Kinetics and avidity of anti-Toxocara antibodies (IgG) in rabbits experimentally infected with Toxocara canis

Cinética e avidez de anticorpos (IgG) anti-Toxocara em coelhos experimentalmente infectados com Toxocara canis

Lundia Luara Cavalcante Bin Vamilton Alvares Santarém Cecília Braga Laposy Guita Rubinsky-Elefant William Henry Roldán Rogério Giuffrida About the authors

Abstract

An evaluation was made of the kinetics and avidity of anti-Toxocara antibodies (IgG) in rabbits experimentally infected with embryonated Toxocara canis eggs. Seventeen four month old New Zealand White rabbits were distributed into two groups. In the experimental group, twelve rabbits were infected orally with 1,000 embryonated T. canis eggs. A second group (n = 5), uninfected, was used as a control. Serum samples were collected for analysis on days 7, 14, 21, 28 and 60 post-infection (DPI). An indirect ELISA test was performed to evaluate the reactivity index (RI) of IgG anti-T. canis antibodies and to calculate the avidity index (AI). The animals showed seroconversion from the 14th DPI, with high AI (over 50%) except for one animal, which presented an intermediate AI. At 60 DPI, all the animals were seropositive and maintained a high AI. The data indicated that specific IgG antibodies formed early (14 DPI) in rabbits infected with T. canis, with a high avidity index that persisted throughout the course of the infection.

Keywords:
Toxocariasis; antibody avidity; immune response; ELISA

Resumo

O objetivo deste estudo foi o de avaliar a cinética e a avidez de anticorpos anti-Toxocara canis, em coelhas infectadas experimentalmente com ovos embrionados de Toxocara canis. Foram utilizados 17 coelhos New Zealand de linhagem branca, com quatro meses de idade, distribuídos em dois grupos. No grupo experimental, doze coelhas foram infectadas, oralmente, com 1.000 ovos larvados de T. canis. Um segundo grupo (n=5), não infectado, foi utilizado como controle. Nos dias 7, 14, 21, 28 e 60 pós-infecção (DPI), foram coletadas amostras de soro para análise. O teste de ELISA indireto foi realizado para avaliar o índice de reatividade (IR) de anticorpos IgG anti-T. canis e para cálculo do índice de avidez (IA). A soroconversão nos animais ocorreu a partir do140 DPI, com verificação de alto IA (superior a 50%), com exceção de um animal, que apresentou médio IA. Aos 60 DPI, todos os animais foram soropositivos e mantiveram alto IA. Os dados mostram que em coelhos infectados por T. canis, anticorpos IgG específicos formam-se precocemente (14 DPI), apresentando alto índice de avidez e que se mantém durante o curso da infecção.

Palavras-chave:
Toxocaríase; avidez de anticorpos; resposta imune; ELISA

Introduction

Toxocariasis, although little recognized as a public health problem, is considered an important worldwide zoonosis (RUBINSKY-ELEFANT et al., 2010Rubinsky-Elefant G, Hirata CE, Yamamoto JH, Ferreira MU. Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. Ann Trop Med Parasitol 2010; 104(1): 3-23. http://dx.doi.org/10.1179/136485910X12607012373957. PMid:20149289.
http://dx.doi.org/10.1179/136485910X1260...
). In humans, the principal route of transmission is by ingestion of embryonated Toxocara canis or T. cati eggs, which are parasites of dogs and cats, respectively (DESPOMMIER, 2003Despommier D. Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clin Microbiol Rev 2003; 16(2): 265-272. http://dx.doi.org/10.1128/CMR.16.2.265-272.2003. PMid:12692098.
http://dx.doi.org/10.1128/CMR.16.2.265-2...
).

Humans act as paratenic hosts of Toxocara spp. After hatching in the intestine, the larvae migrate through the organism and can cause hemorrhage, necrosis and eosinophilic inflammation, or be encapsulated in granulomas, either being destroyed or remaining viable for many years (MAGNAVAL et al., 2001Magnaval JF, Glickman LT, Dorchies P, Morassin B. Highlights of human toxocariasis. Korean J Parasitol 2001; 39(1): 1-11. http://dx.doi.org/10.3347/kjp.2001.39.1.1. PMid:11301585.
http://dx.doi.org/10.3347/kjp.2001.39.1....
).

Human toxocariasis is diagnosed based on clinical, epidemiological and laboratory data, primarily using the ELISA test, whose sensitivity ranges from 78.3% (GLICKMAN et al., 1978Glickman L, Schantz P, Dombroske R, Cypess R. Evaluation of serodiagnostic tests for visceral larva migrans. Am J Trop Med Hyg 1978; 27(3): 492-498. PMid:98065.) to 100% (RUBINSKY-ELEFANT et al., 2006Rubinsky-Elefant G, Shimizu SH, Sanchez MCA, Jacob CMA, Ferreira AW. A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay. J Clin Lab Anal 2006; 20(4): 164-172. http://dx.doi.org/10.1002/jcla.20126. PMid:16874812.
http://dx.doi.org/10.1002/jcla.20126...
; ROLDÁN et al., 2006Roldán W, Cornejo W, Espinoza Y. Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis. Mem Inst Oswaldo Cruz 2006; 101(1): 71-74. http://dx.doi.org/10.1590/S0074-02762006000100013. PMid:16612511.
http://dx.doi.org/10.1590/S0074-02762006...
), and specificity from 86% (JACQUIER et al., 1991Jacquier P, Gottstein B, Stingelin Y, Eckert J. Immunodiagnosis of toxocarosis in humans: evaluation of a new enzyme-linked immunosorbent assay kit. J Clin Microbiol 1991; 29(9): 1831-1835. PMid:1774303.) to 97.4% (SAVIGNY et al., 1979Savigny DH, Voller A, Woodruff AW. Toxocariasis: serological diagnosis by enzyme immunoassay. J Clin Pathol 1979; 32(3): 284-288. http://dx.doi.org/10.1136/jcp.32.3.284. PMid:372253.
http://dx.doi.org/10.1136/jcp.32.3.284...
), for the detection of IgG anti-Toxocara immunoglobulins. In clinical studies on humans, Hubner et al. (2001)Hübner J, Uhlíková M, Leissová M. Diagnosis of the early phase of larval toxocariasis using IgG avidity. Epidemiol Mikrobiol Imunol 2001; 50(2): 67-70. PMid:11329729. and Dziemian et al. (2008)Dziemian E, Zarnowska H, Kołodziej-Sobocińska M, Machnicka B. Determination of the relative avidity of the specific IgG antibodies in human toxocariasis. Parasite Immunol 2008; 30(3): 187-190. http://dx.doi.org/10.1111/j.1365-3024.2007.01010.x. PMid:18179629.
http://dx.doi.org/10.1111/j.1365-3024.20...
reported that, in chronic cases of the disease, there is high IgG avidity, while low avidity is noted in acute infections. However, the persistence of the antibodies over a long period, due to antigenic stimulation caused by the maintenance of the Toxocara spp. larvae in the tissues of paratenic hosts, can make it difficult to classify the infection as acute or chronic (FENOY et al., 1992Fenoy S, Cuéllar C, Aguila C, Guillén JL. Persistence of immune response in human toxocariasis as measured by ELISA. Int J Parasitol 1992; 22(7): 1037-1038. http://dx.doi.org/10.1016/0020-7519(92)90067-U. PMid:1459782.
http://dx.doi.org/10.1016/0020-7519(92)9...
; RUBINSKY-ELEFANT et al., 2006Rubinsky-Elefant G, Shimizu SH, Sanchez MCA, Jacob CMA, Ferreira AW. A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay. J Clin Lab Anal 2006; 20(4): 164-172. http://dx.doi.org/10.1002/jcla.20126. PMid:16874812.
http://dx.doi.org/10.1002/jcla.20126...
).

The literature on the avidity of anti-Toxocara antibodies in hosts is scanty and limited to mice (FENOY et al., 2008Fenoy S, Rodero M, Pons E, Aguila C, Cuéllar C. Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis.Parasitology 2008; 135(6): 725-733. http://dx.doi.org/10.1017/S0031182008004368. PMid:18413003.
http://dx.doi.org/10.1017/S0031182008004...
; SCHOENARDIE et al., 2014Schoenardie ER, Scaini CJ, Avila LF, Sperotto RL, Borsuk S, Felicetti CD, et al. Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis.Rev Bras Parasitol Vet 2014; 23(3): 403-406. http://dx.doi.org/10.1590/S1984-29612014060. PMid:25271464.
http://dx.doi.org/10.1590/S1984-29612014...
). Therefore, studying the avidity of anti-Toxocara spp. antibodies in other animal models may help shed light on the profile of the humoral immune response to toxocariasis.

This study involved an investigation into the kinetics and avidity of anti-Toxocara canis antibodies in experimentally infected rabbits.

Materials and Methods

Animals

Seventeen 4-month old New Zealand White rabbits from the UNOESTE vivarium were used. To confirm the negative diagnosis of enteroparasites, three fecal samples collected in three consecutive weeks prior to infection were analyzed using the Willis-Mollay and Hoffman techniques (HOFFMANN, 1987Hoffmann RP. Diagnóstico de parasitismo veterinário. Porto Alegre: Sulina; 1987.). The project was approved by the Animal Research Ethics Committee of the University of Western São Paulo – UNOESTE in Presidente Prudente, SP, Brazil (under Protocol 132/09).

Experimental groups

The rabbits in this study were divided into two groups. The first group consisted of 12 females artificially infected with 1000 T. canis eggs, and the second comprised five uninfected females, which served as control.

Throughout the experimental period, the animals received commercial rabbit feed pellets and water ad libitum and were housed individually in suspended galvanized wire mesh cages in a biotery.

Obtaining Toxocara canis eggs

T. canis eggs were obtained from adult females which were released from naturally infected puppies kept in the kennel at UNOESTE.

To obtain the eggs, the anterior third of the uteruses of the female nematodes were dissected (FAN et al., 2003Fan CK, Lin YH, Du WY, Su KE. Infectivity and pathogenicity of 14-month-cultured embryonated eggs of in mice. Toxocara canisVet Parasitol 2003; 113(2): 145-155. http://dx.doi.org/10.1016/S0304-4017(03)00046-3. PMid:12695039.
http://dx.doi.org/10.1016/S0304-4017(03)...
) and stored for five weeks at 27 °C for embryonation. After this period, the material was washed three times in saline solution by centrifugation (922 x g) for three minutes. A total of 1000 embryonated eggs were counted in a Neubauer chamber, and diluted in 3.0 mL of phosphate buffered saline (PBS) solution for experimental infection.

Infection of the animals

Prior to experimental infection, the animals were sedated by intraperitonial administration of 30mg/kg of combined tiletamine/zolazepam (Zoletil 5%, Virbac), diluted in saline (KANASHIRO & CASSU, 2008Kanashiro GP, Cassú RN. Anestesia de animais selvagens e de laboratório. In: Andrade SF. Manual de terapêutica veterinária. 3rd ed. Roca: São Paulo; 2008.).

The animals were infected using the procedure described by Pecinali et al. (2005)Pecinali NR, Gomes RN, Amendoeira FC, Bastos AC, Martins MJ, Pegado CS, et al. Influence of murine infection on plasma and bronchoalveolar lavage fluid eosinophil numbers and its correlation with cytokine levels. Toxocara canisVet Parasitol 2005; 134(1-2): 121-130. http://dx.doi.org/10.1016/j.vetpar.2005.06.022. PMid:16168564.
http://dx.doi.org/10.1016/j.vetpar.2005....
, with minor modifications. The animals were orally inoculated with 3.0 mL of the solution containing embryonated eggs, using a gastric gavage. Another 3.0 mL of PBS was administered to the animals via the same route to ensure the passage of the eggs. The control group received only PBS, following the same procedures for the administration of the anesthetic and inoculum.

To obtain the serum, blood samples were collected through puncture of the central auricular artery at the following time points; 0 (pre-infection), 7, 14, 21, 28 and 60 days post-infection (DPI).

Excretory-secretory T. canis antigen (TES) production

The T. canis excretory-secretory larval antigen (TES) for ELISA was prepared as described previously (SAVIGNY, 1975Savigny DH. maintenance of larvae and a simple method for the production of . In vitroToxocara canisToxocara ES antigens for use in serodiagnostic tests for visceral larva migransJ Parasitol 1975; 61(4): 781-782. http://dx.doi.org/10.2307/3279492. PMid:1165568.
http://dx.doi.org/10.2307/3279492...
), with some modifications (RUBINSKY-ELEFANT et al., 2006Rubinsky-Elefant G, Shimizu SH, Sanchez MCA, Jacob CMA, Ferreira AW. A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay. J Clin Lab Anal 2006; 20(4): 164-172. http://dx.doi.org/10.1002/jcla.20126. PMid:16874812.
http://dx.doi.org/10.1002/jcla.20126...
). Briefly, T. canis eggs collected from the uterus of adult female worms were embryonated after incubation in 2% formalin for approximately 1 month at 28 °C. Following, the material was washed by centrifugation (3 minutes at 1,200 g) in 0.9% NaCl until the formalin solution was completely removed. Eggshells were broken by slow homogenisation in an Erlenmeyer flask, and L3 larvae were collected by using a Baermann apparatus in serum-free Eagle’s medium. The supernatant of the larvae culture was weekly collected, and the protease inhibitor phenylmethylsulphonyl fluoride (PMSF, Sigma, 0.2 M, 5.0 µL/mL) added. The collected supernatant was concentrated in Amicon Ultrafiltration units (Millipore, Danvers, MA), dialysed against distilled water, centrifuged (15,000 g for 30 minutes at 4 °C), and filtered using 0.22 µm Millipore membranes. The Lowry method (LOWRY et al., 1951Lowry A, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1): 265-275. PMid:14907713.) was employed to determine the protein content.

ELISA (enzyme-linked immunosorbent assay)

The ELISA test was performed to evaluate the production of anti-T. canis antibodies, using the protocol described by Savigny et al. (1979)Savigny DH, Voller A, Woodruff AW. Toxocariasis: serological diagnosis by enzyme immunoassay. J Clin Pathol 1979; 32(3): 284-288. http://dx.doi.org/10.1136/jcp.32.3.284. PMid:372253.
http://dx.doi.org/10.1136/jcp.32.3.284...
with modifications (RUBINSKY-ELEFANT et al., 2006Rubinsky-Elefant G, Shimizu SH, Sanchez MCA, Jacob CMA, Ferreira AW. A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay. J Clin Lab Anal 2006; 20(4): 164-172. http://dx.doi.org/10.1002/jcla.20126. PMid:16874812.
http://dx.doi.org/10.1002/jcla.20126...
).

To perform the test, 96-well polystyrene microplates (Corning, Costar, New York, NY) were coated (1.9 µg/mL antigen/well) with excretion-secretion antigens produced by the T. canis (L3) larvae (TES) and tested for 2 hours at 37 °C, followed by 18 hours at 4 °C. The microplates were then washed three times for 5 minutes with phosphate buffered saline (PBS) 0.01M, pH 7.2, containing 0.05% Tween-20 (PBS-T). The microplates were blocked with 2.5% skimmed milk (Molico, Nestlé®) in PBS-T (200 µL/well) for 1 hour at 37 °C and then washed three times with PBS-T.

The serum samples (100 µL/well) were diluted at 1:200 and incubated for 40 minutes at 37 °C, in duplicate. After three washing cycles, the plates were incubated with conjugated goat anti-rabbit IgG (100 µL/well; Sigma-Aldrich A0545, USA) at a dilution of 1:40,000 in PBS-T for 40 minutes at 37 °C. After another round of washing, the plates were incubated with chromogenic solution (100µL/well; OPD Fast-Sigma, St Louis, EUA), comprising ortho-phenylenediamine (0.4 mg/mL) and H2O2-urea (0.4 mg/mL) in 0.05M of phosphate-citrate buffer for 15 minutes at 37 °C. The reaction was stopped with H2SO4 2N (50 µL/well), and the optical densities (OD) were measured at 492 nm (Titertek Multiskan MCC/340, Lab-System, Finland).

The cut-off point was calculated from the mean optical densities of sera from 17 uninfected animals (5 animals in the control group and 12 pre-infection animals) plus two standard deviations. The antibody levels were expressed as reactivity indices (RIs), calculated as the ratio between the absorbance value of the optical density of each tested sample and the optical density at the cut-off point (0.292). The samples with RIs above 1 were considered positive.

Avidity of antibodies (IgG)

The dissociative method was used to study avidity, with urea as the denaturing agent (HEDMAN et al., 1989Hedman K, Lappalainen M, Seppäiä I, Mäkelä O. Recent primary infection indicated by a low avidity of specific IgG. ToxoplasmaJ Infect Dis 1989; 159(4): 736-740. http://dx.doi.org/10.1093/infdis/159.4.736. PMid:2926163.
http://dx.doi.org/10.1093/infdis/159.4.7...
). To evaluate the possibility of the denaturing agent acting on the absorbed antigen in the solid phase and interfering in the results, the sensitized and blocked ELISA plate was first treated for 5 minutes with 8M urea prior to performing the assay. To determine the avidity of antibodies, serum samples and conjugate dilutions were the same for ELISA assay, according to the previous description, except that sera were applied in duplicate to the microtiter wells. After incubation for 40 min at 37 °C, one well of the doublet was washed with PBS-T three times for 5 min; likewise, the other well was washed with 8M urea dissolved in PBS-T. The avidity index (AI) was calculated as the ratio between the OD sample treated with urea and the OD sample not treated with urea, multiplied by 100, according to the formula:

AI ( % ) = ( OD with urea/OD without urea ) × 100 (1)

Only the samples with RIs above 1 were considered for AI calculation. The avidity indices were established according to Hedman & Rousseau (1989)Hedman K, Rousseau SA. Measurement of avidity of specific IgG for verification of recent primary rubella. J Med Virol 1989; 27(4): 288-292. http://dx.doi.org/10.1002/jmv.1890270406. PMid:2723615.
http://dx.doi.org/10.1002/jmv.1890270406...
, where: AI lower than 30% indicates predominance of low-avidity antibodies; AI between 30 and 50% indicates intermediate-avidity antibodies; and AI above 50% indicates high-avidity antibodies.

Statistical analysis

The mean values of the avidity and reactivity indices were calculated from the data obtained. The AI and RI of the sera of the females were compared using the paired t-test, while the correlation between the two indices was evaluated via a simple linear regression analysis. The adequacy of the model was tested using residual analysis and adjustment of statistics, with a coefficient of determination (R2). The significance level used in all the comparisons was 5%. All the analyses were performed with the aid of SPSS v. 13.0 software for Windows (FIELD, 2009Field AP. Discovering Statistics Using SPSS. 3rd ed. London: Sage Publications; 2009.).

Results

Seroconversion of the infected animals was observed on the 14th DPI in 41.7%, followed by a gradual increase in RI. Positive RI frequencies of 83.3%; 91.7% and 100% were observed on the 21st, 28th and 60th DPI, respectively.

Only one animal demonstrated seroconversion on the 60th DPI, with a slightly lower RI than that of the other animals (RI = 4.457).

The average RIs were 1.064, 2.264, 3.081 and 4.895 on the 14th, 21st, 28th and 60th DPI, respectively (Figure 1). The average RIs on the 28th and 60th DPI presented a statistically significant difference (p = 0.0019).

Figure 1
Avidity index (AI; %) and Reactivity index (RI) for antibodies (IgG) detected by enzyme linked immunosorbent assay (ELISA) in rabbits experimentally infected with Toxocara canis at different post-infection time points (DPI).

Figure 2 shows the classification of the Avidity Index (AI) of the animals. All the animals that seroconverted had high-avidity antibodies, except for one animal which presented intermediate-avidity antibodies (IA = 46.6%) on the 14th DPI.

Figure 2
Avidity index (%) of IgG anti-Toxocara canis in experimentally infected rabbits, detected by enzyme linked immunosorbent assay (ELISA) at different post-infection time points (DPI). Classification of AI: less than 30: predominance of low avidity; higher than or equal to 30 and less than 50: predominance of intermediate avidity; equal to or higher than 50: predominance of high avidity.

The AI ranged from 46.6% to 86.6% (mean AI = 65.1%) at 14 DPI, 51.0 to 92.6% (mean AI = 67.5%) at 21 DPI, and 55.4% to 90.9% (mean AI = 74.1%) at 28 DPI. At 60 DPI, the AI ranged from 95.7% to 99.9% (mean AI = 97.4%).

The correlation between the RI and AI was evaluated 21, 28 and 60 DPI, since the RIs on the 7th and 14th DPI were inconsistent for calculation. A statistically significant correlation (R= 0.8765; p=0.0096) was observed at 60 DPI (Table 1).

Table 1
Correlation coefficient between reactivity and avidity indices for detection of IgG antibodies by enzyme linked immunosorbent assay (ELISA) in rabbits experimentally infected with Toxocara canis at different time points after infection.

Discussion

The novelty of this study was to evaluate the kinetics and avidity of anti-Toxocara antibodies (IgG) in rabbits experimentally infected with a single dose of embryonated T. canis eggs.

Seroconversion was observed from the 14th DPI, followed by a gradual increase in positive animals over the infection time. Similarly, Morales et al. (2002)Morales OL, Lopez MC, Nicholls RS, Agudelo C. Identification of antigens by Western blot in experimentally infected rabbits. Toxocara canisRev Inst Med Trop Sao Paulo 2002; 44(4): 213-216. http://dx.doi.org/10.1590/S0036-46652002000400006. PMid:12219113.
http://dx.doi.org/10.1590/S0036-46652002...
verified the same response pattern using Western blot for detecting anti-Toxocara antibodies in rabbits (the same strain and age). Variations in seroconversion time have been observed in paratenic hosts experimentally infected with Toxocara spp. In pigs, anti-Toxocara antibodies were observed at 7 (SOMMERFELT et al., 2001Sommerfelt IE, Santillán G, Lopez C, Ribicich M, Franco AJ. Immunological and hematological response in experimental infected pigs. Toxocara canis-Vet Parasitol 2001; 96(2): 127-134. http://dx.doi.org/10.1016/S0304-4017(00)00423-4. PMid:11230919.
http://dx.doi.org/10.1016/S0304-4017(00)...
) and 14 DPI (TAIRA et al., 2003Taira K, Permin A, Kapel CM. Establishment and migration pattern of Toxocara canis larvae in chickens. Parasitol Res 2003; 90(6): 521-523. http://dx.doi.org/10.1007/s00436-003-0894-6. PMid:12827506.
http://dx.doi.org/10.1007/s00436-003-089...
) and in sheep at 21 DPI (SANTARÉM et al., 2011Santarém VA, Chesine PAF, Lamers BEL, Rubinsky-Elefant G, Giuffrida R. Anti- spp. antibodies in sheep from southeastern Brazil. ToxocaraVet Parasitol 2011; 179(1-3): 283-286. http://dx.doi.org/10.1016/j.vetpar.2011.01.050. PMid:21330057.
http://dx.doi.org/10.1016/j.vetpar.2011....
). This phenomenon has also been observed in murines, which are considered the experimental model for human toxocariasis. In mice, seroconversion was observed at 7 (OLLERO et al., 2008Ollero MD, Fenoy S, Cuéllar C, Guillén JL, Del Aguila C. Experimental toxocariosis in BALB/c mice: effect of the inoculation dose on brain and eye involvement. Acta Trop 2008; 105(2): 124-130. http://dx.doi.org/10.1016/j.actatropica.2007.11.001. PMid:18093569.
http://dx.doi.org/10.1016/j.actatropica....
), 14 (KOLBEKOVÁ et al., 2011Kolbeková P, Větvička D, Svoboda J, Skirnisson K, Leissová M, Syrůček M, et al. Toxocara canis larvae reinfecting BALB/c mice exhibit accelerated speed of migration to the host CNS. Parasitol Res 2011; 109(5): 1267-1278. http://dx.doi.org/10.1007/s00436-011-2371-y. PMid:21537988.
http://dx.doi.org/10.1007/s00436-011-237...
) and 23 DPI (LESCANO et al., 2012Lescano SAZ, Nakhle MC, Ribeiro MCSA, Chieffi PP. IgG antibody responses in mice coinfected with and other helminths or protozoan parasites. Toxocara canisRev Inst Med Trop Sao Paulo 2012; 54(3): 145-152. http://dx.doi.org/10.1590/S0036-46652012000300006. PMid:22634886.
http://dx.doi.org/10.1590/S0036-46652012...
).

There are a number of factors that may determine the pattern of immune response and pathogenesis in paratenic hosts, including individual characteristics (CUELLAR et al., 2001Cuéllar C, Fenoy S, Águila C, Guillén JL. Isotype specific immune responses in murine experimental toxocariasis. Mem Inst Oswaldo Cruz 2001; 96(4): 549-553. http://dx.doi.org/10.1590/S0074-02762001000400018. PMid:11391430.
http://dx.doi.org/10.1590/S0074-02762001...
), the infective dose and number of reinfections (KAYES et al., 1985Kayes SG, Omholt PE, Grieve RB. Immune responses of CBA/J mice to graded infections with Toxocara canis.Infect Immun 1985; 48(3): 697-703. PMid:3997242.; OLLERO et al., 2008Ollero MD, Fenoy S, Cuéllar C, Guillén JL, Del Aguila C. Experimental toxocariosis in BALB/c mice: effect of the inoculation dose on brain and eye involvement. Acta Trop 2008; 105(2): 124-130. http://dx.doi.org/10.1016/j.actatropica.2007.11.001. PMid:18093569.
http://dx.doi.org/10.1016/j.actatropica....
). Our findings reinforce the statement that individuality may explain a difference in responsiveness to antigens by each individual animal, since some of the rabbits (41.7%) seroconverted in the early days of infection (14 DPI), followed by observation of seroconversion at different time points, including one registered at 60 DPI.

Experimental studies have shown the influence of infective dose on the immune response to toxocariasis. In pigs experimentally infected with doses of 1000 or 2000 eggs, significant differences were observed in titer values related to dose, with higher titers for the group dosed with 2000 eggs during the period between 2 and 7 weeks post-infection (SOMMERFELT et al., 2001Sommerfelt IE, Santillán G, Lopez C, Ribicich M, Franco AJ. Immunological and hematological response in experimental infected pigs. Toxocara canis-Vet Parasitol 2001; 96(2): 127-134. http://dx.doi.org/10.1016/S0304-4017(00)00423-4. PMid:11230919.
http://dx.doi.org/10.1016/S0304-4017(00)...
). Havasiová-Reiterová et al. (1995)Havasiová-Reiterová K, Tomasovicová O, Dubinský P. Effect of various doses of infective Toxocara canis and Toxocara cati eggs on the humoral response and distribution of larvae in mice. Parasitol Res 1995; 81(1): 13-17. http://dx.doi.org/10.1007/BF00932411. PMid:7724509.
http://dx.doi.org/10.1007/BF00932411...
evaluated the effects of 5 to 2,500 infective Toxocara canis eggs, observing a faster production of antibodies in mice infected with a higher dose of eggs. In our study, the rabbits were infected with a single dose of 1000 eggs, and interestingly, the observed response pattern of the animals was very similar to that described by Morales et al. (2002)Morales OL, Lopez MC, Nicholls RS, Agudelo C. Identification of antigens by Western blot in experimentally infected rabbits. Toxocara canisRev Inst Med Trop Sao Paulo 2002; 44(4): 213-216. http://dx.doi.org/10.1590/S0036-46652002000400006. PMid:12219113.
http://dx.doi.org/10.1590/S0036-46652002...
, using an infective dose of 5000 eggs.

Early antibody response in experimentally infected paratenic hosts may be detected by ELISA (SOMMERFELT et al., 2001Sommerfelt IE, Santillán G, Lopez C, Ribicich M, Franco AJ. Immunological and hematological response in experimental infected pigs. Toxocara canis-Vet Parasitol 2001; 96(2): 127-134. http://dx.doi.org/10.1016/S0304-4017(00)00423-4. PMid:11230919.
http://dx.doi.org/10.1016/S0304-4017(00)...
), however the detection of specific IgG antibodies, does not allow differentiation between acute and chronic phases of toxocariasis (RUBINSKY-ELEFANT et al., 2010Rubinsky-Elefant G, Hirata CE, Yamamoto JH, Ferreira MU. Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. Ann Trop Med Parasitol 2010; 104(1): 3-23. http://dx.doi.org/10.1179/136485910X12607012373957. PMid:20149289.
http://dx.doi.org/10.1179/136485910X1260...
). The use of antibody avidity measurements has been widely employed to distinguish the acute and chronic phases of several parasitic diseases that affect humans, such as schistosomiasis (MOSTAFA et al., 2002Mostafa NE, Awad A, Shalaby M. Low avidity IgG antibodies in diagnosis of recent human schistosomiasis. J Egypt Soc Parasitol 2002; 32(3): 979-985. PMid:12512829.), fascioliasis (ABOU-BASHA et al., 2000Abou-Basha LM, Shehab AY, Osman MM, Farag HF. Specific IgG avidity in acute and chronic human fascioliasis. East Mediterr Health J 2000; 6(5-6): 919-925. PMid:12197349.), neurocysticercosis (MANHANI et al., 2009Manhani MN, Ribeiro VS, Silva DAO, Costa-Cruz JM. Specific IgG avidity in active and inactive human neurocysticercosis. Diagn Microbiol Infect Dis 2009; 65(2): 211-213. http://dx.doi.org/10.1016/j.diagmicrobio.2009.05.018. PMid:19748437.
http://dx.doi.org/10.1016/j.diagmicrobio...
), toxoplasmosis (CANDOLFI et al., 2007Candolfi E, Pastor R, Huber R, Filisetti D, Villard O. IgG avidity assay firms up the diagnosis of acute toxoplasmosis on the first serum sample in immunocompetent pregnant women. Diagn Microbiol Infect Dis 2007; 58(1): 83-88. http://dx.doi.org/10.1016/j.diagmicrobio.
http://dx.doi.org/10.1016/j.diagmicrobio...
; GAY-ANDRIEU et al., 2009Gay-Andrieu F, Fricker-Hidalgo H, Sickinger E, Espern A, Brenier-Pinchart MP, Braun H-B, et al. Comparative evaluation of the ARCHITECT Toxo IgG, IgM, and IgG Avidity assays for anti- antibodies detection in pregnant women sera. ToxoplasmaDiagn Microbiol Infect Dis 2009; 65(3): 279-287. http://dx.doi.org/10.1016/j.diagmicrobio.2009.07.013. PMid:19822270.
http://dx.doi.org/10.1016/j.diagmicrobio...
) and toxocariasis (HUBNER et al. 2001Hübner J, Uhlíková M, Leissová M. Diagnosis of the early phase of larval toxocariasis using IgG avidity. Epidemiol Mikrobiol Imunol 2001; 50(2): 67-70. PMid:11329729.; DZIEMIAN et al., 2008Dziemian E, Zarnowska H, Kołodziej-Sobocińska M, Machnicka B. Determination of the relative avidity of the specific IgG antibodies in human toxocariasis. Parasite Immunol 2008; 30(3): 187-190. http://dx.doi.org/10.1111/j.1365-3024.2007.01010.x. PMid:18179629.
http://dx.doi.org/10.1111/j.1365-3024.20...
).

Regarding human toxocariasis, the chronic phase is characterized by high IgG avidity, while low avidity is observed in acute infections (HUBNER et al., 2001Hübner J, Uhlíková M, Leissová M. Diagnosis of the early phase of larval toxocariasis using IgG avidity. Epidemiol Mikrobiol Imunol 2001; 50(2): 67-70. PMid:11329729.; DZIEMIAN et al., 2008Dziemian E, Zarnowska H, Kołodziej-Sobocińska M, Machnicka B. Determination of the relative avidity of the specific IgG antibodies in human toxocariasis. Parasite Immunol 2008; 30(3): 187-190. http://dx.doi.org/10.1111/j.1365-3024.2007.01010.x. PMid:18179629.
http://dx.doi.org/10.1111/j.1365-3024.20...
). Some authors, however, find it difficult to classify the infection as acute or chronic, due to the persistence of antibodies over a long period as a consequence of the antigenic stimulation caused by Toxocara spp. larvae (GLICKMAN et al., 1986Glickman LT, Schantz PM, Grieve RB. Toxocariasis. In: Walls KW, Schantz PM, editors. Helmintic diseases. New York: Academic Press; 1986. vol. 1. Immunodiagnosis of Parasitic Diseases.; FENOY et al., 1992Fenoy S, Cuéllar C, Aguila C, Guillén JL. Persistence of immune response in human toxocariasis as measured by ELISA. Int J Parasitol 1992; 22(7): 1037-1038. http://dx.doi.org/10.1016/0020-7519(92)90067-U. PMid:1459782.
http://dx.doi.org/10.1016/0020-7519(92)9...
; RUBINSKY-ELEFANT et al., 2006Rubinsky-Elefant G, Shimizu SH, Sanchez MCA, Jacob CMA, Ferreira AW. A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay. J Clin Lab Anal 2006; 20(4): 164-172. http://dx.doi.org/10.1002/jcla.20126. PMid:16874812.
http://dx.doi.org/10.1002/jcla.20126...
). Therefore, experimental studies, particularly in murines, have been carried out to evaluate whether antibody avidity is a useful model for identification of acute and chronic toxocariasis.

In mice infected with a single dose or with multiple infections of T. canis eggs, a high AI (about 50%) was observed 40 to 60 DPI (FENOY et al., 2008Fenoy S, Rodero M, Pons E, Aguila C, Cuéllar C. Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis.Parasitology 2008; 135(6): 725-733. http://dx.doi.org/10.1017/S0031182008004368. PMid:18413003.
http://dx.doi.org/10.1017/S0031182008004...
). In BALB/c mice infected with a single dose of 1000 eggs, a percentage drop (low avidity: 7.25 to 27.5%) was verified at 15 DPI, an intermediate avidity for IgG up to 45 DPI (27.5 to 31.4%), and IgG AI between 31.4 and 58% at 60 DPI (SCHOENARDIE et al., 2014Schoenardie ER, Scaini CJ, Avila LF, Sperotto RL, Borsuk S, Felicetti CD, et al. Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis.Rev Bras Parasitol Vet 2014; 23(3): 403-406. http://dx.doi.org/10.1590/S1984-29612014060. PMid:25271464.
http://dx.doi.org/10.1590/S1984-29612014...
). In our study, however, high avidity IgG was detected early after infection (mean AI = 65.1%) and gradually increased for at least 2 months (mean AI = 97.4%), resulting in a strong association between the Avidity and Reactivity Indices at 60 DPI.

The present study has some limitations. Several experimental studies have evaluated the subclasses of immunoglobulins involved in toxocariasis (KAYES et al., 1985Kayes SG, Omholt PE, Grieve RB. Immune responses of CBA/J mice to graded infections with Toxocara canis.Infect Immun 1985; 48(3): 697-703. PMid:3997242.; OLLERO et al., 2008Ollero MD, Fenoy S, Cuéllar C, Guillén JL, Del Aguila C. Experimental toxocariosis in BALB/c mice: effect of the inoculation dose on brain and eye involvement. Acta Trop 2008; 105(2): 124-130. http://dx.doi.org/10.1016/j.actatropica.2007.11.001. PMid:18093569.
http://dx.doi.org/10.1016/j.actatropica....
). We used the indirect ELISA based on the detection of total IgG anti-Toxocara, which is currently the standard serological method for the diagnosis of human toxocariasis (RUBINSKY-ELEFANT et al., 2010Rubinsky-Elefant G, Hirata CE, Yamamoto JH, Ferreira MU. Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. Ann Trop Med Parasitol 2010; 104(1): 3-23. http://dx.doi.org/10.1179/136485910X12607012373957. PMid:20149289.
http://dx.doi.org/10.1179/136485910X1260...
; MACPHERSON, 2013Macpherson CN. The epidemiology and public health importance of toxocariasis: a zoonosis of global importance. Int J Parasitol 2013; 43(12-13): 999-1008. http://dx.doi.org/10.1016/j.ijpara.2013.07.004. PMid:23954435.
http://dx.doi.org/10.1016/j.ijpara.2013....
). In our study, we evaluated a small sample size to minimize the number of evaluated animals. In terms of challenge, the infection of the rabbits was limited to a single dose of 1000 eggs, which has been used in other studies with murine models (XI & JIN, 1998Xi WG, Jin LZ. A novel method for the recovery of in mice. Toxocara canisJ Helminthol 1998; 72(2): 183-184. http://dx.doi.org/10.1017/S0022149X00016382. PMid:9687601.
http://dx.doi.org/10.1017/S0022149X00016...
; CHO et al., 2007Cho S, Egami M, Ohnuki H, Saito Y, Chinone S, Shichinohe K, et al. Migration behaviour and pathogenesis of five ascarid nematode species in the Mongolian gerbil Meriones unguiculatus.J Helminthol 2007; 81(1): 43-47. http://dx.doi.org/10.1017/S0022149X07212118. PMid:17381866.
http://dx.doi.org/10.1017/S0022149X07212...
; SCHOENARDIE et al., 2014Schoenardie ER, Scaini CJ, Avila LF, Sperotto RL, Borsuk S, Felicetti CD, et al. Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis.Rev Bras Parasitol Vet 2014; 23(3): 403-406. http://dx.doi.org/10.1590/S1984-29612014060. PMid:25271464.
http://dx.doi.org/10.1590/S1984-29612014...
). Thus, future studies are needed, designed to evaluate different subclasses of immunoglobulins and IgG avidity in rabbits subjected to different doses of infection in a long term study, and a larger sample size, to precisely explain the wide individual variations in immune response and to demonstrate the possibility of using rabbits as an animal model for toxocariasis.

Given these considerations and limitations our data suggest that in rabbits infected with T. canis, specific IgG antibodies formed early after infection and a high avidity index was maintained at relatively high levels.

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Publication Dates

  • Publication in this collection
    10 Nov 2015
  • Date of issue
    Jan-Mar 2016

History

  • Received
    02 Mar 2015
  • Accepted
    19 June 2015
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br