Acta Parasitologica

, Volume 57, Issue 2, pp 101–113 | Cite as

Phylogenic analysis of Chinese Leishmania isolates based on small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA)

Original Paper

Abstract

The leishmaniases are zoonotic diseases caused by protozoan parasites of the genus Leishmania. Leishmaniases are still endemic in China, especially in the west and northwest froniter regions. To revalue the preliminary phylogenetic results of Chinese Leishmania isolates, we amplified partial fragment of small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA), then tested the phylogenetic relationships among Chinese Leishmania isolates and their relatives by analyzing SSU rRNA gene sequences and 7SL RNA gene sequences. 19 SSU RNA sequences and 9 7SL RNA sequences were obtained in our study, then analyzed with 42 SSU RNA sequences and 32 7SL RNA sequences retrieved from Genbank, respectively. In the Bayesian analysis of the SSU RNA gene, the isolate MHOM/CN/93/GS7 and the isolate IPHL/CN/77/XJ771 are members of Leishmania donovani complex, while the isolate MHOM/CN/84/JS1 clustered with Leishmania tropica. The other 11 Chinese Leishmania isolates (MHOM/CN/90/WC, MCAN/CN/90/SC11, MHOM/CN/80/XJ801, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/ 89/GS5) form an unclassified group, defined as Leishmania sp., and the most relative species to this group is L. tarentolae. In the Bayesian analysis of the 7SL RNA gene, 9 Chinese Leishmania isolates also formed an unclassified group with L. tarentolae, including canine isolate 10, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/ CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/89/GS5. We concluded that: (1) Chinese Leishmania isolates are non-monophyly group; (2) an unclassified group may exist in China, and the most relative species to this group is L. tarentolae; (3) MHOM/CN/84/JS1, which was previously assigned as L. donovani, was most genetically related to L. tropica strain MHOM/SU/74/K27.

Keywords

Leishmania phylogeny SSU RNA 7SL RNA China 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ababneh F., Jermiin L.S., Ma C., et al. 2006. Matched-pairs tests of homogeneity with applications to homologous nucleotide sequences. Bioinformatics, 22, 1225–1231. DOI: 10.1093/bioinformatics/btl064.PubMedCrossRefGoogle Scholar
  2. Akopyants N.S., Kimblin N., Secundino N., Patrick R., Peters N., Lawyer P., Dobson D.E., Beverley S.M., Sacks D.L. 2009. Demonstration of Genetic Exchange During Cyclical Development of Leishmania in the Sand Fly Vector. Science, 324, 265–268. DOI: 10.1126/science.1169464.PubMedCrossRefGoogle Scholar
  3. Aransay A.M., Scoulica E., Chaniotis B., Tselentis Y. 1999. Typing of sand flies from Greece and Cyprus by DNA polymorphism of 18S rRNA gene. Insect Molecular Biology, 8, 179–184. DOI: 10.1046/j.1365-2583.1999.820179.x.PubMedCrossRefGoogle Scholar
  4. Asato Y., Oshiro M., Myint C.K., et al. 2009. Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing. Experimental Parasitology, 121, 352–361. DOI: 10.1016/j.exppara.2008.12.013.PubMedCrossRefGoogle Scholar
  5. Bañuls A.L., Hide M., Prugnolle F. 2007. Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Advances in Parasitology, 64, 1–109. DOI: 10.1016/S0065-308X(06) 64001-3.PubMedCrossRefGoogle Scholar
  6. Barón S., Martín-Sánchez J., Gállego M., Morales-Yuste M., Boussaa S., Morillas Márquez F. 2008. Intraspecific variability (rDNA ITS and mtDNA Cyt b) of Phlebotomus sergenti in Spain and Morocco. Acta Tropica, 107, 259–267. DOI: 10.1016/j.actatropica.2008.07.003.PubMedCrossRefGoogle Scholar
  7. Barroso P.A., Marco J.D., Kato H., Tarama R., Rueda P., Cajal S.P., Basombrío M.A., Korenaga M., Taranto N.J., Hashiguchi Y. 2007. The identification of sandfly species, froman area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA. Annals of Tropical Medicine and Parasitology, 101, 247–253. DOI: 10.1179/136485907X156988.PubMedCrossRefGoogle Scholar
  8. Baverstock P.R., Illana S., Christy P.E., Robinson B.S., Johnson A.M. 1989. srRNA Evolution and Phylogenetic Relationships of the Genus Naegleria (Protista: Rhizopoda). Molecular Biology and Evolution, 6, 243–257.PubMedGoogle Scholar
  9. Botilde Y., Laurent T., Quispe Tintaya W., et al. 2006. Comparison of molecular markers for strain typing of Leishmania infantum. Infection Genetics and Evolution, 6, 440–446. DOI: 10.1016/j.meegid.2006.02.003.CrossRefGoogle Scholar
  10. Briones M.R., Nelson K., Beverley S.M., Affonso H.T., Camargo E.P., Floeter-Winter L.M. 1992. Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene. Molecular and Biochemical Parasitology, 53, 121–127. DOI: 10.1016/0166-6851(92)90014-B.PubMedCrossRefGoogle Scholar
  11. Bu L.-Y., Hu X.-S., Jing B.-Q., Yi T.-L. 2000. Sequence analysis of SSU rDNA variable regions of Leishmania isolates from hilly foci and plain foci of China. Chinese Journal of Parasitology and Parasitic Disease, 18, 321–324 (in Chinese with English abstract).Google Scholar
  12. Cao D.-P., Guo X.-G., Chen D.-L., Chen J.-P. 2011. Species delimitation and phylogenetic relationships of Chinese Leishmania isolates re-examined using kinetoplast cytochrome oxidase II gene sequences. Parasitology Research, 109, 163–173. DOI: 10.1007/s00436-010-2239-6.PubMedCrossRefGoogle Scholar
  13. Clark C.G., Martin D.S., Diamond L.S. 1995. Phylogenetic relationships among anuran trypanosomes as revealed by riboprinting. Journal of Eukaryotic Microbiology, 42, 92–96. DOI: 10.1111/j.1550-7408.1995.tb01546.x.PubMedCrossRefGoogle Scholar
  14. Croan D., Ellis J. 1996. Phylogenetic relationships between Leishmania, Viannia and Sauroleishmania inferred from comparison of a variable domain within the RNA polymerase II largest subunit gene. Molecular and Biochemical Parasitology, 79, 97–102. DOI: 10.1016/0166-6851(96)02629-1.PubMedCrossRefGoogle Scholar
  15. Croan D.G., Morrison D.A., Ellis J.T. 1997. Evolution of the genus Leishmania revealed by comparison of DNA and RNA polymerase gene sequences. Molecular and Biochemical Parasitology, 89, 149–159. DOI: 10.1016/S0166-6851(97)00111-4.PubMedCrossRefGoogle Scholar
  16. Desjeux P. 2004. Leishmaniasis: current situation and new perspectives. Comparative Immunology, Microbiology and Infectious Diseases, 27, 305–318. DOI: 10.1016/j.cimid.2004.03.004.PubMedCrossRefGoogle Scholar
  17. El Tai N.O., El Fari M., Mauricio I., Miles M.A., Oskam L., El Safi S.H., Presber W.H., Schönian G. 2001. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Experimental Parasitology, 97, 35–44. DOI: 10.1006/expr.2001.4592.PubMedCrossRefGoogle Scholar
  18. Fernandes A.P., Nelson K., Beverley S.M. 1993. Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism. Proceedings of the National Academy of Sciences of the United States of America, 90, 11608–11612. DOI: 10.1073/pnas.90.24.11608.PubMedCrossRefGoogle Scholar
  19. Fraga J., Montalvo A.M., Doncker S.D., Dujardin J.C., der Auwera G.V. 2010. Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infection, Genetics and Evolution, 10, 238–245. DOI: 10.1016/j.meegid.2009.11.007.PubMedCrossRefGoogle Scholar
  20. Fryauff D.J., Hanafi H.A., Klena J.D., Hoel D.F., Appawu M., Rogers W., Puplampu N., Odoom S., Kweku M., Koram K., Wilson M., Raczniak G., Boakye D. 2006. Short report: ITS1-DNA sequence confirmation of Leishmania major as a cause of cutaneous leishmaniasis from an outbreak focus in the HO district, southern Ghana. Tropical Medicine and Hygiene, 75, 502–504.PubMedGoogle Scholar
  21. Grimaldi G., Kreutzer R.D., Hashiguchi Y., Gomez E.A., Mimory T., Tesh R.B. 1992. Description of Leishmania equatorensis sp. n. (Kinetoplastida, Trypanosomatidae), a new parasite infecting arboreal mammals in Ecuador. Memorias do Instituto Oswaldo Cruz, 87, 221–228. DOI: 10.1590/S0074-02761992000200009.CrossRefGoogle Scholar
  22. Guan L.-R., Yang Y.-Q., Ren H.-Y., Shen W.-X. 1995. Discovery and study of Leishmania turanica for the first time in China. Bulletin of World Health Organ, 69, 595–601.Google Scholar
  23. Ho J.W.K., Adams C.E., Lew J.B., et al. 2006. SeqVis: visualization of compositional heterogeneity in large alignments of nucleotides. Bioinformatics, 22, 2162–2163. DOI: 10.1093/bioinformatics/btl283.PubMedCrossRefGoogle Scholar
  24. Hu X.-S., Bu L., Ma Y., Wang Y., Jing B., Yi T. 2002. Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemi foci of visceral leishmaniasis in China. Chinese Medical Journal (Engl.), 115, 1457–1459.Google Scholar
  25. Hu X.-S., Lin F.-Q., Kan B., et al. 1992. Identification of Leishmania donovani isolates from different kala-azar foci in China by kDNA hybridization. Chinese Medical Sciences Journal, 7, 63–66 (in Chinese with English abstract).PubMedGoogle Scholar
  26. Hughes A.L., Piontkivska H. 2003. Phylogeny of Trypanosomatidae and Bodonidae (Kinetoplastida) based on 18S rRNA: evi dence for paraphyly of Trypanosoma and six other genera. Molecular Biology and Evolution, 20, 644–652. DOI: 10.1093/molbev/msg062.PubMedCrossRefGoogle Scholar
  27. Ibrahim M.E., Barker D.C. 2001. The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence. Infection, Genetics and Evolution, 1, 61–68. DOI: 10.1016/S1567-1348(01)00009-0.PubMedCrossRefGoogle Scholar
  28. Johnson A.M., Baverstock P.R. 1989. Rapid ribosomal RNA sequencing and the phylogenetic analysis of protists. Parasitology Today, 5, 102–108. DOI: 10.1016/0169-4758(89) 90046-X.PubMedCrossRefGoogle Scholar
  29. Kuhls K., Mauricio I.L., Pratlong F., Presber W., Schönian G. 2005. Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex. Microbes Infect, 7, 1224–1234. DOI: 10.1016/j.micinf.2005.04.009.PubMedCrossRefGoogle Scholar
  30. Kuwahara K., Kato H., Gomez E.A., et al. 2009. Genetic diversity of ribosomal RNA internal transcribed spacer sequences in Lutzomyia species from areas endemic for New World cutaneous leishmaniasis. Acta Tropica, 112, 131–136. DOI: 10.1016/j.acta-tropica.2009.07.010.PubMedCrossRefGoogle Scholar
  31. Landweber L.F., Gilbert W. 1994. Phylogenetic analysis of RNA editing: A primitive genetic phenomenon. Proceedings of the National Academy of Sciences of the United States of America, 91, 918–921. DOI: 10.1073/pnas.91.3.918.PubMedCrossRefGoogle Scholar
  32. Looker D., Miller L.A., Elwood H.J., Stickel S., Sogin M.L. 1988. Primary structure of the Leishmania donovani small subunit ribosomal RNA coding region. Nucleic Acids Research, 16, 7198. DOI: 10.1093/nar/16.14.7198.PubMedCrossRefGoogle Scholar
  33. Lu D.-M., Hu X.-S., Qiao Z.-D. 2001. Analysis of Leishmania species and strains from China by RAPD technique. Chinese Journal of Parasitology and Parasitic Disease, 19, 290–293 (in Chinese with English abstract).Google Scholar
  34. Lu H.-G., Hu X.-S. 1988. Identification of Leishmania by dot blot hybridization with photobiotin labelled kDNA. Journal of West China University of Medical Science, 19, 222–225 (in Chinese with English abstract).Google Scholar
  35. Lu H.-G., Zhong L., Guan L.-R., Qu J.-Q., Hu X.-S., Chai J.-C., Xu Z.-B., Wang C.-T., Chang K.-P. 1994. Separation of Chinese Leishmania isolates into five genotypes by kinetoplast and chromosomal DNA heterogeneity. American Journal of Tropical Medicine and Hygiene, 50, 763–770.PubMedGoogle Scholar
  36. Lukeš J., Mauricio I. L., Schönian G., et al. 2007. Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy. Proceedings of the National Academy of Sciences of the United States of America, 104, 9375–9380. DOI: 10.1073/pnas.0703678104.PubMedCrossRefGoogle Scholar
  37. Luyo-Acero G.E., Uezato H., Oshiro M., Takei K., Katakara K., Gomez-Landires E., Hashiguchi Y., Nonakas S. 2004. Sequence variation of the Cytochrome b gene of various human infecting members of the genus Leishmania and their phylogeny. Parasitology, 128, 483–491. DOI: 10.1017/S0031182004004792.PubMedCrossRefGoogle Scholar
  38. Marche S., Roth C., Philippe E., Dollet M., Baltz T. 1995. Characterisation and detection of plant trypanosomatids by sequence analysis of the small subunit ribosomal RNA gene. Molecular and Biochemical Parasitology, 71, 15–26. DOI: 10.1016/ 0166-6851(95)00029-Z.PubMedCrossRefGoogle Scholar
  39. Marcili A., Lima L., Valente V.C., et al. 2009. Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering. Infection, Genetics and Evolution, 9, 1265–1274. DOI: 10.1016/j.meegid.2009.07.003.PubMedCrossRefGoogle Scholar
  40. Maslov D.A., Lukes J., Jirku M., Simpson L. 1996. Phylogeny of trypanosomes as inferred from the small and large subunit rRNAs: implications for the evolution of parasitism in the trypanosomatid protozoa. Molecular and Biochemical Parasitology, 75, 197–205. DOI: 10.1016/0166-6851(95)02526-X.PubMedCrossRefGoogle Scholar
  41. Maslov D.A., Simpson L. 1995. Evolution of Parasitism in Kinetoplastid Protozoa. Parasitology Today, 11, 30–32. DOI: 10.1016/0169-4758(95)80106-5.CrossRefGoogle Scholar
  42. Noyes H.A., Arana B.A., Chance M.L., Wingon R. 1997. The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) Complex and the Lizard Leishmania: Their Classification and Evidence for a Neotropical Origin of the Leishmania-Endotrypanum Clade. Journal of Eukaryotic Microbiology, 44, 511–517. DOI: 10.1111/j.1550-7408.1997.tb05732.x.PubMedCrossRefGoogle Scholar
  43. Noyes H.A., Chance M.L., Dedet J.P. 2000. A novel Leishmania from Martinique. School of Biological Sciences, University of Liverpool, Crown St, Liverpool, Merseyside L69 7ZD, UK.Google Scholar
  44. Noyes H.A., Camps A.P., Chance M.L. 1996. Leishmania herreri (Kinetoplastida: Trypanosomatidae) is more closely related to Endotrypanum (Kinetoplastida: Trypanosomatidae) than to Leishmania. Molecular and Biochemical Parasitology, 80, 119–123. DOI: 10.1016/0166-6851(96)02679-5.PubMedCrossRefGoogle Scholar
  45. Piarroux R., Fontes M., Perasso R., Gambarelli F., Joblet C., Dumon H., Auilici M. 1995. Phylogenetic relationships between Old World Leishmania strains revealed by analysis of a repetitive DNA sequence. Molecular and Biochemical Parasitology, 73, 249–252. DOI: 10.1016/0166-6851(95)00097-K.PubMedCrossRefGoogle Scholar
  46. Posada D., Crandall K.A. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics, 14, 817–818. DOI: 10.1093/bioinformatics/14.9.817.PubMedCrossRefGoogle Scholar
  47. Raymond F., Boisvert S., Roy G., Ritt J.F., Legare D., Isnard A., Stanke M., Olivier M., Tremblay J.M., Papadopoulou B., Ouellette M., Corbeil J. 2011. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the incracellular stage of human pathogenic species. Nucleic Acids Research, 2011, 1–17. DOI: 10.1093/nar/gkr834.Google Scholar
  48. Ronquist F., Huelsenbeck J.P. 2003. MrBayes 3: Bayesian phylogenetic inferrence under mixed models. Bioinformatics, 19, 1572–1574. DOI: 10.1093/bioinformatics/btg180.PubMedCrossRefGoogle Scholar
  49. Sambrook J., Russell D.W. 2001. Molecular cloning: a laboratory manual (3rd ed.) Cold Spring Harbor Laboratory Press, New York.Google Scholar
  50. Schönian G., Mauricio I., Cupolillo E. 2010. Is it time to revise the nomenclature of Leishmania? Trends in Parasitology, 26, 466–469. DOI: 10.1016/j.pt.2010.06.013.PubMedCrossRefGoogle Scholar
  51. Slowinski J.B., Page R.D.M. 1999. How should species phylogenies be inferred from sequence data? Systematic Biology, 48, 814–825.PubMedCrossRefGoogle Scholar
  52. Snyder M.J., Maddison D.R. 1997. Molecular phylogeny of glutathione-S-transferases. DNA and Cell Biology, 16, 1373–1384. DOI: 10.1089/dna.1997.16.1373.PubMedCrossRefGoogle Scholar
  53. Sogin M.L., Elwood H.J., Gunderson J.H. 1986. Evolutionary diversity of eukaryotic small subunit rRNA gene. Proceedings of the National Academy of Sciences of the United States of America, 83, 1383–1387. DOI: 10.1073/pnas.83.5.1383.PubMedCrossRefGoogle Scholar
  54. Stevens J.R., Noyes H.A., Schofield C.J., Gibson W. 2001. The molecular evolution of Trypanosomatidae. Advances in Parasitology, 48, 1–56. DOI: 10.1016/S0065-308X(01)48003-1.PubMedCrossRefGoogle Scholar
  55. Suankratay C., Suwanpimolkul G., Wilde H., Siriyasatien P. 2010. Autochthonous visceral leishmaniasis in a human immunodeficiency virus (HIV)-infected patient: the first in Thailand and review of the literature. Tropical Medicine and Hygiene, 82, 4–8. DOI: 10.4269/ajtmh.2010.09-0434.PubMedCrossRefGoogle Scholar
  56. Swofford D.L. 2002. PAUP*. Phylogenetic analysis using parsimony (* and other methods), version 4. Sinauer, Sunderland, MA.Google Scholar
  57. Tamura K., Dudley J., Nei M., Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596–1599. DOI: 10.1093/molbev/msm092.PubMedCrossRefGoogle Scholar
  58. Thompson J.D., Gibson T.J., Plewniak F., et al. 1997. The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882. DOI: 10.1093/nar/25.24.4876.PubMedCrossRefGoogle Scholar
  59. Tian Y., Chen J.-P. 2005. Cloning and sequence analysis of ITS gene of Leishmania donovani isolates from different epidemic Foci in China. Journal of Biomedical Engineering, 22, 540–544 (in Chinese with English abstract).PubMedGoogle Scholar
  60. Trainor K.E., Porter B.F., Logan K.S., Hoffman R.J., Snowden K.F. 2010. Eight cases feline cutaneous leishmaniasis in Texas. Veterinary Pathology, 47, 1076–1081. DOI: 10.1177/0300985810382094.PubMedCrossRefGoogle Scholar
  61. Uliana S.R.B., Affonso M.H.T., Camargo E.P., Floeter-Winter L.M. 1991. Leishmania: genus identification based on a specific sequence of the 18S ribosomal RNA sequence. Experimental Parasitology, 72, 157–163. DOI: 10.1016/0014-4894(91)90 133-H.PubMedCrossRefGoogle Scholar
  62. Van Eys G.J., Schoone G.J., Kroon N.C., et al. 1992. Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Molecular and Biochemical Parasitology, 51, 133–142.PubMedCrossRefGoogle Scholar
  63. Villinski J.T., Klena J.D., Abbassya M., Hoel D.F., Puplampuc N., Mechta S., Boakye D., Raczniak G. 2008. Evidence for a new species of Leishmania associated with a focal disease outbreak in Ghana. Diagnostic Microbiology and Infectious Disease, 60, 323–327. DOI: 10.1016/j.diagmicrobio.2007.09.013.PubMedCrossRefGoogle Scholar
  64. Waters A.P., McCutchan T.F. 1990. Ribosomal RNA: nature’s own polymerase amplified target for diagnosis. Parasitology Today, 6, 56–60. DOI: 10.1016/0169-4758(90)90071-B.PubMedCrossRefGoogle Scholar
  65. Xu Z.-B., Le Blancq S., Evans D.A., Peters W. 1984. The characterization by isoenzyme electrophoresis of Leishmania isolated in the People’s Republic of China. Transaction of the Royal Society of Tropical Medicine and Hygiene, 78, 689–693.CrossRefGoogle Scholar
  66. Yang B.-B., Guo X.-G., Hu X.-S., Zhang J.-G., Liao L., Chen D.-L., Chen J.-P. 2010. Species discrimination and phylogenetic inference of 17 Chinese Leishmania isolates based on internal transcribed spacer 1 (ITS1) sequences. Parasitology Research, 107, 1049–1065. DOI: 10.1007/s00436-010-1969-9.PubMedCrossRefGoogle Scholar
  67. Zheng X.-L., Hu X.-S., Chen J.-P. 1999. Analysis of kinetoplast DNA of Leishmania isolates in China by PCR-SSCP. Chinese Journal of Parasitology and Parasitic Disease, 17, 346–349 (in Chinese with English abstract).Google Scholar
  68. Zelazny A.M., Fedorko D.P., Li L., Neva A.F., Fischer S.H. 2005. Evaluation of 7SL RNA gene sequence for the identification of Leishmania spp. American Journal of Tropical Medicine and Hygiene, 72, 415–420.PubMedGoogle Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2012

Authors and Affiliations

  1. 1.Department of Parasitology, School of Preclinical and Forensic MedicineSichuan UniversityChengduSichuan, China
  2. 2.Department of ParasitologyNorth Sichuan Medical CollegeNanchongSichuan Province, China
  3. 3.Institute of ParasitologyMedical College of Qinghai UmiversityXining City, Qinghai ProvinceChina
  4. 4.Animal Disease Prevention and Food Safety Key Laboratory of Sichuan ProvinceSichuan UniversityChengdu, SichuanChina

Personalised recommendations