Elsevier

Acta Tropica

Volume 202, February 2020, 105245
Acta Tropica

Development and evaluation of a chemiluminescence immunoassay for detecting tropical theileriosis

https://doi.org/10.1016/j.actatropica.2019.105245Get rights and content

Highlights

  • We first developed a CLIA method based on rSVSP450 for detecting tropical theileriosis.

  • The CLIA method required a much shorter assay time.

  • The CLIA showed high sensitivity, specificity, reproductivity and reliability for detecting antibodies against T. annulata.

  • The high seroprevalence of T. annulata varied from 53.3% to 67.3% in Xinjiang, Gansu, Shaanxi and Hubei provinces.

Abstract

Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection.

Introduction

Theileria annulata is a tick-borne apicomplexan parasite causing tropical theileriosis, which predominantly infects cattle in tropical and subtropical areas around the world (Uilenberg et al., 1981; Bishop et al., 2004; Sivakumar et al., 2014). Although numerous Theileria species of tick-transmitted parasites have been identified in wild and domestic ruminants, only Theileria species (T. parva, T. annulata and T. lestoquardi) that have the ability to transform host cells, which are responsible for severe clinical diseases in animals (Mans et al., 2015; Nene and Morrison, 2016; Brown, 1997). In the worldwide, more than 0.25 billion cattle were infected with T. annulata, which led to economic losses of more than 0.8 billion US$ (Brown, 1997; Radositis et al., 1997;Sudan et al., 2014, 2015). To date, T. annulata is the only Theileria species to cause acute lymphoproliferative disease in China where T. parva is not present. The incidence and prevalence of tropical theileriosis ranges from 1.59% to 90% in endemic regions in China based on previous studies (Luo and Lu, 1997; Liu et al., 2015; Aihemaiti et al., 2017). Therefore, the disease causes substantial economic burden in the sustainable development of the livestock industry.

In the late 1970s, the CLIA was developed and widely applied in numerous fields of life sciences (Halmann et al., 1977; Mirasoli and Michelini, 2014). Due to its high sensitivity and signal-to-noise ratio, low background fluorescence signal and wide linear range, the CLIA has been extensively used for diagnosing of various diseases, including hepatitis B virus (HBV) infection (Yang et al., 2016), human immunodeficiency virus (HIV) infection (Alonso et al., 2014; Cui et al., 2015), and foot and mouth disease virus (FMDV) infection (Liu et al., 2018). However, to date, there are no related reports on use of the CLIA method to detect T. annulata infection.

The subtelomeric variable secreted proteins (SVSPs) family is the largest gene family in both T. parva and T. annulata, which may be associated with immune evasion, host transformation and invasion (Schmuckli et al., 2009; Weir et al., 2010; Tretina et al., 2015). The SVSPs are mainly expressed in the T. annulata and T. parva schizont stage, and the majority of the SVSPs of T. annulata are secreted into the host cell cytoplasm (Pain et al., 2005; Shiels et al., 2006; Schmuckli et al., 2009). However, can the genes of this family be used a diagnostic antigen for tropical theileriosis?

In the present study, a recombinant SVSP protein was used to develop a CLIA assay to detect tropical theileriosis, and the specificity and sensitivity of the CLIA were also evaluated. The diagnostic performance of the assay was determined by comparing it with ELISA. In addition, small-scale serological screening of serum samples from four endemic regions was conducted using the CLIA to further identify the reliability.

Section snippets

T. annulata-negative serum samples

Serum samples (n = 302) obtained from domestic cattle in Yuzhong and Wuwei regions of Gansu province in China during 2018 were detected using two indirect ELISA methods based on sporozoite and macroschizont gene 2 (Spm2) (GeneBank accession no: Y15795) protein and major piroplasma surface protein (MPSP) (GeneBank accession no: GQ180193.1) of T. sinensis, respectively (Tian et al., 2018; Zhao et al., 2017). Of these serum samples, 158 samples negative for T. annulata were selected and used to

Identification of the recombinant protein

The SDS-PAGE and western blotting results demonstrated that the recombinant SVSP450 protein had high purity and strong reaction with the anti-histidine tag antibody, which indicated the exact expression of the protein in E. coli (Fig. 1A and 1B). Specific test results showed that the recombinant protein had good immunoreactivity with T. annulata-positive sera, while no cross-reactivity with T. sergenti, T. sinensis, B. bovis, and B. bigemina-positive sera was observed (Fig. 2).

Development and evaluation of the CLIA

Following

Discussion

Although the indirect immunofluorescence assay (IFA) is the gold standard recommended by the OIE (2000) for the detection of T. annulata antibodies in cattle, this method is sophisticated, labor intensive and time-consuming compared with alternative methods. In previous studies, numerous serological assays, including indirect ELISAs and competitive ELISA targeting various antigen genes (Salih et al., 2005; Renneker et al., 2009; Abdo et al., 2010; Rajendran and Ray, 2014; Tian et al., 2018) for

Ethical approval

All of animal experiments in the study were approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. In the present study, all experimental animals used were dealt with according to the Animal Ethics Procedures and Guidelines of the People's Republic of China (SYXK2010-0001).

Declaration of competing interest

All authors declare that they have no competing conflicts of interest.

Acknowledgement

This study was supported by the grants of National Key Research and Development Program of China (2017YFD050403), 973 Program (2015CB150300), ASTIP(CAAS-ASTIP-2016-LVRI), and NBCIS(CARS-37), Central Public-interest Scientific Institution Basal Research Fund (1610312016009), and Jiangsu Co-innovation Center programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses.

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