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Serotyping of naturally Toxoplasma gondii infected meat-producing animals
Veterinary Parasitology 169 (2010) 24–28
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Serotyping of naturally Toxoplasma gondii infected meat-producing animals Susana Sousa a,c,*, Nuno Canada b,c,d, Jose´ Manuel Correia da Costa a,c, Marie-Laure Darde´ e,f a
Centro de Imunologia e Biologia Parasita´ria (CIBP), CSPGF, INSA Porto, Portugal Instituto de Cieˆncias Biome´dicas de Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal c Centro de Estudos de Cieˆncia Animal (CECA), Universidade do Porto, Vaira˜o, Portugal d Laborato´rio Nacional de Investigac¸a˜o Veterina´ria (LNIV), Vaira˜o, Vila do Conde, Portugal e Laboratoire de Parasitologie-Mycologie, EA 3174-NETEC, Faculte´ de Me´decine, Universite´ de Limoges, Limoges, 87025, France f Centre National de Re´fe´rence (CNR) Toxoplasmose/Toxoplasma Biological Resource Center (BRC), Limoges, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2009 Received in revised form 10 December 2009 Accepted 16 December 2009
Serotyping was previously described as a promising method for typing strains of Toxoplasma gondii. The majority of precedent studies utilized serum samples collected from human patients with different T. gondii-associated pathologies. The aim of this work was to study the applicability of the same procedure for serotyping naturally infected meat-producing animals. An ELISA test based on GRA6 and GRA7 C-terminal polymorphic peptides was used. Peptide GRA6II has polymorphisms specific for the archetypal strains type II, GRA6I/III for strains type I and III, GRA7I for strains type I and GRA7III for strains type III. As reference material, and to validate this approach, serum samples from eleven free-range chickens and fifteen pigs used for Toxoplasma genotypes isolation were selected. These strains integrate the Biological Resource Centre (BRC) ToxoBS Bank. Three serum samples from chickens and two from pigs had serotyping results in agreement with genotyping. Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii, from which no isolate was obtained, were also serotyped. Serotype III appeared significantly more frequent among sheep. Our results show that serotyping still need refinement, but may become a valuable tool for typing Toxoplasma strains from animal origin. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Toxoplasma gondii Serotyping Meat-producing-animals
1. Introduction Toxoplasmosis is the parasitic zoonosis with the highest human incidence (EFSA, 2007). There is a widespread distribution of Toxoplasma infections in a variety of livestock and wild animals. Ingestion of environmental oocysts (by drinking water or eating raw vegetables) and
* Corresponding author at: Laborato´rio de Parasitologia, Centro de Sau´de Publica Dr Gonc¸alves Ferreira, Rua Alexandre Herculano no. 321, 4000-055 Porto, Portugal. Tel.: +351 223401100; fax: +351 223401109. E-mail address: [email protected] (S. Sousa). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.12.025
eating raw or undercooked meat containing tissue cysts stages are the main transmission routes in livestock and in humans (Carme et al., 2002; Moura et al., 2006; Heukelbach et al., 2007). Knowledge about genotype of strains infecting meat producing animal is important. Relationship between Toxoplasma genotype and outcome of human toxoplasmosis has been suggested (Darde´, 2008), and severe cases of human toxoplasmosis due to atypical genotypes have been described after ingestion of meat (Carme et al., 2009; Elbez-Rubinstein et al., 2009). Few studies regarding Toxoplasma infection in meatproducing animals were performed in Portugal. Antibodies were found in 27.1% out of 225 free-range chickens (Dubey
S. Sousa et al. / Veterinary Parasitology 169 (2010) 24–28
et al., 2006), and in 15.6% out of 333 free-range pigs (Sousa et al., 2006) from different areas of Portugal. Prevalence of toxoplasmosis in 1467 sheep randomly collected from 160 farms from Northern Portugal, representing approximately 10% of the ovine population was 17.1% (Sousa et al., 2009b). Also, T. gondii was isolated in a rare case of congenital toxoplasmosis in cattle (Canada et al., 2002). The three clonal lineages I, II and III were described in Portugal, although lineages II and III were found to be dominant (Dubey et al., 2006; Sousa et al., 2006). In fact, the distribution of these three clonal lineages differs between the European countries. The few studies conducted in other European countries, revealed a preponderance of genotype II in domestic animals (Dubey et al., 2005; Dume`tre et al., 2006; Owen and Trees, 1999). Genetic characterization is done subsequently to the isolation of the parasite by mouse inoculation, which is a time consuming and labour technique. This is, at our knowledge, the first attempt to apply the serotyping technology to animal reference material. This method, based on the specific antibody–antigen recognition using polymorphic peptides was already described as a reliable method of typing Toxoplasma strains in humans (Kong et al., 2003; Peyron et al., 2006; Morisset et al., 2008; Sousa et al., 2008). Our objective was to test serotyping in serum samples from animal origin, using peptides derived from GRA6 and GRA7 antigens. 2. Materials and methods
25
2.3. ELISA protocol Immobilizer amino plates (Nunc, Denmark) were coated with each peptide diluted to 10 mg/ml in 0.05 M carbonate/bicarbonate buffer pH 9.6 by incubation overnight at 4 8C. Wells were blocked with a solution of 3% BSA in PBS for 1 h at 37 8C in a moisture atmosphere and then washed 3 times with 0.3% Tween-20 in PBS. Serial dilutions of sera and conjugate (data not show) were performed in order to define the optimal work conditions. Chicken sera were diluted at 1/800 in a solution of 3% BSA in PBS with 0.3% Tween-20, pig sera were diluted at 1/100 in a solution of 3% BSA in PBS, sheep sera were diluted at 1/100 in a solution of 3% BSA in PBS with 0.3% Tween-20 and incubated for 2 h at 37 8C in a moist atmosphere. Wells were washed 3 times with PBS/Tween 0.3%. Anti-chicken IgG peroxidase (KPL, USA) was diluted at 1/32,000 in a solution of 3% BSA in PBS with 0.3% Tween-20, anti-swine IgG peroxidase (KPL, USA) was diluted at 1/1000 in a solution of 3% BSA in PBS, anti-sheep/goat IgG peroxidase labeled conjugate (The Binding Site, UK) was diluted at 1/ 10,000 in a solution of 3% BSA in PBS with 0.3% Tween-20 and incubated for 1 h at 37 8C in a moist atmosphere. Wells were washed 3 times with PBS/Tween 0.3% and developed with o-phenylenediamine (OPD) for 15 min at 37 8C. Reaction was stopped with HCl 3 M and absorbance was measured at 490 nm. Optical density (OD) index was calculated by subtracting the OD of the peptide control from the OD of each peptide. Cut off was set on the mean absorbance readings of negative sera plus 2SD (standard deviation).
2.1. Animal serum samples 2.4. Statistical analysis Serum samples from chickens and pigs from Portugal, from which T. gondii strains were previously isolated and genotyped by a multilocus approach (SAG2 and microsatellite analysis) (Ajzenberg et al., 2005), were selected in order to validate serotyping as a typing method of Toxoplasma strains with animal origin. Eleven strains from pigs and eight strains from chickens were type II. Four strains from pigs and three from chickens were type III (Dubey et al., 2006; Sousa et al., 2006). Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii with a modified agglutination test (Dubey et al., 2006; Sousa et al., 2006, 2009b), from which no isolate was obtained, were also studied. Cut-off values for MAT were 1:5 for chickens, 1:10 for pigs and 1:20 for sheep. Serum samples from 13 Toxoplasma negative chickens, 10 Toxoplasma negative pigs and 16 Toxoplasma negative sheep were used to establish cut-off values. 2.2. Peptides Four strain-specific peptides and a control peptide were studied. These peptides were previously described by Sousa et al. (2008, 2009a). Peptide GRA6II has polymorphisms specific for type II strain; peptide GRA6I/III has polymorphisms specific for type I and III strains; peptide GRA7I has polymorphisms specific for type I strain; peptide GRA7III has polymorphisms specific for type III strain.
Statistical analysis was performed using SPSS Version 12.0 for Windows. The Chi-squared test was performed to assess the statistical significance of differences in the prevalence of serotypes for different hosts. p values of less than 0.05 were considered significant. 3. Results Cut-off values were defined for each peptide according animal species. Chickens were considered positive for GRA6II, GRA6I/III, GRA7I and GRA7III when OD index was equal or higher than cut-off established (0.058, 0.051, 0.180 and 0.244 respectively). Pigs were considered positive for GRA6II, GRA6I/III, GRA7I and GRA7III when OD index was equal or higher than cut-off established (0.212, 0.249, 0.231 and 0.228 respectively). Sheep were considered positive for GRA6II, GRA6I/III, GRA7I and GRA7III when OD index was equal or higher than cut-off established (0.452, 0.377, 0.038 and 0.076 respectively). 3.1. Serotyping vs genotyping Serotyping results from 11 chickens and 15 pigs are reported in Tables 1 and 2. Three chickens (GA147, GA163 and GA166) out of eleven had the same result for serotyping and genotyping (Table 1). Chicken GA43 exclusively reacted with peptide GRA6I/III, suggesting a
S. Sousa et al. / Veterinary Parasitology 169 (2010) 24–28
26
Table 1 Serological reactivity of chicken sera with GRA6 and GRA7 specific peptides. Case no.
Strain
GRA6 II
GA19 GA39 GA40 GA43 GA147 GA163 GA164 GA166 GA167 GA170 GA176
TgCkPr1 TgCkPr3 TgCkPr4 TgCkPr5 TgCkPr6 TgCkPr8 TgCkPr9 TgCkPr7 TgCkPr10 TgCkPr11 TgCkPr12
0.023 0.040 0.025 0.038 0.104 0.990 0.170 0.316 0.319 0.026 0.053
GRA6 I/III 0.009 0.005 0.018 0.902 0.004 0.014 0.003 0.005 0.221 0.008 0.013
GRA7 I
GRA7 III
Serotype
Genotypea
0.123 0.049 0.053 0.089 0.012 0.113 0.279 0.070 0.081 0.069 0.041
0.065 0.043 0.092 0.169 0.051 0.181 0.230 0.073 0.116 0.097 0.086
NR NR NR GRA6I/III II II II/I II II/I/III NR NR
III III II III II II II II II II II
GRA7 III
Serotype
Genotypea
0.162 0.186 0.054 0.028 0.134 0.720 0.069 0.048 0.059 0.033 0.036 0.035 0.042 0.020 0.033
NR NR NR NR II II/III GRA6I/III II NR II/I/III II/I/III NR NR NR NR
II III II III II II II II II II II II II III III
OD index above cut off values is in bold. Cut-off value: GRA6II = 0.058, GRA6I/III = 0.051, GRA7I = 0.180 and GRA7III = 0.244. a Genotype defined by the study of SAG2 locus by PCR-RFLP.
Table 2 Serological reactivity of pig sera with GRA6 and GRA7 specific peptides. Case no.
Strain
PV44 PV116 PV214 PV220 PV227 PV231 PV232 PV238 PV266 PV272 PV274 PV282 PV302 PV311 PV316
TgPiPr1 TgPiPr2 TgPiPr3 TgPiPr4 TgPiPr5 TgPiPr6 TgPiPr7 TgPiPr8 TgPiPr9 TgPiPr10 TgPiPr11 TgPiPr12 TgPiPr13 TgPiPr14 TgPiPr15
GRA6 II 0.104 0.008 0.097 0.056 0.824 0.967 0.185 0.615 0.114 0.742 1.074 0.044 0.080 0.023 0.017
GRA6 I/III 0.052 0.089 0.107 0.099 0.080 0.276 0.544 0.020 0.125 0.477 0.576 0.093 0.073 0.080 0.005
GRA7 I 0.017 0.019 0.023 0.011 0.018 0.090 0.044 0.024 0.156 0.032 0.020 0.049 0.019 0.005 0.008
OD index above cut off values is in bold. Cut-off value: GRA6II = 0.212, GRA6I/III = 0.249, GRA7I = 0.231 and GRA7III = 0.228. a Genotype defined by the study of five microsatelites markers (TUB2, TgM A, W35, B17, B18).
possible infection with a strain type I or type III, which is in agreement with the genotyping results (type III). Two chickens reacted with more than one peptide, suggesting a possible mix infection (GA164 and GA167). From serum samples from infected pigs, only two (PV227 and PV238) out of fifteen had the same results for genotyping and serotyping. One pig infected with a strain type II (PV232), reacted with the peptide GRA6I/III (Table 2). Some serum samples had OD index below the established cut-off for all peptides and were considered non-reactive. Five out of eleven from chickens and nine out of fifteen from pigs were non-reactive (Tables 1 and 2). No correlation was found between MAT titers of the sera and response of the peptides. 3.2. Prediction of T. gondii serotypes in naturally infected animals from Portugal Chickens, pigs and sheep positive for Toxoplasma from which no isolate was obtained were serotyped. Different serotype profiles were defined. Serotype I was defined by the reactivity with peptides GRA7I or GRA7I and GRA6I/III. Serotype II was defined by the single reactivity with
peptide GRA6II. Serotype III was defined by the reactivity with peptides GRA7III or GRA7III and GRA6I/III. Serotype GRA6I/III was defined by the single reactivity with peptide GRA6I/III. Cross-reaction (CR) represents serum samples that have reacted with more than one peptide suggesting a possible mixed infection. Non-reactive (NR) represents the serum samples with OD index below cut-off values for all peptides. Five different reactivity profiles were found for chickens, three different profiles for pigs and five for sheep. Serotype III was significantly more frequently found in sheep (p < 0.05) (Table 3). Serotype II was more frequent in chickens and pigs, while cross-reactivity (CR) was more frequent in pigs and sheep. However, these differences were not significant (p > 0.05) (Table 3). The rate of nonreactive (NR) sera was higher than 50% for the three studied animal species, with no significant difference (p > 0.05) (Table 3). 4. Discussion Serotyping using the GRA6 and GRA7 derived peptides were previously described for serotyping human serum
S. Sousa et al. / Veterinary Parasitology 169 (2010) 24–28 Table 3 Frequencies of serotype according to animal species. Serotype
Chickens, n = 35
Pigs, n = 29
Sheep, n = 50
Significancea, p
I II III GRA6I/III Cross-reaction Non-reactive
0 9 (25.7%) 2 (5.7%) 2 (5.7%) 3 (8.6%) 19 (54.3%)
0 6 (20.7%) 0 0 6 (20.7%) 17 (58.6%)
1 (2%) 4 (8%) 10 (20%) 0 10 (20%) 25 (50%)
0.524 0.078 0.011 0.101 0.303 0.756
a
Significant values are in bold.
samples (Sousa et al., 2008, 2009a). In this work, the applicability of the same procedure for serotyping naturally infected meat-producing animals was evaluated. Published data on meat-producing animals from Portugal refers to chickens (Dubey et al., 2006), pigs (Sousa et al., 2006) and bovines (Canada et al., 2002). No data is available about genotypes isolated from sheep. Match rate between genotyping and serotyping results was 36.4% for chickens and 13.3% for pigs. Peptide GRA6II was specifically recognized by type II strains from chickens and pigs, but with low sensitivity (52.6%). Peptide GRA6I/III also has a low sensitivity (only one out of seven serum samples from chickens and pigs infected with type III strains were recognized). Peptide GRA7III does not recognize any serum sample from animals infected with type III strains. Some serum samples from animals infected with type II strains reacted with the peptide GRA6II but also with the peptides specific for strains type I and III. This cross-reactivity may reflect peptides specificity problems, but it can also result from a natural mixed infection that was not detected by bioassay. Serotyping was used in an attempt to determine the genotypes from the infected animals for which no isolate was obtained. For those animals, serotype II prevails in chickens and pigs, while for sheep serotype III seems to be more prevalent. Results obtained for chickens and pigs are in agreement with the genotyping results of Toxoplasma isolates obtained from chickens and pigs from Portugal (Dubey et al., 2006; Sousa et al., 2006). The higher frequency of serotype III in sheep compared to chickens and pigs could suggest a possible strain selection of type III by sheep, but this hypothesis is unlikely as other studies in Europe described the predominance of genotype II in sheep (Dume`tre et al., 2006; Owen and Trees, 1999). This data may suggest that in Portugal genotype III is more frequent than in France. Similar results were obtained with serum samples from human patients, where serotype III was more frequent among Portuguese patients compared to French patients (Sousa et al., 2008). These data demonstrate that serotyping based on these peptides present some limitations for typing strains of animal origin (low sensitivity, cross-reactions). Other peptides from different antigens should be studied. Since animals are a potential source of human infection, through tissue cysts, detection and genetic characterization of Toxoplasma infection in meat producing animals is important. A method that could characterize the Toxoplasma infection in an efficient and rapid way, could serve
27
to control infections with, for example, virulent atypical strains that are responsible for severe cases of human infections. Acknowledgement The authors would like to thank JP Dubey for kindly supplying the anti-chicken and anti-swine conjugates. References Ajzenberg, D., Dume`tre, A., Darde´, M.L., 2005. Multiplex PCR for typing strains of Toxoplasma gondii. J. Clin. Microbiol. 43, 1940–1943. Canada, N., Meireles, C.S., Rocha, A., da Costa, J.M., Erickson, M.W., Dubey, J.P., 2002. Isolation of viable Toxoplasma gondii from naturally infected aborted bovine fetuses. J. Parasitol. 88, 1247–1248. Carme, B., Bissuel, F., Ajzenberg, D., Bouyne, R., Aznar, C., Demar, M., Bichat, S., Louvel, D., Bourbigot, A.M., Peneau, C., Neron, P., Darde´, M.L., 2002. Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J. Clin. Microbiol. 40, 4037– 4044. Carme, B., Demar, M., Ajzenberg, D., Darde´, M.L., 2009. Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana. Emerg. Infect. Dis. 15, 656–658. Darde´, M.L., 2008. Toxoplasma gondii, ‘‘new’’ genotypes and virulence. Parasite 15, 366–371. Dubey, J.P., Edelhofer, R., Marcet, P., Vianna, M.C.B., Kwok, O.C.H., Lehmann, T., 2005. Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria. Vet. Parasitol. 133, 299–306. Dubey, J.P., Vianna, M.C.B., Sousa, S., Canada, N., Meireles, S., Correia da Costa, J.M., Marcet, P.L., Lehmann, T., Darde´, M.L., Thulliez, P., 2006. Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal. J. Parasitol. 92, 184–186. Dume`tre, A., Ajzenberg, D., Rozette, L., Mercier, A., Darde´, M.L., 2006. Toxoplasma gondii infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping by microsatellite analysis. Vet. Parasitol. 142, 376–379. European Food Safety Authority, 2007. Scientific opinion of the panel on biological hazards on a request from EFSA on Surveillance and monitoring of Toxoplasma in humans, foods and animals. EFSA J. 583, 1– 64. Elbez-Rubinstein, A., Ajzenberg, D., Darde´, M.L., Cohen, R., Dume`tre, A., Yera, H., Gondon, E., Janaud, J.C., Thulliez, P., 2009. Congenital Toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J. Infect. Dis. 199, 280–285. Heukelbach, J., Meyer-Cirkel, V., Moura, R.C.S., Gomide, M., Queiroz, J.A.N., Saweljew, P., Liesenfeld, O., 2007. Waterborne toxoplasmosis, Northeastern Brazil. Emerg. Infect. Dis. 13, 287–289. Kong, J.T., Grigg, M.E., Uyetake, L., Parmley, S., Boothroyd, J.C., 2003. Serotyping of Toxoplasma gondii infections in humans using synthetic peptides. J. Infect. Dis. 187, 1484–1495. Morisset, S., Peyron, F., Lobry, J.R., Garweg, J., Ferrandiz, J., Musset, K., Gomez-Marin, J.E., de la Torre, A., Demar, M., Carme, B., Mercier, C., Garin, J.F., Cesbron-Delauw, M.F., 2008. Serotyping of Toxoplasma gondii: striking homogeneous pattern between symptomatic and asymptomatic infections within Europe and South America. Microbes Infect. 10, 742–747. Moura, L., Bahia-Oliveira, L.M., Wada, M.Y., Jones, J.L., Tuboi, S.H., Carmo, E.H., Ramalho, W.M., Camargo, N.J., Trevisan, R., Grac¸a, R.M.T., Silva, A.J., Moura, I., Dubey, J.P., Garrett, D.O., 2006. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg. Infect Dis. 12, 326–329. Owen, M.R., Trees, A.J., 1999. Genotyping of Toxoplasma gondii associated with abortion in sheep. J. Parasitol. 85, 382–384. Peyron, F., Lobry, J.R., Musset, K., Ferrandiz, J., Gomez-Marin, J.E., Petersen, E., Meroni, V., Rausher, B., Mercier, C., Picot, S., Cesbron-Delauw, M.F., 2006. Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America). Microbes Infect. 8, 2333–2340. Sousa, S., Ajzenberg, D., Canada, N., Freire, L., Correia da Costa, J.M., Darde´, M.L., Thulliez, P., Dubey, J.P., 2006. Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal. Vet. Parasitol. 135, 133–136.
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Sousa, S., Ajzenberg, D., Vilanova, M., Costa, J., Darde´, M.L., 2008. Use of GRA6-derived synthetic peptides in an immunoenzymatic assay to serotype Toxoplasma gondii in human serum samples collected from three continents. Clin. Vaccine Immunol. 15, 1380–1386. Sousa, S., Ajzenberg, D., Marle, M., Aubert, D., Villena, I., Correia da Costa, J., Darde´, M.L., 2009a. Selection of polymorphic peptides from GRA6
and GRA7 sequences of Toxoplasma gondii strains to be used in serotyping. Clin. Vaccine Immunol. 16, 1158–1169. Sousa, S., Thompson, G., Silva, E., Freire, L., Lopes, D., Correia da Costa, J.M., Castro, A., Carvalheira, J., Canada, N., 2009b. Determination of the more adequate modified agglutination test cut-off for serodiagnosis of Toxoplasma gondii infection in sheep. Zoonoses Public Health 56, 252–256.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Serotyping of naturally Toxoplasma gondii infected meat-producing animals Susana Sousa a,c,*, Nuno Canada b,c,d, Jose´ Manuel Correia da Costa a,c, Marie-Laure Darde´ e,f a
Centro de Imunologia e Biologia Parasita´ria (CIBP), CSPGF, INSA Porto, Portugal Instituto de Cieˆncias Biome´dicas de Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal c Centro de Estudos de Cieˆncia Animal (CECA), Universidade do Porto, Vaira˜o, Portugal d Laborato´rio Nacional de Investigac¸a˜o Veterina´ria (LNIV), Vaira˜o, Vila do Conde, Portugal e Laboratoire de Parasitologie-Mycologie, EA 3174-NETEC, Faculte´ de Me´decine, Universite´ de Limoges, Limoges, 87025, France f Centre National de Re´fe´rence (CNR) Toxoplasmose/Toxoplasma Biological Resource Center (BRC), Limoges, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2009 Received in revised form 10 December 2009 Accepted 16 December 2009
Serotyping was previously described as a promising method for typing strains of Toxoplasma gondii. The majority of precedent studies utilized serum samples collected from human patients with different T. gondii-associated pathologies. The aim of this work was to study the applicability of the same procedure for serotyping naturally infected meat-producing animals. An ELISA test based on GRA6 and GRA7 C-terminal polymorphic peptides was used. Peptide GRA6II has polymorphisms specific for the archetypal strains type II, GRA6I/III for strains type I and III, GRA7I for strains type I and GRA7III for strains type III. As reference material, and to validate this approach, serum samples from eleven free-range chickens and fifteen pigs used for Toxoplasma genotypes isolation were selected. These strains integrate the Biological Resource Centre (BRC) ToxoBS Bank. Three serum samples from chickens and two from pigs had serotyping results in agreement with genotyping. Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii, from which no isolate was obtained, were also serotyped. Serotype III appeared significantly more frequent among sheep. Our results show that serotyping still need refinement, but may become a valuable tool for typing Toxoplasma strains from animal origin. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Toxoplasma gondii Serotyping Meat-producing-animals
1. Introduction Toxoplasmosis is the parasitic zoonosis with the highest human incidence (EFSA, 2007). There is a widespread distribution of Toxoplasma infections in a variety of livestock and wild animals. Ingestion of environmental oocysts (by drinking water or eating raw vegetables) and
* Corresponding author at: Laborato´rio de Parasitologia, Centro de Sau´de Publica Dr Gonc¸alves Ferreira, Rua Alexandre Herculano no. 321, 4000-055 Porto, Portugal. Tel.: +351 223401100; fax: +351 223401109. E-mail address: [email protected] (S. Sousa). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.12.025
eating raw or undercooked meat containing tissue cysts stages are the main transmission routes in livestock and in humans (Carme et al., 2002; Moura et al., 2006; Heukelbach et al., 2007). Knowledge about genotype of strains infecting meat producing animal is important. Relationship between Toxoplasma genotype and outcome of human toxoplasmosis has been suggested (Darde´, 2008), and severe cases of human toxoplasmosis due to atypical genotypes have been described after ingestion of meat (Carme et al., 2009; Elbez-Rubinstein et al., 2009). Few studies regarding Toxoplasma infection in meatproducing animals were performed in Portugal. Antibodies were found in 27.1% out of 225 free-range chickens (Dubey
S. Sousa et al. / Veterinary Parasitology 169 (2010) 24–28
et al., 2006), and in 15.6% out of 333 free-range pigs (Sousa et al., 2006) from different areas of Portugal. Prevalence of toxoplasmosis in 1467 sheep randomly collected from 160 farms from Northern Portugal, representing approximately 10% of the ovine population was 17.1% (Sousa et al., 2009b). Also, T. gondii was isolated in a rare case of congenital toxoplasmosis in cattle (Canada et al., 2002). The three clonal lineages I, II and III were described in Portugal, although lineages II and III were found to be dominant (Dubey et al., 2006; Sousa et al., 2006). In fact, the distribution of these three clonal lineages differs between the European countries. The few studies conducted in other European countries, revealed a preponderance of genotype II in domestic animals (Dubey et al., 2005; Dume`tre et al., 2006; Owen and Trees, 1999). Genetic characterization is done subsequently to the isolation of the parasite by mouse inoculation, which is a time consuming and labour technique. This is, at our knowledge, the first attempt to apply the serotyping technology to animal reference material. This method, based on the specific antibody–antigen recognition using polymorphic peptides was already described as a reliable method of typing Toxoplasma strains in humans (Kong et al., 2003; Peyron et al., 2006; Morisset et al., 2008; Sousa et al., 2008). Our objective was to test serotyping in serum samples from animal origin, using peptides derived from GRA6 and GRA7 antigens. 2. Materials and methods
25
2.3. ELISA protocol Immobilizer amino plates (Nunc, Denmark) were coated with each peptide diluted to 10 mg/ml in 0.05 M carbonate/bicarbonate buffer pH 9.6 by incubation overnight at 4 8C. Wells were blocked with a solution of 3% BSA in PBS for 1 h at 37 8C in a moisture atmosphere and then washed 3 times with 0.3% Tween-20 in PBS. Serial dilutions of sera and conjugate (data not show) were performed in order to define the optimal work conditions. Chicken sera were diluted at 1/800 in a solution of 3% BSA in PBS with 0.3% Tween-20, pig sera were diluted at 1/100 in a solution of 3% BSA in PBS, sheep sera were diluted at 1/100 in a solution of 3% BSA in PBS with 0.3% Tween-20 and incubated for 2 h at 37 8C in a moist atmosphere. Wells were washed 3 times with PBS/Tween 0.3%. Anti-chicken IgG peroxidase (KPL, USA) was diluted at 1/32,000 in a solution of 3% BSA in PBS with 0.3% Tween-20, anti-swine IgG peroxidase (KPL, USA) was diluted at 1/1000 in a solution of 3% BSA in PBS, anti-sheep/goat IgG peroxidase labeled conjugate (The Binding Site, UK) was diluted at 1/ 10,000 in a solution of 3% BSA in PBS with 0.3% Tween-20 and incubated for 1 h at 37 8C in a moist atmosphere. Wells were washed 3 times with PBS/Tween 0.3% and developed with o-phenylenediamine (OPD) for 15 min at 37 8C. Reaction was stopped with HCl 3 M and absorbance was measured at 490 nm. Optical density (OD) index was calculated by subtracting the OD of the peptide control from the OD of each peptide. Cut off was set on the mean absorbance readings of negative sera plus 2SD (standard deviation).
2.1. Animal serum samples 2.4. Statistical analysis Serum samples from chickens and pigs from Portugal, from which T. gondii strains were previously isolated and genotyped by a multilocus approach (SAG2 and microsatellite analysis) (Ajzenberg et al., 2005), were selected in order to validate serotyping as a typing method of Toxoplasma strains with animal origin. Eleven strains from pigs and eight strains from chickens were type II. Four strains from pigs and three from chickens were type III (Dubey et al., 2006; Sousa et al., 2006). Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii with a modified agglutination test (Dubey et al., 2006; Sousa et al., 2006, 2009b), from which no isolate was obtained, were also studied. Cut-off values for MAT were 1:5 for chickens, 1:10 for pigs and 1:20 for sheep. Serum samples from 13 Toxoplasma negative chickens, 10 Toxoplasma negative pigs and 16 Toxoplasma negative sheep were used to establish cut-off values. 2.2. Peptides Four strain-specific peptides and a control peptide were studied. These peptides were previously described by Sousa et al. (2008, 2009a). Peptide GRA6II has polymorphisms specific for type II strain; peptide GRA6I/III has polymorphisms specific for type I and III strains; peptide GRA7I has polymorphisms specific for type I strain; peptide GRA7III has polymorphisms specific for type III strain.
Statistical analysis was performed using SPSS Version 12.0 for Windows. The Chi-squared test was performed to assess the statistical significance of differences in the prevalence of serotypes for different hosts. p values of less than 0.05 were considered significant. 3. Results Cut-off values were defined for each peptide according animal species. Chickens were considered positive for GRA6II, GRA6I/III, GRA7I and GRA7III when OD index was equal or higher than cut-off established (0.058, 0.051, 0.180 and 0.244 respectively). Pigs were considered positive for GRA6II, GRA6I/III, GRA7I and GRA7III when OD index was equal or higher than cut-off established (0.212, 0.249, 0.231 and 0.228 respectively). Sheep were considered positive for GRA6II, GRA6I/III, GRA7I and GRA7III when OD index was equal or higher than cut-off established (0.452, 0.377, 0.038 and 0.076 respectively). 3.1. Serotyping vs genotyping Serotyping results from 11 chickens and 15 pigs are reported in Tables 1 and 2. Three chickens (GA147, GA163 and GA166) out of eleven had the same result for serotyping and genotyping (Table 1). Chicken GA43 exclusively reacted with peptide GRA6I/III, suggesting a
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Table 1 Serological reactivity of chicken sera with GRA6 and GRA7 specific peptides. Case no.
Strain
GRA6 II
GA19 GA39 GA40 GA43 GA147 GA163 GA164 GA166 GA167 GA170 GA176
TgCkPr1 TgCkPr3 TgCkPr4 TgCkPr5 TgCkPr6 TgCkPr8 TgCkPr9 TgCkPr7 TgCkPr10 TgCkPr11 TgCkPr12
0.023 0.040 0.025 0.038 0.104 0.990 0.170 0.316 0.319 0.026 0.053
GRA6 I/III 0.009 0.005 0.018 0.902 0.004 0.014 0.003 0.005 0.221 0.008 0.013
GRA7 I
GRA7 III
Serotype
Genotypea
0.123 0.049 0.053 0.089 0.012 0.113 0.279 0.070 0.081 0.069 0.041
0.065 0.043 0.092 0.169 0.051 0.181 0.230 0.073 0.116 0.097 0.086
NR NR NR GRA6I/III II II II/I II II/I/III NR NR
III III II III II II II II II II II
GRA7 III
Serotype
Genotypea
0.162 0.186 0.054 0.028 0.134 0.720 0.069 0.048 0.059 0.033 0.036 0.035 0.042 0.020 0.033
NR NR NR NR II II/III GRA6I/III II NR II/I/III II/I/III NR NR NR NR
II III II III II II II II II II II II II III III
OD index above cut off values is in bold. Cut-off value: GRA6II = 0.058, GRA6I/III = 0.051, GRA7I = 0.180 and GRA7III = 0.244. a Genotype defined by the study of SAG2 locus by PCR-RFLP.
Table 2 Serological reactivity of pig sera with GRA6 and GRA7 specific peptides. Case no.
Strain
PV44 PV116 PV214 PV220 PV227 PV231 PV232 PV238 PV266 PV272 PV274 PV282 PV302 PV311 PV316
TgPiPr1 TgPiPr2 TgPiPr3 TgPiPr4 TgPiPr5 TgPiPr6 TgPiPr7 TgPiPr8 TgPiPr9 TgPiPr10 TgPiPr11 TgPiPr12 TgPiPr13 TgPiPr14 TgPiPr15
GRA6 II 0.104 0.008 0.097 0.056 0.824 0.967 0.185 0.615 0.114 0.742 1.074 0.044 0.080 0.023 0.017
GRA6 I/III 0.052 0.089 0.107 0.099 0.080 0.276 0.544 0.020 0.125 0.477 0.576 0.093 0.073 0.080 0.005
GRA7 I 0.017 0.019 0.023 0.011 0.018 0.090 0.044 0.024 0.156 0.032 0.020 0.049 0.019 0.005 0.008
OD index above cut off values is in bold. Cut-off value: GRA6II = 0.212, GRA6I/III = 0.249, GRA7I = 0.231 and GRA7III = 0.228. a Genotype defined by the study of five microsatelites markers (TUB2, TgM A, W35, B17, B18).
possible infection with a strain type I or type III, which is in agreement with the genotyping results (type III). Two chickens reacted with more than one peptide, suggesting a possible mix infection (GA164 and GA167). From serum samples from infected pigs, only two (PV227 and PV238) out of fifteen had the same results for genotyping and serotyping. One pig infected with a strain type II (PV232), reacted with the peptide GRA6I/III (Table 2). Some serum samples had OD index below the established cut-off for all peptides and were considered non-reactive. Five out of eleven from chickens and nine out of fifteen from pigs were non-reactive (Tables 1 and 2). No correlation was found between MAT titers of the sera and response of the peptides. 3.2. Prediction of T. gondii serotypes in naturally infected animals from Portugal Chickens, pigs and sheep positive for Toxoplasma from which no isolate was obtained were serotyped. Different serotype profiles were defined. Serotype I was defined by the reactivity with peptides GRA7I or GRA7I and GRA6I/III. Serotype II was defined by the single reactivity with
peptide GRA6II. Serotype III was defined by the reactivity with peptides GRA7III or GRA7III and GRA6I/III. Serotype GRA6I/III was defined by the single reactivity with peptide GRA6I/III. Cross-reaction (CR) represents serum samples that have reacted with more than one peptide suggesting a possible mixed infection. Non-reactive (NR) represents the serum samples with OD index below cut-off values for all peptides. Five different reactivity profiles were found for chickens, three different profiles for pigs and five for sheep. Serotype III was significantly more frequently found in sheep (p < 0.05) (Table 3). Serotype II was more frequent in chickens and pigs, while cross-reactivity (CR) was more frequent in pigs and sheep. However, these differences were not significant (p > 0.05) (Table 3). The rate of nonreactive (NR) sera was higher than 50% for the three studied animal species, with no significant difference (p > 0.05) (Table 3). 4. Discussion Serotyping using the GRA6 and GRA7 derived peptides were previously described for serotyping human serum
S. Sousa et al. / Veterinary Parasitology 169 (2010) 24–28 Table 3 Frequencies of serotype according to animal species. Serotype
Chickens, n = 35
Pigs, n = 29
Sheep, n = 50
Significancea, p
I II III GRA6I/III Cross-reaction Non-reactive
0 9 (25.7%) 2 (5.7%) 2 (5.7%) 3 (8.6%) 19 (54.3%)
0 6 (20.7%) 0 0 6 (20.7%) 17 (58.6%)
1 (2%) 4 (8%) 10 (20%) 0 10 (20%) 25 (50%)
0.524 0.078 0.011 0.101 0.303 0.756
a
Significant values are in bold.
samples (Sousa et al., 2008, 2009a). In this work, the applicability of the same procedure for serotyping naturally infected meat-producing animals was evaluated. Published data on meat-producing animals from Portugal refers to chickens (Dubey et al., 2006), pigs (Sousa et al., 2006) and bovines (Canada et al., 2002). No data is available about genotypes isolated from sheep. Match rate between genotyping and serotyping results was 36.4% for chickens and 13.3% for pigs. Peptide GRA6II was specifically recognized by type II strains from chickens and pigs, but with low sensitivity (52.6%). Peptide GRA6I/III also has a low sensitivity (only one out of seven serum samples from chickens and pigs infected with type III strains were recognized). Peptide GRA7III does not recognize any serum sample from animals infected with type III strains. Some serum samples from animals infected with type II strains reacted with the peptide GRA6II but also with the peptides specific for strains type I and III. This cross-reactivity may reflect peptides specificity problems, but it can also result from a natural mixed infection that was not detected by bioassay. Serotyping was used in an attempt to determine the genotypes from the infected animals for which no isolate was obtained. For those animals, serotype II prevails in chickens and pigs, while for sheep serotype III seems to be more prevalent. Results obtained for chickens and pigs are in agreement with the genotyping results of Toxoplasma isolates obtained from chickens and pigs from Portugal (Dubey et al., 2006; Sousa et al., 2006). The higher frequency of serotype III in sheep compared to chickens and pigs could suggest a possible strain selection of type III by sheep, but this hypothesis is unlikely as other studies in Europe described the predominance of genotype II in sheep (Dume`tre et al., 2006; Owen and Trees, 1999). This data may suggest that in Portugal genotype III is more frequent than in France. Similar results were obtained with serum samples from human patients, where serotype III was more frequent among Portuguese patients compared to French patients (Sousa et al., 2008). These data demonstrate that serotyping based on these peptides present some limitations for typing strains of animal origin (low sensitivity, cross-reactions). Other peptides from different antigens should be studied. Since animals are a potential source of human infection, through tissue cysts, detection and genetic characterization of Toxoplasma infection in meat producing animals is important. A method that could characterize the Toxoplasma infection in an efficient and rapid way, could serve
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to control infections with, for example, virulent atypical strains that are responsible for severe cases of human infections. Acknowledgement The authors would like to thank JP Dubey for kindly supplying the anti-chicken and anti-swine conjugates. References Ajzenberg, D., Dume`tre, A., Darde´, M.L., 2005. Multiplex PCR for typing strains of Toxoplasma gondii. J. Clin. Microbiol. 43, 1940–1943. Canada, N., Meireles, C.S., Rocha, A., da Costa, J.M., Erickson, M.W., Dubey, J.P., 2002. Isolation of viable Toxoplasma gondii from naturally infected aborted bovine fetuses. J. Parasitol. 88, 1247–1248. Carme, B., Bissuel, F., Ajzenberg, D., Bouyne, R., Aznar, C., Demar, M., Bichat, S., Louvel, D., Bourbigot, A.M., Peneau, C., Neron, P., Darde´, M.L., 2002. Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J. Clin. Microbiol. 40, 4037– 4044. Carme, B., Demar, M., Ajzenberg, D., Darde´, M.L., 2009. Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana. Emerg. Infect. Dis. 15, 656–658. Darde´, M.L., 2008. Toxoplasma gondii, ‘‘new’’ genotypes and virulence. Parasite 15, 366–371. Dubey, J.P., Edelhofer, R., Marcet, P., Vianna, M.C.B., Kwok, O.C.H., Lehmann, T., 2005. Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria. Vet. Parasitol. 133, 299–306. Dubey, J.P., Vianna, M.C.B., Sousa, S., Canada, N., Meireles, S., Correia da Costa, J.M., Marcet, P.L., Lehmann, T., Darde´, M.L., Thulliez, P., 2006. Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal. J. Parasitol. 92, 184–186. Dume`tre, A., Ajzenberg, D., Rozette, L., Mercier, A., Darde´, M.L., 2006. Toxoplasma gondii infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping by microsatellite analysis. Vet. Parasitol. 142, 376–379. European Food Safety Authority, 2007. Scientific opinion of the panel on biological hazards on a request from EFSA on Surveillance and monitoring of Toxoplasma in humans, foods and animals. EFSA J. 583, 1– 64. Elbez-Rubinstein, A., Ajzenberg, D., Darde´, M.L., Cohen, R., Dume`tre, A., Yera, H., Gondon, E., Janaud, J.C., Thulliez, P., 2009. Congenital Toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J. Infect. Dis. 199, 280–285. Heukelbach, J., Meyer-Cirkel, V., Moura, R.C.S., Gomide, M., Queiroz, J.A.N., Saweljew, P., Liesenfeld, O., 2007. Waterborne toxoplasmosis, Northeastern Brazil. Emerg. Infect. Dis. 13, 287–289. Kong, J.T., Grigg, M.E., Uyetake, L., Parmley, S., Boothroyd, J.C., 2003. Serotyping of Toxoplasma gondii infections in humans using synthetic peptides. J. Infect. Dis. 187, 1484–1495. Morisset, S., Peyron, F., Lobry, J.R., Garweg, J., Ferrandiz, J., Musset, K., Gomez-Marin, J.E., de la Torre, A., Demar, M., Carme, B., Mercier, C., Garin, J.F., Cesbron-Delauw, M.F., 2008. Serotyping of Toxoplasma gondii: striking homogeneous pattern between symptomatic and asymptomatic infections within Europe and South America. Microbes Infect. 10, 742–747. Moura, L., Bahia-Oliveira, L.M., Wada, M.Y., Jones, J.L., Tuboi, S.H., Carmo, E.H., Ramalho, W.M., Camargo, N.J., Trevisan, R., Grac¸a, R.M.T., Silva, A.J., Moura, I., Dubey, J.P., Garrett, D.O., 2006. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg. Infect Dis. 12, 326–329. Owen, M.R., Trees, A.J., 1999. Genotyping of Toxoplasma gondii associated with abortion in sheep. J. Parasitol. 85, 382–384. Peyron, F., Lobry, J.R., Musset, K., Ferrandiz, J., Gomez-Marin, J.E., Petersen, E., Meroni, V., Rausher, B., Mercier, C., Picot, S., Cesbron-Delauw, M.F., 2006. Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America). Microbes Infect. 8, 2333–2340. Sousa, S., Ajzenberg, D., Canada, N., Freire, L., Correia da Costa, J.M., Darde´, M.L., Thulliez, P., Dubey, J.P., 2006. Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal. Vet. Parasitol. 135, 133–136.
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