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Kinetic and avidity of IgY anti-Toxocara antibodies in experimentally infected chickens
Experimental Parasitology 171 (2016) 33e41
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Kinetic and avidity of IgY anti-Toxocara antibodies in experimentally infected chickens m b, *, Ricardo da Silva Raposo a, Vamilton Alvares Santare Yslla Fernanda Fitz Balo Merigueti a, Guita Rubinsky-Elefant c, Letícia Maria de Lima Cerazo b, Ludimilla Pereira b, Bianca Pelegi Zampieri b, Aristeu Vieira da Silva d, Cecília Braga Laposy e ~o Post-Graduation Program in Animal Science, Laboratory of Veterinary Parasitology, Veterinary Teaching-Hospital (UNOESTE), Presidente Prudente, Sa Paulo, Brazil b ~o Paulo, Brazil Laboratory of Veterinary Parasitology, Veterinary Teaching-Hospital, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, Sa c ~o Paulo, University of Sa ~o Paulo, Brazil Laboratory of Seroepidemiology and Immunobiology, Instituto de Medicina Tropical de Sa d Zoonosis and Public Health Research Group, State University of Feira de Santana, UEFS, Feira de Santana, Bahia, Brazil e ~o Paulo, Laboratory of Veterinary Clinical Pathology, Veterinary Teaching-Hospital, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, Sa Brazil a
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ELISA for detection of anti-Toxocara IgY and avidity in infected chickens. Experimental infection of chickens with 100, 1000 and 5000 embryonated T. canis eggs. High levels of IgY and high avidity index in the chronic phase of infection. Influence of infective dose on the humoral immune response. ELISA is a useful tool for seroprevalence studies in poultry.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2015 Received in revised form 5 August 2016 Accepted 21 September 2016 Available online 13 October 2016
Toxocariasis is a geohelminth zoonosis with worldwide distribution, mainly transmitted through the ingestion of embryonated eggs of nematodes of the Toxocara genus. The disease can also be transmitted to humans as a result of eating raw or undercooked meat of paratenic hosts, such as chickens. Here, we standardized an enzyme-linked immunosorbent assay (ELISA) for evaluating experimentally the kinetic and avidity index (AI) of IgY in broiler chickens infected with different doses of Toxocara canis eggs (G1:100; G2: 1000; and G3: 5000; n ¼ 12 per group). The test showed 91.7% sensitivity (CI 95%: 77.5 e98.3) and 100% specificity (CI 95%: 92.6e100), and highest efficiency (97.0%) at 60 days post infection. Infection was characterized by the presence of high avidity antibodies in the chronic phase. Our results support that the ELISA can be a highly useful tool for the detection of anti-Toxocara antibodies in chickens. © 2016 Elsevier Inc. All rights reserved.
Keywords: Birds Immune response ELISA Toxocariasis
rio de Parasitologia Veterin rio da Universidade do Oeste Paulista (UNOESTE), Raposo Tavares Km 572, Bairro * Corresponding author. Laborato aria, Hospital Veterina ~o Paulo, Brazil. Limoeiro, Presidente Prudente, CEP, 19067-175 Sa m). E-mail address: [email protected] (V.A. Santare http://dx.doi.org/10.1016/j.exppara.2016.09.009 0014-4894/© 2016 Elsevier Inc. All rights reserved.
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1. Introduction Despite the technology involved in the poultry industry, in many parts of the world, particularly in developing countries, small-scale farms are the dominant producers of chicken products (Matheus and Haley, 2015). Indigenous chicken meat is amongst the main food commodities in the world (FAO, 2013). Traditionally, indigenous chickens are predominantly reared in a semi-intensive or extensive system under subsistence farming, by poor and traditionally disadvantaged household heads, often with high levels of illiteracy or limited education (Mtileni et al., 2009; Henning et al., 2014). Chickens reared in free-range systems are likely to be prone to infection by soil-borne parasites, either by those in which chickens are considered as definitive or intermediate/paratenic hosts. Some of these parasites carry potential risks for humans who ingest raw or undercooked poultry meat. For instance, chickens are considered one of the most important hosts in the epidemiology of Toxoplasma gondii infection and a good indicator of environmental contamination, as they are an efficient source of infection for cats that excrete the environmentally resistant oocysts and because humans may become infected with this parasite after eating undercooked infected chicken meat (Dubey, 2010). According to this last author's review, a very high prevalence of the parasite was found in chickens raised in backyards (up to 100%) and free-range organic (30e50%) establishments. In China, a significantly higher prevalence of antiT. gondii antibodies was observed in free-range compared to caged chickens (Xu et al., 2012). Toxocariasis, a zoonotic parasite of major socioeconomic importance worldwide (Zhu et al., 2015), is another disease associated with the consumption of raw and undercooked poultry meat and entrails (Nagakura et al., 1989; Taira et al., 2004; Morimatsu et al., 2006). The disease is highly prevalent in many developing countries, where its importance is likely to be seriously underestimated (Zhu et al., 2015). The etiologic agents of toxocariasis are one of the two roundworms of the genus Toxocara spp., T. canis and T. cati, parasites of which the dog and cat, respectively, are the definitive hosts (Magnaval et al., 2001; Alvarado-Esquivel, 2013). In humans, who act as paratenic hosts of Toxocara spp., the parasite does not complete the cycle, however, the larvae, originating from ingested eggs present in contaminated soil or tissues of paratenic hosts, cross the intestinal wall and migrate through the circulatory system to various organs (Despommier, 2003). Although toxocariasis is usually asymptomatic, the inflammatory response triggered by the larvae can result in severe infections, with hepatic, pulmonary, ophthalmic and neurological complications (Magnaval et al., 2001; Finsterer and Auer, 2007). Experimental studies have been carried out to evaluate the migratory behavior, infectivity and recovery of Toxocara spp. larvae in bird tissues, including chickens (Galvin, 1964; Pahari and Sasmal, 1990; Taira et al., 2003; Azizi et al., 2007; Dutra et al., 2014). However, information regarding the immune humoral response of chickens against Toxocara spp. infection is scarce. Recently, it was demonstrated a high frequency of anti-Toxocara antibodies in freerange chickens in Southeastern Brazil detected by ELISA test (Campos-da-Silva et al., 2015). The aim of this study was to evaluate the indirect ELISA test to verify the production and avidity of antibodies (IgY) in broiler chickens experimentally infected with different doses of T. canis eggs. 2. Material and methods The present study was approved by the Ethics Committee on Animal Use (CEUA) from the University of Oeste Paulista (Protocol
1677) and the procedures for infection and euthanasia were based on Resolution Number 1000 of the Federal Board of Veterinary Medicine (Brasil, 2012). 2.1. Obtaining Toxocara canis eggs Adult females of T. canis were obtained from naturally infected puppies housed in the Unoeste Kennel, which spontaneously excreted adult parasites after treatment with piperazine (100 mg/ kg). To obtain the eggs, the procedure described by Pecinali et al. (2005) was followed, with modifications. The eggs were extracted directly from the anterior third of the uterus of the adult female T. canis, and subsequently maintained in 2% formalin solution for 3e4 weeks in a temperature controlled environment (28 ± 2 C) for embryonation. After this period, the material was washed three times in 0.9% saline solution by centrifugation (635g, 3 min). Embryogenesis was monitored and larvae within the eggs were evaluated periodically using an optical microscope (10) until the observation of an embryonation minimum rate of 95%. Aliquots of 100, 1000 and 5000 embryonated eggs were obtained in order to perform the experimental infection. The count was performed in a Neubauer chamber, and the eggs diluted in 1.0 mL of phosphate buffered saline (PBS, pH 7.2). Prior the infection, viability of eggs was confirmed by microscopic observation of larval movement, and the doses were carefully adjusted to the desired number of fully embryonated eggs to the inoculation. 2.2. Animals The experiment was conducted in the aviary of the Zootechnical ~o Paulo, Centre, University of Oeste Paulista, Presidente Prudente, Sa Brazil. Forty-eight broiler chickens (Gallus gallus domesticus), female, 14 days old of the Cobb strain were used. The stalls used for the experiment had been previously disinfected by flaming and sweeping with fire before being covered with autoclaved wood shavings (Autoclave Luferco Model. 39224. ~o Paulo, Brazil). Araraquara, Sa The chickens were individually identified with bands “tag type” on the wings, and a 20 day period was allowed for acclimatization to their environment, during which they received commercial grower feed and water ad libitum. The birds were allocated in population densities of 3 chickens/m2, lower than the 13 to 18 chickens/m2 recommended for confined rearing (Brasil, 2007). The cleaning and feeding of the birds were recorded daily. The health management practices included biosecurity in poultry (Brasil, 2007) and immunization of the chickens against Marek's Disease (1 day of life), Gumboro (1, 7, 21 and 56 days) and Newcastle (7, 21 and 56 days). Faecal examinations, using flotation and centrifugal sedimentation (Bowman, 2009) were performed regularly during the adjustment period of the chickens to ensure the absence of coccidia and helminths. 2.3. Experimental infection To evaluate the infectivity of the eggs used to infect the chickens, a preliminarily experiment was conducted in four mice, with a single aliquot of 1000 Toxocara canis eggs. After 72 h, the animals were killed in a CO2 chamber; the livers were extracted and submitted to a modified Baermann technique for recovery of the larvae (Chieffi et al., 1995; Taira et al., 2003). Following the evaluation of the infectivity of the eggs, three, randomly selected, experimental groups were formed with 12 broiler chickens in each group, infected with 100 (G1), 1000 (G2) and 5000 (G3) T. canis eggs, diluted in 1.0 ml of PBS via gavage. The
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control group (G4) received, by the same route, 1.0 ml of PBS, free of eggs. The infection of the chickens was performed at 34 days of life. 2.4. Collection of the samples Blood samples of approximately 2.0 mL were collected from a puncture in the brachial vein of the chicken with the aid of 25 7 mm needles, and stored in sterile test tubes without anticoagulant.
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The samples obtained were centrifuged (1257g, 10 min) to obtain the sera which were stored (30 C) until the moment of processing. The samples were harvested at the following time points; 0 day (pre-infection), and 7, 14, 21, 28, 45 and 60 days post infection (DPI). 2.5. Excretory-secretory Toxocara canis antigen (TES) production Excretory-secretory antigens of T. canis L3 larvae (TES) were
Fig. 1. Optical density of the ELISA for the detection of anti-Toxocara IgY antibodies in sera from chickens inoculated with Toxocara canis eggs, considering different time points (DPI) and experimental infected (G1: 100 eggs; G2: 1000 eggs; G3: 5000 eggs) and control (G4) groups (n ¼ 12 per group). The dotted line indicates the cut-off of the ELISA (OD ¼ 0.368).
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obtained according to the protocol described elsewhere (De Savigny, 1975; Elefant et al., 2006). Briefly, T. canis eggs were collected from the uterus of female adult worms and were embryonated in 2% formalin (28 ± 2 C) for approximately one month. Infective eggs were artificially hatched and the larvae recovered and maintained in vitro (37 C) in a serum-free Eagle's medium. At weekly intervals, the culture supernatant containing the TES was collected in sterile flasks and replaced with fresh culture medium; all the supernatants were treated with 200 mM of the protease inhibitor phenyl-methyl-sulfonyl fluoride (Sigma, St. Louis, MO, USA), concentrated with Amicon Ultrafiltration units (Millipore, Danvers, MA, USA), dialyzed against distilled water, centrifuged (18500g, 60 min, 4 C), and filtered in 0.2 mM Millipore membranes. The protein content was measured using Lowry's method (Lowry et al., 1951). 2.6. Detection of IgY anti-Toxocara canis An ELISA (Enzyme-Linked Immunosorbent Assay) indirect test was standardized to evaluate the production of anti-Toxocara IgY (IgG) antibodies, based on the protocol described elsewhere (De Savigny et al., 1979) with some modifications (Elefant et al., 2006). Polystyrene 96-well microplates (Corning, Costar, New York, NY) were coated (100 mL/well) with TES (37 C, 2 h followed by 4 C, 18 h). After a cycle of three washes (5 min) in PBS-T 20, reaction was blocked (200 mL/well) with bovine serum albumin 1% (BSA, Sigma, St. Louis, MO, USA) in PBS containing 0.05% Tween 20 (PBS-T) (37 C, 1 h). After a cycle of three washes (5 min), serum samples (100 mL/well) diluted 1:200 were incubated in duplicate (37 C, 40 min). After a new cycle of washes (5 min), the plates were incubated (100 mL/well) with rabbit anti-chicken IgY conjugated to peroxidase (A9046-Sigma, Birmingham, AL, USA) diluted at 1:40 000 in PBS-T (37 C, 40 min). After a new cycle of washes (5 min), the chromogenic substrate (100 mL/well) tetramethylbenzidine (TMB-BD®, San Diego, CA, USA), was incubated (37 C, 6 min). The reaction was stopped with 50 mL of 2 N H2SO4 and optical density read at 450 nm (Titertek Multiskan MCC/340, Lab-System, Finland). The cut-off value was defined by an ROC (Receiver Operating Characteristics) curve, based on 48 serum samples (36 prior to infection of the chickens and 12 from the control group), and 36 sera samples of infected chickens (at 45 DPI). Antibody levels were expressed as reactivity indices (RIs), which were calculated as the ratio between the absorbance values of each tested sample and the cut-off value (RI ¼ DO sample/DO cut-off). Samples presenting RIs greater than 1 were considered positive.
2.7. Avidity of IgY Avidity index (AI) of IgY, was performed by a dissociation method, using a 8 M urea solution as the denaturant agent (Hedman et al., 1989). The AI, expressed as a percentage, was defined as the mean optical density (OD) of: [(urea treated/urea untreated) 100], and as has been previously described (Hedman and Rousseau, 1989), where AI values below 30% were classified IgY with low avidity, between 30 and 50% as moderate and >50% as high avidity. 2.8. Statistical analysis The estimates of the sensitivity, specificity, efficiency, positive predictive value (PPV), negative predictive value (NPV), false positives (FP), false negatives (FN) and the kappa index association were calculated at the different time points (Mackinnon, 2000). An ROC curve was obtained for each time point, and others for the phases of infection: 7e21 (initial or acute phase) and 28e60 DPI (final or chronic phase), calculating the area under the curve (AUC) and estimating sensitivity and specificity, considering different cutoff points. The means of the reactivity indices (RI) of IgY detected by the ELISA were compared between groups at the same time point, and between time points for the same group, using one-way analysis of variance (ANOVA) contrasted with the Tukey's test. The correlation between the RI and AI was verified by calculating the Pearson's correlation coefficient. The statistical comparisons were performed with the use of BioEstat Software 5.0 (Ayres et al., 2007). Values of p < 0.05 were considered significant. 3. Results The cut-off defined for the ELISA test for detecting IgY antiToxocara antibodies in the sera of chickens resulted in 0.368. The Fig. 1 represents the OD in different time points according to the experimental groups. It was observed an increase in seroconversion until the final of the observations (60 DPI) in the three experimental groups. The sensitivity, specificity, efficiency as well as PPV, NPV, FP, FN and the kappa index association of the ELISA are expressed in Table 1. According to the results, and based on the ROC curve (Fig. 2), the test sensitivity was low (<50.0%) in the first three weeks post-infection (PI), and increased over the course of the PI period. The specificity of the test, in turn, was greater than 90% at all
Table 1 Performance of the ELISA to detect anti-Toxocara IgY antibodies in sera from chickens (n ¼ 12 per group) inoculated with Toxocara canis (GI1 ¼ 100 eggs; G2 ¼ 1000 eggs; G3 ¼ 5000 eggs), according to the week of sample collection and the phases of infection (Initial: 7e21 days; Final: 28e60 days). Phases
Post-infection time points (days)
Estimates
7
Initial
Sensitivity (%) Specificity (%) Efficiency (%) PPV (%) NPV (%) FP (%) FN (%) Kappa
Final 14
21
28
45
60
Sta.
se
Sta.
se
Sta.
se
Sta.
se
Sta.
se
Sta.
se
0,0 100 25,0 e 25,0 0,0 100 0,0
0,0 0,0 6,3 e 6,3 0,0 0,0 0,0
27,8 100 45,8 100 31,6 0,0 72,2 0,16
7,5 0,0 7,2 0,0 7,5 0,0 7,5 0,06
47,2 100 60,4 100 38,7 0,0 52,8 0,31
8,3 0,0 7,1 0,0 8,8 0,0 8,3 0,09
66,7 91,7 72,9 96,0 47,8 8,3 33,3 0,45
7,9 8,0 6,4 3,9 10,4 8,0 7,9 0,12
91,7 91,7 91,7 97,1 78,6 8,3 8,3 0,79
4,6 8,0 4,0 2,9 11,0 8,0 8,0 0,10
96,8 100 97,0 100 66,7 0,0 3,2 0,78
3,2 0,0 3,0 0,0 27,2 0,0 3,2 0,2
Sta. ¼ Statistics; se ¼ standard error; PPV ¼ Positive Predictive Value; NPV ¼ Negative Predictive Value; FP ¼ False positive and FN ¼ False-negative.
Fig. 2. ROC curve for the values of optical density of the ELISA for the detection of anti-Toxocara IgY antibodies in sera from chickens inoculated with Toxocara canis eggs (G1: 100 eggs; G2: 1000 eggs; G3: 5000 eggs), considering all time points (A), and according to the initial (7e21 DPI; B) and final (28e60 DPI; C) infection phases of the study.
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Table 2 Indices of reactivity of IgY antibodies (mean ± standard deviation) detected using the immunoenzymatic ELISA test, in chickens experimentally infected with Toxocara canis eggs, according to the post-infection time points (n ¼ 12 chicken per group for each timepoint) and experimental groups. Groups
Post-infection time points (days) 0
G1: G2: G3: G4:
7 Aa
0,3 ±0,2 0,2Aa±0,1 0,3Aa±0,1 0,4Aa±0,2
100 eggs 1000 eggs 5000 eggs Control
14 Aa
0,4 ±0,2 0,3Aa±0,1 0,5Aa±0,1 0,4Aa±0,0
21 Aa
0,6 ±0,3 0,7Aa±0,5 0,9Aba±0,5 0,4Aa±0,2
28 ABba
45 ABbc
0,8 ±0,6 1,1Aa±0,6 Ab 1,3 ±0,6 0,4Ba±0,2
60 Adc
1,1 ±0,7 1,6Ab±0,7 Ac 2,0 ±0,8 0,4Bba±0,2
1,6Ad±1,0 2,2Bc±0,9 2,1Ac±1,1 0,8Cc±0,1
1,6 ±0,8 2,2Bcb±0,5 2,5Bc±0,4 0,6Cb±0,2
Means followed by different capital letters indicate significant differences between the groups at the same time point. Means followed by different lowercase letters indicate significant differences between time points for the same group. P value < 0.05. Tukey's test.
assessed moments. The highest efficiency (97.0%) was observed at 60 DPI, with a sensitivity and specificity of 96.8% and 100%, respectively. Taking into account the average RI (Table 2) by group, the seroconversion was verified from day 21 in G2 and G3, while in G1, the antibodies were detected from 28 DPI, with a gradual increase in RI up to 45 DPI. At 45 DPI, it was observed that the average RI of the G2 and G3 groups was significantly higher than that of the G1 animals. In the assessment of avidity (Table 3), the prevalence of antibodies with mean AI at 21 DPI in the G2 and G3 groups was investigated. From the 28th DPI the antibodies presented high AI in all the experimental groups, which was maintained until the end of the study. A correlation was observed between RI and AI at 28 and 60 DPI in the chickens infected with 5000 eggs, and at 60 DPI in those which received 1000 eggs (Table 4). When the analyzes of RI and AI included all the infected animals, regardless of the number of eggs, there was a significant increase in mean (Fig. 3) and significant correlation (p < 0.05) between the indices, when comparing the first three moments of infection (7e21 DPI) with the last three (28e60 DPI). In the preliminarily experiment using a murine model, T. canis larvae were obtained from the liver of all infected mice inoculated with 1000 T. canis eggs (mean ¼ 160 ± 17; range from 142 to 183). During the study, no clinical or behavioral changes were observed in either the infected chickens or the control group.
4. Discussion In the present study, the kinetics and avidity index of IgY in chickens infected with different doses of embryonated T. canis eggs were evaluated from the standardized indirect ELISA. The test showed a low efficiency in the early stage of infection. At 28 DPI, the sensitivity and specificity were close to those observed by Glickman et al. (1986), corresponding to 78% and 92%, respectively. The cutoff defined for the ELISA test for detecting IgY anti-Toxocara antibodies in the sera of chickens (0.368) was slightly higher than the adopted by Campos-da-Silva et al. (2015) to verify the positivity of
Table 3 Avidity indices of IgY (mean ± standard deviation) detected by the immunoenzymatic ELISA test, in chickens experimentally infected with Toxocara canis eggs, according to post-infection time points (n ¼ 12 chickens per group for each timepoint) and experimental groups. Groups
G1: 100 eggs G2: 1000 eggs G3: 5000 eggs
anti-Toxocara antibodies in free-range chickens reared in Brazil. It was observed that the production of IgY was detected at 21 DPI in the groups infected with 1000 and 5000 eggs, and at 28 DPI in the birds which received 100 eggs, demonstrating the influence of the infective dose over time to produce antibodies, as observed in mice (Ollero et al., 2008). In paratenic hosts, seroconversion may be further influenced by the individual characteristics and the species llar et al., 2001). In mice infected with a dose of 1000 of animal (Cue embryonated eggs, for example, seroconversion was observed after 7 (Ollero et al., 2008), 14 (Kolbekov a et al., 2011) and 15 DPI (Schoenardie et al., 2014). Concerned to humoral immune response, it was observed that, over the course of the infection, antibody levels rise and tended to remain stable from 45 DPI. In other species of animals the kinetics of antibodies increases with the course of infection, corroborating the findings of the present study. In pigs, Sommerfelt et al. (2006) verified a peak in the IgG levels between 49 and 56 DPI, followed by a decrease in antibody levels. In mice, the maximum level of IgG was observed at between 42 and 56 DPI (Bowman et al., 1987). The avidity of antibodies has been used to classify the stages of infection in a number of diseases. In the case of human toxocariasis, the acute and chronic phases are characterized by the presence of low and high avidity, respectively (Hübner et al., 2001; Dziemian et al., 2008). In our study, increased AI was observed during the course of infection, with observation of high avidity at 28 DPI in all groups. The increase in AI could be explained by antigenic stimulation caused by the Toxocara spp. larvae which tends to increase with the passage of time of the infection (Dziemian et al., 2008), the chronic phase, as observed in mice infected with 1000 T. canis eggs (Fenoy et al., 1992; Schoenardie et al., 2014). This finding was confirmed by the observation of a significant increase in mean and significant correlation between the RI and AI of the birds, regardless of the infective dose, when comparing the initial or acute phase with the late or chronic phase. In this study, we evaluated fast-growing chickens (short-life cycle), which limited the time for evaluation of humoral response to verify the persistence of antibodies over long periods, as observed in studies with pigs (Sommerfelt et al., 2006) and mice
Table 4 Estimated Pearson correlation coefficient (r) between the reactivity index (RI) and the avidity index (AI; %) of IgY detected by the immunoenzymatic ELISA test, in chickens experimentally infected with different doses of Toxocara canis eggs, according to post-infection time points and experimental groups. Groups
Post-infection time points (days) 7
14
21
28
45
60
G1: 100 eggs G2: 1000 eggs G3: 5000 eggs
NC NC NC
NC NC NC
NC 0,6 0,4
0,6 0,5 0,8a
0,2 0,5 0,5
0,3 0,7a 0,7a
Post-infection time points (days) 7
14
21
28
45
60
NC NC NC
NC NC NC
NC 49,9 ± 15,5 47,9 ± 11,5
56,5 ± 9,2 59,6 ± 11,0 61,9 ± 12,0
60,7 ± 10,3 68,1 ± 13,6 70,3 ± 10,7
62,7 ± 11,8 71,1 ± 11,9 75,1 ± 7,8
NC: Not calculated: Reactivity Index of IgY<1.0.
NC: Not calculated: Reactivity index of IgY<1.0. a Significant correlation at 5%.
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Fig. 3. Reactivity (A) and avidity indices (B), in percentage, of anti-IgY antibodies detected by the immunoenzymatic assay ELISA, in chickens experimentally infected with Toxocara canis eggs (G1; 100 eggs; G2: 1000 eggs; G3: 5000 eggs). Initial (7e21 DPI) and final (28e60 DPI) phases. A ¼ Bars topped with different numbers of asterisks indicate differences between the means of the groups using Tukey's test (P < 0.0001). B ¼ Bars topped with different numbers of asterisks indicate differences between the medians of the groups using the Student t-test (P < 0.0001).
(Bowman et al., 1987; Schoenardie et al., 2014). It was observed that the minimum dose for infection of the birds was 100 eggs. Chieffi et al. (1995) found seropositivity by ELISA before 30 DPI in mice infected with 150 or 200 eggs. Further studies are also necessary to evaluate the persistence and avidity of antibodies are needed to better understand the humoral immune response of the chickens during the chronic phase of infection as well as to evaluate the minimum dose required to stimulate this response in birds. Indirect ELISA test is the standard current method for diagnosing toxocariasis in humans (Rubinsky-Elefant et al., 2010). Absorption of serum samples with Ascaris spp. antigens is employed to remove antibodies elicited by exposure to Ascaris nematoda, which can cross-react with Toxocara antigens (Rubinsky-Elefant et al., 2006). In our study, since animals were kept under controlled condition from the age of one day, and faecal examination were performed before and after infection, we excluded the possibility of cross-infection of chickens by Ascaridia
galli, and, thus, absorption with A. galli antigens was not evaluated. It is important to highlight that in seroepidemiological studies involving naturally infected chickens (or even other birds), the absorption with antigens of ascarids is essential to avoid crossreactivity and false positive results for anti-Toxocara antibodies. Indirect ELISA could be an excellent tool for detecting anti-Toxocara antibodies in birds, serving both for seroepidemiological studies and for evaluating the role of chickens as indicators of environmental contamination through Toxocara spp. eggs. In Southeast Brazil, a study based on an ELISA test with secretory and excretory Toxocara canis antigens, and with A. galli extract to reduce cross-reactivity, showed 58.5% of the free-range chickens for Toxocara spp.; 12.7% had OD over the positive control and may be considered as true infected chickens. In a serosurvey for Toxocara spp. carried out from Northeast Brazil (15 small farms) using our ELISA protocol it was verified an overall prevalence of 93.3% (263 out of 282 samples) in indigeneous chickens reared in semi-
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extensive system. It was also observed in the study that 22.9% (11 out of 48) of the examined soil surrounding the pens were contaminated with Toxocara eggs (data not published). Epidemiologically, our information is important since Toxocara spp. may be transmitted to human through the consumption of raw or undercooked meat and entrails (Nagakura et al., 1989; Morimatsu et al., 2006). In addition, people living in rural areas have little knowledge about the risk of acquiring the zoonosis from raw meat, as observed in a survey on toxoplasmosis carried out in Brazil (Beltrame et al., 2012). Our data demonstrate that the indirect ELISA may be a useful tool for detecting anti-Toxocara antibodies in birds, serving both for seroepidemiological studies and to evaluate the role of chickens as indicators of environmental contamination through Toxocara spp. eggs, as stated by Campos-da-Silva et al. (2015). 5. Conclusion In experimentally infected chickens, production of anti-Toxocara canis antibodies (IgY) increase over the course of infection that was characterized by the presence of high avidity antibodies in the chronic phase. ELISA may be a useful technique for seroepidemiological studies regarding Toxocara spp. infections both in free-range or experimentally infected chickens. Researchers need to bear in mind that is extremely important to take into consideration that in natural infections absorption of serum samples with ascarids antigens is essential to avoid the cross-react with Toxocara antigens. Acknowledgments We would like to thank the coordination Office for Improvement of Higher Education Personnel (CAPES, Brazil) for a PostGraduation grant to R.S. Raposo, and to National Council for Scientific and Technological Development, for part of research funds and Research Productivity Grant to Aristeu V. da Silva. References Alvarado-Esquivel, C., 2013. Toxocariasis in waste pickers: a case control seroprevalence study. PLoS One 8, e54897. Azizi, S., Oryan, A., Sadjjadi, S.M., Zibaei, M., 2007. Histopathologic changes and larval recovery of Toxocara cati in experimentally infected chickens. Parasitol. Res. 102, 47e52. ~es Ayres, M., Ayres Júnior, M., Ayres, D.L., Santos, A., 2007. BioEstat 5.0: aplicaço reas das cie ^ncias biolo gicas e me dicas. Sociedade Civil estatísticas nas a , Bele m, 364 pp. Mamiraua Beltrame, M.A., Pena, H.F., Ton, N.C., Lino, A.J., Gennari, S.M., Dubey, J.P., Pereira, F.E., 2012. Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espírito Santo state, southeastern Brazil. Vet. Parasitol. 188, 225e230. Bowman, D.D., Mika-Grieve, M., Grieve, R.B., 1987. Circulating excretory-secretory antigen levels and specific antibody response in mice infected with Toxocara canis. Am. J. Trop. Med. Hyg. 36, 75e82. Bowman, D.D., 2009. Diagnostic parasitology. In: Bowman, D.D. (Ed.), Georgi's Parasitology for Veterinarians. Saunders-Elsevier, St. Louis, pp. 295e371. Brasil, 2007. Empresa Brasileira de Pesquisa Agropecu aria- EMBRAPA. Centro Nacional de Pesquisa de Suínos e Aves: Boas Pr aticas de Produç~ ao de Frangos de rdia, p. 28. EMBRAPA-CNPSA (Circular Te cnica, 51). Corte, Conco ~e sobre procedimentos e me todos de Brasil, 2012. Resoluç~ ao n. 1000: Dispo sia em animais e da outras provide ^ncias. D.O.U. C.F.M.V. Brasília, p. 2. eutana Campos-da-Silva, D.R., da Paz, J.S., Fortunato, V.R., Beltrame, M.A., Valli, L.C., Pereira, F.E., 2015. Natural infection of free-range chickens with the ascarid nematode Toxocara sp. Parasitol. Res. 114, 4289e4293. ~o, M.M., 1995. PersisChieffi, P.P., Peres, B.A., de Mello, E.O., Kanamura, H., Branda tence of specific antibody response in different experimental infections of mice with Toxocara canis larvae. Rev. Inst. Med. Trop. Sao Paulo 37, 187e190. llar, C., Fenoy, S., del Aguila, C., Guille n, J.L., 2001. Isotype specific immune reCue sponses in murine experimental toxocariasis. Mem. Inst. Oswaldo Cruz 96, 549e553. De Savigny, D.H., 1975. In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigens for use in serodiagnostic tests for visceral larva migrans. J. Parasitol. 61, 781e782.
De Savigny, D.H., Voller, A., Woodruff, A.W., 1979. Toxocariasis: serological diagnosis by enzyme immunoassay. J. Clin. Pathol. 32, 284e288. Despommier, D., 2003. Toxocariasis: clinical aspects, epidemiology, medical ecology and molecular aspects. Clin. Microbiol. Rev. 16, 265e272. Dubey, J.P., 2010. Toxoplasma gondii infections in chickens (Gallus domesticus): prevalence, clinical disease, diagnosis and public health significance. Zoon. Public Hlth 57, 60e73. Dutra, G.F., Pinto, N.S.F., de Avila, L.F.D.C., Dutra, P.C., Telmo, P.D.L., Rodrigues, L.H., Silva, A.M.W.A., Scaini, C.J., 2014. Risk of infection by the consumption of liver of chickens inoculated with low doses of Toxocara canis eggs. Vet. Parasitol. 203, 87e90. ska, M., Machnicka, B., 2008. Dziemian, E., Zarnowska, H., Kołodziej-Sobocin Determination of the relative avidity of the specific IgG antibodies in human toxocariasis. Paras. Immunol. 30, 187e190. Elefant, G.R., Shimizu, S.H., Sanchez, M.C.A., Jacob, C.M.A., Ferreira, A.W., 2006. 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. 20, 164e172. FAO, 2013. World Livestock 2013 Changing Disease Landscapes. Rome. http://www. fao.org/docrep/019/i3440e/i3440e.pdf (access 03.04.14.). n, J., 1992. Persistence of immune response in Fenoy, S., Cuellar, C., Aguila, C., Guille human toxocariasis by ELISA. Int. J. Parasitol. 22, 1037e1038. ~o Paulo 49, Finsterer, J., Auer, H., 2007. Neurotoxocarosis. Rev. Inst. Med. Trop. Sa 279e287. Galvin, T.J., 1964. Experimental Toxocara canis infections in chickens and pigeons. J. Parasitol. 50, 124e127. Glickman, L.T., Schantz, P.M., Grieve, R.B., 1986. Toxocariasis. In: Walls, K.W., Schantz, P.M. (Eds.), Immunodiagnosis of Parasitic Diseases - Helmintic Diseases V. 1. Academic Press, New York, pp. 201e231. Hedman, K., Lappalainen, M., Seppaia, I., Makela, O., 1989. Recent primary Toxoplasma infection indicated by a low avidity of specific IgG. J. Infect. Dis. 159, 736e740. Hedman, K., Rousseau, S.A., 1989. Measurement of avidity of specific IgG for verification of recent primary rubella. J. Med. Virol. 27, 288e292. Henning, J., Hla, T., Meers, J., 2014. Interdisciplinary Communication of Infectious Disease Research - Translating Complex Epidemiological Findings into Understandable Messages for Village Chicken Farmers in Myanmar. SpringerPlus, 3, 726. http://www.springerplus.com/content/3/1/726 (access 03.04.14.). Hübner, J., Uhlikova, M., Leissova, M., 2001. Diagnosis of the early phase of larval toxocariasis using IgG avidity. Epidemiol. Microbiol. Immunol. 50, 67e70. tvi , M., Syr Kolbekov a, P., Ve cka, D., Svoboda, J., Skirnisson, K., Leissova u cek, M., Mare ckov a, H., Kol arov a, L., 2011. Toxocara canis larvae reinfecting BALB/c mice exhibit accelerated speed of migration to the host CNS. Parasitol. Res. 109, 1267e1278. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265e275. Mackinnon, A., 2000. A spread sheet for the calculation of comprehensive statistics for the assessment of diagnostic tests and inter-rater agreement. Comput. Biol. Med. 30, 127e134. Magnaval, J.F., Glickman, L.T., Dorchies, P., Morassin, B., 2001. Highlights of human toxocariasis. Korean J. Parasitol. 39, 1e11. Matheus, K., Haley, M., 2015. Livestock, Dairy, and Poultry Outlook/LDP-m-249/Mar. 16, 2015. Economic Research Service, USDA. http://www.ers.usda.gov/media/ 1801295/ldpm249.pdf (access 27.04.15.). Mtileni, B.J., Muchadeyi, F.C., Maiwashe, A., Phitsane, P.M., Halimani, T.E., Chimonyo, M., Dzama, K., 2009. Characterisation of production systems for indigenous chicken genetic resources of South Africa. Appl. Anim. Husb. Rural. Dev. 2009 (2), 18e22. Morimatsu, Y., Akao, N., Akiyoshi, H., Kawazu, T., Okabe, Y., Aizawa, H., 2006. A familial case of visceral larva migrans after ingestion of raw chicken livers: appearance of specific antibody in bronchoalveolar lavage fluid of the patients. Am. J. Trop. Med. Hyg. 75, 303e306. Nagakura, K., Tachibana, H., Kaneda, Y., Kato, Y., 1989. Toxocariasis possibly caused by ingesting raw chicken. J. Infect. Dis. 160, 735e736. llar, C., Guille n, J.L., Del Aguila, C., 2008. Experimental Ollero, M.D., Fenoy, S., Cue toxocariosis in BALB/c mice: effect of the inoculation dose on brain and eye involvement. Acta Trop. 105, 124e130. Pahari, T.K., Sasmal, N.K., 1990. Infection of Japanese quail with Toxocara canis larvae and establishment of patent infection in pups. Vet. Parasitol. 35, 357e364. Pecinali, N.R., Gomes, R.N., Amendoeira, F.C., Bastos, A.C.M.P., Martins, M.J.Q.A., Pegado, C.S., Bastos, O.M.P., Bozza, P.T., Castro-Faria-Neto, H.C., 2005. Influence of murine Toxocara canis infection on plasma and bronchoalveolar lavage fluid eosinophil numbers and its correlation with cytokine levels. Vet. Parasitol. 134, 121e130. Rubinsky-Elefant, G.R., Hirata, C.E., Yamamoto, J.H., Ferreira, M.U., 2010. Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. Ann. Trop. Med. Parasitol. 104, 3e23. Schoenardie, E.R., Scaini, C.J., Avila, L.F.C., Sperotto, R.L., Borsuk, S., Felicetti, C.D.P., Pepe, M., Berne, M.E.A., 2014. Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis. Braz. J. Vet. Parasitol. 23, 403e406. Sommerfelt, I.E., Santillan, G., Mira, G., Ribicich, M., Betti, A., de Torres, R., 2006. Toxocara canis infections in a pig model: immunological, haematological and blood biochemistry responses. J. Helminthol. 80, 73e77. Taira, K., Permin, A., Kapel, C.M.O., 2003. Establishment and migration pattern of Toxocara canis larvae in chickens. 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Zhu, X.Q., Korhonen, P.K., Cai, H., Young, N.D., Nejsum, P., von SamsonHimmelstjerna, G., Boag, P.R., Tan, P., Li, Q., Min, J., Yang, Y., Wang, X., Fang, X., Hall, R.S., Hofmann, A., Sternberg, P.W., Jex, A.R., Gasser, R.B., 2015. Genetic blueprint of the zoonotic pathogen Toxocara canis. Nat. Commun. 6, 6145. http://dx.doi.org/10.1038/ncomms7145.
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Full length article
Kinetic and avidity of IgY anti-Toxocara antibodies in experimentally infected chickens m b, *, Ricardo da Silva Raposo a, Vamilton Alvares Santare Yslla Fernanda Fitz Balo Merigueti a, Guita Rubinsky-Elefant c, Letícia Maria de Lima Cerazo b, Ludimilla Pereira b, Bianca Pelegi Zampieri b, Aristeu Vieira da Silva d, Cecília Braga Laposy e ~o Post-Graduation Program in Animal Science, Laboratory of Veterinary Parasitology, Veterinary Teaching-Hospital (UNOESTE), Presidente Prudente, Sa Paulo, Brazil b ~o Paulo, Brazil Laboratory of Veterinary Parasitology, Veterinary Teaching-Hospital, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, Sa c ~o Paulo, University of Sa ~o Paulo, Brazil Laboratory of Seroepidemiology and Immunobiology, Instituto de Medicina Tropical de Sa d Zoonosis and Public Health Research Group, State University of Feira de Santana, UEFS, Feira de Santana, Bahia, Brazil e ~o Paulo, Laboratory of Veterinary Clinical Pathology, Veterinary Teaching-Hospital, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, Sa Brazil a
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ELISA for detection of anti-Toxocara IgY and avidity in infected chickens. Experimental infection of chickens with 100, 1000 and 5000 embryonated T. canis eggs. High levels of IgY and high avidity index in the chronic phase of infection. Influence of infective dose on the humoral immune response. ELISA is a useful tool for seroprevalence studies in poultry.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2015 Received in revised form 5 August 2016 Accepted 21 September 2016 Available online 13 October 2016
Toxocariasis is a geohelminth zoonosis with worldwide distribution, mainly transmitted through the ingestion of embryonated eggs of nematodes of the Toxocara genus. The disease can also be transmitted to humans as a result of eating raw or undercooked meat of paratenic hosts, such as chickens. Here, we standardized an enzyme-linked immunosorbent assay (ELISA) for evaluating experimentally the kinetic and avidity index (AI) of IgY in broiler chickens infected with different doses of Toxocara canis eggs (G1:100; G2: 1000; and G3: 5000; n ¼ 12 per group). The test showed 91.7% sensitivity (CI 95%: 77.5 e98.3) and 100% specificity (CI 95%: 92.6e100), and highest efficiency (97.0%) at 60 days post infection. Infection was characterized by the presence of high avidity antibodies in the chronic phase. Our results support that the ELISA can be a highly useful tool for the detection of anti-Toxocara antibodies in chickens. © 2016 Elsevier Inc. All rights reserved.
Keywords: Birds Immune response ELISA Toxocariasis
rio de Parasitologia Veterin rio da Universidade do Oeste Paulista (UNOESTE), Raposo Tavares Km 572, Bairro * Corresponding author. Laborato aria, Hospital Veterina ~o Paulo, Brazil. Limoeiro, Presidente Prudente, CEP, 19067-175 Sa m). E-mail address: [email protected] (V.A. Santare http://dx.doi.org/10.1016/j.exppara.2016.09.009 0014-4894/© 2016 Elsevier Inc. All rights reserved.
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1. Introduction Despite the technology involved in the poultry industry, in many parts of the world, particularly in developing countries, small-scale farms are the dominant producers of chicken products (Matheus and Haley, 2015). Indigenous chicken meat is amongst the main food commodities in the world (FAO, 2013). Traditionally, indigenous chickens are predominantly reared in a semi-intensive or extensive system under subsistence farming, by poor and traditionally disadvantaged household heads, often with high levels of illiteracy or limited education (Mtileni et al., 2009; Henning et al., 2014). Chickens reared in free-range systems are likely to be prone to infection by soil-borne parasites, either by those in which chickens are considered as definitive or intermediate/paratenic hosts. Some of these parasites carry potential risks for humans who ingest raw or undercooked poultry meat. For instance, chickens are considered one of the most important hosts in the epidemiology of Toxoplasma gondii infection and a good indicator of environmental contamination, as they are an efficient source of infection for cats that excrete the environmentally resistant oocysts and because humans may become infected with this parasite after eating undercooked infected chicken meat (Dubey, 2010). According to this last author's review, a very high prevalence of the parasite was found in chickens raised in backyards (up to 100%) and free-range organic (30e50%) establishments. In China, a significantly higher prevalence of antiT. gondii antibodies was observed in free-range compared to caged chickens (Xu et al., 2012). Toxocariasis, a zoonotic parasite of major socioeconomic importance worldwide (Zhu et al., 2015), is another disease associated with the consumption of raw and undercooked poultry meat and entrails (Nagakura et al., 1989; Taira et al., 2004; Morimatsu et al., 2006). The disease is highly prevalent in many developing countries, where its importance is likely to be seriously underestimated (Zhu et al., 2015). The etiologic agents of toxocariasis are one of the two roundworms of the genus Toxocara spp., T. canis and T. cati, parasites of which the dog and cat, respectively, are the definitive hosts (Magnaval et al., 2001; Alvarado-Esquivel, 2013). In humans, who act as paratenic hosts of Toxocara spp., the parasite does not complete the cycle, however, the larvae, originating from ingested eggs present in contaminated soil or tissues of paratenic hosts, cross the intestinal wall and migrate through the circulatory system to various organs (Despommier, 2003). Although toxocariasis is usually asymptomatic, the inflammatory response triggered by the larvae can result in severe infections, with hepatic, pulmonary, ophthalmic and neurological complications (Magnaval et al., 2001; Finsterer and Auer, 2007). Experimental studies have been carried out to evaluate the migratory behavior, infectivity and recovery of Toxocara spp. larvae in bird tissues, including chickens (Galvin, 1964; Pahari and Sasmal, 1990; Taira et al., 2003; Azizi et al., 2007; Dutra et al., 2014). However, information regarding the immune humoral response of chickens against Toxocara spp. infection is scarce. Recently, it was demonstrated a high frequency of anti-Toxocara antibodies in freerange chickens in Southeastern Brazil detected by ELISA test (Campos-da-Silva et al., 2015). The aim of this study was to evaluate the indirect ELISA test to verify the production and avidity of antibodies (IgY) in broiler chickens experimentally infected with different doses of T. canis eggs. 2. Material and methods The present study was approved by the Ethics Committee on Animal Use (CEUA) from the University of Oeste Paulista (Protocol
1677) and the procedures for infection and euthanasia were based on Resolution Number 1000 of the Federal Board of Veterinary Medicine (Brasil, 2012). 2.1. Obtaining Toxocara canis eggs Adult females of T. canis were obtained from naturally infected puppies housed in the Unoeste Kennel, which spontaneously excreted adult parasites after treatment with piperazine (100 mg/ kg). To obtain the eggs, the procedure described by Pecinali et al. (2005) was followed, with modifications. The eggs were extracted directly from the anterior third of the uterus of the adult female T. canis, and subsequently maintained in 2% formalin solution for 3e4 weeks in a temperature controlled environment (28 ± 2 C) for embryonation. After this period, the material was washed three times in 0.9% saline solution by centrifugation (635g, 3 min). Embryogenesis was monitored and larvae within the eggs were evaluated periodically using an optical microscope (10) until the observation of an embryonation minimum rate of 95%. Aliquots of 100, 1000 and 5000 embryonated eggs were obtained in order to perform the experimental infection. The count was performed in a Neubauer chamber, and the eggs diluted in 1.0 mL of phosphate buffered saline (PBS, pH 7.2). Prior the infection, viability of eggs was confirmed by microscopic observation of larval movement, and the doses were carefully adjusted to the desired number of fully embryonated eggs to the inoculation. 2.2. Animals The experiment was conducted in the aviary of the Zootechnical ~o Paulo, Centre, University of Oeste Paulista, Presidente Prudente, Sa Brazil. Forty-eight broiler chickens (Gallus gallus domesticus), female, 14 days old of the Cobb strain were used. The stalls used for the experiment had been previously disinfected by flaming and sweeping with fire before being covered with autoclaved wood shavings (Autoclave Luferco Model. 39224. ~o Paulo, Brazil). Araraquara, Sa The chickens were individually identified with bands “tag type” on the wings, and a 20 day period was allowed for acclimatization to their environment, during which they received commercial grower feed and water ad libitum. The birds were allocated in population densities of 3 chickens/m2, lower than the 13 to 18 chickens/m2 recommended for confined rearing (Brasil, 2007). The cleaning and feeding of the birds were recorded daily. The health management practices included biosecurity in poultry (Brasil, 2007) and immunization of the chickens against Marek's Disease (1 day of life), Gumboro (1, 7, 21 and 56 days) and Newcastle (7, 21 and 56 days). Faecal examinations, using flotation and centrifugal sedimentation (Bowman, 2009) were performed regularly during the adjustment period of the chickens to ensure the absence of coccidia and helminths. 2.3. Experimental infection To evaluate the infectivity of the eggs used to infect the chickens, a preliminarily experiment was conducted in four mice, with a single aliquot of 1000 Toxocara canis eggs. After 72 h, the animals were killed in a CO2 chamber; the livers were extracted and submitted to a modified Baermann technique for recovery of the larvae (Chieffi et al., 1995; Taira et al., 2003). Following the evaluation of the infectivity of the eggs, three, randomly selected, experimental groups were formed with 12 broiler chickens in each group, infected with 100 (G1), 1000 (G2) and 5000 (G3) T. canis eggs, diluted in 1.0 ml of PBS via gavage. The
R. da Silva Raposo et al. / Experimental Parasitology 171 (2016) 33e41
control group (G4) received, by the same route, 1.0 ml of PBS, free of eggs. The infection of the chickens was performed at 34 days of life. 2.4. Collection of the samples Blood samples of approximately 2.0 mL were collected from a puncture in the brachial vein of the chicken with the aid of 25 7 mm needles, and stored in sterile test tubes without anticoagulant.
35
The samples obtained were centrifuged (1257g, 10 min) to obtain the sera which were stored (30 C) until the moment of processing. The samples were harvested at the following time points; 0 day (pre-infection), and 7, 14, 21, 28, 45 and 60 days post infection (DPI). 2.5. Excretory-secretory Toxocara canis antigen (TES) production Excretory-secretory antigens of T. canis L3 larvae (TES) were
Fig. 1. Optical density of the ELISA for the detection of anti-Toxocara IgY antibodies in sera from chickens inoculated with Toxocara canis eggs, considering different time points (DPI) and experimental infected (G1: 100 eggs; G2: 1000 eggs; G3: 5000 eggs) and control (G4) groups (n ¼ 12 per group). The dotted line indicates the cut-off of the ELISA (OD ¼ 0.368).
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obtained according to the protocol described elsewhere (De Savigny, 1975; Elefant et al., 2006). Briefly, T. canis eggs were collected from the uterus of female adult worms and were embryonated in 2% formalin (28 ± 2 C) for approximately one month. Infective eggs were artificially hatched and the larvae recovered and maintained in vitro (37 C) in a serum-free Eagle's medium. At weekly intervals, the culture supernatant containing the TES was collected in sterile flasks and replaced with fresh culture medium; all the supernatants were treated with 200 mM of the protease inhibitor phenyl-methyl-sulfonyl fluoride (Sigma, St. Louis, MO, USA), concentrated with Amicon Ultrafiltration units (Millipore, Danvers, MA, USA), dialyzed against distilled water, centrifuged (18500g, 60 min, 4 C), and filtered in 0.2 mM Millipore membranes. The protein content was measured using Lowry's method (Lowry et al., 1951). 2.6. Detection of IgY anti-Toxocara canis An ELISA (Enzyme-Linked Immunosorbent Assay) indirect test was standardized to evaluate the production of anti-Toxocara IgY (IgG) antibodies, based on the protocol described elsewhere (De Savigny et al., 1979) with some modifications (Elefant et al., 2006). Polystyrene 96-well microplates (Corning, Costar, New York, NY) were coated (100 mL/well) with TES (37 C, 2 h followed by 4 C, 18 h). After a cycle of three washes (5 min) in PBS-T 20, reaction was blocked (200 mL/well) with bovine serum albumin 1% (BSA, Sigma, St. Louis, MO, USA) in PBS containing 0.05% Tween 20 (PBS-T) (37 C, 1 h). After a cycle of three washes (5 min), serum samples (100 mL/well) diluted 1:200 were incubated in duplicate (37 C, 40 min). After a new cycle of washes (5 min), the plates were incubated (100 mL/well) with rabbit anti-chicken IgY conjugated to peroxidase (A9046-Sigma, Birmingham, AL, USA) diluted at 1:40 000 in PBS-T (37 C, 40 min). After a new cycle of washes (5 min), the chromogenic substrate (100 mL/well) tetramethylbenzidine (TMB-BD®, San Diego, CA, USA), was incubated (37 C, 6 min). The reaction was stopped with 50 mL of 2 N H2SO4 and optical density read at 450 nm (Titertek Multiskan MCC/340, Lab-System, Finland). The cut-off value was defined by an ROC (Receiver Operating Characteristics) curve, based on 48 serum samples (36 prior to infection of the chickens and 12 from the control group), and 36 sera samples of infected chickens (at 45 DPI). Antibody levels were expressed as reactivity indices (RIs), which were calculated as the ratio between the absorbance values of each tested sample and the cut-off value (RI ¼ DO sample/DO cut-off). Samples presenting RIs greater than 1 were considered positive.
2.7. Avidity of IgY Avidity index (AI) of IgY, was performed by a dissociation method, using a 8 M urea solution as the denaturant agent (Hedman et al., 1989). The AI, expressed as a percentage, was defined as the mean optical density (OD) of: [(urea treated/urea untreated) 100], and as has been previously described (Hedman and Rousseau, 1989), where AI values below 30% were classified IgY with low avidity, between 30 and 50% as moderate and >50% as high avidity. 2.8. Statistical analysis The estimates of the sensitivity, specificity, efficiency, positive predictive value (PPV), negative predictive value (NPV), false positives (FP), false negatives (FN) and the kappa index association were calculated at the different time points (Mackinnon, 2000). An ROC curve was obtained for each time point, and others for the phases of infection: 7e21 (initial or acute phase) and 28e60 DPI (final or chronic phase), calculating the area under the curve (AUC) and estimating sensitivity and specificity, considering different cutoff points. The means of the reactivity indices (RI) of IgY detected by the ELISA were compared between groups at the same time point, and between time points for the same group, using one-way analysis of variance (ANOVA) contrasted with the Tukey's test. The correlation between the RI and AI was verified by calculating the Pearson's correlation coefficient. The statistical comparisons were performed with the use of BioEstat Software 5.0 (Ayres et al., 2007). Values of p < 0.05 were considered significant. 3. Results The cut-off defined for the ELISA test for detecting IgY antiToxocara antibodies in the sera of chickens resulted in 0.368. The Fig. 1 represents the OD in different time points according to the experimental groups. It was observed an increase in seroconversion until the final of the observations (60 DPI) in the three experimental groups. The sensitivity, specificity, efficiency as well as PPV, NPV, FP, FN and the kappa index association of the ELISA are expressed in Table 1. According to the results, and based on the ROC curve (Fig. 2), the test sensitivity was low (<50.0%) in the first three weeks post-infection (PI), and increased over the course of the PI period. The specificity of the test, in turn, was greater than 90% at all
Table 1 Performance of the ELISA to detect anti-Toxocara IgY antibodies in sera from chickens (n ¼ 12 per group) inoculated with Toxocara canis (GI1 ¼ 100 eggs; G2 ¼ 1000 eggs; G3 ¼ 5000 eggs), according to the week of sample collection and the phases of infection (Initial: 7e21 days; Final: 28e60 days). Phases
Post-infection time points (days)
Estimates
7
Initial
Sensitivity (%) Specificity (%) Efficiency (%) PPV (%) NPV (%) FP (%) FN (%) Kappa
Final 14
21
28
45
60
Sta.
se
Sta.
se
Sta.
se
Sta.
se
Sta.
se
Sta.
se
0,0 100 25,0 e 25,0 0,0 100 0,0
0,0 0,0 6,3 e 6,3 0,0 0,0 0,0
27,8 100 45,8 100 31,6 0,0 72,2 0,16
7,5 0,0 7,2 0,0 7,5 0,0 7,5 0,06
47,2 100 60,4 100 38,7 0,0 52,8 0,31
8,3 0,0 7,1 0,0 8,8 0,0 8,3 0,09
66,7 91,7 72,9 96,0 47,8 8,3 33,3 0,45
7,9 8,0 6,4 3,9 10,4 8,0 7,9 0,12
91,7 91,7 91,7 97,1 78,6 8,3 8,3 0,79
4,6 8,0 4,0 2,9 11,0 8,0 8,0 0,10
96,8 100 97,0 100 66,7 0,0 3,2 0,78
3,2 0,0 3,0 0,0 27,2 0,0 3,2 0,2
Sta. ¼ Statistics; se ¼ standard error; PPV ¼ Positive Predictive Value; NPV ¼ Negative Predictive Value; FP ¼ False positive and FN ¼ False-negative.
Fig. 2. ROC curve for the values of optical density of the ELISA for the detection of anti-Toxocara IgY antibodies in sera from chickens inoculated with Toxocara canis eggs (G1: 100 eggs; G2: 1000 eggs; G3: 5000 eggs), considering all time points (A), and according to the initial (7e21 DPI; B) and final (28e60 DPI; C) infection phases of the study.
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Table 2 Indices of reactivity of IgY antibodies (mean ± standard deviation) detected using the immunoenzymatic ELISA test, in chickens experimentally infected with Toxocara canis eggs, according to the post-infection time points (n ¼ 12 chicken per group for each timepoint) and experimental groups. Groups
Post-infection time points (days) 0
G1: G2: G3: G4:
7 Aa
0,3 ±0,2 0,2Aa±0,1 0,3Aa±0,1 0,4Aa±0,2
100 eggs 1000 eggs 5000 eggs Control
14 Aa
0,4 ±0,2 0,3Aa±0,1 0,5Aa±0,1 0,4Aa±0,0
21 Aa
0,6 ±0,3 0,7Aa±0,5 0,9Aba±0,5 0,4Aa±0,2
28 ABba
45 ABbc
0,8 ±0,6 1,1Aa±0,6 Ab 1,3 ±0,6 0,4Ba±0,2
60 Adc
1,1 ±0,7 1,6Ab±0,7 Ac 2,0 ±0,8 0,4Bba±0,2
1,6Ad±1,0 2,2Bc±0,9 2,1Ac±1,1 0,8Cc±0,1
1,6 ±0,8 2,2Bcb±0,5 2,5Bc±0,4 0,6Cb±0,2
Means followed by different capital letters indicate significant differences between the groups at the same time point. Means followed by different lowercase letters indicate significant differences between time points for the same group. P value < 0.05. Tukey's test.
assessed moments. The highest efficiency (97.0%) was observed at 60 DPI, with a sensitivity and specificity of 96.8% and 100%, respectively. Taking into account the average RI (Table 2) by group, the seroconversion was verified from day 21 in G2 and G3, while in G1, the antibodies were detected from 28 DPI, with a gradual increase in RI up to 45 DPI. At 45 DPI, it was observed that the average RI of the G2 and G3 groups was significantly higher than that of the G1 animals. In the assessment of avidity (Table 3), the prevalence of antibodies with mean AI at 21 DPI in the G2 and G3 groups was investigated. From the 28th DPI the antibodies presented high AI in all the experimental groups, which was maintained until the end of the study. A correlation was observed between RI and AI at 28 and 60 DPI in the chickens infected with 5000 eggs, and at 60 DPI in those which received 1000 eggs (Table 4). When the analyzes of RI and AI included all the infected animals, regardless of the number of eggs, there was a significant increase in mean (Fig. 3) and significant correlation (p < 0.05) between the indices, when comparing the first three moments of infection (7e21 DPI) with the last three (28e60 DPI). In the preliminarily experiment using a murine model, T. canis larvae were obtained from the liver of all infected mice inoculated with 1000 T. canis eggs (mean ¼ 160 ± 17; range from 142 to 183). During the study, no clinical or behavioral changes were observed in either the infected chickens or the control group.
4. Discussion In the present study, the kinetics and avidity index of IgY in chickens infected with different doses of embryonated T. canis eggs were evaluated from the standardized indirect ELISA. The test showed a low efficiency in the early stage of infection. At 28 DPI, the sensitivity and specificity were close to those observed by Glickman et al. (1986), corresponding to 78% and 92%, respectively. The cutoff defined for the ELISA test for detecting IgY anti-Toxocara antibodies in the sera of chickens (0.368) was slightly higher than the adopted by Campos-da-Silva et al. (2015) to verify the positivity of
Table 3 Avidity indices of IgY (mean ± standard deviation) detected by the immunoenzymatic ELISA test, in chickens experimentally infected with Toxocara canis eggs, according to post-infection time points (n ¼ 12 chickens per group for each timepoint) and experimental groups. Groups
G1: 100 eggs G2: 1000 eggs G3: 5000 eggs
anti-Toxocara antibodies in free-range chickens reared in Brazil. It was observed that the production of IgY was detected at 21 DPI in the groups infected with 1000 and 5000 eggs, and at 28 DPI in the birds which received 100 eggs, demonstrating the influence of the infective dose over time to produce antibodies, as observed in mice (Ollero et al., 2008). In paratenic hosts, seroconversion may be further influenced by the individual characteristics and the species llar et al., 2001). In mice infected with a dose of 1000 of animal (Cue embryonated eggs, for example, seroconversion was observed after 7 (Ollero et al., 2008), 14 (Kolbekov a et al., 2011) and 15 DPI (Schoenardie et al., 2014). Concerned to humoral immune response, it was observed that, over the course of the infection, antibody levels rise and tended to remain stable from 45 DPI. In other species of animals the kinetics of antibodies increases with the course of infection, corroborating the findings of the present study. In pigs, Sommerfelt et al. (2006) verified a peak in the IgG levels between 49 and 56 DPI, followed by a decrease in antibody levels. In mice, the maximum level of IgG was observed at between 42 and 56 DPI (Bowman et al., 1987). The avidity of antibodies has been used to classify the stages of infection in a number of diseases. In the case of human toxocariasis, the acute and chronic phases are characterized by the presence of low and high avidity, respectively (Hübner et al., 2001; Dziemian et al., 2008). In our study, increased AI was observed during the course of infection, with observation of high avidity at 28 DPI in all groups. The increase in AI could be explained by antigenic stimulation caused by the Toxocara spp. larvae which tends to increase with the passage of time of the infection (Dziemian et al., 2008), the chronic phase, as observed in mice infected with 1000 T. canis eggs (Fenoy et al., 1992; Schoenardie et al., 2014). This finding was confirmed by the observation of a significant increase in mean and significant correlation between the RI and AI of the birds, regardless of the infective dose, when comparing the initial or acute phase with the late or chronic phase. In this study, we evaluated fast-growing chickens (short-life cycle), which limited the time for evaluation of humoral response to verify the persistence of antibodies over long periods, as observed in studies with pigs (Sommerfelt et al., 2006) and mice
Table 4 Estimated Pearson correlation coefficient (r) between the reactivity index (RI) and the avidity index (AI; %) of IgY detected by the immunoenzymatic ELISA test, in chickens experimentally infected with different doses of Toxocara canis eggs, according to post-infection time points and experimental groups. Groups
Post-infection time points (days) 7
14
21
28
45
60
G1: 100 eggs G2: 1000 eggs G3: 5000 eggs
NC NC NC
NC NC NC
NC 0,6 0,4
0,6 0,5 0,8a
0,2 0,5 0,5
0,3 0,7a 0,7a
Post-infection time points (days) 7
14
21
28
45
60
NC NC NC
NC NC NC
NC 49,9 ± 15,5 47,9 ± 11,5
56,5 ± 9,2 59,6 ± 11,0 61,9 ± 12,0
60,7 ± 10,3 68,1 ± 13,6 70,3 ± 10,7
62,7 ± 11,8 71,1 ± 11,9 75,1 ± 7,8
NC: Not calculated: Reactivity Index of IgY<1.0.
NC: Not calculated: Reactivity index of IgY<1.0. a Significant correlation at 5%.
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Fig. 3. Reactivity (A) and avidity indices (B), in percentage, of anti-IgY antibodies detected by the immunoenzymatic assay ELISA, in chickens experimentally infected with Toxocara canis eggs (G1; 100 eggs; G2: 1000 eggs; G3: 5000 eggs). Initial (7e21 DPI) and final (28e60 DPI) phases. A ¼ Bars topped with different numbers of asterisks indicate differences between the means of the groups using Tukey's test (P < 0.0001). B ¼ Bars topped with different numbers of asterisks indicate differences between the medians of the groups using the Student t-test (P < 0.0001).
(Bowman et al., 1987; Schoenardie et al., 2014). It was observed that the minimum dose for infection of the birds was 100 eggs. Chieffi et al. (1995) found seropositivity by ELISA before 30 DPI in mice infected with 150 or 200 eggs. Further studies are also necessary to evaluate the persistence and avidity of antibodies are needed to better understand the humoral immune response of the chickens during the chronic phase of infection as well as to evaluate the minimum dose required to stimulate this response in birds. Indirect ELISA test is the standard current method for diagnosing toxocariasis in humans (Rubinsky-Elefant et al., 2010). Absorption of serum samples with Ascaris spp. antigens is employed to remove antibodies elicited by exposure to Ascaris nematoda, which can cross-react with Toxocara antigens (Rubinsky-Elefant et al., 2006). In our study, since animals were kept under controlled condition from the age of one day, and faecal examination were performed before and after infection, we excluded the possibility of cross-infection of chickens by Ascaridia
galli, and, thus, absorption with A. galli antigens was not evaluated. It is important to highlight that in seroepidemiological studies involving naturally infected chickens (or even other birds), the absorption with antigens of ascarids is essential to avoid crossreactivity and false positive results for anti-Toxocara antibodies. Indirect ELISA could be an excellent tool for detecting anti-Toxocara antibodies in birds, serving both for seroepidemiological studies and for evaluating the role of chickens as indicators of environmental contamination through Toxocara spp. eggs. In Southeast Brazil, a study based on an ELISA test with secretory and excretory Toxocara canis antigens, and with A. galli extract to reduce cross-reactivity, showed 58.5% of the free-range chickens for Toxocara spp.; 12.7% had OD over the positive control and may be considered as true infected chickens. In a serosurvey for Toxocara spp. carried out from Northeast Brazil (15 small farms) using our ELISA protocol it was verified an overall prevalence of 93.3% (263 out of 282 samples) in indigeneous chickens reared in semi-
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extensive system. It was also observed in the study that 22.9% (11 out of 48) of the examined soil surrounding the pens were contaminated with Toxocara eggs (data not published). Epidemiologically, our information is important since Toxocara spp. may be transmitted to human through the consumption of raw or undercooked meat and entrails (Nagakura et al., 1989; Morimatsu et al., 2006). In addition, people living in rural areas have little knowledge about the risk of acquiring the zoonosis from raw meat, as observed in a survey on toxoplasmosis carried out in Brazil (Beltrame et al., 2012). Our data demonstrate that the indirect ELISA may be a useful tool for detecting anti-Toxocara antibodies in birds, serving both for seroepidemiological studies and to evaluate the role of chickens as indicators of environmental contamination through Toxocara spp. eggs, as stated by Campos-da-Silva et al. (2015). 5. Conclusion In experimentally infected chickens, production of anti-Toxocara canis antibodies (IgY) increase over the course of infection that was characterized by the presence of high avidity antibodies in the chronic phase. ELISA may be a useful technique for seroepidemiological studies regarding Toxocara spp. infections both in free-range or experimentally infected chickens. Researchers need to bear in mind that is extremely important to take into consideration that in natural infections absorption of serum samples with ascarids antigens is essential to avoid the cross-react with Toxocara antigens. Acknowledgments We would like to thank the coordination Office for Improvement of Higher Education Personnel (CAPES, Brazil) for a PostGraduation grant to R.S. Raposo, and to National Council for Scientific and Technological Development, for part of research funds and Research Productivity Grant to Aristeu V. da Silva. References Alvarado-Esquivel, C., 2013. Toxocariasis in waste pickers: a case control seroprevalence study. PLoS One 8, e54897. Azizi, S., Oryan, A., Sadjjadi, S.M., Zibaei, M., 2007. Histopathologic changes and larval recovery of Toxocara cati in experimentally infected chickens. Parasitol. Res. 102, 47e52. ~es Ayres, M., Ayres Júnior, M., Ayres, D.L., Santos, A., 2007. BioEstat 5.0: aplicaço reas das cie ^ncias biolo gicas e me dicas. Sociedade Civil estatísticas nas a , Bele m, 364 pp. Mamiraua Beltrame, M.A., Pena, H.F., Ton, N.C., Lino, A.J., Gennari, S.M., Dubey, J.P., Pereira, F.E., 2012. Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espírito Santo state, southeastern Brazil. Vet. Parasitol. 188, 225e230. Bowman, D.D., Mika-Grieve, M., Grieve, R.B., 1987. Circulating excretory-secretory antigen levels and specific antibody response in mice infected with Toxocara canis. Am. J. Trop. Med. Hyg. 36, 75e82. Bowman, D.D., 2009. Diagnostic parasitology. In: Bowman, D.D. (Ed.), Georgi's Parasitology for Veterinarians. Saunders-Elsevier, St. Louis, pp. 295e371. Brasil, 2007. Empresa Brasileira de Pesquisa Agropecu aria- EMBRAPA. Centro Nacional de Pesquisa de Suínos e Aves: Boas Pr aticas de Produç~ ao de Frangos de rdia, p. 28. EMBRAPA-CNPSA (Circular Te cnica, 51). Corte, Conco ~e sobre procedimentos e me todos de Brasil, 2012. Resoluç~ ao n. 1000: Dispo sia em animais e da outras provide ^ncias. D.O.U. C.F.M.V. Brasília, p. 2. eutana Campos-da-Silva, D.R., da Paz, J.S., Fortunato, V.R., Beltrame, M.A., Valli, L.C., Pereira, F.E., 2015. Natural infection of free-range chickens with the ascarid nematode Toxocara sp. Parasitol. Res. 114, 4289e4293. ~o, M.M., 1995. PersisChieffi, P.P., Peres, B.A., de Mello, E.O., Kanamura, H., Branda tence of specific antibody response in different experimental infections of mice with Toxocara canis larvae. Rev. Inst. Med. Trop. Sao Paulo 37, 187e190. llar, C., Fenoy, S., del Aguila, C., Guille n, J.L., 2001. Isotype specific immune reCue sponses in murine experimental toxocariasis. Mem. Inst. Oswaldo Cruz 96, 549e553. De Savigny, D.H., 1975. In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigens for use in serodiagnostic tests for visceral larva migrans. J. Parasitol. 61, 781e782.
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