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Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil
Veterinary Parasitology 169 (2010) 190–192
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil Lilian S. Catenacci a, Juliana Griese a, Rodrigo C. da Silva b, Helio Langoni b,* a
Instituto de Estudos Socioambientais do sul da Bahia (IESB), Rua Major Homem d’El Rey, 147 Cidade Nova, Ilhe´us, CEP 45652-180, BA, Brazil Departamento de Higiene Veterina´ria e Sau´de Pu´blica (DHVSP), Faculdade de Medicina Veterina´ria e Zootecnia (FMVZ), Universidade Estadual Paulista (UNESP), Campus de Botucatu, Distrito de Rubia˜o Ju´nior, s/n, Botucatu, CEP 18618-000, SP, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 July 2009 Received in revised form 12 December 2009 Accepted 14 December 2009
The zoonoses toxoplasmosis and leishmaniasis are important worldwide and also affect wild animals. Thus, the present study aimed to assess the prevalence of Leishmania spp. and Toxoplasma gondii antibodies in 52 serum samples from captive crab-eating foxes (Cerdocyon thous) kept in 17 zoos in Sa˜o Paulo State, Brazil. Modified agglutination test (MAT, for toxoplasmosis) and indirect fluorescent antibody test (IFAT, for toxoplasmosis and leishmaniasis) were employed with heterologous anti-dog immunoglobulin. Antibodies to T. gondii were found in 19.2% animals, with an almost perfect concordance (k = 0.86; standard error = 9.31%; CI95% = 68.25–104.76%; P < 0.0001) and a strong correlation coefficient (rs = 0.87; P < 0.0001), which allows the use of heterologous anti-dog immunoglobulin to perform IFAT for toxoplasmosis in crab-eating foxes. No sample was positive for Leishmania spp. Toxoplasmosis infection occurs in wild animals from the studied Brazilian zoos, which indicates a probable environmental contamination, highlighting the importance of appropriate zoo management and the action of the parasite as a sentinel to human infection. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Cerdocyon thous Leishmania spp. Toxoplasma gondii MAT IFAT Heterologous anti-serum immunoglobulin
1. Introduction Toxoplasma gondii and Leishmania spp. are parasite protozoans that infect wild and domestic animals worldwide. They also affect humans, especially immunosuppressed patients. T. gondii can be detected in several wild Canidae (Silva et al., 1997; Gennari et al., 2004). In addition, wild animals are good indicators of environmental contamination by T. gondii, acting as sentinels for infections in humans and production animals, which requires the adoption of epidemiological and sanitary control measures (Gennari et al., 2004). The crab-eating fox Cerdocyon thous has been considered an important sentinel for T. gondii infection in humans and a potential reservoir of Leishmania spp., showing high
* Corresponding author. Tel.: +55 14 38116270; fax: +55 14 38116075. E-mail address: [email protected] (H. Langoni). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.12.019
prevalence in both cases (Deane and Deane, 1955; Gennari et al., 2004) together with lesions caused by L. chagasi (Silva et al., 2000) or L. infantum (Courtenay et al., 2002a,b). In wild environments, many crab-eating foxes are hunted and commercialized for animal or human consumption, being frequently ingested undercooked. Studies with Leishmania spp. in wild animals has increased in Brazil due to the importance of these species in the life cycle of leishmaniasis, as detected by Luppi et al. (2008). The importance of crab-eating foxes as reservoirs depends on their ability to successfully transmit the infection to sand flies rather than on their infection rate; it also depends on their capability of (re)introducing the pathogen into Leishmania-free dog populations, associated with epidemiological captive conditions of zoos favoring the infection (Dressen, 1990). Thus, the present study aimed to determine the prevalence of T. gondii and Leishmania spp. infection in C. thous kept in captivity in 17 zoos from Sa˜o Paulo State, Brazil.
L.S. Catenacci et al. / Veterinary Parasitology 169 (2010) 190–192
2. Materials and methods This study was conducted in 17 zoos (A–Q) from Sa˜o Paulo State, as well as in Sa˜o Paulo State University (UNESP), Botucatu Campus, Sa˜o Paulo State, Brazil. A total of 52 crab-eating foxes (C. thous), 25 males and 27 females, kept under captive conditions in zoos were included, except animals younger than 6 months and pregnant or lactating females with average weight of 6.6 kg (9.3 4.2 kg). The study was approved by the Ethics Committee in Animal Experimentation of the School of Veterinary Medicine and Animal Science, UNESP, Botucatu Campus. The animals were anesthetized by means of intramuscular injection. Atropine (0.05 mg/kg, Prado1), and ketamine (10 mg/kg, Fort Dodge1): xylazine (1 mg/kg Sedazina, Fort Dodge1) were used for pre-anesthesia and induction of anesthesia, respectively. Blood samples were collected from the jugular vein of all animals, sent to the Zoonosis Research Center (NUPEZO, UNESP), centrifuged at 1600 g during 10 min, and kept at 20 8C until the serological tests. All serum samples were researched for T. gondii antibodies through modified agglutination test (MAT) (Desmonts and Remington, 1980), a gold-standard test, and indirect fluorescent antibody test (IFAT) (Camargo, 1974); for Leishmania spp. antibody research, only IFAT was employed. In IFAT, both for toxoplasmosis and for leishmaniasis, a heterologous (anti-dog) anti-serum conjugated to fluorescein isothiocyanate, kindly supplied by the Zoonosis Control Center of Sa˜o Paulo, was used. To determine the impact of using this heterologous immunoglobulin, we compared MAT and IFAT results for toxoplasmosis. For MAT, a clear-cut button-shaped deposit of parasite suspension at the bottom of the well was interpreted as negative, and a carpet of agglutinated organisms, ranging from 50 to 100%, was considered to be positive. IFAT was positive when up to 50% of tachyzoite or promastigote forms per field presented complete fluorescence. The software BioEstat 5.0 was used to determine the concordance (kappa index: k) between MAT and IFAT (Rosner, 2006), whereas Spearman’s correlation coefficient (rs) was used to determine the concordance between titers, adopting a significance level of 5%. Sensitivity (S), specifity (E), and positive (PPV) and negative predictive values (NPV) in IFAT for toxoplasmosis were manually calculated. 3. Results and discussion T. gondii antibodies were detected in 10/52 (19.2%) serum samples from 6/17 (35.3%) zoos (Table 1). Eight samples (15.39%) presented a MAT titer of 16, four of which (50.0%) were from the same zoo; two of these four animals were kept in the same enclosure, one in a neighboring enclosure and the fourth in a somewhat distant enclosure. Nine serum samples were positive to IFAT, seven of which (13.5%) had titer 16 and two (3.8%) had titer 64. T. gondii serological results are shown in Table 2. IFAT presented high sensitivity (88.89%), specificity (97.62%), and positive (88.89%) and negative predictive values (97.62), indicating an almost perfect concordance with MAT (k = 0.86; standard
191
Table 1 Distribution of IgG Toxoplasma gondii antibodies of Cerdocyon thous sera samples from six positive Sa˜o Paulo state zoos. Zoos
Positive/tested; percentage
A B C D E F
1/2; 1/2; 2/3; 1/2; 1/2; 4/6;
Total
10/52; 19.23
50.00 50.00 66.67 50.00 50.00 66.67
error = 9.31%; CI95% = 68.25–104.76%; P < 0.0001) (Siegel and Castellan, 1988), although the latter detected more positive samples with titer 16. The correlation between titers of both tests was positive, presenting a strong correlation coefficient (rs = 0.87; P < 0.0001). All serum samples assayed for Leishmania spp. antibodies through IFAT were negative. The frequency of T. gondii antibodies was low (19.2%) in 6/17 zoos, compared to the results of Gennari et al. (2004), who detected 9/15 (60%) infected C. thous in Sa˜o Paulo and Parana´ States, Brazil. In spite of the low titers, 50% positive serum samples were detected in animals from the same zoo (Table 1). This fact could reflect an error in the institution management, facilitating the contact with the infective agent by ingesting oocysts from the environment, food and water, as well as bradyzoites from tissue cysts of intermediate hosts with chronic infection, or tachyzoites present inside immune cells located in cavity liquids (Tenter et al., 2000). Wild carnivores are sentinels for T. gondii infection in humans, especially considering environmental contamination (Dubey and Beattie, 1988). Zoo animals are susceptible to this infection since domestic cats are frequently present in these places (Franti et al., 1979; Tenter et al., 2000), along with synanthropic animals, which constitute potential prey for carnivores (Zarnke et al., 2000). Thus, appropriate food and water management is important to prevent and control T. gondii infection in zoos (Tenter et al., 2000). IFAT using heterologous immunoglobulin to detect T. gondii antibodies had high sensitivity and specificity, showing an almost perfect concordance and a strong correlation coefficient. These results allow the use of heterologous immunoglobulin for wild animals, especially dog immunoglobulin for C. thous antibody research, confirming the use of MAT as an epidemiological tool to assay this wild species for toxoplasmosis since it is a fast test that does not require expensive reagents or equipment (Desmonts and Remington, 1980). On the other hand, no animal was positive to Leishmania spp. Crab-eating fox involvement in visceral leishmaniasis Table 2 Frequency of Toxoplasma gondii antibodies by indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT). MAT results
IFAT results Negative
16
64
Negative 16
41; 80.4% 1; 2.0%
1; 2.0% 6; 11.8%
0; 0.0% 2; 3.9%
192
L.S. Catenacci et al. / Veterinary Parasitology 169 (2010) 190–192
epidemiology has been reported in several Brazilian studies (Deane and Deane, 1955; Mello et al., 1988; Lainson et al., 1990; MacDonald and Courtenay, 1993; MacDonald et al., 1994; Curi et al., 2006), especially in tropical areas due to the presence of sand flies, Lutzomyia longipalpis, and infected animals, mainly dogs and some other carnivores (Lainson et al., 1990). As some of the visited zoos are located in leishmaniasis occurrence areas such as Arac¸atuba City, Sa˜o Paulo State (Lindoso and Goto, 2006), the possibility of infection must be considered. Luppi et al. (2008) reported the presence of one infected C. thous in a Brazilian zoo and several wild and synanthropic animals living in the same area, including large populations of opossums and pigeons. The presence of these animals, especially birds, represents a risk factor of visceral leishmaniasis (Diniz et al., 2008), serving as nutrition source for Lutzomyia longipalpis. Silva et al. (2000) reported this infection in a free-ranging C. thous in Minas Gerais State, Brazil; the animal had a lesion caused by L. chagasi in its muzzle. Besides domestic dogs, crab-eating foxes have been identified as reservoirs for human visceral leishmaniasis in Brazil (Courtenay et al., 1996; Deane and Deane, 1962; Lainson et al., 1969). However, the crab-eating fox populations do not maintain a transmission cycle independently of domestic dogs; they are unable to introduce the parasite into Leishmania-free dog populations (Courtenay et al., 2002a,b). Wild animals can be infected, presenting clinical signs and transmitting the parasite under captive conditions in endemic areas. Thus, C. thous presents great importance under free and captive conditions; however, further studies on the environmental transmission of T. gondii, the epidemiology of Leishmania in C. thous and the comparison between MAT and IFAT using heterologous immunoglobulin are needed to understand the epidemiological role of C. thous and its importance for public health and control in zoos. Acknowledgements This study was supported by Training Special Program (PET – SESu/MEC). We thank Dr. Mariaˆngela Lozano Cruz and Rodrigo L. Maruccio for helping with field procedures, and Andre´ Peres Barbosa de Castro, Moˆnika Vogl Sampaio, Juliana Satie Carvalho Akaboshi for helping in the laboratory. We also thank UNESP and all zoos for logistic support. References Camargo, M.E., 1974. Introduc¸a˜o a`s te´cnicas de imunofluoresceˆncia. Rev. Bras. Patol. Clin. 10, 143–169. Courtenay, O., Quinnell, R.J., Garcez, L.M., Dye, C., 2002. Low infectiousness of a wildlife host of Leishmania infantum: the crab-eating fox is not important for transmission. Parasitolology 125, 407–414. Courtenay, O., Quinnell, R.J., Garcez, L.M., Shaw, J.J., Dye, C., 2002. Infectiousness in a cohort of Brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission. J. Infect. Dis. 186, 1314–1320.
Courtenay, O., Santana, E.W., Johnson, P., Vasconcelos, I.A.B., Vasconcelos, A.W., 1996. Visceral leishmaniasis in the hoary zorro Dusicyon vetulus: a case of mistaken identity. Trans. R. Soc. Trop. Med. Hyg. 90, 498– 502. Curi, N.H.A., Miranda, I., Talamoni, S.A., 2006. Serologic evidence of leishmania infection in free-ranging wild and domestic canids around a Brazilian national park. Mem. Inst. Oswaldo Cruz 101, 99–101. Deane, M.P., Deane, L.M., 1955. Observac¸o˜es preliminares sobre a importaˆncia comparativa do homen, do ca˜o e da raposa (Lycalopex vetulus) como reservato´rios da Leishmania donovani, em a´reas endeˆmicas de calazar no Ceara´. O Hospital XLVIII 61–76. Deane, M.P., Deane, L.M., 1962. Visceral leishmaniasis in Brazil: geographical distribution and transmission. Rev. Inst. Med. Trop. 4, 198–212. Desmonts, G., Remington, J.S., 1980. Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity and specificity. J. Clin. Microbiol. 11, 562–568. Diniz, S.A., Silva, F.L., Carvalho Neta, A.V., Bueno, R., Guerra, R.M.S.N.C., Abreu-Silva, A.L., Santos, R.L., 2008. Animal reservoirs for visceral leishmaniasis in densely populated urban areas. J. Infect. Dev. Countries 2, 24–33. Dressen, D.W., 1990. Toxoplasma gondii infections in wildlife. J. Am. Vet. Med. Assoc. 196, 274–276. Dubey, J.P., Beattie, C.P., 1988. Toxoplasmosis in dogs. Canine Pract. 12, 7– 28. Franti, C.E., Riemann, H.P., Behymer, D.E., Suther, D., Howarth, J.A., Ruppanner, R., 1979. Prevalence of Toxoplasma gondii antibodies in wild and domestic animals in northern California. J. Am. Vet. Med. Assoc. 169, 901–906. Gennari, S.M., Canno´n-Franco, W.A., Yai, L.E.O., De Souza, S.L.P., Santos, L.C., Farias, N.A.R., Ruas, J., Rossi, F.W., Gomes, A.B., 2004. Seroprevalence of Toxoplasma gondii antibodies from wild canids from Brazil. Vet. Parasitol. 121, 337–340. Lainson, R., Dye, C., Shaw, J.J., MacDonald, D.W., Courtenay, O., Souza, A.A.A., Silveira, F.T., 1990. Amazonian visceral leishmaniases—distribution of the vector Lutzomya longipalpis (Lutz & Neiva) in relation to the fox Cerdocyon thous (Linn) and the efficiency of this reservoir host as a source of infection. Mem. Inst. Oswaldo Cruz 85, 135–137. Lainson, R., Shaw, J.J., Lins, Z.C., 1969. Leishmaniasis in Brazil: IV. The Fox, Cerdocyon thous (L.) as a reservoir of Leishmania donovani in Para state, Brazil. Trans. R. Soc. Trop. Med. Hyg. 63, 741–745. Lindoso, J.A.L., Goto, H., 2006. Leishmaniose visceral: Situac¸a˜o atual e perspectivas futuras. BEPA 26, 7–11. Luppi, M.M., Malta, M.C.C., Silva, T.M.A., Silva, F.L., Motta, R.O.C., Miranda, I., Ecco, R., Santos, R.L., 2008. Visceral leishmaniasis in captive wild canids in Brazil. Vet. Parasitol. 155, 146–151. MacDonald, D.W., Courtenay, A., 1993. Wild and domestic canids as reservoirs of American visceral leishmaniasis in Amazonia. Symp. Zool. Soc. Lond. 65, 465–479. MacDonald, D.W., Courtenay, A., Lainson, R., Shaw, J.J., Dye, C., 1994. Epidemiology of canine leishmaniasis: a comparative serological study of the dogs and foxes in Amazon Brazil. Parasitology 109, 273–279. Mello, D.A., Rego Jr., F.A., Osmozo, E., Nunes, V.L.B., 1988. Cerdocyon thous (L.) (Carnivora, Canidae) naturally infected with L. donovani chagasi (Cunha & Chagas, 1973) in Corumba´ (Mato Grosso do Sul state, Brazil). Mem. Inst. Oswaldo Cruz 83, 259. Rosner, B., 2006. Fundamentals of Biostatistics, 6th ed. Duxbury Press, Boston, MA, USA, p. 868. Siegel, S., Castellan, N., 1988. Nonparametric Statistics for the Behavioral Sciences, 2nd ed. McGraw-Hill, New York, NY, USA, pp. 284–285. Silva, D.A.O., Cabral, D.D., Bernardina, B.L.D., Souza, M.A., Mineo, J.R., 1997. Detection of Toxoplasma gondii-specific antibodies in dogs. A comparative study of immunoenzymatic, immunofluorescent and haemagglutination titers. Mem. Inst. Oswaldo Cruz 92, 785–789. Silva, E.S., Pirmez, C., Gontijo, C.M., Fernandes, O., Brazil, R.P., 2000. Visceral leishmaniasis in the crab-eating fox (Cerdocyon thous) in South-East Brazil. Vet. Rec. 147, 421–422. Tenter, A.M., Heckeroth, A.R., Weiss, L.M., 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30, 1217–1258. Zarnke, R.L., Dubey, J.P., Kwok, O.C., Ver Hoef, J.M., 2000. Serologic survey for Toxoplasma gondii in selected wildlife species from Alaska. J. Wildl. Dis. 36, 219–224.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil Lilian S. Catenacci a, Juliana Griese a, Rodrigo C. da Silva b, Helio Langoni b,* a
Instituto de Estudos Socioambientais do sul da Bahia (IESB), Rua Major Homem d’El Rey, 147 Cidade Nova, Ilhe´us, CEP 45652-180, BA, Brazil Departamento de Higiene Veterina´ria e Sau´de Pu´blica (DHVSP), Faculdade de Medicina Veterina´ria e Zootecnia (FMVZ), Universidade Estadual Paulista (UNESP), Campus de Botucatu, Distrito de Rubia˜o Ju´nior, s/n, Botucatu, CEP 18618-000, SP, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 July 2009 Received in revised form 12 December 2009 Accepted 14 December 2009
The zoonoses toxoplasmosis and leishmaniasis are important worldwide and also affect wild animals. Thus, the present study aimed to assess the prevalence of Leishmania spp. and Toxoplasma gondii antibodies in 52 serum samples from captive crab-eating foxes (Cerdocyon thous) kept in 17 zoos in Sa˜o Paulo State, Brazil. Modified agglutination test (MAT, for toxoplasmosis) and indirect fluorescent antibody test (IFAT, for toxoplasmosis and leishmaniasis) were employed with heterologous anti-dog immunoglobulin. Antibodies to T. gondii were found in 19.2% animals, with an almost perfect concordance (k = 0.86; standard error = 9.31%; CI95% = 68.25–104.76%; P < 0.0001) and a strong correlation coefficient (rs = 0.87; P < 0.0001), which allows the use of heterologous anti-dog immunoglobulin to perform IFAT for toxoplasmosis in crab-eating foxes. No sample was positive for Leishmania spp. Toxoplasmosis infection occurs in wild animals from the studied Brazilian zoos, which indicates a probable environmental contamination, highlighting the importance of appropriate zoo management and the action of the parasite as a sentinel to human infection. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Cerdocyon thous Leishmania spp. Toxoplasma gondii MAT IFAT Heterologous anti-serum immunoglobulin
1. Introduction Toxoplasma gondii and Leishmania spp. are parasite protozoans that infect wild and domestic animals worldwide. They also affect humans, especially immunosuppressed patients. T. gondii can be detected in several wild Canidae (Silva et al., 1997; Gennari et al., 2004). In addition, wild animals are good indicators of environmental contamination by T. gondii, acting as sentinels for infections in humans and production animals, which requires the adoption of epidemiological and sanitary control measures (Gennari et al., 2004). The crab-eating fox Cerdocyon thous has been considered an important sentinel for T. gondii infection in humans and a potential reservoir of Leishmania spp., showing high
* Corresponding author. Tel.: +55 14 38116270; fax: +55 14 38116075. E-mail address: [email protected] (H. Langoni). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.12.019
prevalence in both cases (Deane and Deane, 1955; Gennari et al., 2004) together with lesions caused by L. chagasi (Silva et al., 2000) or L. infantum (Courtenay et al., 2002a,b). In wild environments, many crab-eating foxes are hunted and commercialized for animal or human consumption, being frequently ingested undercooked. Studies with Leishmania spp. in wild animals has increased in Brazil due to the importance of these species in the life cycle of leishmaniasis, as detected by Luppi et al. (2008). The importance of crab-eating foxes as reservoirs depends on their ability to successfully transmit the infection to sand flies rather than on their infection rate; it also depends on their capability of (re)introducing the pathogen into Leishmania-free dog populations, associated with epidemiological captive conditions of zoos favoring the infection (Dressen, 1990). Thus, the present study aimed to determine the prevalence of T. gondii and Leishmania spp. infection in C. thous kept in captivity in 17 zoos from Sa˜o Paulo State, Brazil.
L.S. Catenacci et al. / Veterinary Parasitology 169 (2010) 190–192
2. Materials and methods This study was conducted in 17 zoos (A–Q) from Sa˜o Paulo State, as well as in Sa˜o Paulo State University (UNESP), Botucatu Campus, Sa˜o Paulo State, Brazil. A total of 52 crab-eating foxes (C. thous), 25 males and 27 females, kept under captive conditions in zoos were included, except animals younger than 6 months and pregnant or lactating females with average weight of 6.6 kg (9.3 4.2 kg). The study was approved by the Ethics Committee in Animal Experimentation of the School of Veterinary Medicine and Animal Science, UNESP, Botucatu Campus. The animals were anesthetized by means of intramuscular injection. Atropine (0.05 mg/kg, Prado1), and ketamine (10 mg/kg, Fort Dodge1): xylazine (1 mg/kg Sedazina, Fort Dodge1) were used for pre-anesthesia and induction of anesthesia, respectively. Blood samples were collected from the jugular vein of all animals, sent to the Zoonosis Research Center (NUPEZO, UNESP), centrifuged at 1600 g during 10 min, and kept at 20 8C until the serological tests. All serum samples were researched for T. gondii antibodies through modified agglutination test (MAT) (Desmonts and Remington, 1980), a gold-standard test, and indirect fluorescent antibody test (IFAT) (Camargo, 1974); for Leishmania spp. antibody research, only IFAT was employed. In IFAT, both for toxoplasmosis and for leishmaniasis, a heterologous (anti-dog) anti-serum conjugated to fluorescein isothiocyanate, kindly supplied by the Zoonosis Control Center of Sa˜o Paulo, was used. To determine the impact of using this heterologous immunoglobulin, we compared MAT and IFAT results for toxoplasmosis. For MAT, a clear-cut button-shaped deposit of parasite suspension at the bottom of the well was interpreted as negative, and a carpet of agglutinated organisms, ranging from 50 to 100%, was considered to be positive. IFAT was positive when up to 50% of tachyzoite or promastigote forms per field presented complete fluorescence. The software BioEstat 5.0 was used to determine the concordance (kappa index: k) between MAT and IFAT (Rosner, 2006), whereas Spearman’s correlation coefficient (rs) was used to determine the concordance between titers, adopting a significance level of 5%. Sensitivity (S), specifity (E), and positive (PPV) and negative predictive values (NPV) in IFAT for toxoplasmosis were manually calculated. 3. Results and discussion T. gondii antibodies were detected in 10/52 (19.2%) serum samples from 6/17 (35.3%) zoos (Table 1). Eight samples (15.39%) presented a MAT titer of 16, four of which (50.0%) were from the same zoo; two of these four animals were kept in the same enclosure, one in a neighboring enclosure and the fourth in a somewhat distant enclosure. Nine serum samples were positive to IFAT, seven of which (13.5%) had titer 16 and two (3.8%) had titer 64. T. gondii serological results are shown in Table 2. IFAT presented high sensitivity (88.89%), specificity (97.62%), and positive (88.89%) and negative predictive values (97.62), indicating an almost perfect concordance with MAT (k = 0.86; standard
191
Table 1 Distribution of IgG Toxoplasma gondii antibodies of Cerdocyon thous sera samples from six positive Sa˜o Paulo state zoos. Zoos
Positive/tested; percentage
A B C D E F
1/2; 1/2; 2/3; 1/2; 1/2; 4/6;
Total
10/52; 19.23
50.00 50.00 66.67 50.00 50.00 66.67
error = 9.31%; CI95% = 68.25–104.76%; P < 0.0001) (Siegel and Castellan, 1988), although the latter detected more positive samples with titer 16. The correlation between titers of both tests was positive, presenting a strong correlation coefficient (rs = 0.87; P < 0.0001). All serum samples assayed for Leishmania spp. antibodies through IFAT were negative. The frequency of T. gondii antibodies was low (19.2%) in 6/17 zoos, compared to the results of Gennari et al. (2004), who detected 9/15 (60%) infected C. thous in Sa˜o Paulo and Parana´ States, Brazil. In spite of the low titers, 50% positive serum samples were detected in animals from the same zoo (Table 1). This fact could reflect an error in the institution management, facilitating the contact with the infective agent by ingesting oocysts from the environment, food and water, as well as bradyzoites from tissue cysts of intermediate hosts with chronic infection, or tachyzoites present inside immune cells located in cavity liquids (Tenter et al., 2000). Wild carnivores are sentinels for T. gondii infection in humans, especially considering environmental contamination (Dubey and Beattie, 1988). Zoo animals are susceptible to this infection since domestic cats are frequently present in these places (Franti et al., 1979; Tenter et al., 2000), along with synanthropic animals, which constitute potential prey for carnivores (Zarnke et al., 2000). Thus, appropriate food and water management is important to prevent and control T. gondii infection in zoos (Tenter et al., 2000). IFAT using heterologous immunoglobulin to detect T. gondii antibodies had high sensitivity and specificity, showing an almost perfect concordance and a strong correlation coefficient. These results allow the use of heterologous immunoglobulin for wild animals, especially dog immunoglobulin for C. thous antibody research, confirming the use of MAT as an epidemiological tool to assay this wild species for toxoplasmosis since it is a fast test that does not require expensive reagents or equipment (Desmonts and Remington, 1980). On the other hand, no animal was positive to Leishmania spp. Crab-eating fox involvement in visceral leishmaniasis Table 2 Frequency of Toxoplasma gondii antibodies by indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT). MAT results
IFAT results Negative
16
64
Negative 16
41; 80.4% 1; 2.0%
1; 2.0% 6; 11.8%
0; 0.0% 2; 3.9%
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epidemiology has been reported in several Brazilian studies (Deane and Deane, 1955; Mello et al., 1988; Lainson et al., 1990; MacDonald and Courtenay, 1993; MacDonald et al., 1994; Curi et al., 2006), especially in tropical areas due to the presence of sand flies, Lutzomyia longipalpis, and infected animals, mainly dogs and some other carnivores (Lainson et al., 1990). As some of the visited zoos are located in leishmaniasis occurrence areas such as Arac¸atuba City, Sa˜o Paulo State (Lindoso and Goto, 2006), the possibility of infection must be considered. Luppi et al. (2008) reported the presence of one infected C. thous in a Brazilian zoo and several wild and synanthropic animals living in the same area, including large populations of opossums and pigeons. The presence of these animals, especially birds, represents a risk factor of visceral leishmaniasis (Diniz et al., 2008), serving as nutrition source for Lutzomyia longipalpis. Silva et al. (2000) reported this infection in a free-ranging C. thous in Minas Gerais State, Brazil; the animal had a lesion caused by L. chagasi in its muzzle. Besides domestic dogs, crab-eating foxes have been identified as reservoirs for human visceral leishmaniasis in Brazil (Courtenay et al., 1996; Deane and Deane, 1962; Lainson et al., 1969). However, the crab-eating fox populations do not maintain a transmission cycle independently of domestic dogs; they are unable to introduce the parasite into Leishmania-free dog populations (Courtenay et al., 2002a,b). Wild animals can be infected, presenting clinical signs and transmitting the parasite under captive conditions in endemic areas. Thus, C. thous presents great importance under free and captive conditions; however, further studies on the environmental transmission of T. gondii, the epidemiology of Leishmania in C. thous and the comparison between MAT and IFAT using heterologous immunoglobulin are needed to understand the epidemiological role of C. thous and its importance for public health and control in zoos. Acknowledgements This study was supported by Training Special Program (PET – SESu/MEC). We thank Dr. Mariaˆngela Lozano Cruz and Rodrigo L. Maruccio for helping with field procedures, and Andre´ Peres Barbosa de Castro, Moˆnika Vogl Sampaio, Juliana Satie Carvalho Akaboshi for helping in the laboratory. We also thank UNESP and all zoos for logistic support. References Camargo, M.E., 1974. Introduc¸a˜o a`s te´cnicas de imunofluoresceˆncia. Rev. Bras. Patol. Clin. 10, 143–169. Courtenay, O., Quinnell, R.J., Garcez, L.M., Dye, C., 2002. Low infectiousness of a wildlife host of Leishmania infantum: the crab-eating fox is not important for transmission. Parasitolology 125, 407–414. Courtenay, O., Quinnell, R.J., Garcez, L.M., Shaw, J.J., Dye, C., 2002. Infectiousness in a cohort of Brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission. J. Infect. Dis. 186, 1314–1320.
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