Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (2): 415-424.doi: 10.3864/j.issn.0578-1752.2022.02.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Isolation and Identification of Brucella melitensis from Ticks on Cattle Surface in Hulunbuir Area

HUANG TianPeng1(),GUO Xu2,SUN ChangYun3,CHEN JingDiao3,CHAOMULIGE 1,WU Jie3,GERILETU 1()   

  1. 1College of Veterinary Medicine, Inner Mongolia Agricultural University,Hohhot 010018
    2Erguna animal disease prevention and control center,Erguna 022250, Inner Mongolia
    3Guangdong Provincial Center for Disease Control and Prevention,Guangzhou 511430
  • Received:2020-11-16 Accepted:2021-03-16 Online:2022-01-16 Published:2022-01-26
  • Contact: GERILETU E-mail:1401180252@qq.com;geriletusy@imau.edu.cn

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Abstract:

【Objective】 The aim of this study was to elucidate the relationship between Dermacentor nuttalli and brucellosis in cattle and sheep. 【Method】 The species of ticks collected in Hulunbuir were identified by traditional morphological and molecular biology methods; the ticks collected from the cattle surface were used as materials, and the suspicious bacteria were isolated from their bodies. The isolated bacterial species were determined by methods, such as bacterial isolation and identification, morphological observation, serological detection and molecular biology. 【Result】 The dominant tick species were identified as D. nuttalli in this area, and four strains of suspicious bacteria were successfully isolated in their bodies. The isolated bacteria were gram-negative Brevibacterium by Gram staining, and the bcsp31 and omp22 gene sequences of Brucella were successfully cloned by PCR method. After gene sequencing and BLAST analysis, it was found that the similarity with the Brucella reference sequence was above 99.0%. The phylogenetic tree was successfully constructed by using Mega 7.0 software. The isolated strains were clustered in the same branch with the known sequences of Brucella. Using AMOS-PCR and Brucella typing test, four isolated strains were identified as Brucella melitensis. 【Conclusion】 In this experiment, four strains of B. melitensis were isolated from D. nuttalli collected from cattle in Hulunbuir area, which indicated that as a blood sucking arthropod, D. nuttalli might play an important role in the transmission of brucellosis among different hosts. This paper provided basic data for the investigation and control of Brucella in Inner Mongolia.

Key words: Dermacentor nuttalli, molecular biology, Brucella, Bcsp31, Omp22

Table 1

Primers sequences for PCR of mitochondria"

扩增片段 Amplified fragments 引物名称 Primer name 引物序列 Primer sequence (5'-3') 片段大小 Fragment size (bp)
16S rRNA 16Sf TGACTATACRAAGGTATTG,（R=A或G） 346
16Sr GGACGGACTCGTAGCCTG
CO І LCO1490 GGTCAACAAATCATAAAGATATTGG 658
HCO2198 TAAACTTCAGGGTGACCAAAAAATCA

Table 2

The primer sequence"

扩增片段 Amplified fragments 引物名称 Primer name 引物序列 Primer sequence (5'-3') 片段大小 Fragment size (bp)
Bcsp31 B4F TGGCTCGGTTGCCAATATCAA 223
B5R CGCGCTTGCCTTTCAGGTCTG
Omp22 Omp22F TGATGGGAGGGACCGACTA 498
Omp22R TGGTTCTTCAGGTTGTTACGC

Table 3

The primer sequence of AMOS-PCR"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 片段大小
Fragment size (bp)
A GAC GAA CGG AAT TTT TCC AAT CCC 498
M AAA TCG CGT CCTTGC TGG TCT GA 731
O GCG CGG TTT TCT GAA GGT TCAGG 961
S GCG CGG TTT TCT GAA GGT TCAGG 285
IS711 TGC CGA TCA CTT AAG GGC CTT CAT

Fig. 1

Morphology of identification D.nuttalli specimens A:Female ticks; B:Nymphs; C:Male ticks a1、a2:Palp articles; b1、b2:Coxae; c1、c2:Valve plate; d1、d2:Festoon"

Fig. 2

Evolutionary relationships by NJ methods analysis in 16S rRNA gene sequences"

Fig. 3

Evolutionary relationships by NJ methods analysis in CO І gene sequences"

Fig. 4

Colony morphology of isolated bacteria A: Brucella colony in selective medium; B: Brucella colony in blood plate"

Fig. 5

Gram staining and Kirschner staining microscopic examination of isolated bacteria（×100） A:The results of microscopic examination with Gram staining;B:The results of microscopic examination with Kirschner's staining"

Fig. 6

phylogenetic tree based on Omp22 of Brucella"

Fig. 7

Identificated of Brucella by AMOS-PCR M: DL2000 DNA marker; 1-4: B. melitensis IMHT 5-B. melitensis IMHT8; 5: B. melitensis 16M strain; 6: B. abortus 544A strain; 7: B. suis 1330s strain N: negative control"

Table 4

The results of classification and identification of Brucella"

试验与结果
Experiment and result		 B. melitensis NMT5 B. melitensis NMT6 B. melitensis NMT7 B. melitensis NMT8 参考标准株 International reference strains
16 M 1330S 544A
宿主Host 牛Cattle 牛Cattle 牛Cattle 牛Cattle
染料抑菌
Dye inhibition		 硫瑾 Thionin + + + + + + +
碱性品红
Basic fuchsin		 + + + + + + +
噬菌体裂解
Lysis by phage at RTD		 Tb RTD
104 Tb RTD
BK2		 -
-
+		 -
-
+		 -
-
+		 -
-
+		 -
-
+		 -
-
+		 +
+
+
特异性血清
Agglutination on
reaction with
specific serum		 A
M
R		 +
-
-		 +
+
-		 +
+
-		 +
-
-		 -
+
-		 +
-
-		 +
-
-
CO2需求
CO2 requirement		 - - - - - - -
产H2S
H2S production		 - - - - + + +
结果
Result		 种型
Species biotype		 羊种2型
布鲁氏菌
Brucella
melitensis 2		 羊种3型
布鲁氏菌
Brucella
melitensis 3		 羊种3型
布鲁氏菌
Brucella
melitensis 3		 羊种2型
布鲁氏菌
Brucella
melitensis 2		 羊种1型
布鲁氏菌
Brucella
melitensis 1		 猪种1型
布鲁氏菌
Brucella
suis 1		 牛种1型
布鲁氏菌
Brucella
abortus 1
[1] PAPPAS G, PAPADIMITRIOU P, AKRITIDIS N, CHRISTOU L, TSIANOS E V. The new global map of human brucellosis. The Lancet Infectious Diseases, 2006, 6(2):91-99. doi: 10.1016/S1473-3099(06)70382-6.
doi: 10.1016/S1473-3099(06)70382-6
[2] AVILA-CALDERÓN E D, LOPEZ-MERINO A, SRIRANGANATHAN N, BOYLE S M, CONTRERAS-RODRÍGUEZ A. A history of the development of Brucella vaccines. BioMed Research International, 2013, 2013:743509. doi: 10.1155/2013/743509.
[3] 崔步云, 姜海. 2005—2016年全国布鲁氏菌病监测数据分析. 疾病监测, 2018, 33(3):188-192. doi: 10.3784/j.issn.1003-9961.2018.03.005.
CUI B Y, JIANG H. Surveillance data of brucellosis in China, 2005-2016. Disease Surveillance, 2018, 33(3):188-192. doi: 10.3784/j.issn.1003-9961.2018.03.005. (in Chinese)
[4] 范蒙光, 塔娜, 郭威, 尉瑞平, 米景川, 宋利桃, 李晓燕, 张文强. 2010—2014年内蒙古自治区人间布鲁氏菌病监测结果分析. 疾病监测, 2016, 31(11):945-948. doi: 10.3784/j.issn.1003-9961.2016.11.013.
FAN M G, TANA, GUO W, YU R P, MI J C, SONG L T, LI X Y, ZHANG W Q. Evaluation on surveillance for human brucellosis in Inner Mongolia Autonomous Region, 2010-2014. Disease Surveillance, 2016, 31(11):945-948. doi: 10.3784/j.issn.1003-9961.2016.11.013. (in Chinese)
[5] 梁晗玮, 塔娜, 米景川, 尉瑞平, 郭威, 张文强. 2009—2018年内蒙古自治区人间布鲁氏菌病流行时空分布特征. 疾病监测, 2019, 34(12):1058-1063.
LIANG H W, TANA, MI J C, YU R P, GUO W, ZHANG W Q. Spatial and temporal distribution of human brucellosis in Inner Mongolia, 2009-2018. Disease Surveillance, 2019, 34(12):1058-1063. (in Chinese)
[6] SOHN A H, PROBERT W S, GLASER C A, GUPTA N, BOLLEN A W, WONG J D, GRACE E M, MCDONALD W C. Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp. Emerging Infectious Diseases, 2003, 9(4):485-488. doi: 10.3201/eid0904.020576.
doi: 10.3201/eid0904.020576
[7] TESSARO S V. The existing and potential importance of brucellosis and tuberculosis in Canadian wildlife: a review The Canadian Veterinary Journal = La Revue Veterinaire Canadienne, 1986, 27(3):119-124.
[8] DADAR M, SHAHALI Y, FAKHRI Y, GODFROID J. The global epidemiology of Brucella infections in terrestrial wildlife: a meta-analysis. Transboundary and Emerging Diseases, 2021, 68(2):715-729. doi: 10.1111/tbed.13735.
doi: 10.1111/tbed.v68.2
[9] GRANTINA-IEVINA L, AVSEJENKO J, CVETKOVA S, KRASTINA D, STREIKISA M, STEINGOLDE Z, VEVERE I, RODZE I. Seroprevalence of Brucella suis in eastern Latvian wild boars (Sus scrofa). Acta Veterinaria Scandinavica, 2018, 60(1):19. doi: 10.1186/s13028-018-0373-9.
doi: 10.1186/s13028-018-0373-9
[10] PRITULIN P. On the transmission of brucellosis by the pasture ticks Dermacentor nuttallia and Hyalomma marginatum. Veterinariya, 1954(7):31-33.
[11] ZOTOVA A A, BALDITSINA K S. An expereicne in infecting ticks with brucellosis under laboratory conditions. Series Zoology Alma-Ata, 1943(1):73-79.
[12] ZOTOVA A, BOLDICINA K. The experimental infection of ticks with brucellosis under laboratory conditions. DTIC Document, 1951.
[13] 邓国藩, 姜在阶. 中国经济昆虫志. 第三十九册,蜱螨亚纲､硬蜱科, 北京: 科学出版社, 1991:343-349.
DENG G F, JIANG Z J. Economic Entomology of China. Vol. 39, acari, Ixodidae. Beijing: Science Press, 1991:43-349.(in Chinese)
[14] HORAK I G, BRAACK L E, FOURIE L J, WALKER J B. Parasites of domestic and wild animals in South Africa. XXXVIII. Ixodid ticks collected from 23 wild carnivore species. The Onderstepoort Journal of Veterinary Research, 2000, 67(4):239-250.
[15] 巴音查汗, 徐显曾. 森林革蜱与草原革蜱的超微形态学比较. 中国兽医科技, 2001, 31(6):27-30. doi: 10.16656/j.issn.1673-4696.2001. 06.016.
BAYINCHAHAN, XU X Z. Ultramorphologic comparison between Dermacentor silvarum and D. nuttalli. Chinese Journal of Veterinary Science and Technology, 2001, 31(6):27-30. doi: 10.16656/j.issn.1673-4696.2001.06.016. (in Chinese)
[16] FOLMER O, BLACK M, HOEH W, LUTZ R, VRIJENHOEK R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 1994, 3(5):294-299.
[17] QUEIPO-ORTUÑO M I, COLMENERO J D, REGUERA J M, GARCÍA-ORDOÑEZ M A, PACHÓN M E, GONZALEZ M, MORATA P. Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clinical Microbiology and Infection, 2005, 11(9):713-718. doi: 10.1111/j.1469-0691.2005.01202.x.
doi: 10.1111/j.1469-0691.2005.01202.x
[18] 唐莉娟, 陈创夫, 王远志, 张辉. 畜产品中布鲁氏菌巢氏PCR快速诊断方法研究. 上海畜牧兽医通讯, 2008(4):39-41. doi: 10.3969/ j.issn.1000-7725.2008.04.016.
TANG L J, CHEN C F, WANG Y Z, ZHANG H. Rapid detection of Brucella in animal products by nested PCR. Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2008(4):39-41. doi: 10.3969/j.issn.1000-7725.2008.04.016. (in Chinese)
[19] 姜海, 崔步云, 赵鸿雁, 朴东日, 李兰玉. AMOS-PCR对布鲁氏菌种型鉴定的应用. 中国人兽共患病学报, 2009, 25(2):107-109. doi: 10.3969/j.issn.1002-2694.2009.02.004.
JIANG H, CUI B Y, ZHAO H Y, PIAO D R, LI L Y. Use of AMOS-PCR assay for the species identification of Brucella. Chinese Journal of Zoonoses, 2009, 25(2):107-109. doi: 10.3969/j.issn.1002-2694.2009.02.004. (in Chinese)
[20] 王建军, 郭威, 刘俊, 张忠兵. 内蒙古部分蜱类标本的鉴定及其医学意义. 寄生虫与医学昆虫学报, 2014, 21(1):54-58.
WANG J J, GUO W, LIU J, ZHANG Z B. Ticks checklist and tick-borne diseases in Inner Mongolia. Acta Parasitologica et Medica Entomologica Sinica, 2014, 21(1):54-58.(in Chinese)
[21] 黄天鹏. 内蒙古优势蜱种—草原革蜱的生物学特性及其潜在传播人和动物布鲁斯菌病的风险分析[D]. 呼和浩特: 内蒙古农业大学, 2017.
HUANG T P. Biological characteristics of dominant ticks- Dermacentor nuttalli in Inner Mongolia and their potential risk of transmission of human and animal brucellosis[D]. Hohhot: Inner Mongolia Agricultural University, 2017. (in Chinese)
[22] GUGLIELMONE A A, VENZAL J M, GONZÁLEZ-ACUÑA D, NAVA S, HINOJOSA A, MANGOLD A J. The phylogenetic position of Ixodes stilesi Neumann, 1911 (Acari: Ixodidae): morphological and preliminary molecular evidences from 16S rDNA sequences. Systematic Parasitology, 2006, 65(1):1-11. doi: 10.1007/s11230-005-9024-4.
doi: 10.1007/s11230-005-9024-4
[23] 王云冲, 黄天鹏, 格日勒图. 内蒙古额济纳旗亚洲璃眼蜱的鉴定及其基因多态性分析. 中国兽医学报, 2020, 40(12):2337-2341, 2373. doi: 10.16303/j.cnki.1005-4545.2020.12.11.
WANG Y C, HUANG T P, GERILETU. Identification and genetic polymorphism of Hyalomma asiaticum in Ejinaqi, Inner Mongolia. Chinese Journal of Veterinary Science, 2020, 40(12):2337-2341, 2373. doi: 10.16303/j.cnki.1005-4545.2020.12.11. (in Chinese)
[24] HOSSEINI-CHEGENI A, TAVAKOLI M, TELMADARRAIY Z, SEDAGHAT M M, FAGHIHI F. Detection of a Brucella-like (alphaproteobacteria) bacterium in boophilus spp. (Acari: Ixodidae) from Iran. Journal of Medical Microbiology and Infectious Diseases, 2017, 5(3):66-68. doi: 10.29252/jommid.5.3.4.66.
doi: 10.29252/JoMMID.5.3.4.66
[25] HALLING S M, DETILLEUX P G, TATUM F M, JUDGE B A, MAYFIELD J E. Deletion of the BCSP31 gene of Brucella abortus by replacement. Infection and Immunity, 1991, 59(11):3863-3868. doi: 10.1128/iai.59.11.3863-3868.1991.
doi: 10.1128/iai.59.11.3863-3868.1991
[26] LIU Z G, WANG M, ZHAO H Y, PIAO D R, JIANG H, LI Z J. Investigation of the molecular characteristics of Brucella isolates from Guangxi Province, China. BMC Microbiology, 2019, 19(1):292. doi: 10.1186/s12866-019-1665-6.
doi: 10.1186/s12866-019-1665-6
[27] MATOPE G, BHEBHE E, MUMA J B, SKJERVE E, DJØNNE B. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR. BMC Research Notes, 2009, 2:261. doi: 10.1186/1756-0500-2-261.
doi: 10.1186/1756-0500-2-261
[28] DAUGALIYEVA А, SULTANOV A, USSERBAYEV B, BARAMOVA S, MODESTO P, ADAMBAYEVA A, ACUTIS P L, PELETTO S. Genotyping of Brucella melitensis and Brucella abortus strains in Kazakhstan using MLVA-15. Infection, Genetics and Evolution, 2018, 58:135-144. doi: 10.1016/j.meegid.2017.12.022.
doi: 10.1016/j.meegid.2017.12.022
[29] EWALT D R, BRICKER B J. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. Journal of Clinical Microbiology, 2000, 38(8):3085-3086. doi: 10.1128/jcm.38.8.3085-3086.2000.
doi: 10.1128/JCM.38.8.3085-3086.2000
[30] 刘志国, 王妙, 刘日宏, 尚修建, 崔步云. 内蒙古自治区乌兰察布市羊种布鲁氏菌HOOF基因分型特征. 中华流行病学杂志, 2017(7):954-958. doi: 10.3760/cma.j.issn.0254-6450.2017.07.021.
LIU Z G, WANG M, LIU R H, SHANG X J, CUI B Y. HOOF genotyping characteristics of Brucella melitensis strains isolated in Ulanqab of Inner Mongolia Autonomous Region. Chinese Journal of Epidemiology, 2017(7):954-958. doi: 10.3760/cma.j.issn.0254-6450.2017.07.021. (in Chinese)
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