Accepted Manuscript Title: The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis Author: Lucyna Holec-G˛asior Bartłomiej Ferra El˙zbieta Hiszczy´nska-Sawicka J´ozef Kur PII: DOI: Reference:
S0304-4017(14)00503-2 http://dx.doi.org/doi:10.1016/j.vetpar.2014.09.018 VETPAR 7398
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
7-10-2013 13-9-2014 20-9-2014
Please cite this article as: Holec-G˛asior, L., Ferra, B., Hiszczy´nska-Sawicka, E., Kur, J.,The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights 1. Recombinant antigens provide a new tool in the diagnosis of toxoplasmosis. 2. GRA1+P22+ROP1 mixture is the most effective for diagnosis of ovine toxoplasmosis.
Cocktail of recombinant proteins can replace the native antigens.
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The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1)
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for diagnosis of ovine toxoplasmosis
Gdańsk University of Technology, Chemical Faculty, Department of Microbiology,
Narutowicza 11/12 Str., 80-233 Gdańsk, POLAND
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AgResearch, Lincoln Science Centre, Private Bag 4749, Christchurch, NEW ZEALAND
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Lucyna Holec-Gąsior1*, Bartłomiej Ferra1, Elżbieta Hiszczyńska-Sawicka2, Józef Kur1
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Keywords: sheep; Toxoplasma gondii; recombinant antigen; GRA1; P22; ROP1; ELISA
*Corresponding author: Tel.: +48 58 3472406; fax: +48 58 3471822, e-mail address: [email protected] (L. Holec-Gąsior)
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Abstract Toxoplasmosis, caused by Toxoplasma gondii, is the major parasitic disease affecting sheep. Infection not only results in significant reproductive losses in these animals, but has
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public health implications since consumption of infected meat can facilitate zoonotic transmission. Although several serological tests are currently used for diagnosis of ovine
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toxoplasmosis, production of reliable reagents is a constraint and therefore there is a need to develop new diagnostic tools. In this paper, we assess for the first time, the preliminary
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diagnostic utility of nineteen T. gondii recombinant antigens (GRA1, GRA2ex2, GRA4, GRA5, GRA6, GRA9, SAG1, SAG4, BSR4, P22, ROP1, P36, MIC1ex2, MIC1ex34, MIC3,
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MAG1, BAG1, LDH1, and LDH2) in immunoglobulin G (IgG) enzyme-linked
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immunosorbent assays (IgG ELISAs). Following an initial evaluation, eight recombinant antigens (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, ROP1) were chosen for
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subsequent testing and comparison against the native Toxoplasma lysate antigen (TLA) in
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IgG ELISAs using 88 sera from naturally infected sheep and 20 sera from healthy animals. The reactivity of these antigens was variable with the best results for GRA1, P22, ROP1 and
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TLA. High sensitivity and specificity (100%) was noted for GRA1, ROP1 and TLA; P22 showed a slightly lower sensitivity (98.9%) but the same high specificity (100%). Four different combinations of these antigens (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1; M4: GRA1+P22+ROP1) were tested against the same pool of ovine sera; all IgG-positive serum samples were detected by all of the mixtures. However, the most effective for diagnosis of toxoplasmosis in sheep, based on the highest absorbance values, was the mixture M4 containing three proteins. High sensitivity and specificity (100%) was observed from tests containing either M4 or TLA antigens with a new pool of sera (93 seropositive and 35 seronegative). Thus, the present study shows that a cocktail of GRA1+P22+ROP1
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recombinant proteins can be used to diagnose T. gondii infection in sheep, and consequently
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will assist in epidemiological studies.
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1. Introduction Toxoplasmosis, a zoonotic infection caused by the protozoan parasite, Toxoplasma gondii, has economic relevance to both veterinary and human medicine. T. gondii has a worldwide
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distribution and is a major cause of congenital disease, abortion and stillbirth in humans and farm animals, especially sheep (Hill et al, 2005). Human infection is post-natal by ingesting
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tissue cysts from undercooked meat, or by consuming food and water contaminated with
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oocysts (Tenter et al., 2000). The ingestion of undercooked infected lamb meat is considered an important source of T. gondii infection for humans (Cook et al., 2000). In healthy
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individuals, a primary infection with T. gondii usually causes relatively mild flu-like symptoms, whereas in immunocompromised patients it can cause opportunistic life-
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threatening infections. Moreover, toxoplasmosis acquired by women during pregnancy poses a significant threat to the fetus. Mother to child transmission of the parasite occurs only when
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infection is acquired for the first time in the months just before or during pregnancy. The risk
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of transmission rises steeply with gestational age at maternal infection (from 1% to 90%), but
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the severity of disease in the fetus decreases (Dunn et al., 1999). Congenital infection may cause a spontaneous abortion or severe fetal abnormalities. Most children with congenital toxoplasmosis develop normally (Salt et al., 2005) but up to 4% die or have evidence of permanent neurological damage or bilateral visual impairment during the first years of life (Guerina et al., 1994; Gras et al., 2005). Congenital infection with T. gondii is also an important cause of abortion in sheep worldwide with millions of productive lambs lost due to parasite infection (Dubey, 2009). Sheep play a significant role in the economy of many countries since they are important sources of meat and milk products. Control of T. gondii infection in sheep and other domesticated animals used for meat production is important not only for reproductive reasons, but primarily for public health as Toxoplasma is recognized as food-borne risk (Jones and 5 Page 5 of 25
Holland, 2010). Seroprevalence of anti-T. gondii antibodies in sheep across the world range from 3% to 95% (Tenter at al., 2000; Dubey, 2009). Different climate conditions that interfere with the maintenance and viability of infective oocysts in the environment, and different types
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of sanitary management are the main reasons for the differences in seropositivity between countries (Robert-Gangneux and Darde, 2012). Furthermore, recent research has shown that
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the prevalence of antibodies in ewes was more than twice that in lambs, depending on the age of lambs sampled (Dubey, 2009).
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Serological techniques play a major role in the diagnosis of toxoplasmosis in humans and animals. Assays for the detection of T. gondii-specific antibodies are usually based on crude
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antigen extracts from T. gondii cultures, otherwise known as TLA or Toxoplasma lysate
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antigen. Alternatively, purified native proteins of the parasite are used. Production of these native parasitic antigens is limited, and their quantity in the antigen mixture are difficult to
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standardize. Furthermore the methods and source of native antigens involve the maintenance
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of parasite tissue cultures which can prove to be hazardous to laboratory staff handling the samples. Recombinant proteins, cloned and expressed in suitable bacterial or eukaryotic
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expression systems, could alleviate these issues and be a potential alternative source of antigens for diagnostic assays (Holec-Gąsior, 2013). The major advantages of recombinant antigens for the diagnosis of T. gondii infections are: 1) the precise antigen composition of the test is known, 2) more than one defined antigen can be used, 3) the method can easily be standardized and 4) the molecular methods used to produce recombinant antigens avoid the biohazard, time, labor and cost issues that accompany the production of native antigen. In the past few decades, several studies have reported the use of recombinant antigens for the serological detection of T. gondii infections particularly in humans (Holec-Gąsior, 2013). Recombinant antigens have been used to distinguish between infections that may have happened years ago and those which have been recently acquired. However, the use of these
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recombinant antigens in serodiagnosis of toxoplasmosis in livestock animals, has not been well-studied. Given that the proteome of T. gondii is well known and accurately described (Xia et
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al., 2008), there is significant potential for the biotechnological production of various parasitic antigens. T. gondii native proteins are localized in the outer membrane, cytosol, secretory
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organelles (micronemes, rhoptries) and the dense granules. During host cell invasion different Toxoplasma proteins are released to the forming parasitophorous vacuole (PV) or to the tissue
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cyst. The microneme proteins (MIC) are released first, upon contact with the host cells. These proteins are involved in host cell recognition and attachment (Carruthers et al., 1999). The
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rhoptry proteins (ROP) are released next, and they may facilitate formation of the PV and
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mediate its clustering with host cell organelles (Sam-Yellowe, 1996). The dense granular proteins (GRA) are exocytosed both during and after invasion into the PV. Following
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secretion, most of the GRA antigens appear as both a soluble and a membrane-associated
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form in the vacuole and they are thought to modify the environment within the PV, thereby functioning for intracellular survival and replication (Mercier et. al., 2002; 2005). Moreover,
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these proteins constitute an important fraction of antigens that circulate in the bloodstream during the first hours following infection (Hughes and Van, 1982). The surface of T. gondii is covered with a family of glycosylphosphatidylinositol (GPI)-anchored antigens (SAGs) and SAG-related sequence (SRS) proteins, most of which are members of the SAG1 or SAG2 families (Lekutis et al., 2001). These proteins appear to play a role in host cell invasion, immune modulation and/or virulence attenuation, although they may also provide protection needed by the parasite to survive in the environment (Lekutis et al., 2001). Genes encoding the aforementioned antigenic proteins of the parasite are good molecular targets for cloning and production of recombinant antigens of diagnostic significance.
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Considering the negative impact of toxoplasmosis on sheep reproduction and the risk of human transmission from the ingestion of undercooked infected meat, there is a real need for diagnosis of ovine toxoplasmosis and the development of new diagnostic methods for
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detection of T. gondii infection in sheep. This study investigates the usefulness of recombinant antigenic proteins for the serodiagnosis of ovine toxoplasmosis and proposes a
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new assay based on three reactive proteins.
Materials and methods
2.1.
Toxoplasma gondii recombinant antigens and TLA
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Nineteen T. gondii recombinant antigens: GRA1 (amino acids 24–190), GRA2ex2 (amino
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acids 51–185), GRA4 (amino acids 20–279), GRA5 (amino acids 26–94), GRA6 (amino acids 30–231), GRA9 (amino acids 21–328), SAG1 (amino acids 49–313), SAG4 (amino acids 26–
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150), BSR4 (amino acids 33–356), P22 (amino acids 30–231), ROP1 (amino acids 85–396),
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P36 (ROP9, amino acids 30–363), MIC1ex2 (amino acids 25–182), MIC1ex34 (amino acids 183–456), MIC3 (amino acids 67–359), MAG1 (amino acids 30–222), BAG1 (amino acids 1–
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229), LDH1 (amino acids 1–329), and LDH2 (amino acids 1–326) containing a cluster of six histidine residues for the purification by metal-affinity chromatography at the N- and Ctermini were obtained as previously described (Hiszczyńska-Sawicka et al., 2003; Hiszczyńska et al., 2005; Holec et al., 2007; Holec et al., 2008; Holec-Gąsior et al., 2009; Holec-Gąsior i Kur, 2010). The recombinant proteins were analyzed by SDS–PAGE on 12% acrylamide gels and stained with Coomassie blue. The concentration of purified proteins was determined by the Bradford method (Bradford, 1976) using bovine serum albumin (BSA) as a standard. Toxoplasma lysate antigen (TLA) from tachyzoites (strain RH) was prepared according to the method previously described (Holec-Gąsior et al., 2010). 8 Page 8 of 25
2.2.
Test sera
A total number of 260 ovine sera were received from the Veterinary Hygiene Station (Gdańsk, Poland). These serum samples have been obtained from epidemiological studies
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conducted on a population of nearly two thousand sheep from northern region of Poland. All serum samples were analyzed and divided into two groups in accordance with the results
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obtained using the agglutination test (Toxo-Screen DA, bioMérieux) and immunofluorescence test, with the use of slides coated with T. gondii antigen (bioMérieux), and fluorescein
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(FITC)-conjugated rabbit anti-sheep IgG (JacksonImmuno Research). Group I (IgG anti-T. gondii positive in the two assays) contained 183 sera from naturally infected animals, and
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group II (IgG anti-T. gondii negative in the two assays) contained 77 sera from seronegative
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animals. All 260 ovine sera were also seronegative for the specific anti-Neospora caninum antibodies with the use a commercial competitive-inhibition enzyme-linked immunosorbent
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assay (cELISA) (VMRD, Inc.). Fifteen serum samples, seronegative for T. gondii and
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seropositive for N. caninum, were used for estimating the recombinant antigens cross
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reactivity with specific N. caninum antibodies. 2.3.
IgG ELISA
MaxiSorp multiwells plates (Nunc, Denmark) were coated with single recombinant antigens, mixtures of recombinant proteins, or with a Toxoplasma lysate antigen (TLA) at a final concentration of 2.5 µg/ml for each recombinant protein and 1 µg/ml for the TLA in a coating buffer (0.05 M carbonate buffer, pH 9.6). After an overnight incubation at 4°C, the plates were washed three times with PBS-0.1% Triton X-100 and blocked for 1 h at 37°C in blocking solution (1% bovine serum albumin, 0.5% Triton X-100 in PBS). The cells were then washed three times and incubated for 1 h at 37°C, with the sheep serum diluted 1:100 in blocking solution. Next, the plates were washed three times with washing buffer and incubated with rabbit anti-sheep IgG peroxidase-labeled conjugates (JacksonImmuno 9 Page 9 of 25
Research) solution for 1 h at 37°C, after which o-phephenylenediamine dihydrochloride (Sigma) chromogenic substrate was added. After 45 min at 37°C incubation in darkness, the reaction was stopped by the addition of 2 M sulfuric acid and the optical density (OD) was
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measured using a microtiter plate reader (Multiscan FC; Thermo scientific) at 492 nm. Each serum sample was examined twice. The results were determined for each sample by
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calculating the mean value of the OD reading for duplicate wells. A positive result was defined as being of any value higher than the average OD reading plus two standard
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deviations (cutoff) obtained with 20 serum samples from the control group II, which consisted
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of seronegative serum samples. Statistical analysis
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Means, standard deviation and ranges of the absorbance measurements for the sera used in the individual studies are given. Spearman’s rank correlation coefficient (r) was used to test
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3. Results
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correlation between the results obtained with TLA and recombinant antigens.
3.1.
Preliminary evaluation of single recombinant antigens in IgG ELISA
Each of nineteen tested antigens was coated separately in IgG ELISA and examined with four sheep serum samples; two sera positive for anti-T. gondii antibodies randomly selected from group I and two sera negative for specific IgG, randomly selected from group II. Twenty sera from group II were used to obtain the relative absorbance and cutoff values for each serum sample. Absorbance values obtained in IgG ELISA assays with the use of different recombinant antigens were variable (Table 1). Eight of the tested proteins (GRA1, GRA9, SAG1, SAG4, MIC1ex2, MIC3, P22, ROP1) in IgG ELISA assay showed high absorbance values for the sera containing specific anti-T. gondii antibodies (samples no. 13 and 78) while these antigens did not react with sera from seropositive sheep (samples no. 6 10 Page 10 of 25
and 71) and gave absorbance values below the calculated cutoffs. Some of recombinant antigens (e.g. GRA5, BAG1), were not reliable antigen markers for T. gondii infection as the absorbance values obtained for the positive and negative sera were either below or above the
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cutoff value, respectively. For GRA2ex2, GRA4, GRA5, GRA6, P36 and BAG1, one of the negative serum samples gave a false positive reading, e.g. absorbance value was higher than
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the cutoff value. One false negative test was recorded for each of GRA4, BSR4 and MIC1ex2, where one of the positive serum sample yielded an absorbance value that was lower than the
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cutoff value. Neither MAG1 nor LDH2 recombinant proteins were recognized by specific IgG antibodies from ovine seropositive sera, as two false negative results were recorded in the
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preliminary evaluation of their reactivity.
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As a consequence of a preliminary evaluation eight recombinant proteins (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, and ROP1) have been selected for more expanded
Evaluation of most promising single recombinant antigens in IgG ELISA
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study.
In order to determine further the usefulness of selected recombinant antigens for the
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detection of IgG anti-T. gondii antibodies in sera from infected sheep, the pool of 88 serum samples from group I was used. Twenty sera from group II were used to obtain the range of absorbance values for each negative serum sample and to determine the cutoff values. An additional twenty sera from this group were used to determine the specificity of IgG ELISAs. All of these positive and negative sera were tested in IgG ELISA assays using the native antigen preparation (TLA). None of the negative serum samples reacted above the cutoff values for five of the recombinant antigens (GRA1, SAG1, SAG4, P22 and ROP1) and the native antigen, TLA, resulting in a specificity of 100%. For the remaining three proteins, the specificity was lower; 90% for GRA9 and 95% for both MIC1ex2 and MIC3 (Table 2). The sensitivity of IgG ELISAs calculated from 88 positive serum samples (group I) differed
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depending on the antigen preparations used. Recombinant antigens, GRA1 and ROP1, had the same high sensitivity (100%) as the native antigen (TLA). Relatively high sensitivities of IgG ELISAs were also observed for P22, SAG1, MIC3 and MIC1ex2 recombinant proteins
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(98.9%, 90.9%, 86.4 and 83%, respectively), whereas much lower sensitivities (40.9% and 62.5%) were noted for recombinant GRA9 and SAG4, respectively (Table 2). These results
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showed that the recombinant proteins have potential as diagnostic antigens for the detection of
reactive recombinant antigens (GRA1, P22 and ROP1). 3.3.
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T. gondii infection in sheep. This formed the basis for a detailed study using the three most
Evaluation of novel IgG ELISAs based on mixtures of recombinant antigens
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Four separate IgG ELISAs were developed using different mixtures of three selected
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recombinant proteins (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1; M4: GRA1+P22+ROP1) as a coating antigen to evaluate their potential for the serodiagnosis of
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ovine toxoplasmosis. We used the same pool of positive and negative sera, which was tested
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against the single antigens and TLA, to test the sensitivity and specificity of the mixtures and to obtain the relative absorbance of negative serum samples and the cutoff values (Table 3), as
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described previously. The sensitivity of IgG ELISAs calculated from all positive serum samples tested in this part of the study (n=88) was 100% for all tested antigen combinations, whereas the specificity obtained for two mixtures (M2: GRA1+P22 and M3: P22+ROP1) was slightly lower (95%) than for the other two which were 100% (M1: GRA1+ROP1 and M4: GRA1+P22+ROP1) (Table 3). Although the reactivity of all examined mixtures was equal 100% (all sera reacted over cutoff absorbance), the highest absorbance values were observed for the mixture of three recombinant proteins (M4). Furthermore, the difference between absorbance values for the positive and negative sera was greatest for this mixture. Based on the above experiments, the mixture of three recombinant antigens (GRA1+P22+ROP1) was
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chosen for further evaluation of a novel IgG ELISA assay for the detection of T. gondii infection in sheep.
Evaluation of IgG ELISA assay based on GRA1+P22+ROP1
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3.4.
This part of the study evaluated the diagnostic utility of IgG ELISAs containing three
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selected recombinant antigens (M4: GRA1+P22+ROP1) coated together in the same microplate well. A new pool of 128 sheep sera (93 from group I and 35 from group II) was
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used. The results were compared with the results of the IgG ELISA test based on crude native antigen (TLA). The relative sensitivity and specificity of the IgG ELISAs for
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GRA1+P22+ROP1 mixture and TLA were 100% (Fig. 1). None of the 35 negative serum
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samples from group II were found to score above the cutoff (range absorbance: 0.207-0.471 and 0.174-0.311 for the mixture and TLA, respectively), while all the sera from group I
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reacted above this value (range absorbance: 0.725-3.523 and 0.313-1.194 for the mixture and
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TLA, respectively). However, reactivity of the three recombinant antigen mixture with specific IgG antibodies from positive sera was higher (mean absorbance value 1.604) than for
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TLA (mean absorbance value 0.578). Moreover, a weak positive correlation was found when the results obtained with TLA were compared to those from the mixture (r = 0.145). In order to evaluate the cross reactivity of the selected recombinant antigens, a GRA1+P22+ROP1 mixture was tested using 15 sheep serum samples containing specific anti-N. caninum antibodies. None of these sera were found to score above the cutoff (range absorbance: 0.1690.334, mean absorbance 0.251). 4. Discussion
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In this study we developed an IgG ELISA assay based on a mixture of three recombinant T. gondii antigens (GRA1+P22+ROP1) that gave 100% sensitivity and specificity with sheep sera.
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Recombinant antigens of T. gondii provide a new tool in the diagnosis of toxoplasmosis both in humans and farm animals. To date, recombinant antigens are not frequently used in
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serodiagnosis of toxoplasmosis in sheep. ELISA assays based on recombinant parasite polypeptides to measure T. gondii-specific antibodies in sera of sheep and cats were
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developed for the first time by Tenter et al. (1992). Compared with an ELISA test based on the native antigen (TLA), an assay using recombinant proteins for sheep sera had a sensitivity
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of 79% and specificity of 100%. In 2010, Tumurjav et al. showed the usefulness of TgMAG1
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recombinant antigen for the epidemiological investigation of ovine toxoplasmosis in Mongolia. The results of an ELISA assay based on TgMAG1 were compared with those for
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the commercialized latex agglutination test (LAT). Of 175 ovine sera analyzed, 42 (24.00%)
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and 29 (16.57%) samples were positive by the ELISA and LAT, respectively. Thus, these results revealed the high sensitivity of ELISA assay based on TgMAG1 in the detection of
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specific IgG antibodies and suggest that this recombinant protein could be used as a reliable antigen for serodiagnosis of ovine toxoplasmosis (Tumurjav et al., 2010). Here we evaluated the usefulness of T. gondii recombinant proteins for the detection of specific IgG antibodies in sera from infected sheep. Out of the 19 antigens tested in IgG ELISA (GRA1, GRA2ex2, GRA4, GRA5, GRA6, GRA9, SAG1, SAG4, BSR4, P22, ROP1, P36, MIC1ex2, MIC1ex34, MIC3, MAG1, BAG1, LDH1, and LDH2), eight separate antigens were selected for further evaluation (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, and ROP1). The use of 88 seropositive sheep sera allowed the afore-mentioned recombinant proteins to be assessed as a diagnostic tool. Two IgG ELISAs based on GRA1 or ROP1 were able to detect all positive ovine sera, just as the native antigen (TLA), resulting in
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a sensitivity of 100%. The remaining recombinant antigens (P22, SAG1, MIC3, MIC1ex2, GRA9, and SAG4) showed varying sensitivity in IgG ELISA tests, from high (98.9% for P22) to low (40.9% for GRA9). There are at least two explanations for these variable results.
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Firstly, recombinant proteins produced in bacterial expression systems such as E. coli, often lose their antigenic properties due to incorrect folding. For this reason, some of the epitopes
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characteristic for native proteins are not present in the recombinant antigen and therefore cannot be recognized by specific antibodies. It is commonly known that the humoral immune
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response of the host varies with the stage of infection. Some IgG specific antibodies may be present at one stage of infection but absent in another stage. This means that multiple epitopes
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from various antigens must be present in an immunoassay to detect the range of antibodies
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presented throughout the disease process. For this reason, we selected three recombinant proteins (GRA1, P22 and ROP1) which were the most reactive as single antigens in IgG
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ELISAs. Any combination of two (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1) or
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three (M4: GRA1+P22+ROP1) T. gondii recombinant antigens was able to detect all IgGpositive serum samples, resulting in a sensitivity of 100%. However, for the
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GRA1+P22+ROP1 mixture, the highest absorbance values and the most substantial difference between values for seropositive and seronegative sera were observed. In our opinion, these higher absorbance values were obtained by the addition of P22 antigen to the GRA1+ROP1 combination. The addition of this surface protein would probably increase the number of epitopes that are able to recognize antibodies present in serum from acute and/or chronic T. gondii infection. In addition, higher absorbance values obtained for the mixture of three proteins, in comparison to the two antigen mixtures, demonstrated a higher detection level of the specific antibodies. Therefore, in order to develop a new diagnostic assay for the detection of T. gondii infection in sheep we decided to evaluate an IgG ELISA based on a mixture of the three recombinant antigens,GRA1+P22+ROP1, with the use of a new pool of ovine sera.
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The results are very promising; the sensitivity and specificity of the mixture was 100%, identical to TLA. In addition, considerably higher absorbance values were recorded for each positive serum when compared to TLA. Hence, the mixture of three antigens is better at
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binding with anti-T. gondii antibodies in sera of infected animals than the native antigen. Furthermore, this mixture did not react with sera containing specific antibodies against N.
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caninum (a parasite closely related to T. gondii), indicating its high specificity.
Control strategies for the management of toxoplasmosis should be based on systematical
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serological investigations at the farm level. The development of more sensitive methods to detect T. gondii in slaughtered animals would facilitate such studies and thereby minimize
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consumer exposure to this parasite. The main goal of this study was to develop a new
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serodiagnostic test using T. gondii recombinant antigens for diagnosis of ovine toxoplasmosis. Our results suggest a high diagnostic usefulness of IgG ELISA assay based on a mixture
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of GRA1+P22+ROP1 recombinant antigens for the detection of anti-T. gondii antibodies in
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sheep sera, and consequently in epidemiological studies. This cocktail of recombinant proteins can replace the native antigens in serological tests for detection of ovine
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toxoplasmosis. The main advantages of this new assay are: 1) high sensitivity and specificity; 2) high reactivity with specific antibodies as evidenced by the high absorbance values and 3) a lower cost of and safer production.
Furthermore, the use of recombinant antigens based assays instead of commercial kits, which are usually based on antigen preparations derived from tachyzoites, can reduce the cost of the analysis while providing excellent diagnostic results.
Acknowledgements
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This study was financed from budgetary means for science in the years 2012-2014 (grant No. IP2011 017571). We thank Nicky Richards (AgResearch) for reviewing and improving this
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article. The authors declare that they have no conflict of interest.
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Dubey, J.P., 2009. Toxoplasmosis in sheep – The last 20 years. Vet. Parasitol. 163, 1-14. Dunn, D., Wallon, M., Peyron, F., Petersen, E., Peckham, C., Gilbert, R., 1999. Mother-tochild transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 353, 1829-1833.
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Regional Toxoplasma Working Group. 1994. Neonatal serologic screening and early treatment for congenital Toxoplasma gondii infection. N. Engl. J. Med. 330, 1858-1863. Hill, D.E., Chirukandoth, S., Dubey, J.P., 2005. Biology and epidemiology of Toxoplasma
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gondii in man and animals. Anim. Health Res. Rev. 6, 41-61. Hiszczyńska-Sawicka, E., Brillowska-Dąbrowska, A., Dąbrowski, S., Pietkiewicz, H., Myjak,
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P., Kur, J., 2003. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli. Protein Exp. Purif. 27, 150-157.
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Hiszczyńska-Sawicka, E., Kur, J., Pietkiewicz, H., Holec, L., Gąsior A., Myjak, P., 2005. Efficient production of the Toxoplasma gondii GRA6, p35 and SAG2 recombinant
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antigens and their applications in the serodiagnosis of toxoplasmosis. Acta Parasitol. 50,
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249-254.
Holec, L., Hiszczyńska-Sawicka, E., Gąsior, A., Brillowska-Dąbrowska, A., Kur, J., 2007.
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Use of MAG1 recombinant antigen for detection of Toxoplasma gondii infection in
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humans. Clin. Vaccine Immunol. 14, 220-225. Holec, L., Gąsior, A., Brillowska-Dąbrowska, A., Kur, J., 2008. Toxoplasma gondii: Enzyme-
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linked immunosorbent assai usign different fragments of recombinant microneme protein (MIC1) for detection of immunoglobulin G antibodies. Exp. Parasitol. 119, 1-6.
Holec-Gąsior, L., Kur, J., 2010. Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. Exp. Parasitol. 124, 272-279.
Holec-Gąsior, L., Kur, J., Hiszczyńska-Sawicka, E., 2009. GRA2 and ROP1 recombinant antigens
as
potential
markers
for
detection
of
Toxoplasma
gondii-specific
immunoglobulin G in human with acute toxoplasmosis. Clin. Vaccine Immunol. 16, 510514.
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Holec-Gasior, L., Kur, J., Hiszczyńska-Sawicka, E., Drapała, D., Dominiak-Górski, B., Pejsak, Z., 2010. Application of recombinant antigens in serodiagnosis of swine toxoplasmosis and prevalence of Toxoplasma gondii infection among pigs in Poland. Pol.
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J. Vet. Sci. 13, 457-464. Holec-Gąsior, L., 2013. Toxoplasma gondii antigens as tool for serodiagnosis of human
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toxoplasmosis – the current status of studies. Clin. Vaccine Immunol. 20, 1343-1351.
Hughes, H.P., Van, K.F., 1982. Characterization of a secretory antigen from Toxoplasma
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gondii and its role in circulating antigen production. Int. J. Parasitol. 12, 433-437. Jones, J. L. and Holland, G. N., 2010. Annual burden of ocular toxoplasmosis in the US. Am.
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J. Trop. Med. Hyg. 82, 464-465.
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Lekutis, C., Ferguson, D.J.P., Grigg, M.E., Camps M., Boothroyd, J.C., 2001. Surface antigens of Toxoplasma gondii: variations of a theme. Inter. J. Parasitol. 31, 1285-1292.
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Mercier, C, Adjogble, K.D., Däubener, W., Delauw, M.F., 2005. Dense granules: are they key
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organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? Int. J. Parasitol. 35, 829-849.
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Mercier, C., Dubremetz, J.F., Rauscher, B., Lecordier, L., Sibley, L.D., Cesbron-Delauw, M.F. ,2002. Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Mol. Biol. Cell. 13, 2397-2409.
Robert-Gangneux, F., Dardé, M., 2012. Epidemiology of and Diagnostic Strategies for Toxoplasmosis. Clin. Microbiol. Rev. 25:264.
Sam-Yellowe, T.Y., 1996. Rhoptry organelles of the Apicomplexa: their role in host cell invasion and intracellular survival. Parasitol. Today. 12, 308-316. Tenter, A.M., Heckeroth, A.R., Weiss, L.M., 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30, 1217-1258.
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Tenter, A.M., Vietmeyer, C., Johnson, A.M., 1992. Development of ELISAs based on recombinant antigens for the detection of Toxoplasma gondii-specific antibodies in sheep and cats. Vet. Parasitol. 43, 189-201.
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Tumurjav, B., Terkawi, M.A., Zhang, H., Zhang, G., Jia, H., Goo, Y.K., Yamagishi, J., Nishikawa, Y., Igarashi, I., Sugimoto, C., Xuan, X., 2010. Serodiagnosis of ovine
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toxoplasmosis in Mongolia by an enzyme-linked immunosorbent assay with recombinant Toxoplasma gondii matrix antigen 1. Jpn. J. Vet. Res. 58, 111-119.
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Xia, D., Sanderson, S. J., Jones, A. R., Prieto, J. H., Yates, J. R., Bromley, E., Wastling, J. M., 2008. The proteome of Toxoplasma gondii: integration with the genome provides novel
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insights into gene expression and annotation. Genome Biology, 9, R116.
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Table 1. Absorbance values obtained in IgG ELISA assays for two seropositive and two
Cutoff value
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0.301 0.890 0.551 0.445 0.356 0.564 0.519 0.426 0.389 0.569 0.264 0.458 0.265 0.563 0.459 0.515 0.315 0.379 0.245
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GRA1 GRA2ex2 GRA4 GRA5 GRA6 GRA9 SAG1 SAG4 BSR4 P22 ROP1 P36 MIC1ex2 MIC1ex34 MIC3 MAG1 BAG1 LDH1 LDH2
Absorbance value (492 nm) Positive sera Negative sera No. 13 No. 78 No. 6 No. 71 1.551 2.416 0.245 0.182 1.177 1.553 0.799 0.985 0.940 0.443 0.274 0.635 1.552 2.747 0.379 0.830 1.571 0.901 0.615 0.186 1.281 0.937 0.398 0.427 0.738 0.994 0.225 0.188 0.885 1.120 0.376 0.321 0.325 1.497 0.387 0.257 1.409 2.221 0.257 0.171 0.666 0.907 0.167 0.252 0.697 0.551 0.629 0.327 0.842 1.611 0.204 0.249 0.536 1.755 0.464 0.458 2.135 1.035 0.278 0.231 0.450 0.472 0.496 0.378 0.380 1.399 0.904 0.184 0.378 1.450 0.290 0.295 0.154 0.210 0.133 0.113
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Recombinant antigen*
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seronegative serum samples with nineteen T. gondii recombinant antigens.
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*Antigens indicated in bold type were selected for further testing.
21 Page 21 of 25
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MIC3) and the TLA with using a pool of 108 ovine sera.
Recombinant
No. (%) of
Mean
antigen
positive sera
Pool of seronegative sera (n=20)
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Pool of seropositive sera (n=88)
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Table 2. The results of IgG ELISA obtained for eight T. gondii recombinant proteins (GRA1, GRA9, SAG1, SAG4, P22, ROP1, MIC1ex2,
Range
No. (%) of
Mean
Range
absorbance
absorbance
negative sera
absorbance
absorbance
value
values
value
values
Cutoff value
88 (100%)
0.734
0.308-1.713
20 (100%)
0.201
0.115-0.293
0.301
GRA9
36 (40.9%)
0.563
0.250-1.091
18 (90%)
0.340
0.174-0.591
0.564
SAG1
80 (90.9%)
0.825
0.463-1.570
20 (100%)
0.352
0.212-0.494
0.519
SAG4
55 (62.5%)
0.558
0.215-1.347
20 (100%)
0.263
0.147-0.409
0.426
P22
87 (98.9%)
1.419
0.569-2.400
20 (100%)
0.356
0.223-0.553
0.569
73 (83%)
0.457
0.177-1.447
19 (95%)
0.178
0.110-0.267
0.265
MIC3
76 (86.4%)
0.769
0.168-1.885
19 (95%)
0.254
0.110-0.557
0.459
ROP1
88 (100%)
0.905
0.293-2.936
20 (100%)
0.171
0.114-0.241
0.264
TLA
88 (100%)
0.681
0.332-1.450
20 (100%)
0.232
0.174-0.285
0.312
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MIC1ex2
ed
GRA1
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Table 3. The results of IgG ELISA obtained for four mixtures of recombinant antigens (GRA1+ROP1, GRA1+P22, P22+ROP1 and
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GRA1+P22+ROP1) with using a pool of 108 ovine sera. Pool of seropositive sera (n=88) Mixtures of recombinant
No. (%) of
Mean
antigens
positive sera
Pool of seronegative sera (n=20) No. (%) of
Mean
Range
absorbance
absorbance
negative sera
absorbance
absorbance
value
values
value
values
88 (100%)
1.059
GRA1+P22
88 (100%)
P22+ROP1
88 (100%)
GRA1+P22+ROP1
88 (100%)
Cutoff value
0.422-3.062
20 (100%)
0.233
0.165-0.320
0.323
1.294
0.557-2.353
19 (95%)
0.319
0.190-0.488
0.480
1.373
0.592-3.048
19 (95%)
0.535
0.297-0.553
0.535
1.468
0.557-3.017
20 (100%)
0.339
0.258-0.475
0.478
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GRA1+ROP1
ed
Range
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Fig. 1. Immunoreactivities of a recombinant antigens mixture (M4: GRA1+P22+ROP1) and
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the TLA using 93 sera from infected sheep () and 35 sera form healthy animals (▲). The cutoff value is indicated for each experiment by horizontal line (0.478 for mixture and 0.312
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for TLA).
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Figure 1
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S0304-4017(14)00503-2 http://dx.doi.org/doi:10.1016/j.vetpar.2014.09.018 VETPAR 7398
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
7-10-2013 13-9-2014 20-9-2014
Please cite this article as: Holec-G˛asior, L., Ferra, B., Hiszczy´nska-Sawicka, E., Kur, J.,The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights 1. Recombinant antigens provide a new tool in the diagnosis of toxoplasmosis. 2. GRA1+P22+ROP1 mixture is the most effective for diagnosis of ovine toxoplasmosis.
Cocktail of recombinant proteins can replace the native antigens.
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3.
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The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1)
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for diagnosis of ovine toxoplasmosis
Gdańsk University of Technology, Chemical Faculty, Department of Microbiology,
Narutowicza 11/12 Str., 80-233 Gdańsk, POLAND
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AgResearch, Lincoln Science Centre, Private Bag 4749, Christchurch, NEW ZEALAND
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Lucyna Holec-Gąsior1*, Bartłomiej Ferra1, Elżbieta Hiszczyńska-Sawicka2, Józef Kur1
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Keywords: sheep; Toxoplasma gondii; recombinant antigen; GRA1; P22; ROP1; ELISA
*Corresponding author: Tel.: +48 58 3472406; fax: +48 58 3471822, e-mail address: [email protected] (L. Holec-Gąsior)
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Abstract Toxoplasmosis, caused by Toxoplasma gondii, is the major parasitic disease affecting sheep. Infection not only results in significant reproductive losses in these animals, but has
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public health implications since consumption of infected meat can facilitate zoonotic transmission. Although several serological tests are currently used for diagnosis of ovine
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toxoplasmosis, production of reliable reagents is a constraint and therefore there is a need to develop new diagnostic tools. In this paper, we assess for the first time, the preliminary
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diagnostic utility of nineteen T. gondii recombinant antigens (GRA1, GRA2ex2, GRA4, GRA5, GRA6, GRA9, SAG1, SAG4, BSR4, P22, ROP1, P36, MIC1ex2, MIC1ex34, MIC3,
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MAG1, BAG1, LDH1, and LDH2) in immunoglobulin G (IgG) enzyme-linked
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immunosorbent assays (IgG ELISAs). Following an initial evaluation, eight recombinant antigens (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, ROP1) were chosen for
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subsequent testing and comparison against the native Toxoplasma lysate antigen (TLA) in
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IgG ELISAs using 88 sera from naturally infected sheep and 20 sera from healthy animals. The reactivity of these antigens was variable with the best results for GRA1, P22, ROP1 and
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TLA. High sensitivity and specificity (100%) was noted for GRA1, ROP1 and TLA; P22 showed a slightly lower sensitivity (98.9%) but the same high specificity (100%). Four different combinations of these antigens (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1; M4: GRA1+P22+ROP1) were tested against the same pool of ovine sera; all IgG-positive serum samples were detected by all of the mixtures. However, the most effective for diagnosis of toxoplasmosis in sheep, based on the highest absorbance values, was the mixture M4 containing three proteins. High sensitivity and specificity (100%) was observed from tests containing either M4 or TLA antigens with a new pool of sera (93 seropositive and 35 seronegative). Thus, the present study shows that a cocktail of GRA1+P22+ROP1
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recombinant proteins can be used to diagnose T. gondii infection in sheep, and consequently
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will assist in epidemiological studies.
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1. Introduction Toxoplasmosis, a zoonotic infection caused by the protozoan parasite, Toxoplasma gondii, has economic relevance to both veterinary and human medicine. T. gondii has a worldwide
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distribution and is a major cause of congenital disease, abortion and stillbirth in humans and farm animals, especially sheep (Hill et al, 2005). Human infection is post-natal by ingesting
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tissue cysts from undercooked meat, or by consuming food and water contaminated with
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oocysts (Tenter et al., 2000). The ingestion of undercooked infected lamb meat is considered an important source of T. gondii infection for humans (Cook et al., 2000). In healthy
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individuals, a primary infection with T. gondii usually causes relatively mild flu-like symptoms, whereas in immunocompromised patients it can cause opportunistic life-
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threatening infections. Moreover, toxoplasmosis acquired by women during pregnancy poses a significant threat to the fetus. Mother to child transmission of the parasite occurs only when
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infection is acquired for the first time in the months just before or during pregnancy. The risk
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of transmission rises steeply with gestational age at maternal infection (from 1% to 90%), but
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the severity of disease in the fetus decreases (Dunn et al., 1999). Congenital infection may cause a spontaneous abortion or severe fetal abnormalities. Most children with congenital toxoplasmosis develop normally (Salt et al., 2005) but up to 4% die or have evidence of permanent neurological damage or bilateral visual impairment during the first years of life (Guerina et al., 1994; Gras et al., 2005). Congenital infection with T. gondii is also an important cause of abortion in sheep worldwide with millions of productive lambs lost due to parasite infection (Dubey, 2009). Sheep play a significant role in the economy of many countries since they are important sources of meat and milk products. Control of T. gondii infection in sheep and other domesticated animals used for meat production is important not only for reproductive reasons, but primarily for public health as Toxoplasma is recognized as food-borne risk (Jones and 5 Page 5 of 25
Holland, 2010). Seroprevalence of anti-T. gondii antibodies in sheep across the world range from 3% to 95% (Tenter at al., 2000; Dubey, 2009). Different climate conditions that interfere with the maintenance and viability of infective oocysts in the environment, and different types
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of sanitary management are the main reasons for the differences in seropositivity between countries (Robert-Gangneux and Darde, 2012). Furthermore, recent research has shown that
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the prevalence of antibodies in ewes was more than twice that in lambs, depending on the age of lambs sampled (Dubey, 2009).
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Serological techniques play a major role in the diagnosis of toxoplasmosis in humans and animals. Assays for the detection of T. gondii-specific antibodies are usually based on crude
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antigen extracts from T. gondii cultures, otherwise known as TLA or Toxoplasma lysate
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antigen. Alternatively, purified native proteins of the parasite are used. Production of these native parasitic antigens is limited, and their quantity in the antigen mixture are difficult to
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standardize. Furthermore the methods and source of native antigens involve the maintenance
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of parasite tissue cultures which can prove to be hazardous to laboratory staff handling the samples. Recombinant proteins, cloned and expressed in suitable bacterial or eukaryotic
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expression systems, could alleviate these issues and be a potential alternative source of antigens for diagnostic assays (Holec-Gąsior, 2013). The major advantages of recombinant antigens for the diagnosis of T. gondii infections are: 1) the precise antigen composition of the test is known, 2) more than one defined antigen can be used, 3) the method can easily be standardized and 4) the molecular methods used to produce recombinant antigens avoid the biohazard, time, labor and cost issues that accompany the production of native antigen. In the past few decades, several studies have reported the use of recombinant antigens for the serological detection of T. gondii infections particularly in humans (Holec-Gąsior, 2013). Recombinant antigens have been used to distinguish between infections that may have happened years ago and those which have been recently acquired. However, the use of these
6 Page 6 of 25
recombinant antigens in serodiagnosis of toxoplasmosis in livestock animals, has not been well-studied. Given that the proteome of T. gondii is well known and accurately described (Xia et
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al., 2008), there is significant potential for the biotechnological production of various parasitic antigens. T. gondii native proteins are localized in the outer membrane, cytosol, secretory
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organelles (micronemes, rhoptries) and the dense granules. During host cell invasion different Toxoplasma proteins are released to the forming parasitophorous vacuole (PV) or to the tissue
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cyst. The microneme proteins (MIC) are released first, upon contact with the host cells. These proteins are involved in host cell recognition and attachment (Carruthers et al., 1999). The
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rhoptry proteins (ROP) are released next, and they may facilitate formation of the PV and
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mediate its clustering with host cell organelles (Sam-Yellowe, 1996). The dense granular proteins (GRA) are exocytosed both during and after invasion into the PV. Following
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secretion, most of the GRA antigens appear as both a soluble and a membrane-associated
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form in the vacuole and they are thought to modify the environment within the PV, thereby functioning for intracellular survival and replication (Mercier et. al., 2002; 2005). Moreover,
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these proteins constitute an important fraction of antigens that circulate in the bloodstream during the first hours following infection (Hughes and Van, 1982). The surface of T. gondii is covered with a family of glycosylphosphatidylinositol (GPI)-anchored antigens (SAGs) and SAG-related sequence (SRS) proteins, most of which are members of the SAG1 or SAG2 families (Lekutis et al., 2001). These proteins appear to play a role in host cell invasion, immune modulation and/or virulence attenuation, although they may also provide protection needed by the parasite to survive in the environment (Lekutis et al., 2001). Genes encoding the aforementioned antigenic proteins of the parasite are good molecular targets for cloning and production of recombinant antigens of diagnostic significance.
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Considering the negative impact of toxoplasmosis on sheep reproduction and the risk of human transmission from the ingestion of undercooked infected meat, there is a real need for diagnosis of ovine toxoplasmosis and the development of new diagnostic methods for
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detection of T. gondii infection in sheep. This study investigates the usefulness of recombinant antigenic proteins for the serodiagnosis of ovine toxoplasmosis and proposes a
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new assay based on three reactive proteins.
Materials and methods
2.1.
Toxoplasma gondii recombinant antigens and TLA
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Nineteen T. gondii recombinant antigens: GRA1 (amino acids 24–190), GRA2ex2 (amino
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acids 51–185), GRA4 (amino acids 20–279), GRA5 (amino acids 26–94), GRA6 (amino acids 30–231), GRA9 (amino acids 21–328), SAG1 (amino acids 49–313), SAG4 (amino acids 26–
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150), BSR4 (amino acids 33–356), P22 (amino acids 30–231), ROP1 (amino acids 85–396),
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P36 (ROP9, amino acids 30–363), MIC1ex2 (amino acids 25–182), MIC1ex34 (amino acids 183–456), MIC3 (amino acids 67–359), MAG1 (amino acids 30–222), BAG1 (amino acids 1–
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229), LDH1 (amino acids 1–329), and LDH2 (amino acids 1–326) containing a cluster of six histidine residues for the purification by metal-affinity chromatography at the N- and Ctermini were obtained as previously described (Hiszczyńska-Sawicka et al., 2003; Hiszczyńska et al., 2005; Holec et al., 2007; Holec et al., 2008; Holec-Gąsior et al., 2009; Holec-Gąsior i Kur, 2010). The recombinant proteins were analyzed by SDS–PAGE on 12% acrylamide gels and stained with Coomassie blue. The concentration of purified proteins was determined by the Bradford method (Bradford, 1976) using bovine serum albumin (BSA) as a standard. Toxoplasma lysate antigen (TLA) from tachyzoites (strain RH) was prepared according to the method previously described (Holec-Gąsior et al., 2010). 8 Page 8 of 25
2.2.
Test sera
A total number of 260 ovine sera were received from the Veterinary Hygiene Station (Gdańsk, Poland). These serum samples have been obtained from epidemiological studies
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conducted on a population of nearly two thousand sheep from northern region of Poland. All serum samples were analyzed and divided into two groups in accordance with the results
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obtained using the agglutination test (Toxo-Screen DA, bioMérieux) and immunofluorescence test, with the use of slides coated with T. gondii antigen (bioMérieux), and fluorescein
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(FITC)-conjugated rabbit anti-sheep IgG (JacksonImmuno Research). Group I (IgG anti-T. gondii positive in the two assays) contained 183 sera from naturally infected animals, and
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group II (IgG anti-T. gondii negative in the two assays) contained 77 sera from seronegative
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animals. All 260 ovine sera were also seronegative for the specific anti-Neospora caninum antibodies with the use a commercial competitive-inhibition enzyme-linked immunosorbent
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assay (cELISA) (VMRD, Inc.). Fifteen serum samples, seronegative for T. gondii and
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seropositive for N. caninum, were used for estimating the recombinant antigens cross
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reactivity with specific N. caninum antibodies. 2.3.
IgG ELISA
MaxiSorp multiwells plates (Nunc, Denmark) were coated with single recombinant antigens, mixtures of recombinant proteins, or with a Toxoplasma lysate antigen (TLA) at a final concentration of 2.5 µg/ml for each recombinant protein and 1 µg/ml for the TLA in a coating buffer (0.05 M carbonate buffer, pH 9.6). After an overnight incubation at 4°C, the plates were washed three times with PBS-0.1% Triton X-100 and blocked for 1 h at 37°C in blocking solution (1% bovine serum albumin, 0.5% Triton X-100 in PBS). The cells were then washed three times and incubated for 1 h at 37°C, with the sheep serum diluted 1:100 in blocking solution. Next, the plates were washed three times with washing buffer and incubated with rabbit anti-sheep IgG peroxidase-labeled conjugates (JacksonImmuno 9 Page 9 of 25
Research) solution for 1 h at 37°C, after which o-phephenylenediamine dihydrochloride (Sigma) chromogenic substrate was added. After 45 min at 37°C incubation in darkness, the reaction was stopped by the addition of 2 M sulfuric acid and the optical density (OD) was
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measured using a microtiter plate reader (Multiscan FC; Thermo scientific) at 492 nm. Each serum sample was examined twice. The results were determined for each sample by
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calculating the mean value of the OD reading for duplicate wells. A positive result was defined as being of any value higher than the average OD reading plus two standard
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deviations (cutoff) obtained with 20 serum samples from the control group II, which consisted
2.4.
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of seronegative serum samples. Statistical analysis
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Means, standard deviation and ranges of the absorbance measurements for the sera used in the individual studies are given. Spearman’s rank correlation coefficient (r) was used to test
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3. Results
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correlation between the results obtained with TLA and recombinant antigens.
3.1.
Preliminary evaluation of single recombinant antigens in IgG ELISA
Each of nineteen tested antigens was coated separately in IgG ELISA and examined with four sheep serum samples; two sera positive for anti-T. gondii antibodies randomly selected from group I and two sera negative for specific IgG, randomly selected from group II. Twenty sera from group II were used to obtain the relative absorbance and cutoff values for each serum sample. Absorbance values obtained in IgG ELISA assays with the use of different recombinant antigens were variable (Table 1). Eight of the tested proteins (GRA1, GRA9, SAG1, SAG4, MIC1ex2, MIC3, P22, ROP1) in IgG ELISA assay showed high absorbance values for the sera containing specific anti-T. gondii antibodies (samples no. 13 and 78) while these antigens did not react with sera from seropositive sheep (samples no. 6 10 Page 10 of 25
and 71) and gave absorbance values below the calculated cutoffs. Some of recombinant antigens (e.g. GRA5, BAG1), were not reliable antigen markers for T. gondii infection as the absorbance values obtained for the positive and negative sera were either below or above the
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cutoff value, respectively. For GRA2ex2, GRA4, GRA5, GRA6, P36 and BAG1, one of the negative serum samples gave a false positive reading, e.g. absorbance value was higher than
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the cutoff value. One false negative test was recorded for each of GRA4, BSR4 and MIC1ex2, where one of the positive serum sample yielded an absorbance value that was lower than the
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cutoff value. Neither MAG1 nor LDH2 recombinant proteins were recognized by specific IgG antibodies from ovine seropositive sera, as two false negative results were recorded in the
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preliminary evaluation of their reactivity.
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As a consequence of a preliminary evaluation eight recombinant proteins (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, and ROP1) have been selected for more expanded
Evaluation of most promising single recombinant antigens in IgG ELISA
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study.
In order to determine further the usefulness of selected recombinant antigens for the
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detection of IgG anti-T. gondii antibodies in sera from infected sheep, the pool of 88 serum samples from group I was used. Twenty sera from group II were used to obtain the range of absorbance values for each negative serum sample and to determine the cutoff values. An additional twenty sera from this group were used to determine the specificity of IgG ELISAs. All of these positive and negative sera were tested in IgG ELISA assays using the native antigen preparation (TLA). None of the negative serum samples reacted above the cutoff values for five of the recombinant antigens (GRA1, SAG1, SAG4, P22 and ROP1) and the native antigen, TLA, resulting in a specificity of 100%. For the remaining three proteins, the specificity was lower; 90% for GRA9 and 95% for both MIC1ex2 and MIC3 (Table 2). The sensitivity of IgG ELISAs calculated from 88 positive serum samples (group I) differed
11 Page 11 of 25
depending on the antigen preparations used. Recombinant antigens, GRA1 and ROP1, had the same high sensitivity (100%) as the native antigen (TLA). Relatively high sensitivities of IgG ELISAs were also observed for P22, SAG1, MIC3 and MIC1ex2 recombinant proteins
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(98.9%, 90.9%, 86.4 and 83%, respectively), whereas much lower sensitivities (40.9% and 62.5%) were noted for recombinant GRA9 and SAG4, respectively (Table 2). These results
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showed that the recombinant proteins have potential as diagnostic antigens for the detection of
reactive recombinant antigens (GRA1, P22 and ROP1). 3.3.
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T. gondii infection in sheep. This formed the basis for a detailed study using the three most
Evaluation of novel IgG ELISAs based on mixtures of recombinant antigens
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Four separate IgG ELISAs were developed using different mixtures of three selected
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recombinant proteins (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1; M4: GRA1+P22+ROP1) as a coating antigen to evaluate their potential for the serodiagnosis of
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ovine toxoplasmosis. We used the same pool of positive and negative sera, which was tested
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against the single antigens and TLA, to test the sensitivity and specificity of the mixtures and to obtain the relative absorbance of negative serum samples and the cutoff values (Table 3), as
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described previously. The sensitivity of IgG ELISAs calculated from all positive serum samples tested in this part of the study (n=88) was 100% for all tested antigen combinations, whereas the specificity obtained for two mixtures (M2: GRA1+P22 and M3: P22+ROP1) was slightly lower (95%) than for the other two which were 100% (M1: GRA1+ROP1 and M4: GRA1+P22+ROP1) (Table 3). Although the reactivity of all examined mixtures was equal 100% (all sera reacted over cutoff absorbance), the highest absorbance values were observed for the mixture of three recombinant proteins (M4). Furthermore, the difference between absorbance values for the positive and negative sera was greatest for this mixture. Based on the above experiments, the mixture of three recombinant antigens (GRA1+P22+ROP1) was
12 Page 12 of 25
chosen for further evaluation of a novel IgG ELISA assay for the detection of T. gondii infection in sheep.
Evaluation of IgG ELISA assay based on GRA1+P22+ROP1
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3.4.
This part of the study evaluated the diagnostic utility of IgG ELISAs containing three
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selected recombinant antigens (M4: GRA1+P22+ROP1) coated together in the same microplate well. A new pool of 128 sheep sera (93 from group I and 35 from group II) was
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used. The results were compared with the results of the IgG ELISA test based on crude native antigen (TLA). The relative sensitivity and specificity of the IgG ELISAs for
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GRA1+P22+ROP1 mixture and TLA were 100% (Fig. 1). None of the 35 negative serum
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samples from group II were found to score above the cutoff (range absorbance: 0.207-0.471 and 0.174-0.311 for the mixture and TLA, respectively), while all the sera from group I
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reacted above this value (range absorbance: 0.725-3.523 and 0.313-1.194 for the mixture and
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TLA, respectively). However, reactivity of the three recombinant antigen mixture with specific IgG antibodies from positive sera was higher (mean absorbance value 1.604) than for
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TLA (mean absorbance value 0.578). Moreover, a weak positive correlation was found when the results obtained with TLA were compared to those from the mixture (r = 0.145). In order to evaluate the cross reactivity of the selected recombinant antigens, a GRA1+P22+ROP1 mixture was tested using 15 sheep serum samples containing specific anti-N. caninum antibodies. None of these sera were found to score above the cutoff (range absorbance: 0.1690.334, mean absorbance 0.251). 4. Discussion
13 Page 13 of 25
In this study we developed an IgG ELISA assay based on a mixture of three recombinant T. gondii antigens (GRA1+P22+ROP1) that gave 100% sensitivity and specificity with sheep sera.
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Recombinant antigens of T. gondii provide a new tool in the diagnosis of toxoplasmosis both in humans and farm animals. To date, recombinant antigens are not frequently used in
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serodiagnosis of toxoplasmosis in sheep. ELISA assays based on recombinant parasite polypeptides to measure T. gondii-specific antibodies in sera of sheep and cats were
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developed for the first time by Tenter et al. (1992). Compared with an ELISA test based on the native antigen (TLA), an assay using recombinant proteins for sheep sera had a sensitivity
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of 79% and specificity of 100%. In 2010, Tumurjav et al. showed the usefulness of TgMAG1
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recombinant antigen for the epidemiological investigation of ovine toxoplasmosis in Mongolia. The results of an ELISA assay based on TgMAG1 were compared with those for
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the commercialized latex agglutination test (LAT). Of 175 ovine sera analyzed, 42 (24.00%)
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and 29 (16.57%) samples were positive by the ELISA and LAT, respectively. Thus, these results revealed the high sensitivity of ELISA assay based on TgMAG1 in the detection of
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specific IgG antibodies and suggest that this recombinant protein could be used as a reliable antigen for serodiagnosis of ovine toxoplasmosis (Tumurjav et al., 2010). Here we evaluated the usefulness of T. gondii recombinant proteins for the detection of specific IgG antibodies in sera from infected sheep. Out of the 19 antigens tested in IgG ELISA (GRA1, GRA2ex2, GRA4, GRA5, GRA6, GRA9, SAG1, SAG4, BSR4, P22, ROP1, P36, MIC1ex2, MIC1ex34, MIC3, MAG1, BAG1, LDH1, and LDH2), eight separate antigens were selected for further evaluation (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, and ROP1). The use of 88 seropositive sheep sera allowed the afore-mentioned recombinant proteins to be assessed as a diagnostic tool. Two IgG ELISAs based on GRA1 or ROP1 were able to detect all positive ovine sera, just as the native antigen (TLA), resulting in
14 Page 14 of 25
a sensitivity of 100%. The remaining recombinant antigens (P22, SAG1, MIC3, MIC1ex2, GRA9, and SAG4) showed varying sensitivity in IgG ELISA tests, from high (98.9% for P22) to low (40.9% for GRA9). There are at least two explanations for these variable results.
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Firstly, recombinant proteins produced in bacterial expression systems such as E. coli, often lose their antigenic properties due to incorrect folding. For this reason, some of the epitopes
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characteristic for native proteins are not present in the recombinant antigen and therefore cannot be recognized by specific antibodies. It is commonly known that the humoral immune
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response of the host varies with the stage of infection. Some IgG specific antibodies may be present at one stage of infection but absent in another stage. This means that multiple epitopes
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from various antigens must be present in an immunoassay to detect the range of antibodies
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presented throughout the disease process. For this reason, we selected three recombinant proteins (GRA1, P22 and ROP1) which were the most reactive as single antigens in IgG
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ELISAs. Any combination of two (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1) or
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three (M4: GRA1+P22+ROP1) T. gondii recombinant antigens was able to detect all IgGpositive serum samples, resulting in a sensitivity of 100%. However, for the
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GRA1+P22+ROP1 mixture, the highest absorbance values and the most substantial difference between values for seropositive and seronegative sera were observed. In our opinion, these higher absorbance values were obtained by the addition of P22 antigen to the GRA1+ROP1 combination. The addition of this surface protein would probably increase the number of epitopes that are able to recognize antibodies present in serum from acute and/or chronic T. gondii infection. In addition, higher absorbance values obtained for the mixture of three proteins, in comparison to the two antigen mixtures, demonstrated a higher detection level of the specific antibodies. Therefore, in order to develop a new diagnostic assay for the detection of T. gondii infection in sheep we decided to evaluate an IgG ELISA based on a mixture of the three recombinant antigens,GRA1+P22+ROP1, with the use of a new pool of ovine sera.
15 Page 15 of 25
The results are very promising; the sensitivity and specificity of the mixture was 100%, identical to TLA. In addition, considerably higher absorbance values were recorded for each positive serum when compared to TLA. Hence, the mixture of three antigens is better at
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binding with anti-T. gondii antibodies in sera of infected animals than the native antigen. Furthermore, this mixture did not react with sera containing specific antibodies against N.
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caninum (a parasite closely related to T. gondii), indicating its high specificity.
Control strategies for the management of toxoplasmosis should be based on systematical
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serological investigations at the farm level. The development of more sensitive methods to detect T. gondii in slaughtered animals would facilitate such studies and thereby minimize
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consumer exposure to this parasite. The main goal of this study was to develop a new
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serodiagnostic test using T. gondii recombinant antigens for diagnosis of ovine toxoplasmosis. Our results suggest a high diagnostic usefulness of IgG ELISA assay based on a mixture
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of GRA1+P22+ROP1 recombinant antigens for the detection of anti-T. gondii antibodies in
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sheep sera, and consequently in epidemiological studies. This cocktail of recombinant proteins can replace the native antigens in serological tests for detection of ovine
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toxoplasmosis. The main advantages of this new assay are: 1) high sensitivity and specificity; 2) high reactivity with specific antibodies as evidenced by the high absorbance values and 3) a lower cost of and safer production.
Furthermore, the use of recombinant antigens based assays instead of commercial kits, which are usually based on antigen preparations derived from tachyzoites, can reduce the cost of the analysis while providing excellent diagnostic results.
Acknowledgements
16 Page 16 of 25
This study was financed from budgetary means for science in the years 2012-2014 (grant No. IP2011 017571). We thank Nicky Richards (AgResearch) for reviewing and improving this
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article. The authors declare that they have no conflict of interest.
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Carruthers, V.B., Giddings, O.K., Sibley, L.D., 1999. Secretion of micronemal proteins is
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Holec-Gąsior, L., Kur, J., Hiszczyńska-Sawicka, E., 2009. GRA2 and ROP1 recombinant antigens
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Holec-Gasior, L., Kur, J., Hiszczyńska-Sawicka, E., Drapała, D., Dominiak-Górski, B., Pejsak, Z., 2010. Application of recombinant antigens in serodiagnosis of swine toxoplasmosis and prevalence of Toxoplasma gondii infection among pigs in Poland. Pol.
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Xia, D., Sanderson, S. J., Jones, A. R., Prieto, J. H., Yates, J. R., Bromley, E., Wastling, J. M., 2008. The proteome of Toxoplasma gondii: integration with the genome provides novel
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Table 1. Absorbance values obtained in IgG ELISA assays for two seropositive and two
Cutoff value
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0.301 0.890 0.551 0.445 0.356 0.564 0.519 0.426 0.389 0.569 0.264 0.458 0.265 0.563 0.459 0.515 0.315 0.379 0.245
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GRA1 GRA2ex2 GRA4 GRA5 GRA6 GRA9 SAG1 SAG4 BSR4 P22 ROP1 P36 MIC1ex2 MIC1ex34 MIC3 MAG1 BAG1 LDH1 LDH2
Absorbance value (492 nm) Positive sera Negative sera No. 13 No. 78 No. 6 No. 71 1.551 2.416 0.245 0.182 1.177 1.553 0.799 0.985 0.940 0.443 0.274 0.635 1.552 2.747 0.379 0.830 1.571 0.901 0.615 0.186 1.281 0.937 0.398 0.427 0.738 0.994 0.225 0.188 0.885 1.120 0.376 0.321 0.325 1.497 0.387 0.257 1.409 2.221 0.257 0.171 0.666 0.907 0.167 0.252 0.697 0.551 0.629 0.327 0.842 1.611 0.204 0.249 0.536 1.755 0.464 0.458 2.135 1.035 0.278 0.231 0.450 0.472 0.496 0.378 0.380 1.399 0.904 0.184 0.378 1.450 0.290 0.295 0.154 0.210 0.133 0.113
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Recombinant antigen*
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seronegative serum samples with nineteen T. gondii recombinant antigens.
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*Antigens indicated in bold type were selected for further testing.
21 Page 21 of 25
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MIC3) and the TLA with using a pool of 108 ovine sera.
Recombinant
No. (%) of
Mean
antigen
positive sera
Pool of seronegative sera (n=20)
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Pool of seropositive sera (n=88)
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Table 2. The results of IgG ELISA obtained for eight T. gondii recombinant proteins (GRA1, GRA9, SAG1, SAG4, P22, ROP1, MIC1ex2,
Range
No. (%) of
Mean
Range
absorbance
absorbance
negative sera
absorbance
absorbance
value
values
value
values
Cutoff value
88 (100%)
0.734
0.308-1.713
20 (100%)
0.201
0.115-0.293
0.301
GRA9
36 (40.9%)
0.563
0.250-1.091
18 (90%)
0.340
0.174-0.591
0.564
SAG1
80 (90.9%)
0.825
0.463-1.570
20 (100%)
0.352
0.212-0.494
0.519
SAG4
55 (62.5%)
0.558
0.215-1.347
20 (100%)
0.263
0.147-0.409
0.426
P22
87 (98.9%)
1.419
0.569-2.400
20 (100%)
0.356
0.223-0.553
0.569
73 (83%)
0.457
0.177-1.447
19 (95%)
0.178
0.110-0.267
0.265
MIC3
76 (86.4%)
0.769
0.168-1.885
19 (95%)
0.254
0.110-0.557
0.459
ROP1
88 (100%)
0.905
0.293-2.936
20 (100%)
0.171
0.114-0.241
0.264
TLA
88 (100%)
0.681
0.332-1.450
20 (100%)
0.232
0.174-0.285
0.312
ce pt
Ac
MIC1ex2
ed
GRA1
23
Page 22 of 25
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Table 3. The results of IgG ELISA obtained for four mixtures of recombinant antigens (GRA1+ROP1, GRA1+P22, P22+ROP1 and
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GRA1+P22+ROP1) with using a pool of 108 ovine sera. Pool of seropositive sera (n=88) Mixtures of recombinant
No. (%) of
Mean
antigens
positive sera
Pool of seronegative sera (n=20) No. (%) of
Mean
Range
absorbance
absorbance
negative sera
absorbance
absorbance
value
values
value
values
88 (100%)
1.059
GRA1+P22
88 (100%)
P22+ROP1
88 (100%)
GRA1+P22+ROP1
88 (100%)
Cutoff value
0.422-3.062
20 (100%)
0.233
0.165-0.320
0.323
1.294
0.557-2.353
19 (95%)
0.319
0.190-0.488
0.480
1.373
0.592-3.048
19 (95%)
0.535
0.297-0.553
0.535
1.468
0.557-3.017
20 (100%)
0.339
0.258-0.475
0.478
Ac
ce pt
GRA1+ROP1
ed
Range
24
Page 23 of 25
Fig. 1. Immunoreactivities of a recombinant antigens mixture (M4: GRA1+P22+ROP1) and
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the TLA using 93 sera from infected sheep () and 35 sera form healthy animals (▲). The cutoff value is indicated for each experiment by horizontal line (0.478 for mixture and 0.312
Ac ce p
te
d
M
an
us
cr
for TLA).
25 Page 24 of 25
Ac
ce
pt
ed
M
an
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cr
i
Figure 1
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