Basic research
Lack of association between ABO histo-blood groups, secretor and non-secretor phenotypes, and anti-Toxoplasma gondii antibodies among pregnant women from the northwestern region of São Paulo State, Brazil

Introduction
Toxoplasma gondii infects human hosts utilizing the gastrointestinal tract as one route with its adherence to specific receptors being an important factor in the pathogenesis of toxoplasmosis [1, 2]. Experimental trials have demonstrated that this protozoan expresses sugar-binding proteins on its rhoptries which are able to bind N-acetylglucosamine and galactose [3]. These monosaccharides are present in glycoproteins and glycolipids of the gastrointestinal tract with the expression of these molecules being related, at least in part, to the profile of ABH glycoconjugates secreted by carriers of secretor phenotype. Carriers of non-secretor phenotype are incapable of secreting these glycoconjugates [4]. The secretor and non-secretor phenotypes are controlled by the FUT2 gene (19q13.3) [4], and consequently the ABH glycoconjugate profile results from the epistatic interactions between this gene and the ABO (9q34.1) gene [5]. Therefore, each glycoconjugate of the profile may act as a potential receptor and influence the susceptibility or resistance to infection by micro-organisms [6]. As the entrance of T. gondii and the expression of ABH glycoconjugates occur in the gastrointestinal tract, there may be some link between them. Previous reports have explored possible associations between ABO blood groups and T. gondii infection. Four of them reported that individuals of the B and AB blood groups present high susceptibility to infections by this protozoan as seen by the detection of specific IgG class antibodies [7-10]. These studies also indicated that the B antigen may act as a potential candidate receptor for T. gondii in the gastrointestinal tract. Two other similar studies did not find an association between ABO blood groups and anti-T. gondii antibodies [11, 12]. The results of these reports remain inconclusive as they are based only on the identification of ABO blood group phenotypes and did not include an analysis of the FUT2 gene as a determinant of the profile of ABH glycoconjugates expressed by secretor and non-secretor phenotype carriers. The aim of this study was to test the hypothesis that the ABH glycoconjugate profile in the gastrointestinal tract is associated with infection by T. gondii.
Material and methods

Selection of pregnant women
A total of 367 pregnant Caucasian and non-Caucasian women, who attended the High-Risk Pregnancy Clinic of Hospital de Base linked to the Medicine School in Sa~o José do Rio Preto, were enrolled in this study between October 2005 and February 2007. The number of pregnant women selected is sufficient to demonstrate the difference for the B blood group as reported in the previous paper [7] with power higher than 90%. Self-definition was used as the means of identifying their ethnic groups taking into account two generations of progenitors. Under 18-year olds and individuals suffering from other infectious or parasitic diseases were not included in the study.
Ethical considerations and blood sampling
The study was approved by the Research Ethics Committee of FAMERP (case 089/2005). After receiving written consent forms from each participant, two blood samples were drawn and stored in vacuum tubes, only one of which contained anticoagulant.
ABO phenotyping
The ABO blood groups were determined by the haemagglutination method using commercial anti-sera for direct typing and commercial standard red blood cells for reverse typing (Biotest, Sao Paulo, Brazil). A drop of a suspension of red blood cells in 5% sterile saline solution (0.9% NaCl) prepared for each blood sample was mixed with a drop of each of the anti-A, anti-B and anti-A/B anti-sera to define the erythrocytic antigens. Two drops of blood plasma from each sample were mixed in a tube to one drop of each of the 5% standard red blood suspensions of groups A and B to identify anti-A and anti-B antibodies. The tubes were centrifuged at 3400 rpm for 1.5 min with interpretation of the results being based on the presence or absence of haemagglutination. In all procedures, the recommendations of the manufacturers of the reagents used were strictly followed.
Extraction of genomic DNA
The genomic DNA was extracted by a salting-out procedure [13]. The isolated leukocytes were rinsed three times in buffered saline solution (PBS) and incubated overnight at 37°C in 20 µl of 10% K-proteinase solution. The DNA was precipitated in 2 ml of 6 M NaCl, rinsed three times in absolute ethanol and three times in 70% ethanol followed by dissolution in 300 µl MilliQ water and stored at –20°C until use.
FUT2 genotyping
The FUT2 genotypes resulting from homozygosis or heterozygosis of the G428A substitution were identified by PCR-RFLP according to the protocol of Svensson and co-workers [14]. Amplification reactions used the sense, 5’ CGC TCC TTC AGC TGG GCA CTG GA 3’ and antisense, 5’ CGG CCT CTC AGG TGA ACC AAG AAG CT 3’, primers to differentiate the G and A alleles at the 428 position of the FUT2 gene. Each reaction used a final volume of 25 µl containing 10 mM TRIS-HCL, 50 mM KCl, 1.5 mM MgCl2, 20 mM of each dNTP [dATP, dTTP, dCTP, dGTP], 10 pM of each primer, 0.5 U of Taq and 5 ng of genomic DNA. The conditions of amplification involved pre-desnaturation at 94°C for 5 min, followed by 35 cycles (94°C for 1 min, 63°C for 1 min and 72°C for 1 min) and an additional extension at 72°C for 5 min. The fragment containing 1033 base pairs after digestion with the Ava II enzyme was cleaved in a variable number of fragments according to the alleles: 459, 295, 149 and 130 base pairs for the G allele; and 459, 425 and 149 base pairs for the A allele. Separation of these fragments was achieved by electrophoresis in 2% agarose gel stained using ethidium bromide and viewed under UV light. Thus, GG and GA individuals were considered secretors and AA individuals non-secretors of ABH glycoconjugates (Figure 1).
Diagnosis of Toxoplasma gondii infection
Anti-T. gondii antibodies were detected by commercial haemagglutination serodiagnosis test kits (IMUNO-HAI-TOXO – Wama Diagnóstica, Sa~o Carlos, Brazil). The instructions of the manufacturer were strictly followed. The results are expressed as “seropositive” and “seronegative” for the presence and absence of anti-T. gondii antibodies, respectively.
Statistical analysis
The data were entered into the Graphpad Instat computer program and analyzed using Pearson’s c2 test and Fisher’s exact test. The level of significance adopted was 5%.
Results
Of the 367 participating pregnant women, 155 (42.2%) were Caucasian, 170 (46.3%) were half-castes, 39 (10.6%) were Afro-descendents, two (0.54%) were Native Indians and one (0.27%) was Oriental. The average age of the participants was 26.4 years (range 18 to 44 years). The overall distribution of ABO blood group phenotypes was 34.3% (A), 12.8% (B), 3.8% (AB) and 48.7% (O). The overall frequencies of genotypes that define the secretor phenotype (GG and GA) and non-secretor phenotype (AA) were 76.3% (280/367) and 23.7% (87/367), respectively. Overall, 49.6% (182/367) were seropositive and 50.4% (185/367) were seronegative for anti-T. gondii antibodies. The participants were divided into two groups according to the presence or absence of anti-T. gondii antibodies but did not reveal statistically significant differences in relation to the frequencies of the ABO blood groups (P value =0.20, c2=4.567) or the secretor and non-secretor phenotypes (P value =0.41, c2 =0.6786), when considered in isolation or in combination (Table I).
Discussion
Investigations of T. gondii infection have paid special attention to the prevalence of positive serological tests in pregnant women because of the risk of congenital transmission and the resulting sequelae in newborn babies [15-18]. Parallel to this, there has been growing interest in the biology of this protozoan, especially in its ability to utilize glycosylated molecules expressed in the gastrointestinal tract as receptors [1, 2]. The previous demonstration that the rhoptries of tachyzoites of T. gondii express sugar-binding proteins which bind N-acetylglucosamine and galactose [3], the use of the gastrointestinal tract as a route of infection [1, 2] and the action of the FUT2 gene controlling the secretor and non-secretor phenotypes [4] prompted us to test the hypothesis that the ABH glycoconjugate profile in the gastrointestinal tract is associated with infection by T. gondii. The background of the casuistic approach used in this study is representative of the northwestern region of Sao Paulo State, in Brazil, concerning the ethnicity, ABO blood group phenotypes, secretor and non-secretor phenotypes, and prevalence of infection by T. gondii [19-22]. According to the results of this study all the pregnant women seem to be equally susceptible to T. gondii infection, this fact being confirmed by the presence of specific anti-T. gondii antibodies in both secretor and non-secretor individuals of all the ABO blood groups. However, the absence of anti-T. gondii antibodies may be due to the lack of exposure to the parasite or even the action of other resistance factors not associated with the ABO blood groups and the secretor and non-secretor phenotypes. The differences not being statistically significant among the frequencies of ABO blood groups, secretor and non-secretor phenotypes when considered in isolation or in combination, in the presence of seropositive and seronegative tests for anti-T. gondii antibodies, suggests that the hypothesis that the ABH glycoconjugate profile in the gastrointestinal tract is associated with infection by T. gondii is not valid. The results of this paper are in disagreement with those that stated that the B and AB blood group carriers are more susceptible to T. gondii infection and the B antigen may act as a receptor for this protozoan [7-10]. However, they are in agreement, at least in part, with other reports which also did not find any association with ABO blood groups and anti-T. gondii antibodies [11, 12]. Regrettably, all these reports evaluated only the ABO erythrocytic phenotypes, but the protozoan T. gondii does not infect red blood cells. Besides, these studies did not consider the influence of the FUT2 gene on the control of the expression of ABH glycoconjugates in the gastrointestinal tract. The proposition that B antigen may act as a potential candidate receptor for T. gondii [7, 8] is attractive due to the fact that some micro-organisms are able to bind carbohydrates such as those present in the ABO blood group structures [6, 23]. ABH glycoconjugate expression in the human gastrointestinal tract depends on the presence of at least one functional allele of the FUT2 gene [4]. Homozygosity and heterozygosity for the G allele lead to expression of the FUTII enzyme, which is capable of incorporating a fucose molecule to the galactose terminal of type 1 oligosaccharide precursors (Galb1→3GlcNAcb1 →R) to form the H type 1 antigen ([Fuca1→2]Galb1→ 3GlcNAcb1→R). This antigen, when glycosylated by a-3-D-N-acetylgalactosaminyltransferase or a-3- D-galactosyltransferase enzymes coded by A and B alleles of the ABO gene, results in A type 1 (NAcGala1→ 3[Fuca1→2]Galb1→3GlcNAcb1→R) or B type 1 (Gala1→3[Fuca1→2]Galb1→3GlcNAcb1→R) antigens [5, 14]. These glycoconjugates are derived from a common precursor but present with variations in their spatial structures and chemical compositions [5, 6] which may not influence the selective binding of the tachyzoites from T. gondii to epithelial cells of the human gastrointestinal tract. Experimental trials have demonstrated that T. gondii is capable of binding different monosaccharides expressed on the surface of the vertebrate cells, including those present on the ABH glycoconjugates composition, but this ability seems not to be exclusive for galactose, which defines the specificity of the B antigen [3, 24]. Various factors may have contributed to the disagreement between the data of this study with those proposing that B and AB blood group carriers are more susceptible to T. gondii infection [7-10]. It is possible that the ABH glycoconjugate profile containing the B antigen constitutes a small risk for T. gondii infection. Besides, the Brazilian genetic background and the elevated prevalence of infection by this protozoan in the population living in the northwestern region of Sao Paulo State obscures its importance for susceptibility. Additionally, due to the variability of T. gondii strains that infect the Brazilian population [25], it is possible that only some may utilize specific ABH glycoconjugates as receptors in the gastrointestinal tract. Another aspect to be considered is that this study only analyzed a female cohort and so the influence of gender in the relationship between humans and anti-T. gondii antibodies was not considered [12]. As individuals of all ABO blood groups, both secretors and non-secretors, seem to be equally susceptible to T. gondii infection, in principle there is no reason to believe that ABH glycoconjugate profile may influence infection by this protozoan. However, the presence or absence of the FUTII enzyme, although necessary to create the differentiation of ABH glycoconjugate profiles in the gastrointestinal tract, is not sufficient to influence the susceptibility or resistance to T. gondii infection among females. In conclusion, the ABH glycoconjugate profile in the human gastrointestinal tract controlled by the FUT2 and ABO genes is not associated with the presence or absence of anti-T. gondii antibodies and thus it does not seem to be a crucial factor in increased or decreased risk of infection.
Acknowledgments
The authors wish to thank the pregnant women who participated in this study, Valéria Daltibari Fraga, Luciana Moran Conceiça~o, Fernanda da Silva and Angelita Feltrin for their technical assistance in analyses, Prof. Dr. José Antônio Cordeiro for help with the statistical analysis and David Hewitt for the English version. Work carried out in the Immunogenetics Laboratory and Micro-organism Investigation Centre of FAMERP. Financial support: BAP–FAMERP 2005/2006 & CNPq # 131228/2007-2. CCBM is a Master’s student of the Postgraduate Course in Genetics of the Sao Paulo State University and received a grant from the Ministry of Science and Technology – CNPq (National Council for Scientific and Technological Development), Brazil.
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