肠艾美耳球虫重组微线蛋白2对家兔免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2024.06.030

Abstract

Abstract:

The aim of this study was to evaluate the immunoprotective effect of recombinant Eimeria intestinalis microneme protein 2 (rEiMIC2) in rabbits and to provide a reference for the study of subunit recombinant vaccine against Eimeria intestinalis. EiMIC2 gene was identified in E. intestinalis transcriptome data, then cloning, prokaryotic expression and protein purification were conducted. The immunoreactivity of the recombinant protein was also detected using immunoblotting. Thirty-two 35-day-old coccidia-free New Zealand young rabbits were randomly divided into 4 groups (unchallenged group, challenged group, pET-32a(+) vector group and rEiMIC2 immunization group), with 8 rabbits in each group. In the unchallenged and challenged groups, 1 mL sterile PBS was subcutaneously injected into the neck of each rabbit. While in the pET-32a(+) vector group and rEiMIC2 immunization group, each rabbits were inoculated with 100 μg pET-32a(+) protein or rEiMIC2. A second immunization was conducted after 2 weeks of the first immunization. Fourteen days after the second immunization, excluding the unchallenged group, each of the remaining three groups of New Zealand rabbits was orally inoculated with 5×10⁴ sporulated oocysts of Eimeria intestinalis; All New Zealand rabbits were euthanized 14 days post-infection. After immunizing rabbits with recombinant protein, the immune protection effect of rEiMIC2 was comprehensively evaluated by observing clinical symptoms, evaluating intestinal lesions, and measuring relative weight gain rate, oocyst reduction rate, anticoccidial index (ACI), feed-to-meat ratio, serum IgG, IL-2, IL-4, IL-10, IFN-γ. The results showed that, the open reading frame length of EiMIC2 gene was 1 605 bp, encoded a protein with a molecular mass of approximately 57.68 ku. Western blot showed that rEiMIC2 could be recognized by positive sera, indicating its good immunoreactivity. The immune protection experiment showed that after the challenge, the symptoms of depression and diarrhea appeared in each group, and one rabbit died in each challenged group and the rEiMIC2 immunization group; the relative weight gain rate of the rEiMIC2 immunization group was 73.97%, and the oocyst reduction rate was 69.98%, the ACI was 136.97, and the feed-to-meat ratio was 3.98∶1, which were significantly different from the challenged group (P < 0.05). Compared with the intestinal lesions in the challenged group, the intestinal lesions in the rEiMIC2 immunization group were milder, and the level of the IgG in serum was significantly higher than that in the challenged group (P < 0.05). In summary, after immunizing rabbits with rEiMIC2, it can effectively reduce the excretion of oocysts and the loss of weight gain, and reduce intestinal damage, which providing a certain protective effect of the rabbits.

Key words: Eimeria intestinalis, microneme proteins 2, rabbit, immune protective

CLC Number:

S852.723

Hao CHEN, Ge HAO, Jiayan PU, Jie XIAO, Changming XIONG, Wei HE, Yuhua ZHU, Liwen XU, Qing JIANG, Guangyou YANG. Evaluation of the Immune Protective Effect of Recombinant MIC2 from Eimeria intestinalis in Rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2588-2598.

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References 43

1	XIE Y , XIAO J , ZHOU X , et al. Global transcriptome landscape of the rabbit protozoan parasite Eimeria stiedae[J]. Parasit Vectors, 2021, 14 (1): 308. doi: 10.1186/s13071-021-04811-5
2	PAKANDL M . Coccidia of rabbit: a review[J]. Folia Parasitol (Praha), 2013, 56 (3): 153- 166.
3	SHI T Y , TAO G R , BAO G L , et al. Stable transfection of Eimeria intestinalis and investigation of its life cycle, reproduction and immunogenicity[J]. Front Microbiol, 2016, 7, 807.
4	ABD EI-GHANY W A . Coccidiosis: a parasitic disease of significant importance in rabbits[J]. World Vet J, 2020, 10 (4): 499- 507.
5	闫文朝, 王天奇, 索勋, 等. 家兔球虫病的研究进展[J]. 中国兽医科学, 2010, 40 (11): 1200- 1205.
	YAN W C , WANG T Q , SUO X , et al. Advances in coccidiosis of domestic rabbits[J]. Chinese Veterinary Science, 2010, 40 (11): 1200- 1205.
6	罗跃军, 任永军, 白鑫, 等. 斯氏艾美耳球虫重组表面抗原SAG13和SAG14对兔的免疫保护效果初步观察[J]. 畜牧兽医学报, 2022, 53 (3): 883- 893.
	LUO Y J , REN Y J , BAI X , et al. Preliminary observation on the Immunoprotective effects of Recombinant surface antigens SAG13 and SAG14 of Eimeria stiedae in rabbits[J]. Acta Veterinaria Et Zootechnica Sinica, 2022, 53 (3): 883- 893.
7	XIAO J , CHEN H , ZHENG R Y , et al. Recombinant GMA56 and ROP17 of Eimeria magna conferred protection against infection by homologous species[J]. Front Immunol, 2023, 13, 1037949. doi: 10.3389/fimmu.2022.1037949
8	DUBOIS D J , SOLDATI-FAVRE D . Biogenesis and secretion of micronemes in Toxoplasma gondii[J]. Cell Microbiol, 2019, 21 (5): e13018. doi: 10.1111/cmi.13018
9	OLAJIDE J S , QU Z G , YANG S L , et al. Eimeria proteins: order amidst disorder[J]. Parasit Vectors, 2022, 15 (1): 38. doi: 10.1186/s13071-022-05159-0
10	田秀玲, 汤新明, 秦梅, 等. 基于IFA技术检测微线体蛋白2在艾美耳属球虫的空间分布[J]. 寄生虫与医学昆虫学报, 2015, 21 (4): 240- 243.
	TIAN X L , TANG X M , QIN M , et al. Analysis on the location of microneme 2 protein in sporozoites of Eimeria spp. by indirect immunofluorescence assay[J]. Acta Parasitology et Medica Entomologica Sinica, 2015, 21 (4): 240- 243.
11	FOROUTAN M , ZAKI L , GHAFFARIFAR F . Recent progress in microneme-based vaccines development against Toxoplasma gondii[J]. Clin Exp Vaccine Res, 2018, 7 (2): 93- 103. doi: 10.7774/cevr.2018.7.2.93
12	YAN M , CUI X X , ZHAO Q P , et al. Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella[J]. Parasite, 2018, 25, 60. doi: 10.1051/parasite/2018061
13	ZHANG Z C , LIU L R , HUANG J W , et al. The molecular characterization and immune protection of microneme 2 of Eimeria acervulina[J]. Vet Parasitol, 2006, 215, 96- 105.
14	XIAO J , HE W , XIONG C M , et al. Protective efficacy of recombinant proteins AMA1 and IMP1 in rabbits infected with Eimeria intestinalis[J]. Vet Parasitol, 2023, 320, 109985. doi: 10.1016/j.vetpar.2023.109985
15	WEI W R , SHEN N X , XIAO J , et al. Expression analysis and serodiagnostic potential of microneme proteins 1 and 3 in Eimeria stiedai[J]. Genes (Basel), 2020, 11 (7): 725. doi: 10.3390/genes11070725
16	BORTOLUZZI C , PARAS K L , APPLEGATE T J , et al. Comparison between McMaster and Mini-FLOTAC methods for the enumeration of Eimeria maxima oocysts in poultry excreta[J]. Vet Parasitol, 2018, 254, 21- 25. doi: 10.1016/j.vetpar.2018.02.039
17	JOHNSON J , REID W M . Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens[J]. Exp Parasitol, 1970, 28 (1): 30- 36. doi: 10.1016/0014-4894(70)90063-9
18	SHEN N X , WEI W R , CHEN Y H , et al. An antibody persistent and protective two rSsCLP-based subunit cocktail vaccine against Sarcoptes scabiei in a rabbit model[J]. Vaccines (Basel), 2020, 8 (1): 129. doi: 10.3390/vaccines8010129
19	FANG S F , GU X L , EL-ASHRAM S , et al. Immune protection provided by a precocious line trivalent vaccine against rabbit Eimeria[J]. Vet Parasitol, 2019, 275, 108927. doi: 10.1016/j.vetpar.2019.108927
20	SOUTTER F , WERLING D , NOLAN M , et al. A novel whole yeast-based subunit oral vaccine against Eimeria tenella in Chickens[J]. Front Immunol, 2022, 13, 809711. doi: 10.3389/fimmu.2022.809711
21	LIU Q , CHEN Z T , SHI W Y , et al. Preparation and initial application of monoclonal antibodies that recognize Eimeria tenella microneme proteins 1 and 2[J]. Parasitol Res, 2014, 113 (11): 4151- 4161. doi: 10.1007/s00436-014-4087-2
22	BROSSIER F , DAVID SIBLEY L . Toxoplasma gondii: microneme protein MIC2[J]. Int J Biochem Cell Biol, 2005, 37 (11): 2266- 2272. doi: 10.1016/j.biocel.2005.06.006
23	BANDINI G , LEON D R , HOPPE C M , et al. O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites[J]. J Biol Chem, 2019, 294 (6): 1967- 1983. doi: 10.1074/jbc.RA118.005179
24	SASAI K , FETTERER R H , LILLEHOJ H , et al. Characterization of monoclonal antibodies that recognize the Eimeria tenella Microneme protein MIC2[J]. J Parasitol, 2008, 94 (6): 1432- 1434. doi: 10.1645/GE-1558.1
25	CARRUTHERS V B, TOMLEY F M. Microneme proteins in apicomplexans[M]//BURLEIGH B A, SOLDATI-FAVRE D. Molecular Mechanisms of Parasite Invasion. New York: Springer, 2008: 33-45.
26	HOAN T D , ZHANG Z C , HUANG J W , et al. Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti[J]. Exp Parasitol, 2016, 162, 7- 17. doi: 10.1016/j.exppara.2015.12.015
27	HUANG J W , ZHANG Z C , LI M H , et al. Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge[J]. Parasitol Int, 2015, 64 (5): 408- 416. doi: 10.1016/j.parint.2015.06.002
28	AKPO Y , KPODÉKON M T , DJAGO Y , et al. Vaccination of rabbits against coccidiosis using precocious lines of Eimeria magna and Eimeria media in Benin[J]. Vet Parasitol, 2012, 184 (1): 73- 76.
29	SADEK BACHENE M , TEMIM S , AINBAZIZ H , et al. A vaccination trial with a precocious line of Eimeria magna in Algerian local rabbits Oryctolagus cuniculus[J]. Vet Parasitol, 2018, 261, 73- 76.
30	PAKANDL M , SEWALD B , DROUET-VIARD F . Invasion of the intestinal tract by sporozoites of Eimeria coecicola and Eimeria intestinalis in naive and immune rabbits[J]. Parasitol Res, 2006, 98 (4): 310- 316.
31	DROUET-VIARD F , LICOIS D , PROVÔT F , et al. The invasion of the rabbit intestinal tract by Eimeria intestinalis sporozoites[J]. Parasitol Res, 1994, 80 (8): 706- 707.
32	YUAN X , LIU J , HU X F , et al. Alterations in the jejunal microbiota and fecal metabolite profiles of rabbits infected with Eimeria intestinalis[J]. Parasit Vectors, 2022, 15 (1): 231.
33	LU C Y , YAN Y Q , JIAN F C , et al. Coccidia-Microbiota interactions and their effects on the host[J]. Front Cell Infect Microbiol, 2021, 11, 751481.
34	CHAPMAN H D . Milestones in avian coccidiosis research: a review[J]. Poult Sci, 2014, 93 (3): 501- 511.
35	DONG X J , ABDELNABI G H , LEE S H , et al. Enhanced egress of intracellular Eimeria tenella sporozoites by splenic lymphocytes from coccidian-infected chickens[J]. Infect Immun, 2011, 79 (8): 3465- 3470.
36	SONG X K , HUANG X M , YAN R F , et al. Efficacy of chimeric DNA vaccines encoding Eimeria tenella 5401 and chicken IFN-γ or IL-2 against coccidiosis in chickens[J]. Exp Parasitol, 2015, 156, 19- 25.
37	HOAN T D , THAO D T , GADAHI J A , et al. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine[J]. Exp Parasitol, 2014, 144, 65- 72.
38	ARENDT M , ELISSA J , SCHMIDT N , et al. Investigating the role of interleukin 10 on Eimeria intestinal pathogenesis in broiler chickens[J]. Vet Immunol Immunopathol, 2019, 218, 109934.
39	GAZZINELLI-GUIMARÃES A C , GAZZINELLI-GUIMARÃES P H , NOGUEIRA D S , et al. IgG induced by vaccination with Ascaris suum extracts is protective against infection[J]. Front Immunol, 2018, 9, 2535.
40	XIONG L , CHEN L , CHEN Y X , et al. Evaluation of the immunoprotective effects of eight recombinant proteins from Baylisascaris schroederi in mice model[J]. Parasit Vectors, 2023, 16 (1): 254.
41	ZHAO P F , LI Y C , ZHOU Y Q , et al. In vivo immunoprotective comparison between recombinant protein and DNA vaccine of Eimeria tenella surface antigen 4[J]. Vet Parasitol, 2020, 278, 109032.
42	ZHANG Z C , LIU X C , YANG X C , et al. The molecular characterization and immunity identification of microneme 3 of Eimeria acervulina[J]. J Eukaryot Microbiol, 2016, 63 (6): 709- 721.
43	CHEN H , PU J Y , XIAO J , et al. Evaluation of the immune protective effects of rEmMIC2 and rEmMIC3 from Eimeria magna in rabbits[J]. Parasitol Res, 2023, 122 (2): 661- 669.

组别 Group	动物数量 Rabbits number	免疫原及剂量 Immunogen and dosage	免疫方式 Immunization route	免疫次数 Immunization times	攻虫 Challenge dosage
未免疫未攻虫组 Unchallenged group	8	PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	未感染
未免疫攻虫组 Challenged group	8	PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	口服5×10⁴孢子化卵囊
pET-32a(+)组 pET-32a(+) vector group	8	100 μg载体蛋白+1 mg皂素溶于无菌PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	口服5×10⁴孢子化卵囊
rEiMIC2免疫组 rEiMIC2 immunized group	8	100 μg rEiMIC2+ 1mg皂素溶于无菌PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	口服5×10⁴孢子化卵囊

评分 Score	小肠病变记分的判定标准 Criteria for scoring intestinal lesions
0	空肠、回肠未见明显病理改变，表现正常
1	肠壁可见少量白色结节或少量出血点，未见增厚
2	出血点或白色结节较多，肠壁稍增厚
3	可见大量出血点或白色结节，肠壁增厚
4	动物死亡；肠壁广泛出血，白色结节连成斑块；肠壁极度扩张增厚

试验组卵囊数占感染非免疫组比例 The proportion of oocyst in the experimental group to unimmunized and challenged group	卵囊值 Oocyst value
0~1%	0
2%~25%	1
26%~50%	10
51%~75%	20
76%~100%	40

范围 Range	抗球虫效果 Anti coccidiosis effect
ACI＜120	无
120≤ACI＜160	中等
160≤ACI≤180	良好
ACI＞180	优秀

组别 Group	攻虫前平均增重/g Average body weight gain before challenge	攻虫后平均增重/g Average body weight gain after challenge	相对增重率/% Relative weight gain	OPG/10⁵	卵囊减少率/% Oocyst decrease ratio	料肉比 Feed conversion ratio	平均病变评分 Mean lesion scores	ACI
未免疫未攻虫组	510.00±91.92^a	716.50±98.48^a	—	—	—	3.00∶1	—	200
Unchallenged group
未免疫攻虫组	481.62±66.25^a	438.28±81.60^b	61.17	9.45±8.79^a	—	4.71∶1	2.03±1.39	88.37
Challenged group
pET-32a(+)组	511.18±90.82^a	419.37±80.64^b	58.53	9.32±10.01^a	-1.4	4.95∶1	2.67±0.75	91.83
pET-32a(+) vector group
rEiMIC2免疫组	471.87±82.97^a	530.00±90.18^c	73.97	2.83±3.22^b	69.98	3.98∶1	1.45±1.19	136.97
rEiMIC2 immunization group

Evaluation of the Immune Protective Effect of Recombinant MIC2 from Eimeria intestinalis in Rabbits

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