Introduction
Neurocysticercosis (NCC) is caused by the presence of Taenia solium metacestodes in the central nervous system (CNS). The infection begins with the ingestion of parasite eggs, which release oncospheres that invade body tissues, such as muscles, eyes and frequently the brain, causing complications that may lead to severe infection (Garcia et al., Reference Garcia, Nash and Del Brutto2014). The disease is responsible for most cases of epilepsy worldwide, affecting about 30% of people in endemic areas (WHO, 2019). NCC diagnosis involves neuroimaging techniques, such as computed tomography (CT) and magnetic resonance imaging, which are considered gold standards in research and laboratory environments (Del Brutto et al., Reference Del Brutto, Nash, White, Rajshekhar, Wilkins, Singh, Vasquez, Salgado, Gilman and Garcia2017). Furthermore, immunological tests, such as enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB), have been used to increase the diagnostic accuracy of NCC (Gabriël et al., Reference Gabriël, Blocher, Dorny, Abatih, Schmutzhard, Ombay, Mathias and Winkler2012; Hernández-González, et al., Reference Hernández-González, Noh, Perteguer, Gárate and Handali2017).
Nevertheless, detection of circulating immune complexes (CIC) can be used for NCC diagnosis, since it has been successfully applied in helminth diagnosis approaches (Faria et al., Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019; Lopes et al., Reference Lopes, de Faria, de Sousa, Borges, Ribeiro, Bueno, Rodrigues Ávila, Ferreira-Júnior and Costa-Cruz2019). This method provides clarity to understand changes in the organism before and after disease treatment, since CIC can be present in plasma, and also be found attached to lymphocytes. In addition, this method is capable of developing reproducible immunoassays that might be used in biological and clinical research (Barnett, Reference Barnett1986).
Regarding the laborious obtention of suitable antigens from metacestodes of naturally infected porcines, the search for other antigenic sources has been proposed, such as Taenia crassiceps metacestodes. The simple obtention of T. crassiceps extracts, allied to their cross-reactivity with T. solium antigens, turns them an interesting alternative antigenic source for NCC immunodiagnosis (Larralde et al., Reference Larralde, Sotelo, Montoya, Palencia, Padilla, Govezensky, Diaz and Sciutto1990; Bueno et al., Reference Bueno, Vaz, Machado, Livramento and Mielle2000).
The application of antigenic fractions in human NCC diagnosis, from total antigen extracts of T. solium and T. crassiceps metacestodes, has shown efficient results in ELISA, WB and immunofluorescence antibody test (IFAT). The heterologous antigen obtained from T. crassiceps has been used in NCC immunodiagnostic techniques through the detection of IgG (Machado et al., Reference Machado, Santiago, Mineo and Costa-Cruz2007; Gonçalves et al., Reference Gonçalves, Machado, Oliveira, Rezende, Mineo and Costa-Cruz2010). However, the prevalence of cross-reactions in some studies, as well as false-negative results, due to the number and different metacestode forms, has turned the diagnosis difficult (Gripper and Welburn, Reference Gripper and Welburn2017). These results indicate the need to test an alternative antibody with greater sensitivity and specificity in antigen detection assays.
Owing to the phylogenetic and evolutionary divergence of avians from mammals, it results to increase the probability of raising antibodies against mammalian antigens and circumvents problems associated with antigen self-tolerance (Lee et al., Reference Lee, Syed Atif, Tan and Leow2017). The main advantages of using polyclonal antibodies are the feasibility, reduction of animals use and cost-effective antibody production (Kousted et al., Reference Kousted, Kalliokoski, Christensen, Winther and Hau2017).
This study developed for the first time a high-specific serological platform using specific immunoglobulin Y (IgY) antibodies produced against T. crassiceps hydrophobic antigenic fractions, to be tested on human NCC diagnosis. Therefore, this study aimed to improve the immunodiagnostic tests of human NCC, considering the difficulty of obtaining an accurate diagnosis in patients with low parasite load and inactive forms of the disease. The specific IgY antibodies were also used to improve the antigen recognition in parasite tissues and immune complexes from NCC serum samples.
Materials and methods
Human serum samples
A panel of 114 human sera from samples bank of the Laboratório de Diagnóstico de Parasitoses from the Federal University of Uberlândia (UFU) were divided into three groups: Group 1 (n = 44) – serum samples from patients with confirmed NCC by means of clinical, epidemiological, serological and neuroimaging data, obtained by analysis of medical files. These samples were selected based on NCC diagnostic guidelines proposed by Del Brutto (Reference Del Brutto2012) and subdivided into active (n = 22) and inactive (n = 22) NCC, according to Sotelo et al. (Reference Sotelo, Guerrero and Rubio1985). Group 2 (n = 35) – samples from healthy individuals, based on clinical status, from an endemic area for cysticercosis, with no household contact with T. solium infection and negative parasitological examinations of three fecal samples by Baermann (Reference Baermann1917), Moraes (Reference Moraes1948) and Lutz (Reference Lutz1919). Group 3 (n = 35) – samples from patients with other parasitic diseases, such as hookworms (n = 4), Ascaris lumbricoides (n = 3), Echinococcus granulosus (n = 5), Entamoeba histolytica/dispar (n = 1), Enterobius vermicularis (n = 4), Giardia lamblia (n = 3), Hymenolepis nana (n = 4), Schistosoma mansoni (n = 3), Strongyloides stercoralis (n = 2), Taenia spp. (n = 3), Trichuris trichiura (n = 1) and coinfections: E. histolytica/dispar + G. lamblia (n = 1) and hookworm + A. lumbricoides (n = 1). Samples of this group were obtained from patients who carried out parasitological exams of feces with three samples from each individual, according to Ritchie (Reference Ritchie1948), they came from an endemic area for cysticercosis and had no history of infection with T. solium (taeniasis or cysticercosis). In the case of patients infected with E. granulosus, the diagnosis was made by ultrasound, CT, serology and confirmation by cystic fluid parasitological examination after surgery. All cases underwent surgery for hydatid disease.
Mice infection and larvae obtention
Female BALB/c mice (Mus musculus) were intraperitoneally infected with T. crassiceps (ORF strain) metacestodes at the Animal Experimentation Center (CBEA) from the UFU, located at the city of Uberlândia, after 3 months post-infection, time required for parasite replication. The metacestodes were obtained from the peritoneal cavity of the animals after euthanasia using an anaesthetic overdose (ketamine hydrochloride 10% and xylazine hydrochloride 2%), and stored in 0.01 M phosphate-buffered saline (PBS), pH 7.2, at −20 °C.
Antigen fractionation
The hydrophobic antigen (hFTc) was obtained from the total saline extract of T. crassiceps metacestodes (TeTc), using Triton X-114 (TX-114) fractionation method as described by Silva et al. (Reference Silva, Nunes, Sousa, Gonçalves-Pires, Levenhagen and Costa-Cruz2017). The fractionation process started with a protein mass of 14 mg mL−1 from TeTc. Antigenic fractions with a final concentration of 2 mg mL−1 were obtained and evaluated by 12% SDS-PAGE under non-reducing conditions. Protein concentration was determined by Lowry et al. (Reference Lowry, Rosebrough, Farr and Randall1951).
Hen immunization and collection of blood and eggs
Four laying hens (Gallus gallus domesticus) were used, two as controls and two were immunized with hFTc antigen to elicit the production of specific anti-hFTc IgY antibodies according to the methodology proposed by Schwarzkopf et al. (Reference Schwarzkopf, Staak, Behn, Erhard, Schade, Behn, Erhard, Hlinak and Staak2001). Hens were first immunized using Freund's complete adjuvant and then boosted three times at 14-day intervals using incomplete Freund's adjuvant (Fig. 1). Blood and egg collection were carried out for 8 weeks (0–8).
Fig. 1. Hen immunization timeline with a hydrophobic fraction of T. crassiceps metacestodes (hFTc) (GI) and phosphate-buffered saline (PBS) (GII) at 14-day intervals.
IgY fractionation
Fractionation of polyclonal antibodies was performed according to Akita and Nakai (Reference Akita and Nakai1993) method, with modifications. Briefly, pooled egg yolks of each week were diluted and homogenized in ultrapure water and the solution was centrifuged at 800 × g for 40 min at 4 °C. The supernatant was collected, filtered and the pH adjusted to 7.4 with 0.1 M NaOH. The lipid precipitate was stored at −20 °C. Precipitation of IgY antibodies was performed by addition of 20% (m/v) ammonium sulphate to the supernatant, under slow agitation at 4 °C for 45 min. The solution was centrifuged at 2000 × g for 20 min at 4 °C, and the precipitate obtained was resuspended in PBS (pH 7.4) at 1:10 initial volume.
IgY kinetics and avidity test
Indirect ELISA tests were performed to determine the production kinetics and maturation avidity of anti-hFTc IgY antibodies from egg yolks. Hen serum samples were also collected for seroconversion evaluation. Low-affinity (Greiner, BIoOne, Austria) and high-binding (Corning Costar, Sigma-Aldrich, Corning, New York, USA) 96-well microplates were used for kinetic and avidity tests, respectively. Microplates were coated with hFTc antigens (10 µg) (50 µL well−1) in carbonate-bicarbonate buffer (0.06 M, pH 9.6), overnight at 4 °C. Secondary antibodies (anti-IgY) were added at 1: 15 000 dilution for both assays. The cut-off from egg yolk and hen serum samples was calculated by the mean optical density (OD) from control animals (nine samples each, weeks 0–8), plus three standard deviations. The ELISA procedures, as well as indexes calculation, ELISA index (EI) and avidity index (AI %), were carried out as previously described (Faria et al., Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019). The antigen–antibody binding strength was defined as low avidity (AI < 40%) and high avidity (AI > 60%), and the result was expressed as a percentage.
IgY purification
Specific IgY antibodies obtained from week 6 and week 8 egg yolks were pooled and purified (Fig. 2). These two weeks were selected due to higher production indexes and reactivity in kinetic and avidity tests. The pooled antibodies were dialysed and concentrated in an ultrafiltration system (EMD Millipore, USA) with a 30 kDa membrane (AMICON YM 30, USA). Purification was performed at HiTrap IgY Purification HP column, 5 mL (GE Healthcare, USA), previously equilibrated with 0.02 M sodium phosphate (Na3PO4) and 0.5 M potassium sulphate (K2SO4) buffers. Antibodies were eluted in a flow rate of 1 mL min−1 using a linear gradient of 0.02 M sodium-phosphate buffer (pH 7.5) in the ÄKTA prime plus liquid chromatography complete system (GE Healthcare). Afterwards, the pooled antibodies were dialysed with ultrapure water and concentrated using an AMICON Ultra-15 centrifugal filter unit (Sigma-Aldrich) at 3000 × g for 20 min at 24 °C. The purified IgY antibodies were collected and the protein concentration was determined at 280 nm by spectrophotometer (Biodrop Ulite, UK).
Fig. 2. Purification scheme of specific anti-hFTc IgY antibodies.
Evaluation of fractionated and purified IgY
The quality of fractionated and purified IgY antibodies was assessed by 12% SDS-PAGE under reducing conditions, according to Laemmli (Reference Laemmli1970). Protein aliquots (10 µg) were diluted in sample buffer with 5% β-mercaptoethanol (Sigma-Aldrich). Electrophoresis was performed using SE-300 miniVE vertical electrophoresis system (Hoefer, Inc., San Francisco, USA), with Coomassie Brilliant blue G-250 (Sigma-Aldrich) staining.
Western blotting assay
The hFTc antigenic recognition using weekly fractionated and the purified IgY antibodies was investigated by WB. Primary, an hFTc aliquot (250 µg) was submitted to 12% SDS-PAGE under non-reducing conditions. Then, proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories Inc., USA), which were incubated with anti-hFTc IgY antibodies (6 µg). The reaction was performed as described by Faria et al. (Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019).
Antigenic recognition in histological sections of Taenia crassiceps and Taenia solium
The T. crassiceps strain was kept at CBEA from UFU, and T. solium metacestodes were collected in slaughterhouses at the city of Uberlândia, from skeletal muscles of naturally infected porcines. The IFAT was carried out to test the antigenic recognition in tissue sections of T. crassiceps and T. solium metacestodes using purified anti-hFTc IgY antibodies. The antibodies (780 µg mL−1) were diluted in PBS (50 µL) and the reaction was performed according to Faria et al. (Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019). The images were obtained using an inverted fluorescence microscopy system (EVOS, AMG Microscopy Group, USA). The control of the secondary antibody was performed with PBS (50 µL well−1).
Detection of immune complexes in human sera
Purified and specific IgY antibodies were assessed for the detection of antigen–antibody complex (CIC) in human sera with confirmed NCC. A sandwich ELISA was carried out on high-binding 96-well microplates (Santa Cruz Biotechnology, CA, USA) coated with anti-hFTc IgY (10 µg) diluted in carbonate-bicarbonate buffer (0.06 M, pH 9.6) and incubated overnight at 4 °C. Then, microplates were washed three times with PBS-T. Human serum samples (NCC positive, healthy individuals and other parasitic diseases patients) were diluted in 1:200 PBS-T, and a final volume of 50 µL well−1 was added to the microplates. Then, the plates were incubated for 45 min at 37 °C. After another step of washing, secondary antibody produced in goat anti-human IgG, Fc-specific peroxidase-conjugate (Sigma-Aldrich) diluted 1:2000 in PBS-T was added. After incubation for 45 min at 37 °C, the plates were washed as described above and developed according to ELISA kinetic test. Absorbance was determined at 492 nm in Epoch microplate reader (Biotek, UK).
Statistical analysis
Statistical analyses were performed using GraphPad Prism software version 6.0 (GraphPad Software, San Diego, CA, USA). The variables were assessed by D'Agostino and Pearson, Shapiro–Wilk and Kolmogorov–Smirnov tests according to the data distribution. Sensitivity, specificity, positive and negative likelihood ratios (LR+ and LR−), area under the curve (AUC) and cut-off values for sandwich ELISA were set using receiver operating characteristic (ROC) curve. One-way ANOVA with Tukey post-test (P < 0.0001) were performed to compare the variance differences of means between pairs of human serum groups. Values of P < 0.05 were considered to be statistically significant.
Results
Antigenic characterization of Taenia crassiceps protein extracts
Proteins from TeTc (14 mg mL−1) and hFTc (2 mg mL−1) were characterized by 12% SDS-PAGE. A total of 12 and 14 protein bands were identified for TeTc and hFTc, respectively (Fig. 3A).
Fig. 3. Electrophoretic profile of total extract (TeTc) and a hydrophobic fraction (hFTc) from T. crassiceps metacestodes obtained by TX-114 fractionation. Silver nitrate was used as staining for protein bands (A). Electrophoresis under reducing conditions using anti-hFTc IgY antibodies obtained from fractionation and purification processes (B). Y: egg yolk solution obtained before fractionation; w6: fractionated antibodies from week 6; w8: fractionated antibodies from week 8; p: pooled IgY antibodies obtained from week 6 and week 8 after purification with sepharose resin. The arrow indicates ovalbumin bands. The brackets indicate IgY heavy and light chains. MW, molecular weight standard; kDa, kilodalton.
Analysis of fractionated and purified IgY
Aliquots from fractionated and purified IgY were used to assess the quality of protein fractions. Anteriorly to the fractionation step, a fraction of egg yolk homogenate (Y) was obtained from week 6 and week 8 (Fig. 3B). The total protein yield obtained from each week ranged from 2852 µg mL−1 (week 0) to 9186 µg mL−1 (week 8). Purification was confirmed after application of the purified antibodies (20 µL) to a nitrocellulose membrane (0.45 µm) for a Dot-blot assay (Fig. S1). Then, the protein profile of the purified IgY was analysed and compared with the previously obtained protein fractions from the fractionation process. The antibodies from week 6 and week 8, after treatment with 5% β-mercaptoethanol, showed equal patterns, with heavy (65–70 kDa) and light (20–22 kDa) chains. Ovalbumin (~42 kDa) was detected in antibody samples before the purification process. The purified IgY (p) showed only heavy chains (66–70 kDa) (Fig. 3B).
Evaluation of production, avidity and antigen recognition by IgY
Polyclonal IgY antibodies obtained after egg yolk fractionation were evaluated for production kinetics and avidity maturation by indirect ELISA. The egg yolk antibodies started the production at week 2, showing higher concentration at week 6 and week 8. Seroconversion of the antibodies started at week 2 and the production was constant from week 4 (Fig. 4A). Avidity maturation test was performed with polyclonal IgY samples showing EI > 1. Fractionated egg yolk antibodies since week 2 were selected for testing. The avidity indexes (AI %) ranged from 66.1% (week 2) to 95.08% (week 8), showing higher values at week 6 and week 8, with 88.17 and 95.08%, respectively (Fig. 4B). Antigen recognition was tested by WB using fractionated anti-hFTc IgY from each week (0–8) and the purified IgY. Two pairs of bands (80 and 70 kDa) and (70 and 65 kDa) reacted from the third week for T. crassiceps (Fig. 5A) and T. solium (Fig. 5B) WB assays. Immunofluorescence was performed in histological sections of T. crassiceps and T. solium metacestodes. In both assays, purified anti-hFTc IgY recognized tegument features of the metacestodes (see arrows). PBS was used as a control (Fig. 6).
Fig. 4. Production kinetics of anti-hFTc IgY antibodies fractionated from egg yolk and hen serum (A). Avidity maturation of egg yolk IgY antibodies obtained throughout the experiment (B). Urea 6 M was used as a chaotropic agent. Dotted lines show the cut-off (ELISA index, EI > 1) and avidity index (AI > 60%). Weeks with AI > 85% were selected for later assays. Week 0 = pre-immunization; *immunization.
Fig. 5. Western blotting assay showing the protein profile of hydrophobic fraction obtained from Taenia crassiceps (hFTc) (A) and Taenia solium (hFTs) metacestodes using fractionated and purified anti-hFTc IgY antibodies (B). Membranes were stained with 3,3’ Diaminobenzidine (DAB). MW, molecular weight standard; w, week; p, pooled IgY; kDa, kilodalton.
Fig. 6. Immunofluorescence test using specific anti-hFTc IgY antibodies in histological sections of Taenia crassiceps and Taenia solium metacestodes. Taenia solium was used as a positive control. Arrows indicate the metacestode tegument features. Fluorescein isothiocyanate-conjugated anti-IgY (FITC) was used as staining. Images were obtained by inverted fluorescence microscopy (EVOS, AMG Microscopy Group).
Application of specific IgY antibodies in human NCC diagnosis
Purified anti-hFTc IgY antibodies were tested for immune complexes detection in human serum samples (NCC positive, healthy individuals and other parasitic diseases patients). Immune complexes were detected in 93.2% (41/44) of serum samples from NCC-positive patients. All patients with active NCC (n = 22) and 86.36% with inactive NCC (19/22) presented immune complexes. Healthy patients showed 8.57% (3/35) of detection. Positivity rates in patients with other parasitic diseases reached 11.43%: E. granulosus (1/5), H. nana (1/4), S. mansoni (1/3) and T. trichiura (1/1) (Fig. 7A). The test showed high diagnostic performance (Fig. 7B). One-way ANOVA was performed to compare the variance difference between OD means of the human sera groups. Significant differences were shown between group means (F = 97.84, R = 0.7274, P < 0.0001, CI 95%). Multiple comparisons with Tukey post-test were used to evaluate the variance difference of OD means between group pairs. Significant difference between NCC-positive patients and their subgroups (active and inactive NCC) with the healthy and the other parasitic diseases group (P < 0.0001) was shown. There was no significant difference between active and inactive NCC subgroups and between healthy and other parasitic diseases group.
Fig. 7. Sandwich ELISA for immune complexes detection using specific anti-hFTc IgY antibodies in human serum samples from NCC positive (NCC active, n = 22; NCC inactive, n = 22), healthy (n = 35) and other parasitic diseases (n = 35) patients (A). ROC curve showing cut-off, sensitivity (Se), specificity (Sp), area under the curve (AUC), P value, positive and negative (LR+/LR−) likelihood ratios (B). Samples with EI > 1 were considered to be positive. ****One-way ANOVA, with Tukey post-test (P < 0.0001).
Discussion
Improved neuroimaging techniques and immunological tests increase the accuracy of NCC diagnosis by detecting metacestodes at different evolutionary stages in CNS and characterizing host-specific antibodies (Del Brutto et al., Reference Del Brutto, Nash, White, Rajshekhar, Wilkins, Singh, Vasquez, Salgado, Gilman and Garcia2017; Carpio et al., Reference Carpio, Fleury, Romo and Abraham2018).
This study used the hFTc antigen to produce specific IgY antibodies obtained from immunized hens. Antigens of T. crassiceps metacestodes efficiently replace those from T. solium in human NCC diagnosis (Larralde et al., Reference Larralde, Montoya, Sciutto, Diaz, Govezensky and Coltorti1989; Vaz et al., Reference Vaz, Nunes, Piazza, Livramento, Silva, Nakamura and Ferreira1997).
The higher sensitivity of T. crassiceps antigens to detect antibodies and antigens in sera and CSF samples from patients presenting no signals of inflammatory process or with calcifications was previously demonstrated (Bueno et al., Reference Bueno, Vaz, Machado, Livramento and Mielle2000; Pardini et al., Reference Pardini, Vaz, dos Ramos Machado and Livramento2001). Considering the presence of common antigenic determinants shared with T. solium metacestodes, along with the minimal cost of antigen production in laboratory, our results indicate the hydrophobic fraction of T. crassiceps as a potential diagnostic tool for NCC diagnosis. Antigenic fractions of T. crassiceps have been successfully tested in CSF samples of NCC-positive patients, showing high values of Se and Sp in immunoassays using the hydrophobic antigen (Silva et al., Reference Silva, Nunes, Sousa, Gonçalves-Pires, Levenhagen and Costa-Cruz2017).
In an attempt to improve tests based on the detection of specific antigens, the polyclonal IgY antibodies emerge as promising for human NCC diagnosis. Avian IgY antibodies are excellent for use in immunological assays involving mammalian sera due to the different properties of IgY compared to IgG, since IgY does not react with rheumatoid factor, does not activate the complement system and does not bind to the mammalian Fc receptor. These characteristics allow the development of more efficient immunodiagnostic and immunotherapeutic applications (Hodek et al., Reference Hodek, Trefil, Simunek, Hudecek and Stiborova2013; Amro et al., Reference Amro, Al-Qaisi and Al-Razem2018).
Polyclonal antibodies have been widely used in laboratory and biotechnological diagnostic studies in human and veterinary medicine by immunizing hens with E. granulosus (Gottstein and Hemmeler, Reference Gottstein and Hemmeler1985), Ascaris suum (Schniering et al., Reference Schniering, Schade and Hiepe1996), T. solium (Manhani et al., Reference Manhani, Ribeiro, Cardoso, Ueira-Vieira, Goulart and Costa-Cruz2011), Schistosoma japonicum (Cai et al., Reference Cai, Guo, Chen, Tian, Steinmann, Chen, Li, Ai and Chen2012), Trichinella spiralis (Wang et al., Reference Wang, Fu, Jing, Jin, Ren, Jiang and Cui2012), Toxoplasma gondii (Ferreira-Junior et al., Reference Ferreira-Junior, Santiago, Silva, Ferreira, MacedoJunior, Mota, Faria, Silva-Filho, Silva, Cunha-Junior, Mineo and Mineo2012) and Strongyloides venezuelensis (Faria et al., Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019). Immunization of hens with parasitic antigens has been promising for the understanding and characterization of polyclonal antibodies. The production of specific IgY antibodies is capable of enhancing immunodiagnostic and therapeutic techniques (Schade et al., Reference Schade, Calzado, Sarmiento, Chacana, Porankiewicz-Asplund and Terzolo2005; Lee et al., Reference Lee, Syed Atif, Tan and Leow2017).
The specific IgY antibodies produced in this study showed increasing production kinetics during the course of immunization, which was observed in other studies. Da Silva Raposo et al. (Reference da Silva Raposo, Santarém, Merigueti, Rubinsky-Elefant, de Lima Cerazo, Pereira, Zampieri, da Silva and Laposy2016) observed a constant increase in IgY levels in hens immunized with embryonated eggs of Toxocara canis. In this study, the kinetic of antibody production in egg yolks started on 15-day post-immunization (dpi), which coincided with seroconversion, corroborating with da Silva Raposo et al. (Reference da Silva Raposo, Santarém, Merigueti, Rubinsky-Elefant, de Lima Cerazo, Pereira, Zampieri, da Silva and Laposy2016). Ferreira-Junior et al. (Reference Ferreira-Junior, Santiago, Silva, Ferreira, MacedoJunior, Mota, Faria, Silva-Filho, Silva, Cunha-Junior, Mineo and Mineo2012) also observed high antibody levels after 14 dpi with T. gondii in hens. In this study, the antibodies reached higher production levels from 29 dpi, remaining constant from 43 dpi, complying with the findings of da Silva Raposo et al. (Reference da Silva Raposo, Santarém, Merigueti, Rubinsky-Elefant, de Lima Cerazo, Pereira, Zampieri, da Silva and Laposy2016), which showed constant levels of antibodies from 45 dpi.
Regarding the avidity indexes (AI %), the levels of antibodies increased from 15 dpi and showed the highest indexes (AI > 85%) at 43 and 57 dpi, contrasting with da Silva Raposo et al. (Reference da Silva Raposo, Santarém, Merigueti, Rubinsky-Elefant, de Lima Cerazo, Pereira, Zampieri, da Silva and Laposy2016), who had higher AI after the final phase of immunization (28 dpi, AI > 56%). Ferreira-Junior et al. (Reference Ferreira-Junior, Santiago, Silva, Ferreira, MacedoJunior, Mota, Faria, Silva-Filho, Silva, Cunha-Junior, Mineo and Mineo2012) also observed a high maturation level of IgY treated with 6 M urea at 42 dpi, as well as a slight decrease in AI levels at 49 dpi, complying with this study. Moreover, the increased avidity along time after immunization was already shown in recent studies with helminths. Faria et al. (Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019) presented high avidity indexes for specific anti-S. venezuelensis parthenogenetic female IgY (anti-pF IgY), and anti-S. venezuelensis filariform larvae (anti-iL3 IgY), ranging from 74.0% (week 2) to 95.4% (week 10) and from 68.0% (week 2) to 82.8% (week 10). Lopes et al. (Reference Lopes, de Faria, de Sousa, Borges, Ribeiro, Bueno, Rodrigues Ávila, Ferreira-Júnior and Costa-Cruz2019), immunizing hens with A. suum antigenic extract, obtained high AI levels of IgY after the second immunization, ranging from 64% (week 2) to 93% (week 10). In this study, we showed high avidity indexes in only 8 weeks of experimentation, ranging from 66.1% (week 2) to 95.08% (week 8).
Successful application of specific IgY antibodies for antigen recognition by WB and IFAT has been recently performed in other studies with helminths, such as S. venezuelensis (Faria et al., Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019) and A. suum (Lopes et al., Reference Lopes, de Faria, de Sousa, Borges, Ribeiro, Bueno, Rodrigues Ávila, Ferreira-Júnior and Costa-Cruz2019).
In this study, all 22 samples with active NCC were positive and only three out of 22 samples with inactive NCC were negative, with 93.2% (Se) and 94.3% (Sp). Garcia et al. (Reference Garcia, Castillo, Gonzales, Bustos, Saavedra, Jacob, Del Brutto, Wilkins, Gonzalez and Gilman2018) tested human serum samples with viable (n = 45) and calcified (n = 45) NCC, as well as sera from patients with other cestodes (hymenolepiasis, n = 45 and hydatidosis, n = 45) and evaluated the detection of anti-T. solium IgG in two commercial ELISA kits. Low sensitivity values were detected for both clinical forms of NCC (viable and calcified), 44.4 and 22.2% for viable, and 6.7 and 4.5% for calcified form. Furthermore, high levels of cross-reactivity with hydatidosis (84.4 and 55.6%) and hymenolepiasis (11.1 and 2.2%) were observed. These results highlight the efficiency of specific IgY antibodies produced from T. crassiceps hydrophobic antigen for NCC immunodiagnosis compared to human IgG.
Machado et al. (Reference Machado, Santiago, Mineo and Costa-Cruz2007) tested human serum samples from NCC-positive, healthy and other parasitic diseases patients, and obtained similar diagnostic values, showing 92.5% (Se) and 93.3% (Sp), using T. solium hydrophobic antigen. Among the samples tested in the group with other parasitic diseases, patients infected with E. granulosus showed 70% (7/10) reactivity. In this study, only one sample with E. granulosus reacted (1/5), attesting the efficiency of anti-hFTc IgY antibodies. Gonçalves et al. (Reference Gonçalves, Machado, Oliveira, Rezende, Mineo and Costa-Cruz2010), tested human serum samples with anti-T. solium IgG against T. saginata hydrophobic antigen, and obtained 95% (Se) and 82.6% (Sp), showing cross-reactivity with the other parasitic diseases group (n = 45) in all samples with E. granulosus (6/45) and three with H. nana (4/45). In this study, only one sample with E. granulosus and one with H. nana reacted. These results denote the efficiency and specificity of anti-hFTc IgY antibodies compared to human IgG against T. solium and T. saginata hydrophobic antigens.
The application of specific anti-hFTc IgY antibodies in human NCC diagnosis showed similar diagnostic performance for immune complexes detection in human serum samples when compared with specific IgY antibodies produced against S. venezuelensis (Faria et al., Reference Faria, de Souza, Ribeiro, Sousa, Borges, Ávila, Ferreira-Júnior, Goulart and Costa-Cruz2019). On the other hand, higher diagnostic performances are shown when compared to specific IgY antibodies produced against A. suum (80% Se; 90% Sp) (Lopes et al., Reference Lopes, de Faria, de Sousa, Borges, Ribeiro, Bueno, Rodrigues Ávila, Ferreira-Júnior and Costa-Cruz2019).
In conclusion, our results demonstrated that specific IgY antibodies produced from immunization of hens with T. crassiceps hydrophobic fraction were capable to recognize antigens from parasite tissue and CIC. Furthermore, the first application of specific anti-hFTc IgY antibodies in this study presented to be a promising and innovative tool, with low-cost and high efficiency in human NCC diagnosis.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182019001446
Acknowledgements
We thank the professor Dr Jair Pereira da Cunha Júnior for the supply of centrifugal filters used in the purification process of IgY antibodies.
Financial support
This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – 303843/2015-2 and CNPq – 404816/2016-9) (JMCC) and (CNPq – 131474/2017-0) (GCMP).
Conflict of interest
None.
Ethical standards
The authors assert that all procedures contributing to this study comply with the Comitê de Ética em Pesquisas com Seres Humanos (CEP) of the Universidade Federal de Uberlândia (CAAE: 65800116.4.0000.5152/No.2088856) and Comitê de Ética na Utilização de Animais (CEUA) – Universidade Federal de Uberlândia (protocol number: 094/16).
