Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China

Chen, Youlu; Ma, Yijuan; Fu, Yong; Mohamed, Mohamed; Sun, Tiancong; Shen, Xiuying; Duo, Hong; Guo, Zhihong; 张, 学勇; Jiunai, Gengqiu

doi:10.9775/kvfd.2020.25067

Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China

Hong DUO, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Yong FU, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Xiuying SHEN, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Zhihong GUO, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Xueyong ZHANG, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Yijuan MA, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Mohamed YOSRI, (Al-Azhar University, The Regional Center for Mycology and Biotechnology, Egypt)

Tiancong SUN, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Youlu CHEN, (Qinghai University, State Key Laboratory of Plateau Ecology and Agriculture, Qinghai Academy of Animal Science and Veterinary Medicine, China)

Gengqiu JIUNAI (Animal Husbandry and Veterinary Station, Zhenqin Township, China)

Kafkas Üniversitesi Veteriner Fakültesi Dergisi

2 1

Yıl: 2021 Cilt: 27 Sayı: 2 Sayfa Aralığı: 203 - 208 Metin Dili: İngilizce DOI: 10.9775/kvfd.2020.25067 İndeks Tarihi: 03-10-2021

Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China

Öz:

The unique geographical and ecological conditions of the Qinghai-Tibetan Plateau (QTP) constitute the environmental conditions of the natural foci of cystic echinococcosis (CE). It is a rare and highly endemic area in the world. The main host of the pathogen, Tibetan sheep, is widely distributed, and the population density is on the rise in recent years, which increases the pressure on the transmission and risk of the natural epidemic source. Currently, the developments of serological diagnosis test for Echinococcus granulosus infection on the QTP Tibetan sheep are scanty, which seriously restricts the epidemiological investigation of CE in Tibetan sheep on the QTP. EgAgB8/2 is one of the most important targets of host antibody response in CE and has better diagnostic performance in livestock. This study expressed the recombinant EgAgB8/2 antigen of E. granulosus from Tibetan sheep in prokaryotic expression vector, and also preliminarily evaluated its potential value for diagnosing CE in Tibetan sheep using indirect ELISA. Our preliminary results shown that the recombinant EgAgB8/2 antigen had good immunogenicity and exhibited high sensitivity (95%) and high specificity (100%) and no cross-reacted with both Taenia multiceps and Taenia hydatigena. Further studies are needed to collect more Tibetan sheep sera from E. granulosus and other parasitic infections, which may evaluate further sensitivity, specificity and cross-reactive of recombinant EgAgB8/2 antigen in Tibetan sheep positive for CE of the QTP, China.

Anahtar Kelime:

Çin’in Qinghai-Tibet Platosu’ndaki Tibet Koyunlarında Kistik Ekinokokkozisin Tanısında Rekombinant EgAgB8/2 Antijeninin Değerlendirilmesi

Öz:

Qinghai-Tibet Platosu’nun eşsiz coğrafyası ve ekolojisi, kistik ekinokokkozisin (KE) doğal odaklarının çevresel koşullarını oluşturur. Burası dünyadanadir bulunan ve oldukça endemik bir bölgedir. Patojenin ana konakçısı olan Tibet koyununun yaygınlığı ve popülasyon yoğunluğu son yıllardaartmaktadır ve bu durum doğal salgın kaynağı riskini ve bulaş üzerindeki baskıyı artırmaktadır. Günümüzde, Qinghai-Tibet Platosu’ndaki Tibetkoyunlarında Echinococcus granulosus enfeksiyonunun serolojik tanı testlerindeki gelişmeler yetersizdir ve bu, bölgede Tibet koyunlarında KE’ayönelik epidemiyolojik araştırmaları ciddi şekilde kısıtlamaktadır. EgAgB8/2, KE’da konakçı antikor yanıtının en önemli hedefl erinden birisidir ve çiftlikhayvanlarında enfeksiyonun teşhisinde daha iyi bir performansa sahiptir. Bu çalışmada, prokaryotik vektör kullanılarak Tibet koyunlarından elde edilenE. granulosus’un rekombinant EgAgB8/2 antijeninin ekspresyonu sağlandı ve bu antijenin Tibet koyunlarında KE’un teşhisinde kullanım potansiyelineyönelik ön değerlendirmesi indirekt ELISA ile gerçekleştirildi. Çalışmamıza ait ön bulgular, rekombinant EgAgB8/2 antijeninin iyi bir immünojeniteyesahip olduğunu, yüksek duyarlılık (%95) ve özgüllük (%100) sergilediğini ve hem Taenia multiceps hem de Taenia hydatigena ile çapraz reaksiyonvermediğini gösterdi. E. granulosus ve diğer parazitik etkenlerle enfekte daha fazla Tibet koyunundan toplanacak serumlarla, rekombinant EgAgB8/2antijeninin duyarlılık, özgüllük ve çapraz reaksiyonlarının değerlendirmesine yönelik daha fazla çalışma yapılmasına ihtiyaç vardır.

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

1. Deplazes P, Rinaldi L, Rojas CAA, Torgerson PR, Harandi MF, Romig T, Antolova D, Schurer JM, Lahmar S, Cringoli G, Magambo J, Thompson RCA, Jenkins EJ: Global distribution of alveolar and cystic echinococcosis. Adv Parasitol, 95, 315-493, 2017. DOI: 10.1016/ bs.apar.2016.11.001
2. Zhang W, Zhang Z, Wu W, Shi B, Li J, Zhou X, Wen H, McManus DP: Epidemiology and control of echinococcosis in central Asia, with particular reference to the People’s Republic of China. Acta Trop, 141, 235- 243, 2015. DOI: 10.1016/j.actatropica.2014.03.014
3. Wang Q, Huang Y, Huang L, Yu W, He W, Zhong B, Li W, Zeng X, Vuitton DA, Giraudoux P, Craig PS, Wu W: Review of risk factors for human echinococcosis prevalence on the Qinghai-Tibet Plateau, China: A prospective for control options. Infect Dis Poverty, 3:3, 2014. DOI: 10.1186/2049-9957-3-3
4.Qian MB, Abela-Ridder B, Wu WP, Zhou XN: Combating echinococcosis in China: Strengthening the research and development. Infect Dis Poverty, 6:161, 2017. DOI: 10.1186/s40249-017-0374-3
5. Han XM, Cai QG, Wang W, Wang H, Zhang Q, Wang YS: Childhood suffering: Hyper endemic echinococcosis in Qinghai-Tibetan primary school students, China. Infect Dis Poverty, 7:71, 2018. DOI: 10.1186/ s40249-018-0455-y
6. Li K, Zhang L, Zhang H, Lei Z, Luo H, Mehmood K, Shahzad M, Lan Y, Wang M, Li J: Epidemiological investigation and risk factors of Echinococcus granulosus in yaks (Bos grunniens), Tibetan pigs and Tibetans on Qinghai Tibetan plateau. Acta Trop, 173, 147-152, 2017. DOI: 10.1016/j. actatropica.2017.06.019
7. Thompson RCA: Biology and systematics of echinococcus. Adv Parasitol, 95, 65-109, 2017. DOI: 10.1016/bs.apar.2016.07.001
8. Hernández-González A, Sánchez-Ovejero C, Manzano-Román R, Sánchez MG, Delgado JM, Pardo-García T, Soriano-Gálvez F, Akhan O, Cretu CM, Vutova K, Tamarozzi F, Mariconti M, Brunetti E, Vola A, Fabiani M, Casulli A, Siles-Lucas M: Evaluation of the recombinant antigens B2t and 2B2t, compared with hydatid fluid, in IgG-ELISA and immunostrips for the diagnosis and follow up of CE patients. PLoS Negl Trop Dis, 12 (9): e0006741, 2018. DOI: 10.1371/journal.pntd.0006741
9. Rialch A, Raina OK, Tigga MN, Anandanarayanan A, Ganaie ZA, Aftab A, Lalrinkima H, Singh MN, Varghese A, Samanta S, Banerjee PS, Singh P, Verma MR: Evaluation of Echinococcus granulosus recombinant EgAgB8/1, EgAgB8/2 and EPC1 antigens in the diagnosis of cystic echinococcosis in buffaloes. Vet Parasitol, 252, 29-34, 2018. DOI: 10.1016/j. vetpar.2018.01.020
10. Yang YR, McManus DP, Huang Y, Heath DD: Echinococcus granulosus infection and options for control of cystic echinococcosis in Tibetan communities of Western Sichuan Province, China. PLoS Negl Trop Dis, 3 (4): e426, 2009. DOI: 10.1371/journal.pntd.0000426
11. Hu XJ, Yang J, Xie XL, Lv FH, Cao YH, Li WR, Liu MJ, Wang YT, Li JQ, Liu YG, Ren YL, Shen ZQ, Wang F, Hehua E, Han JL, Li MH: The genome landscape of Tibetan sheep reveals adaptive introgression from Argali and the history of early human settlements on the QinghaiTibetan Plateau. Mol Biol Evol, 36 (2): 283-303, 2019. DOI: 10.1093/ molbev/msy208
12. Lightowlers MW, Lawrence SB, Gauci CG, Young J, Ralston MJ, Maas D, Heath DD: Vaccination against hydatidosis using a defined recombinant antigen. Parasit Immunol, 18 (9): 457-462, 1996. DOI: 10.1111/ j.1365-3024.1996.tb01029.x
13. Mamuti W, Sako Y, Nakao M, Xiao N, Nakaya K, Ishikawa Y, Yamasaki H, Lightowlers MW, Ito A: Recent advances in characterization of Echinococcus antigen B. Parasitol Int, 55, S57-S62, 2006. DOI: 10.1016/j. parint.2005.11.008
14. Haag KL, Alves-Junior L, Zaha A, Ayala FJ: Contingent, nonneutral evolution in a multicellular parasite: Natural selection and gene conversion in the Echinococcus granulosus antigen B gene family. Gene, 333, 157-167, 2004. DOI: 10.1016/j.gene.2004.02.027
15. Rott MB, Fernandez V, Farias S, Ceni J, Ferreira HB, Haag KL, Zaha A: Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: Gene sequences, expression in Escherichia coli and serological evaluation. Acta Trop, 75 (3): 331-340, 2000. DOI: 10.1016/ s0001-706x(00)00069-3
16. Virginio VG, Hernandez A, Rott MB, Monteiro KM, Zandonai AF, Nieto A, Zaha A, Ferreira HB: A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease. Clin Exp Immunol, 132 (2): 309-315, 2003. DOI: 10.1046/j.1365-2249.2003.02123.x
17. Golassa L, Abebe T, Hailu A: Evaluation of crude hydatid cyst fluid antigens for the serological diagnosis of hydatidosis in cattle. J Helminthol, 85 (1): 100-108, 2011. DOI: 10.1017/S0022149X10000349
18. Moro P, Verastegui M, Gilman RH, Falcon N, Bernal T, Gavidia C, Gonzalez A, Malqui V, Moro MH, Dueger E: Enzyme-linked immunoelectrotransfer blot assay for diagnosis of hydatidosis (Echinococcus granulosus) in sheep. Vet Rec, 140 (23): 605-606, 1997.
19. Ibrahem MM, Rafiei A, Dar FK, Azwai SM, Carter SD, Craig PS: Serodiagnosis of cystic echinococcosis in naturally infected camels. Parasitology, 125, 245-251, 2002. DOI: 10.1017/s0031182002002044
20. Bashiri S, Nemati Mansoor F, Valadkhani Z: Expansion of a highly sensitive and specific ELISA test for diagnosis of hydatidosis using recombinant EgB8/2 protein. Iran J Basic Med Sci, 22 (2): 134-139, 2019. DOI: 10.22038/ijbms.2018.29024.7021
21. Hernández-González A, Santivañez S, García HH, Rodríguez S, Muñoz S, Ramos G, Orduña A, Siles-Lucas M: Improved serodiagnosis of cystic echinococcosis using the new recombinant 2B2t antigen. PLoS Negl Trop Dis, 6 (7): e1714, 2012. DOI: 10.1371/journal.pntd.0001714
22. Wang J, Wang N, Hu D, Zhong X, Wang S, Gu X, Peng X, Yang G: Genetic diversity of Echinococcus granulosus in Southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene. Sci World J, 2014:867839, 2014. DOI: 10.1155/2014/867839
23. Song X, Yan M, Hu D, Wang Y, Wang N, Gu X, Peng X, Yang G: Molecular characterization and serodiagnostic potential of a novel dithiol glutaredoxin 1 from Echinococcus granulosus. Parasit Vectors, 9:456, 2016. DOI: 10.1186/s13071-016-1741-9
24. Wu M, Yan M, Xu J, Yin X, Dong X, Wang N, Gu X, Xie Y, Lai W, Jing B, Peng X, Yang G: Molecular characterization of triosephosphate isomerase from Echinococcus granulosus. Parasitol Res, 117 (10): 3169- 3176, 2018. DOI: 10.1007/s00436-018-6015-3
25. Jacobson RH: Validation of serological assays for diagnosis of infectious diseases. Rev Sci Tech, 17 (2): 469-526, 1998. DOI: 10.20506/ rst.17.2.1119
26. Brunetti E, Kern P, Vuitton DA, Writing Panel for the WHO-IWGE: Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Trop, 114 (1): 1-16, 2010. DOI: 10.1016/j. actatropica.2009.11.001
27. Kordafshari S, Hosseini SH, Jalousian F, Rajabibazl M, Jones M, Etebar F: Evaluation of dot immunogold filtration assay (DIGFA) by recombinant protein EPC1 for anti-Echinococcus granulosus IgG antibody. Iran J Parasitol, 10 (1): 30-38, 2015.
28. Mcmanus DP: Immunodiagnosis of sheep infections with Echinococcus granulosus: In 35 years where have we come? Parasit Immunol, 36 (3), 125- 130, 2013. DOI: 10.1111/pim.12072
29. Fathi S, Jalousian F, Hosseini SH, Najafi A, Darabi E, Koohsar F: Design and construction of a new recombinant fusion protein (2b2t+EPC 1) and its assessment for serodiagnosis of cystic echinococcosis. Apmis, 126 (5): 428-439, 2018. DOI: 10.1111/apm.12838
30. Bart JM, Knapp J, Gottstein B, El-Garch F, Giraudoux P, Glowatzki ML, Berthoud H, Maillard S, Piarroux R: EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis. Infect Genet Evol, 6 (5): 390-400, 2006. DOI: 10.1016/j.meegid.2006.01.006
31. Torgerson PR, Heath DD: Transmission dynamics and control options for Echinococcus granulosus. Parasitology, 127, S143-S158, 2003. DOI: 10.1017/s0031182003003810
32. Manzano-Roman R, Sanchez-Ovejero C, Hernandez-Gonzalez A, Casulli A, Siles-Lucas M: Serological diagnosis and follow-up of human cystic echinococcosis: A new hope for the future? Biomed Res Int, 2015:428205, 2015. DOI: 10.1155/2015/428205
33. McManus DP: Immunodiagnosis of sheep infections with Echinococcus granulosus: In 35 years where have we come? Parasite Immunol, 36 (3): 125-130, 2014. DOI: 10.1111/pim.12072
34. Juyi L, Yan J, Xiufang W, Zhaoqing Z, Junliang L, Mingxing Z, Wei Z: Analysis of the chemical components of hydatid fluid from Echinococcus granulosus. Rev Soc Bras Med Trop, 46 (5): 605-610, 2013. DOI: 10.1590/0037-8682-0154-2013
35. Lightowlers MW, Gottstein B: Echinococcosis/hydatidosis: Antigens, immunological and molecular diagnosis. Cab International, 1995.
36. Maddison SE, Slemenda SB, Schantz PM, Fried JA, Wilson M, Tsang VCW: A specific diagnostic antigen of Echinococcus granulosus with an apparent molecular weight of 8 kDa. Am J Trop Med Hyg, 40 (4): 377-383, 1989. DOI: 10.4269/ajtmh.1989.40.377
37. Abdi J, Kazemi B, Mohebali M, Bandehpour M, Rahimi MT, Rokni MB: Gene cloning, expression and serological evaluation of the 12-kDa antigen-B subunit from Echinococcus granulosus. Ann Trop Med Parasitol, 104 (5): 399-407, 2010. DOI: 10.1179/136485910X12743554760261
38.Chen X, Chen X, Lu X, Feng X, Wen H: The production and comparative evaluation of native and recombinant antigens for the fast serodiagnosis of cystic echinococcosis with dot immunogold filtration assay. Parasit Immunol, 37 (1): 10-15, 2015. DOI: 10.1111/pim.12151
39. Khalilpour A, Sadjjadi SM, Moghadam ZK, Yunus MH, Zakaria ND, Osman S, Noordin R: Lateral flow test using Echinococcus granulosus native antigen B and comparison of IgG and IgG4 dipsticks for detection of human cystic echinococcosis. Am J Trop Med Hyg, 91 (5): 994-999, 2014. DOI: 10.4269/ajtmh.14-0170
40. Golassa L, Abebe T, Hailu A: Evaluation of crude hydatid cyst fluid antigens for the serological diagnosis of hydatidosis in cattle. J Helminthol, 85 (1): 100-108, 2011. DOI: 10.1017/S0022149X10000349
41. Zhong X, Wang N, Hu D, Wang J, Liu T, Gu X, Wang S, Peng X, Yang G: Sequence analysis of cytb gene in Echinococcus granulosus from Western China. Korean J Parasitol, 52 (2): 205-209, 2014. DOI: 10.3347/ kjp.2014.52.2.205
42. Hu D, Song X, Wang N, Zhong X, Wang J, Liu T, Jiang Z, Dawa T, Gu X, Peng X, Yang G: Molecular identification of Echinococcus granulosus on the Tibetan Plateau using mitochondrial DNA markers. Genet Mol Res, 14 (4): 13915-13923, 2015. DOI: 10.4238/2015.October.29.12
43. Liu CN, Lou ZZ, Li L, Yan HB, Blair D, Lei MT, Cai JZ, Fan YL, Li JQ, Fu BQ, Yang YR, McManus DP, Jia WZ: Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus using a multiplex PCR assay. PLoS Negl Trop Dis, 9 (9): e0004084, 2015. DOI: 10.1371/journal. pntd.0004084
44. Ma J, Wang H, Lin G, Zhao F, Li C, Zhang T, Ma X, Zhang Y, Hou Z, Cai H, Liu P, Wang Y: Surveillance of Echinococcus isolates from Qinghai, China. Vet Parasitol, 207 (1-2): 44-48, 2015. DOI: 10.1016/j. vetpar.2014.11.012
45. Yang YR, Rosenzvit MC, Zhang LH, Zhang JZ, McManus DP: Molecular study of Echinococcus in west-central China. Parasitology, 131, 547-555, 2005. DOI: 10.1017/S0031182005007973
46. Yan N, Nie HM, Jiang ZR, Yang AG, Deng SJ, Guo L, Yu H, Yan YB, Tsering D, Kong WS, Wang N, Wang JH, Xie Y, Fu Y, Yang DY, Wang SX, Gu XB, Peng XR, Yang GY: Genetic variability of Echinococcus granulosus from the Tibetan plateau inferred by mitochondrial DNA sequences. Vet Parasitol, 196 (1-2): 179-183, 2013. DOI: 10.1016/j.vetpar.2013.02.010
47. Tawfeek GM, Elwakil HS, El-Hoseiny L, Thabet HS, Sarhan RM, Awad NS, Anwar WA: Comparative analysis of the diagnostic performance of crude sheep hydatid cyst fluid, purified antigen B and its subunit (12 Kda), assessed by ELISA, in the diagnosis of human cystic echinococcosis. Parasitol Res, 108 (2): 371-376, 2011. DOI: 10.1007/ s00436-010-2074-9

APA	Duo H, Fu Y, Shen X, Guo Z, 张学, Ma Y, Mohamed M, Sun T, Chen Y, Jiunai G (2021). Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. , 203 - 208. 10.9775/kvfd.2020.25067
Chicago	Duo Hong,Fu Yong,Shen Xiuying,Guo Zhihong,张学勇,Ma Yijuan,Mohamed Mohamed,Sun Tiancong,Chen Youlu,Jiunai Gengqiu Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. (2021): 203 - 208. 10.9775/kvfd.2020.25067
MLA	Duo Hong,Fu Yong,Shen Xiuying,Guo Zhihong,张学勇,Ma Yijuan,Mohamed Mohamed,Sun Tiancong,Chen Youlu,Jiunai Gengqiu Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. , 2021, ss.203 - 208. 10.9775/kvfd.2020.25067
AMA	Duo H,Fu Y,Shen X,Guo Z,张学,Ma Y,Mohamed M,Sun T,Chen Y,Jiunai G Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. . 2021; 203 - 208. 10.9775/kvfd.2020.25067
Vancouver	Duo H,Fu Y,Shen X,Guo Z,张学,Ma Y,Mohamed M,Sun T,Chen Y,Jiunai G Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. . 2021; 203 - 208. 10.9775/kvfd.2020.25067
IEEE	Duo H,Fu Y,Shen X,Guo Z,张学,Ma Y,Mohamed M,Sun T,Chen Y,Jiunai G "Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China." , ss.203 - 208, 2021. 10.9775/kvfd.2020.25067
ISNAD	Duo, Hong vd. "Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China". (2021), 203-208. https://doi.org/10.9775/kvfd.2020.25067

APA	Duo H, Fu Y, Shen X, Guo Z, 张学, Ma Y, Mohamed M, Sun T, Chen Y, Jiunai G (2021). Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 27(2), 203 - 208. 10.9775/kvfd.2020.25067
Chicago	Duo Hong,Fu Yong,Shen Xiuying,Guo Zhihong,张学勇,Ma Yijuan,Mohamed Mohamed,Sun Tiancong,Chen Youlu,Jiunai Gengqiu Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 27, no.2 (2021): 203 - 208. 10.9775/kvfd.2020.25067
MLA	Duo Hong,Fu Yong,Shen Xiuying,Guo Zhihong,张学勇,Ma Yijuan,Mohamed Mohamed,Sun Tiancong,Chen Youlu,Jiunai Gengqiu Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, vol.27, no.2, 2021, ss.203 - 208. 10.9775/kvfd.2020.25067
AMA	Duo H,Fu Y,Shen X,Guo Z,张学,Ma Y,Mohamed M,Sun T,Chen Y,Jiunai G Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2021; 27(2): 203 - 208. 10.9775/kvfd.2020.25067
Vancouver	Duo H,Fu Y,Shen X,Guo Z,张学,Ma Y,Mohamed M,Sun T,Chen Y,Jiunai G Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2021; 27(2): 203 - 208. 10.9775/kvfd.2020.25067
IEEE	Duo H,Fu Y,Shen X,Guo Z,张学,Ma Y,Mohamed M,Sun T,Chen Y,Jiunai G "Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China." Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 27, ss.203 - 208, 2021. 10.9775/kvfd.2020.25067
ISNAD	Duo, Hong vd. "Evaluation of the Recombinant EgAgB8/2 Antigen for the Diagnosisof Cystic Echinococcosis in Tibetan Sheep on the Qinghai-TibetanPlateau, China". Kafkas Üniversitesi Veteriner Fakültesi Dergisi 27/2 (2021), 203-208. https://doi.org/10.9775/kvfd.2020.25067