title: Molecular identification and antigenic characterization of a merozoite surface antigen and a secreted antigen of Babesia canis (BcMSA1 and BcSA1).
abstract: BACKGROUND
  Babesia canis is an apicomplexan tick-transmitted hemoprotozoan responsible for causing canine babesiosis in Europe and west Asia. Despite its importance, there is no known rapid diagnostic kit detection of B. canis infection in dogs. The present study identified two novel antigens of B. canis and used the recombinant antigens to establish a rapid, specific and sensitive serodiagnostic technique for detection of B. canis infection.
  
  
  METHODS
  A complementary DNA (cDNA) expression library was constructed from the mRNA of B. canis and immunoscreened using B. canis-infected dog sera. The cDNAs encoding a merozoite surface antigen and a secreted antigen protein were identified and designated as BcMSA1 and BcSA1, respectively. The recombinant BcMSA1 and BcSA1 (rBcMSA1 and rBcSA1) expressed in Escherichia coli were purified and injected into mice for production of anti-sera. The native proteins were characterized by Western blot analysis and immunofluorescence. Furthermore, indirect enzyme-linked immunosorbent assays (iELISA) and rapid immunochromatographic tests (ICT) based on rBcMSA1 or rBcSA1 were established and evaluated to test specific antibodies in consecutive plasma samples from two B. canis-infected dogs.
  
  
  RESULTS
  Antiserum raised against rBcMSA1 and rBcSA1 recognized the 39 kDa and 44 kDa native proteins by Western blot analysis, respectively. In addition, immunofluorescence and confocal microscopic observations revealed that BcMSA1 was found on the surface of parasites. However, BcSA1 localized in the matrix of the merozoites. The ELISA and ICT based on rBcMSA1 or rBcSA1 could detect specific antibodies in consecutive plasma samples from two B. canis-infected dogs. They showed no cross-reactions against the serum samples collected from dogs experimentally infected with closely related parasites.
  
  
  CONCLUSIONS
  Taken together, the current results indicated that the rBcMSA1 and rBcSA1 are promising serodiagnostic antigens for developing iELISA and ICT to detect B. canis infection. To our knowledge, this study is the first to report BcMSA1 and BcSA1 as potential antigenic proteins for serodiagnosis of B. canis infection in dogs.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Mo
authors.initials: M
authors.lastname: Zhou
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.|Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Maduan Street 427, Nangang District, Harbin, 150001, PR China.
authors.forename: Shinuo
authors.initials: S
authors.lastname: Cao
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Yuzi
authors.initials: Y
authors.lastname: Luo
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Mingming
authors.initials: M
authors.lastname: Liu
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Guanbo
authors.initials: G
authors.lastname: Wang
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Paul Franck Adjou
authors.initials: PF
authors.lastname: Moumouni
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Charoonluk
authors.initials: C
authors.lastname: Jirapattharasate
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Iguchi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Patrick
authors.initials: P
authors.lastname: Vudriko
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Mohamad Alaa
authors.initials: MA
authors.lastname: Terkawi
authors.affiliation: Megacor Diagnostik GmbH, Hoerbranz, Vorarlberg, A-6912, Austria.
authors.forename: Mario
authors.initials: M
authors.lastname: Löwenstein
authors.affiliation: Megacor Diagnostik GmbH, Hoerbranz, Vorarlberg, A-6912, Austria.
authors.forename: Angela
authors.initials: A
authors.lastname: Kern
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nishikawa
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. gen@obihiro.ac.jp.
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000378:metabolism*; D001403:Babesia / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D015723:Gene Library; D020066:Merozoite Surface Protein 1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data
chemical_list: D000953:Antigens, Protozoan; D018076:DNA, Complementary; D020066:Merozoite Surface Protein 1
keywords: Babesia canis; BcMSA1; BcSA1; Canine babesiosis; ELISA; Immunochromatographic tests