Elsevier

Experimental Parasitology

Volume 130, Issue 2, February 2012, Pages 178-182
Experimental Parasitology

Research Brief
A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model

https://doi.org/10.1016/j.exppara.2011.10.012Get rights and content

Abstract

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.

Highlights

► A sandwich ELISA targeting secreted antigen 1 of Babesia microti (BmSA1) was developed. ► The sandwich ELISA detected circulating BmSA1 earlier than antibody detection by indirect ELISA. ► The kinetics of circulating BmSA1 coincided with the profile of parasitemia. ► This assay was more sensitive than the microscopic examination of Giemsa-stained blood smears. ► BmSA1 could be a potential marker for surveillance of human babesiosis.

Introduction

Babesia microti is a tick-transmitted intraerythrocytic protozoon of the genus Babesia. The parasite has recently emerged as lethal opportunistic pathogens in immunocompromised patients (Homer et al., 2000, Vannier and Krause, 2009). Human babesiosis is caused by some babesial species that have distinct geographic distributions on the basis of the presence of their hosts (Vannier and Krause, 2009). B. microti has initially been identified to be prevalent in diverse regions of the United States (Scholtens et al., 1968, Krause et al., 2003) and recently emerged in different parts of the world including Europe (Herwaldt et al., 2003, Hildebrandt et al., 2007) and Asia (Shih et al., 1997, Wei et al., 2001). The infection usually causes asymptomatic presentation to flu-like symptoms with fever, chills, sweat, headache, fatigue, and anemia (Homer et al., 2000, Gubernot et al., 2009a). Severe or fatal illness mainly occurs in elderly, splenectomized and immunocompromised people (Rosner et al., 1984, Krause et al., 2008, Gubernot et al., 2009b). In human, B. microti is known to be mainly transmitted in nature by Ixodes scapularis tick. However, a large number of infections have been documented to be transmitted via blood transfusion (Saito-Ito et al., 2000, Leiby, 2006, Gubernot et al., 2009b). The lack of specific symptoms in asymptomatic individuals exhibiting low parasitemia can be a potential threat for blood transfusion (Krause et al., 1998). Therefore, effective diagnostic tests are urgently needed for detection of the parasite carriers and differentiation of the infection stage.

Microscopic examination of Giemsa-stained blood smear has been considered as the gold standard for Babesia diagnosis, but it is problematic for early and chronic stages of infection when the parasitemia is very low. Alternatively, polymerase chain reaction (PCR) has been used for detecting B. microti infection yielding high sensitivity and specificity (Persing et al., 1992). However, this method requires expensive laboratory equipments and well-trained personnel, thus these problems have limited the application of the assay. Inoculation of susceptible animals is reliable, but not practical for routine diagnosis (Kjemtrup and Conrad, 2000). On the other hand, serological methods, including immunofluorescent antibody test (IFAT), indirect enzyme-linked immunosorbent assay (iELISA), and immunochromatographic test (ICT) have been developed for antibody detection (Krause et al., 1994, Homer et al., 2003, Luo et al., 2011). However, these tests are incapable of detecting the infection at an early stage. Furthermore, antibody level may only indicate past exposure to the parasite rather than current infection and even potential disease. This is because the antibody level can remain for a long time, even after the parasites have been eliminated (Gubernot et al., 2009a). Secreted antigens or circulating antigens that are released into plasma by the parasites could be potential targets for antigen detection necessary for defining the current infection status in the host. Indeed, double antibody sandwich ELISA (DAS-ELISA) for detecting circulating antigens has been successfully applied for serological diagnosis of many protozoan infections (Montealegre et al., 1987, De Arruda et al., 2004, Dondorp et al., 2005, Chen et al., 2008). We believe that a combined iELISA and DAS-ELISA approaches could provide powerful tools for accurate diagnosis of B. microti infection. Therefore, we have attempted to develop a DAS-ELISA based on the antibodies to the recombinant B. microti secreted antigen 1 (rBmSA1). Our data might provide a new avenue for diagnosis of Babesia infection in human and animals.

Section snippets

Parasites and experimental animals

B. microti human Gray strain (US type, American Type Culture Collection, Catalog No. 30221) was maintained in specific pathogen-free (SPF) Golden Syrian hamsters (Clea, Japan) by intraperitoneal injection with 107 B. microti-infected erythrocytes. Infection was monitored by microscopic examination of Giemsa-stained blood smears. Blood was collected during different stages of infection, and then the serum fraction was separated by centrifuging the collected blood at 3500g for 30 min, and then

Development of DAS- ELISA

Circulating BmSA1 was previously identified by immunoscreening of a B. microti cDNA expression library using antisera generated using plasma of hamsters infected with B. microti (Luo et al., 2011). In order to confirm the secretory nature of BmSA1, Western blotting was carried out using sera of B. microti-infected hamster and SPF hamster probed with rabbit anti-rBmSA1 serum. As shown in Fig. 1, authentic BmSA1 was detected in serum of B. microti-infected hamster and the molecular weight was

Discussion

Diagnosis of human babesiosis can be achieved either directly by detecting parasites by microscopy and PCR, or indirectly by detecting the antibody to the parasitic pathogen using IFAT and iELISA (Hunfeld et al., 2008). However, these tests are not always sensitive and specific enough to detect the parasitic infection and to differentiate the stage of infection. Thus, an alternative diagnostic strategy is extremely desirable for the detection of this disease. During the parasite infection, a

Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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