4611 Ty Ie nhiem virus cum A tucmg dot cac 6 cac 4). Ty Ie nhiem virus cum A tren ga, vit va moi miia trong nam, miia kho la 77,78% (28/36) va miia tnrang tucmg doi cao lan luot vcri ty le la 87,5%
mua la 66,67% (24/36) (P>0.05) (bang 3). Theo tac (21/24), 66,67% (16/24)va 62;5% (15/24), trong gia va Manh Cuong va cs (2013), cho thiy ty Ic do mot so miu ga va vit phat hien ducmg tinh voi luu hanh d6i voi cum gia dm type A la eao quanh subtype H,, N^. Ty le mau duong tinh virus ciim nam. Chiing toi ghi nhan duoe 7/36 (19,44%) mlu A Cf hai miia deu eao, miia kho 77,78% (28/36) ya duong tinh subtype H^ trong do 5 mlu duong tinh mtia mua la 66,67% (24/36), trong do mot s6 mlu subtype N (13,89%), dSu dien ra trong miia mua. duong tinh vcri subtype Hj, N, duac ghi nhan vao Trong miia kho kbong phat hien mau nao duang miia mua.
tinh vai eae subtype ciia virus cum A. 4,2. Di nghi
4. KET LUAN Tiep tue cac chucmg trinh giam sat doi vdi ciim 4.1. Kit luan gia clm A/H^N,; A/H^N^; A/H^N^ tren gia cam va Ty le nhiSm virus ciim A cao tir 61,11% - *uy clm ban t?i cac chp dlu m6i tinh Lam D6ng 83,33%/ nam, trong do mot s6 mlu duong tinh voi vao eac nam tiep theo. Tien hanh nghien cuu eac subtype H„ N, duoc phat hien vao thang 11 (dot subtype H va N khac cua vi nit ciim type A tren
dan gia cam va thiiy cam ban tai chg dau mdi.
SURVEY ON PREVALENCE OF AVIAN ENFLUENZA VIRUSES TYPES A/H^N,, A/H,N, AND.
A/H,N, AT WHOLESALE MARKETS IN DUC TRONG DISTRICT, LAM DONG PROVINCE
Dang Manh Hung^, Hoang Thi Anh PhuongS Dinh Nam Lam' Received Date: 01/9/2018; Revised Date: 15/9/2018; Accepted for Publication: 30/9/2018
SUMMARY
The study was conducted to survey the prevalence of influenza virus oftypesA/HjN|, A/H^N^andA/
H^N, in poultry at wholesale markets in Due Trong district, Lam Dong province. The research took one year with four periods of collecting samples, including April (first stage). May (second stage) October (third stage) and November (fourth stage). A total of 72 swab samples frorapharynxof chickens, ducks and environment were collected and examined with A influenza virus of subtypes H^, N,, N^, H,, and N, with Real-time PCR method. The findings show a highly positive percentage with A influenza vims 72,22% (52/72). The positive rates with A influenza virus in chickens, ducks and environment are 87,5% (21/24), 66,67% (16/24) and 62,5% (15/24), respectively The A-intiuenza infection proportion is high in both seasons, in dry season with 77,78% (28/36) and in rainy season with 66,67% (24/36).
Approximately 7% (5/72) is positive to type A/H5N6 influenza virus. Type A/H7N9 was not detected in samples of living poultry on sale at the markets and in environment samples during the study period.
Keywords: avian fluenza. virus, H^^, H^^, H/^^ PCR.
TAI LIEU THAM KHAO
Can Xuan Minh (2012), "Mgt so dgc diem dich ti hgc benh ciim gia cdm vd gidm sal khdng the khdng virus cdm A/H5N1 a ddn gd nudi tgi mgt sd huyen ngoai thdnh Hd Ndi ". Luan van Thac sy Ndng nghiep, Trudng Dai hoc Ndng nghiep Ha Ngi, 46 - 51.
Cue Thu y (2007), "So tay chdn dodn cum gia cdm "
NguySn Tien Dung, Nguyin Thuy Dien, D6 Quy Phirong, Dao Thanh Van, Biii NghTa Vugng, Nguyin Th6 Vinh (2005) "Gidm sdt benh cum gia cdm tai Thai Binh ". Tap chi Khoa hoc ky thuat Thii y XII, 2 , 6 - 1 2 .
QIAGEN (2017) "Sdtay Qiagen Rneasy Mini Kit".
Vd Manh Cuong, Nguyen Thi Thanh Thuy, Dinh Nam Lara (2013), "Ddnh gid luu hdnh virus cum A/
H5NI trin thiiy cdm sdng tai cdc cha a mgt sd linh miin Bdc bdng phuang phdp Realtime RT - PCR ", Tap chi Y hgc du phdng. XXIII, 12, 77 - 83.
4 MSc, y Regional Veterinary Office;
5 MSc, Falculty of Animal Science and Veterinary Medicine, Tay Nguven University, 6 PhD, Falculty of Animal Science and Veterinary Medicine. Tay Nguyen University;
Corresponding author- Dang Manh Hung; Tel: 01674779779: Email, [email protected].
