title: Development of recombinant BgP12 based enzyme linked immunosorbent assays for serodiagnosis of Babesia gibsoni infection in dogs.
abstract: Indirect ELISA and dot-ELISA using recombinant BgP12 (rBgP12) were developed for the diagnosis of Babesia gibsoni infected dogs. The complete open reading frame of BgP12 gene (378bp) was cloned in pET-32a(+) expression vector and expressed in Escherichia coli as a soluble thioredoxin (Trx) fusion protein. The purified rBgP12 was used for production of anti-rBgP12 rabbit serum, which recognized a native 12-kDa protein in B. gibsoni infected erythrocyte by Western blot analysis. To evaluate the potential of rBgP12 for the serodiagnosis of B. gibsoni, a panel of serum/plasma samples from dogs infected with B. gibsoni (n=13), uninfected sera (n=13) and sera from dogs infected with other haemoparasites viz., Babesia canis vogeli (n=3), Ehrlichia canis (n=3), Hepatozoon canis (n=1) and Dirofilaria immitis (n=1) were used in ELISA formats. In addition, the performance of rBgP12 based indirect ELISA and dot-ELISA were evaluated using 75 serum/plasma samples collected from suspected dogs, in respect to the nested PCR as reference test. The diagnostic sensitivities of indirect ELISA and dot-ELISA were 94.59% and 89.18%, respectively, while their specificities were 84.21% and 81.57%, respectively. Moreover, both the assays using rBgP12 showed no cross reaction with sera from dogs infected with other common haemoparasites indicating their high specificity. High kappa values of indirect ELISA and dot-ELISA indicated the potentials of these assays with substantial agreement at 95% confidence level. It is concluded that indirect ELISA and dot ELISA using rBgP12 might be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs.
authors.affiliation: Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Mrityunjay
authors.initials: M
authors.lastname: Mandal
authors.affiliation: Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India. Electronic address: banerjeeparth62@gmail.com.
authors.forename: Partha S
authors.initials: PS
authors.lastname: Banerjee
authors.affiliation: Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Saroj
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Rajat
authors.initials: R
authors.lastname: Garg
authors.affiliation: Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Hira
authors.initials: H
authors.lastname: Ram
authors.affiliation: Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Opinder K
authors.initials: OK
authors.lastname: Raina
mesh_terms: D000818:Animals; D001404:Babesiosis / Q000175:diagnosis*; D004283:Dog Diseases / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D017125:Genes, Protozoan; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests / Q000662:veterinary
chemical_list: C000611522:BgP12 protein, Babesia gibsoni; D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: Babesia gibsoni; Diagnosis; Dot-ELISA; Indirect ELISA; Recombinant BgP12