Monoclonal Antibody against
            <i>Babesia equi</i>
            : Characterization and Potential Application of Antigen for Serodiagnosis

Avarzed, Abgaandorjiin; Igarashi, Ikuo; Waal, Theo de; Kawai, Shinya; Oomori, Yukio; Inoue, Noboru; Maki, Yoshiyuki; Omata, Yoshitaka; Saito, Atsushi; Nagasawa, Hideyuki; Toyoda, Yutaka; Suzuki, Naoyoshi

doi:10.1128/jcm.36.7.1835-1839.1998

Cited by 18 publications

(1 citation statement)

References 22 publications

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“…Five plasmids, respectively containing an insert of 18S rRNA sequence representing each T. equi genotype, were prepared and used for specificity testing of the developed PCR assays. A long 18S rRNA fragment (1591 bp) of genotype A was amplified from DNA extracted from an in vitro culture (T. equi USDA strain) [32], using the forward primer Nbab_1F and reverse primer TB-rev [33,34], as described previously [24]. The 18S rRNA of genotype B was amplified from a DNA template synthesized based on a Sudanese sequence (GenBank accession number AB515312).…”

Section: Plasmids Containing 18s Rrna Representing Each T Equi Genotypementioning

confidence: 99%

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

Ahedor,

Otgonsuren,

Zhyldyz

et al. 2023

Parasites Vectors

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Background Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. Methods A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. Results Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. Conclusions The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes. Graphical abstract

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Section: Plasmids Containing 18s Rrna Representing Each T Equi Genotypementioning

confidence: 99%

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

Ahedor,

Otgonsuren,

Zhyldyz

et al. 2023

Parasites Vectors

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Babesia caballi and Babesia equi: Implications of host sialic acids in erythrocyte infection

Okamura

Yokoyama

Wickramathilaka

et al. 2005

Experimental Parasitology

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Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni

El-Shehabi¹,

Vermeire²,

Yoshino³

et al. 2009

Experimental Parasitology

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A Schistosoma mansoni G-protein coupled receptor (SmGPCR) was previously cloned and shown to be activated by the biogenic amine, histamine. Here we report a first investigation of the receptor’s subunit organization, tissue distribution and expression levels in different stages of the parasite. A polyclonal antibody was produced in rabbits against the recombinant third intracellular loop (il3) of SmGPCR. Western blot studies of the native receptor and recombinant protein expressed in HEK293 cells showed that SmGPCR exists both as a monomer (65 kDa) and an apparent dimer of ≈130 kDa These species were verified by immunoprecipitation of SmGPCR from S. mansoni extracts, using antibody that was covalently attached to agarose beads. Further investigation determined that the SmGPCR dimer was resistant to treatment with various detergents, 4 M urea and 0.1 M DTT but could be made to dissociate at acidic pH, suggesting the dimer is non-covalent in nature. Confocal immunofluorescence studies revealed significant SmGPCR immunoreactivity in sporocysts, schistosomula and adult worms but not miracidia. SmGPCR was found to be most widely expressed in the schistosomula, particularly the tegument, the subtegumental musculature and the acetabulum. In the adult stage we detected SmGPCR immunofluorescence mainly in the tubercles of male worms and, to a lesser extent, the body wall musculature. Localization in sporocysts was mainly confined to the tegument and cells within parenchymal matrices. A realtime quantitative reverse-transcription PCR analysis revealed that SmGPCR is upregulated at the mRNA level in the parasitic stages compared to the free-living miracidium and cercariae, and it is particularly elevated during early sporocyst and schistosomula development. The results identify SmGPCR as an important parasite receptor with potential functions in muscle and the tegument of S. mansoni.

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Monoclonal Antibody against Babesia equi : Characterization and Potential Application of Antigen for Serodiagnosis

Cited by 18 publications

References 22 publications

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

Babesia caballi and Babesia equi: Implications of host sialic acids in erythrocyte infection

Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni

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