J. Parasitol., 95(1), 2009, pp. 235–237 ᭧ American Society of Parasitologists 2009

Toxoplasma gondii Isolates From Free-Range Chickens From the Northeast Region of

L. N. de Oliveira, L. M. Junior*, C. F. de Melo†, J. C. Ramos ‡, C. M. L. Bevilaqua§, S. S. Azevedo§, V. Muradian, D. A. F. V. Arau´jo࿣, J. P. Dubey#, and S. M. Gennari, Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universita´ria, CEP 05508-270, Sa˜o Paulo, Sa˜o Paulo, Brazil; *Centro de Cieˆncias Agra´rias e Ambientais, Universidade Federal do Maranha˜o, Chapadinha, Maranha˜o, Brazil; †Faculdade de Agronomia e Medicina Veterina´ria, Universidade de Brası´lia, UNB, Brası´lia, Distrito Federal , Brazil; ‡Departamento de Medicina Veterina´ria, Universidade Federal Rural de , , Pernambuco, Brasil; §Faculdade de Veterina´ria, Universidade Estadual do Ceara´, , Ceara´, Brazil; ࿣Unidade Acadeˆmica de Medicina Veterina´ria, Centro de Sau´de e Tecnologia Rural, Universidade Federal de Campina Grande, Patos, Paraı´ba, Brasil; #To whom correspondence should be addressed. Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, United States Department of , Beltsville, Maryland 20705-2350. e-mail: [email protected]

ABSTRACT: The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the strains prevalent in their environment chickens is a good indicator of the prevalence of T. gondii oocysts in (Ruiz and Frenkel, 1980; Dubey, Applewaite et al., 2007). Recently, the soil because chickens feed from the ground. The prevalence of T. this animal species was used to collect T. gondii isolates from soil gondii in 152 free-range chickens (Gallus domesticus) from 22 munic- worldwide (Dubey, 2009), particularly from Brazil. We previously sur- ipalities in 7 northeastern states (Pernambuco, do Norte, veyed FR chickens from the southeastern (Dubey et al., 2002; Dubey, Maranha˜o, , Ceara´, , and ) of Brazil was deter- Graham et al., 2003), south (Dubey, Navarro et al., 2003; Dubey, Sundar mined. Antibodies to T. gondii were assayed by the modified aggluti- et al., 2007), and Amazon and northern areas (Dubey et al., 2006, Dub- nation test (MAT); 81 (53.3 %) chickens had titers of 1:5 in 26, 1:10 ey, Sundar et al., 2007) of Brazil. The aim of the present study was to in 9, 1:20 in 4, 1:40 in 1, 1:80 in 6, 1:160 in 6, 1:320 in 13, 1:640 in biologically characterize T. gondii isolates from chickens from the 6, 1:1,280 in 3, 1:2,560 in 6, and 1:5,120 or higher in 1. Hearts and northeastern region of Brazil. brains of 81 seropositive chickens were bioassayed individually in mice. From October 2006 to August 2007, 152 chickens were obtained Toxoplasma gondii was isolated from 23 chickens with MAT titers of from 22 different from 7 states located in the northeastern 1:5 or higher; the isolates were designated TgCKBr165-187. Five iso- region of Brazil (Table I). Heart, head, and serum from each chicken lates killed all infected mice. Results indicate widespread contamination were transported under refrigeration, by air to the Veterinary Faculty, of rural environment in Brazil with T. gondii oocysts. University of Sa˜o Paulo, Sa˜o Paulo, Brazil, for T. gondii examination. Sera of chickens were tested for T. gondii antibodies using modified Toxoplasma gondii infections are widely prevalent in human beings agglutination test (MAT) starting in a 1:5 dilution and end titrated using and other animals worldwide (Dubey and Beattie, 1988; Tenter et al., a 2-fold serial dilution as described by Dubey and Desmonts (1987). 2000). One of the most prevalent regions of T. gondii in humans and Brains and hearts of all seropositive chickens (MAT 1:5 or higher) animals is Brazil, with infection levels that can reach 100% in some were pooled, digested in pepsin, and bioassayed in Swiss Webster mice areas (Bahia-Oliveira et al., 2003; Sobral et al., 2005; Cavalcante et al., for T. gondii infection (Dubey et al., 2002). The inoculated mice were 2006; de Moura et al., 2006). examined for T. gondii infection as described previously (Dubey et al., Because free-range (FR) chickens become infected mostly by feeding 2002); they were considered infected with T. gondii when tachyzoites from ground contaminated with oocysts, prevalence of T. gondii in or tissue cysts were found in tissues.

TABLE I. Isolation of Toxoplasma gondii from free-range chickens from northeastern region of Brazil by of origin.

Municipality State No. examined No. seopositive T. gondii isolated Strain designation

Caruaru Pernambuco 3 2 1 TgCkBr165 Gravata´ Pernambuco 2 2 1 TgCkBr166 Camaragibe Pernambuco 12 5 0 Paudalho Pernambuco 3 1 0 Ouro Branco 27 4 1 TgCkBr167 Rosado Rio Grande do Norte 4 1 0 Caraı´bas Rio Grande do Norte 3 3 0 Apodi Rio Grande do Norte 3 2 0 Felipe Guerra Rio Grande do Norte 1 1 1 TgCkBr168 Serra do Mel Rio Grande do Norte 4 3 1 TgCkBr169 Alexandria Rio Grande do Norte 1 1 0 Barau´na Rio Grande do Norte 4 2 1 TgCkBr170 Chapadinha Maranha˜o 20 14 2 TgCkBr171, TgCkBr172 Jacobina Bahia 13 4 1 TgCkBr173 Boa Vista Bahia 2 1 0 Cae´m Bahia 5 5 3 TgCkBr174, TgCkBr175, TgCkBr176 Cascavel Ceara´ 15 11 3 TgCkBr177, TgCkBr178, TgCkBr179 Quixada Ceara´ 10 6 3 TgCkBr180, TgCkBr181, TgCkBr182 Sergipe 7 4 1 TgCkBr183 Sergipe 5 1 0 Porto Real Alagoas 3 3 2 TgCkBr184, TgCkBr185 Penedo Alagoas 5 5 2 TgCkBr186, TgCkBr187 Total 152 81 23

235 236 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 1, FEBRUARY 2009

TABLE II. Prevalence of antibodies and viable Toxoplasma gondii in al., 2002; Pena et al., 2008). The results of the present study affirm that 152 free-range chickens from the northeastern region of Brazil. T. gondii infections are widely prevalent in FR chickens in Brazil, in- dicating contamination of soil with oocysts. We thank Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo, No. of chickens Sa˜o Paulo, for the fellowship to L. N. de Oliveira and CNPq, Brazil, Antibody titer Bioassayed T. gondii isolated % for fellowship to S. M. Gennari and C. M. L. Bevilaqua. We also thank Dr. Eugenia M. D. Oliveira (in memorium), from Universidade Federal 5 26 1 3.8 da Bahia, for the chickens from Bahia. 10 9 1 11.1 20 4 2 50.0 LITERATURE CITED 40 1 0 0.0 BAHIA-OLIVEIRA, L. M. G., J. L. JONES,J.AZEVEDO-SILVA,C.C.F. 80 6 1 16.7 ALVES,F.ORE´ FICE, AND D. G. ADDISS. 2003. Highly endemic, wa- 160 6 2 33.3 terborne toxoplasmosis in north State, Brazil. 320 13 8 61.5 Emerging Infectious Diseases 9: 55–62. 640 6 3 50.0 CAVALCANTE, G. T., D. M. AGUIAR,D.CHIEBAO,J.P.DUBEY,V.L.A. 1,280 3 1 33.3 RUIZ,R.A.DIAS,L.M.A.CAMARGO,M.B.LABRUNA, AND S. M. 2,560 6 4 66.7 GENNARI. 2006. Seroprevalence of Toxoplasma gondii antibodies 5,120 1 0 0.0 in cats and pigs from rural western Amazon, Brazil. Journal of Total 81 23 28.4 Parasitology 92: 863–864. DE MOURA, L., L. M. G. BAHIA-OLIVEIRA,M.Y.WADA,J.L.JONES,S. H. TUBOI,E.H.CARMO,W.M.RAMALHO,N.J.CAMARGO,R.TREV- ISAN,R.M.T.GRAC¸A ET AL. 2006. Waterborne outbreak of toxo- Antibodies to T. gondii were found in 81 (53.3%) of the 152 exam- plasmosis, Brazil, from field to gene. Emerging Infectious Diseases ined chickens, with titers from 5 to 5,120 (Table II). In all studied 12: 326–329. municipalities, at least 1 T. gondii seropositive chicken was detected. DUBEY, J. P. 2009. Toxoplasma gondii infections in chickens (Gallus Viable T. gondii was isolated from 23 chickens (Table II). The number domesticus): Prevalence, clinical disease, diagnosis, and public of isolates increased with the antibody titer seroprevalence as found health significance. Journal of Zoonosis and Public Health. (In previously with tissues of many species of animals, including cats from press). Brazil (Pena et al., 2006). It is of interest that viable T. gondii was ———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man. isolated from a chicken with a low MAT titer of 1:5, confirming earlier CRC Press, Boca Raton, Florida, 220 p. findings (Dubey et al., 2002). ———, AND G. DESMONTS. 1987. Serological responses of equids fed Five of the 23 isolates killed all T. gondii-exposed mice (Table III); Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337– the mice died of toxoplasmic pneumonia between 12 and 25 days post- 339. inoculation (PI). These results confirm our previous findings that mouse ———, L. APPLEWHAITE,N.SUNDAR,G.V.VELMURUGAN,L.A.BAN- virulent T. gondii circulate in asymptomatic hosts in Brazil (Dubey et DINI,O.C.H.KWOK,R.HILL, AND C. SU. 2007. Molecular and

TABLE III. Isolation of Toxoplasma gondii from free-range chickens from the northeastern region of Brazil.

Mouse bioassay No.ofmice No. died/no. Mortality Day of death State Chicken no. Antibody titer per group infected (%) (PI)

Pernambuco TgCkBr165 160 3 0/2 0 * TgCkBr166 640 5 0/5 0 * Rio Grande do Norte TgCkBr167 640 3 0/1 0 * TgCkBr168 5 3 0/2 0 * TgCkBr169 2,560 3 2/2 100.0 17, 18 TgCkBr170 320 3 0/3 0 * Maranha˜o TgCkBr171 10 2 0/1 0 * TgCkBr172 320 1 0/1 0 * Bahia TgCkBr173 2,560 3 0/3 0 * TgCkBr174 320 3 0/2 0 * TgCkBr175 1,280 3 0/2 0 * TgCkBr176 320 3 0/3 0 * Ceara´ TgCkBr177 320 3 3/3 100.0 15, 16, 18 TgCkBr178 20 3 1/3 33.3 31 TgCkBr179 320 3 0/3 0 * TgCkBr180 80 3 0/1 0 * TgCkBr181 2,560 3 3/3 100.0 14, 20, 21 TgCkBr182 640 3 3/3 100.0 21, 25, 25 Sergipe TgCkBr183 20 2 0/2 0 * Alagoas TgCkBr184 320 3 0/3 0 * TgCkBr185 160 3 0/2 0 * TgCkBr186 2,560 3 3/3 100.0 13, 13, 13 TgCkBr187 320 3 0/3 0 *

PI ϭ postinoculation. * Survived. RESEARCH NOTES 237

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