检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2023.05.027

Abstract

Abstract: This study aimed at establishing an indirect-ELISA method for the titer detection of Mycoplasma gallisepticum (MG) antibodies in chicken, which could provide a set of effective technology for large-scale clinical monitoring of MG infection combined with HI test established. Prokaryotic-expressed protein VlhA3.03 (rVlhA) was obtained as envelope antigen. The linear equation lg (antibody titer)=1.257×lg (S/P value at 1:500) +3.709 of serum antibody titer and S/P value at 1:500 was established by a single serum dilution test and the correlation coefficient (R²) was 0.917 6. The cutoff value of negative and positive S/P was determined to be 0.32, and the corresponding titer is 1 200. The rVlhA-ELISA showed good specificity and repeatability. The detection limit of MG positive sera is 1:2 000. An HI assay was also established using domestic MG isolate SH/2020-1 as hemagglutination inhibition test antigen. The combined rVlhA-ELISA method and HI test, and IDEXX-MG were compared in serum sample detection, with RT-qPCR method to monitor MG infection. The results showed that the trend of serum antibody monitored by rVlhA-ELISA and IDEXX-MG was consistent with the result of HI assay. The coincidence rate between combined rVlhA-ELISA and HI, and IDEXX-MG was 91.53%. These results indicated that the rVlhA-ELISA and HI test established in this study were beneficial to clinical application, which can be applied to large-scale and rapid clinical screening of MG infection and recheck the results.

Key words: Mycoplasma gallisepticum, variable lipoprotein haemagglutinin, antibody detection, indirect enzyme-linked immunosorbent assay

CLC Number:

S852.62

CHEN Yang, MENG Linchun, GUO Mengjiao, ZHANG Chengcheng, BO Zongyi, CHU Dianfeng, CAO Yongzhong, WU Yantao, ZHANG Xiaorong. Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2062-2072.

References 29

[1]	LEVISOHN S, KLEVEN S H. Avian mycoplasmosis (Mycoplasma gallisepticum)[J]. Rev Sci Tech, 2000, 19(2):425-442.
[2]	ARMOUR N K, FERGUSON-NOEL N. Evaluation of the egg transmission and pathogenicity of Mycoplasma gallisepticum isolates genotyped as ts-11[J]. Avian Pathol, 2015, 44(4):296-304.
[3]	向勇, 曹伟胜. 禽支原体检测技术进展[J]. 养禽与禽病防治, 2018(4):2-9.XIANG Y, CAO W S. Advances in detection technology of avian mycoplasma[J]. Poultry Raising and Disease Control, 2018(4):2-9. (in Chinese)
[4]	BRADBURY J M. Avian mycoplasma infections:prototype of mixed infections with mycoplasmas, bacteria and viruses[J]. Ann Microbiol (Paris), 1984, 135(1):83-89.
[5]	YADAV J P, BATRA K, SINGH Y, et al. Comparative evaluation of indirect-ELISA and DOT blot assay for serodetection of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry[J]. J Microbiol Methods, 2021, 189:106317.
[6]	ANSARI A A, TAYLOR R F, CHANG T S. Application of enzyme-linked immunosorbent assay for detecting antibody to Mycoplasma gallisepticum infections in poultry[J]. Avian Dis, 1983, 27(1):21-35.
[7]	任娟. 间接夹心ELISA检测鸡毒霉形体研究[D]. 武汉:华中农业大学, 2009.REN J. Research of indirect sandwich ELISA for mycoplasma gallisepticum[D]. Wuhan:Huazhong Agricultural University, 2009. (in Chinese)
[8]	甘孟侯. 中国禽病学[M]. 北京:中国农业出版社, 1999.GAN M H. Avian epidemiology in China[M]. Beijing:China Agricultural Publishing House, 1999. (in Chinese)
[9]	周云雷, 李健, 魏飞龙, 等. 以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立[J]. 中国预防兽医学报, 2011, 33(9):713-717.ZHOU Y L, LI J, WEI F L, et al. Development of an indirect ELISA for detecting antibodies against Mycoplasma gallisepticum based on recombinant PvpA protein[J]. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(9):713-717. (in Chinese)
[10]	王莉莉. 鸡毒支原体抗体间接ELISA检测方法的建立及初步应用[D]. 北京:中国农业科学院, 2018.WANG L L. Establishment and preliminary application of indirect ELISA for detecting antibody to Mycoplasma gallisepticum[D]. Beijing:Chinese Academy of Agricultural Sciences, 2018. (in Chinese)
[11]	BENCINA D. Haemagglutinins of pathogenic avian mycoplasmas[J]. Avian Pathol, 2002, 31(6):535-547.
[12]	MARDASSI B B A, BÉJAOUI A, OUSSAEIF L, et al. A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis infections[J]. Avian Dis, 2008, 52(2):214-221.
[13]	陈玥彤, 祁晶晶, 胡增金, 等. 鸡毒支原体VLHA3. 03蛋白的原核表达及免疫原性分析[J]. 中国动物传染病学报, 2022, 30(2):134-141.CHEN Y T, QI J J, HU Z J, et al. Prokaryotic expression and immunogenicity analysis of VLHA3. 03 protein in Mycoplasma gallisepticum[J]. Chinese Journal of Animal Infectious Diseases, 2022, 30(2):134-141. (in Chinese)
[14]	SNYDER D B, MARQUARDT W W, MALLINSON E T, et al. Rapid serological profiling by enzyme-linked immunosorbent assay. I. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution[J]. Avian Dis, 1983, 27(1):161-170.
[15]	SNYDER D B, MARQUARDT W W, RUSSEK E. Rapid serological profiling by enzyme-linked immunosorbent assay. II. Comparison of computational methods for measuring antibody titer in a single serum dilution[J]. Avian Dis, 1983, 27(2):474-484.
[16]	MEHLHORN H. World organization for animal health[M]//MEHLHORN H. Encyclopedia of Parasitology. Berlin:Springer, 2016:3037.
[17]	何吉鑫. 检测滑液囊支原体抗体的冻干血凝抑制试验抗原的研制和间接ELISA方法的建立[D]. 扬州:扬州大学, 2020.HE J X. Development of lyophilized hemagglutination inhibition test antigen and establishment of indirect ELISA method for detecting Mycoplasma synoviae antibodies[D]. Yangzhou:Yangzhou University, 2020. (in Chinese)
[18]	陶丽寒. 滑液支原体血凝抑制试验与荧光定量PCR检测方法的建立与应用[D]. 合肥:安徽农业大学, 2018.TAO L H. Development and application of hemagglutinin inhibition assay and SYBR green Ⅰ real-time PCR assay for detection of Mycoplasma synoviae[D]. Hefei:Anhui Agricultural University, 2018. (in Chinese)
[19]	SPRYGIN A V, ANDREYCHUK D B, KOLOTILOV A N, et al. Development of a duplex real-time TaqMan PCR assay with an internal control for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from commercial and backyard poultry[J]. Avian Pathol, 2010, 39(2):99-109.
[20]	任妮, 邵建忠, 何闪. 监控种鸡群沙门氏菌、支原体和禽流感的流行情况[J]. 国外畜牧学(猪与禽), 2018, 38(7):9-13.REN N, SHAO J Z, HE S. Monitoring breeder flocks for salmonella, mycoplasma and avian influenza[J]. Animal Science Abroad (Pigs and Poultry), 2018, 38(7):9-13. (in Chinese)
[21]	LEY D H, MCLAREN J M, MILES A M, et al. Transmissibility of live Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from vaccinated layer pullets to sentinel poultry[J]. Avian Dis, 1997, 41(1):187-194.
[22]	STEINLAGE S J T, FERGUSON N, SANDER J E, et al. Isolation and characterization of a 6/85-like Mycoplasma gallisepticum from commercial laying hens[J]. Avian Dis, 2003, 47(2):499-505.
[23]	PRADHAN S K, KAMBLE N M, PILLAI A S, et al. Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry[J]. J Virol Methods, 2014, 209:1-6.
[24]	MOHAN C M, DEY S, RAI A, et al. Recombinant haemagglutinin neuraminidase antigen-based single serum dilution ELISA for rapid serological profiling of Newcastle disease virus[J]. J Virol Methods, 2006, 138(1-2):117-122.
[25]	RAMADASS P, PARTHIBAN M, THIAGARAJAN V, et al. Development of single serum dilution ELISA for detection of infectious bursal disease virus antibodies[J]. Veterinarski Arhiv, 2008, 78(1):23-30.
[26]	ARAVIND S, PATIL B R, DEY S, et al. Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis[J]. J Virol Methods, 2012, 185(2):234-238.
[27]	DEY S, MOHAN C M, KUMAR T M A S, et al. Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis[J]. Vet Microbiol, 2004, 103(1-2):99-106.
[28]	AVAKIAN A P, KLEVEN S H, GLISSON J R. Evaluation of the specificity and sensitivity of two commercial enzyme-linked immunosorbent assay kits, the serum plate agglutination test, and the hemagglutination-inhibition test for antibodies formed in response to Mycoplasma gallisepticum[J]. Avian Dis, 1988, 32(2):262-272.
[29]	STIPKOVITS L, CZIFRA G, SUNDQUIST B. Indirect ELISA for the detection of a specific antibody response against Mycoplasma gallisepticum[J]. Avian Pathol, 1993, 22(3):481-494.

Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody

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