Identification and Expression of a 50-Kilodalton Surface Antigen of
            <i>Babesia gibsoni</i>
            and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

Fukumoto, Shinya; Xuan, Xuenan; Nishikawa, Yoshifumi; Inoue, Noboru; Igarashi, Ikuo; Nagasawa, Hideyuki; Fujisaki, Kozo; Mikami, Takeshi

doi:10.1128/jcm.39.7.2603-2609.2001

Cited by 50 publications

(58 citation statements)

References 25 publications

(21 reference statements)

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“…In the previous studies, we have identified and cloned a novel gene encoding a surface antigen with a molecular mass of 50 kDa (P50) by immunoscreening of cDNA library prepared from mRNA of B. gibsoni merozoites with canine infected serum, and demonstrated that P50 is an immunodominant antigen [6,7]. The ELISA with GST-P50f expressed in E. coli was able to diagnose both acute and chronic B. gibsoni infections in dogs [8].…”

Section: Discussionmentioning

confidence: 99%

“…The NRCPD strain of B. gibsoni was experimentally infected and maintained in Beagles as described previously [6,12].…”

Section: Parasitementioning

confidence: 99%

“…Cloning of a truncated P50 gene: The truncated P50 (P50t) gene without sequences encoding highly hydrophobic signal peptide (aa 1-19) and C-terminus (aa 447-466) using a plasmid containing entire P50 gene [6] and following primers: 5'-ACG AAT TCT TAC CAC TCC CCT TCT -3' and 5'-ACG AAT TCC ACC ATC CTC ACC TTG -3', which both contains introduced EcoRI sites to facilitate cloning. The PCR product was cut with EcoRI and cloned into the EcoRI site of a bacterial expression vector, pGEX-4T-3 (Amersham Pharmacia Biotech, U.S.A.).…”

Section: Parasitementioning

confidence: 99%

“…Recently, Fukumoto et al have identified a novel gene encoding an immunodominat antigen, P50 of B. gibsoni, and evaluated its diagnostic potential in ELISA [6][7][8]. The ELISA with recombinant full-length P50 (P50f) expressed in Escherichia coli was able to differentiate clearly between B. gibsoni-infected do gs and B. canis-infected do gs or noninfected dogs [8].…”

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confidence: 99%

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Serodiagnosis of Babesia gibsoni Infection in Dogs by an Improved Enzyme-Linked Immunosorbent Assay with Recombinant Truncated P50

Verdida

Hara

Xuan

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-len gth), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliabl e reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.

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Section: Discussionmentioning

confidence: 99%

“…The NRCPD strain of B. gibsoni was experimentally infected and maintained in Beagles as described previously [6,12].…”

Section: Parasitementioning

confidence: 99%

Section: Parasitementioning

confidence: 99%

mentioning

confidence: 99%

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Serodiagnosis of Babesia gibsoni Infection in Dogs by an Improved Enzyme-Linked Immunosorbent Assay with Recombinant Truncated P50

Verdida

Hara

Xuan

et al. 2004

The Journal of Veterinary Medical Science

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“…Apicomplexa parasites (e.g. B. bovis) have been demonstrated to produce a high level of protein-free GPI that could interfere with the immune response against parasites (32,36).…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Characterization of Bc28.1, Major Erythrocyte-binding Protein from Babesia canis Merozoite Surface

Yang

Murciano

Moubri

et al. 2012

Journal of Biological Chemistry

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Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of protozoa parasites from the genus Babesia. Like Plasmodium falciparum, the agent of malaria, or Toxoplasma gondii, responsible for human toxoplasmosis, Babesia belongs to the Apicomplexa family. Babesia canis is the agent of the canine babesiosis in Europe. Clinical manifestations of this disease range from mild to severe and possibly lead to death by multiple organ failure. The identification and characterization of parasite surface proteins represent major goals, both for the understanding of the Apicomplexa invasion process and for the vaccine potential of such antigens. Indeed, we have already shown that Bd37, the major antigenic adhesion protein from Babesia divergens, the agent of bovine babesiosis, was able to induce complete protection against various parasite strains. The major merozoite surface antigens of Babesia canis have been described as a 28-kDa membrane protein family, anchored at the surface of the merozoite. Here, we demonstrate that Bc28.1, a major member of this multigenic family, is expressed at high levels at the surface of the merozoite. This protein is also found in the parasite in vitro culture supernatants, which are the basis of effective vaccines against canine babesiosis. We defined the erythrocyte binding function of Bc28.1 and determined its high resolution solution structure using NMR spectroscopy. Surprisingly, although these proteins are thought to play a similar role in the adhesion process, the structure of Bc28.1 from B. canis appears unrelated to the previously published structure of Bd37 from B. divergens. Site-directed mutagenesis experiments also suggest that the mechanism of the interaction with the erythrocyte membrane could be different for the two proteins. The resolution of the structure of Bc28 represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.The phylum Apicomplexa, a large group of single-celled eukaryotic intracellular parasites, includes some of the most important pathogenic parasites of humans and animals, the deadliest of which is the malaria parasite Plasmodium falciparum, responsible for one million human deaths per year and with almost half of the human population at risk of contracting malaria. No vaccine currently exists against P. falciparum, and parasites are becoming increasingly resistant to pharmaceuticals. This is also true for other pathogenic Apicomplexa, such as Toxoplasma, which, together with Plasmodium, challenge global human health improvement programs, and coccidiosis and babesiosis are detrimental for agri-business in both industrialized and developing countries. Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of an Apicomplexa from the Babesia genus. Hemolytic anemia due to parasite development leads to major symptoms, such as hemoglobinury, fever, asthenia, and renal failure. Amo...

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Protective efficacy of Babesia gibsoni culture-derived exoantigens against the challenge infection in dogs

et al. 2014

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The aim of this study is to determine the efficacy of exoantigens derived from Babesia gibsoni cultures to induce protective immunity against challenge exposure of virulent organisms. An attenuated B. gibsoni Oita strain was maintained in vitro by the microaerophilus stationary phase (MASP) method, and exoantigens-containing supernatant fluids were collected for preparation of the immunization. Two dogs received three subcutaneous immunizations with a 20-day interval of B. gibsoni exoantigens plus 0.5 mg saponin (Quil A). On day 68 after the prime immunization, the immunized dogs and control dogs were challenged intravenously with 2 × 10(8) virulent parasites of a homologous B. gibsoni strain. The results showed that exoantigens could induce a high degree of protection against virulent homologous challenge exposure. Two dogs immunized with exoantigens showed a lower parasitemia, accompanied by a slight decrease in the PCV that returned to normal values. Control dogs developed typical acute clinical signs, including severe anemia and hyperthermia. The immunization elicited humoral immune responses. In dogs immunized with exoantigens, the maximum antibody titer was 2,560 and 5,120 by indirect fluorescent antibody test (IFAT), respectively. Preliminary Western blot analysis of the immunogen revealed five dominant proteins of molecular weights of 18, 37, 43, 50, and 57 kDa. These results suggested that the culture-derived exoantigens were candidates for non-viable vaccine.

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Identification and Expression of a 50-Kilodalton Surface Antigen of Babesia gibsoni and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

Cited by 50 publications

References 25 publications

Serodiagnosis of Babesia gibsoni Infection in Dogs by an Improved Enzyme-Linked Immunosorbent Assay with Recombinant Truncated P50

Serodiagnosis of Babesia gibsoni Infection in Dogs by an Improved Enzyme-Linked Immunosorbent Assay with Recombinant Truncated P50

Structural and Functional Characterization of Bc28.1, Major Erythrocyte-binding Protein from Babesia canis Merozoite Surface

Protective efficacy of Babesia gibsoni culture-derived exoantigens against the challenge infection in dogs

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