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Occurrence of Toxoplasma gondii antibodies in birds from the Atlantic Forest, state of São Paulo, Brazil
Veterinary Parasitology 200 (2014) 193–197
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Occurrence of Toxoplasma gondii antibodies in birds from the Atlantic Forest, state of São Paulo, Brazil夽 S.M. Gennari a,∗ , M. Ogrzewalska a , H.S. Soares a , D.G. Saraiva a , A. Pinter b , M.B. Labruna a , J.P. Dubey c a Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP CEP 05508-270, Brazil b Superintendência de Controle de Endemias (Sucen), São Paulo, Brazil c United States Department of Agriculture, Agricultural Research Service, Beltsville Agriculture Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 4 October 2013 Accepted 7 October 2013 Keywords: Toxoplasma gondii Birds Atlantic Forest Brazil
a b s t r a c t Avian are considered important intermediate hosts for Toxoplasma gondii because they serve as source of infection for Felidae, which shed environmentally resistant oocysts after ingesting infected tissues. Little is known of epidemiology of toxoplasmosis in wild birds. In the present study, antibodies to T. gondii were determined in 202 wild birds of 37 species captured in seven small areas of the Atlantic Forest, in the state of São Paulo, Brazil, and provided information on possible associated risk factors. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 73 (36.1%) of 202 birds with titers of 1:5 in 16 samples, 1:10 in 26 samples, 1:20 in 17 samples, 1:40 in 10 samples, 1:80 in three samples, and 1:160 in one sample. No association was observed between T. gondii seropositivity and the local where the birds were collected. Seropositivity was higher in birds that lived on the forest floor (p < 0.001; U = 1230.0), and in omnivorous birds (p = 0.007; U = 3939.0). T. gondii antibodies were reported for the first time in 23 species of birds enlarging the host range of this parasite. Notably, T. gondii antibodies were found in 83.3% (15/18) of the Rufous-bellied Thrush (Turdus rufiventris). © 2013 Elsevier B.V. All rights reserved.
1. Introduction Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including birds (Dubey, 2010). Avian are considered important intermediate hosts for T. gondii because they serve as source of infection for Felidae, which shed environmentally resistant oocysts after ingesting infected tissues. It has been
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel: +55 11 30917654; fax: +55 11 30917928. E-mail address: [email protected] (S.M. Gennari). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.10.003
estimated that cats prey billions of birds annually (Loss et al., 2013). Additionally, ground-feeding birds are considered sentinels for soil contamination with T. gondii oocysts (Ruiz and Frenkel, 1980); additionally, toxoplasmosis is also cause of mortality in certain avian species (Dubey, 2002, 2010). In many countries, including Brazil, most surveys for toxoplasmosis have been focused on domestic birds and little information is available on wild birds (Leite et al., 2007; Dubey, 2010; Soares et al., 2010; Vitaliano et al., 2010). Aims of the present study were to investigate the occurrence of T. gondii antibodies in wild birds captured in seven areas of the Atlantic Forest, in the state of São Paulo, Brazil, and provide information on possible associated risk factors.
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2. Materials and methods 2.1. Ethics
Table 1 Number of birds analyzed and positive (%) for the presence of T. gondii antibody per examined locality. Municipality
Protocol that agrees with Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation was followed. The use of the wild animals was approved by the Brazilian Institute of Natural Resources (IBAMA number 22212-2). 2.2. Study areas In all seven localities, wild birds were sampled inside the forest fragment: three small forest areas in the southern part of the São Paulo metropolitan area (SPMA), within the municipalities of São Bernardo do Campo, Diadema and Santo André; three small forest areas in the northern part of the SPMA (municipalities of Arujá, Mairiporã and Nazaré Paulista) and one large forest area (Cantareira State Park) in the northern part of the SPMA, within the São Paulo municipality. The detailed characteristics of the study areas are described elsewhere (Ogrzewalska et al., 2012). Sampled areas are located between 765 and 1000 m above sea level and have a subtropical climate. Summer is warm and rainy, while winter is mild. The average annual temperature is around 18 ◦ C. The coldest month is July (average 14 ◦ C) and the warmest is February (average 22 ◦ C). The annual rainfall index is approximately 1400 mm.
Number of birds Sampled
S. Bernardo do Campo Diadema Santo André Arujá Mairiporã Nazaré Paulista São Paulo Total
Positive
Occurrence (%)
22 13 21 37 40 44 25
8 6 10 13 11 14 11
36.4a 46.1a 47.6a 35.1a 27.5a 31.8a 44.0a
202
73
36.1
a
Same letters in the occurrence column means the proportion has no significant difference (p > 0.05).
naturally infected chickens (Gallus domesticus) with this level of seropositivity (Dubey, 2010). There is no easy way to establish sensitivity and specificity for rare wild species (as sampled here), therefore we state all titers. 2.5. Statistical analysis Association among bird preferential forest strata and different feeding behavior with the presence of antibodies anti-T. gondii were analyzed by non-parametric tests. All associations were analyzed by Mann–Whitney test; furthermore the association of infection with the sampled place were previously analyzed by Kruskal–Wallis test.
2.3. Capture evenness 3. Results and discussion From each of the seven study areas samples were collected four times, at 3-month intervals. The first visit was in autumn (April–May) 2010, followed by collections in winter (July–August), spring (September–October) and summer (November–January 2011). Birds were caught using 10 mist nets (12 m long × 2 m wild, 36 mm mesh) displayed along animal trails (two work days per area) inside the forest. In each area, mist nets were left open from 6:00 am to 6:00 pm on the first day, and from 6:00 am to 12:00 pm on the second day, resulting in 5040 net-hours for the whole study. Mist nets were checked every 40 min. Captured birds were identified to species following Sigrist (2007). Blood collection were obtained from a jugular vein and placed in a serum collection tube until clotted and then centrifuged. Sera were stored at −20 ◦ C until analysis. The birds were released after sample collection. 2.4. Serological test Sera were tested for the presence of antibodies to T. gondii by modified agglutination test (MAT) as previously described (Dubey and Desmonts, 1987). Sera were first screened at 1:5 dilution and the positive samples were tested at two-fold serial dilutions. Positive and negative control sera were included in each test. The MAT is considered as one of the most sensitive test for the detection of T. gondii antibodies in human or animal sera (Dubey, 2010). The low serum dilution (1:5) was used to screen sera because viable T. gondii was isolated occasionally from
Serum samples were obtained from 202 birds of 37 species, belonging to the orders: Galbuliformes (1 family, 3 samples), Piciformes (1 family, 1 sample) and Passeriformes (15 families, 198 samples). From those, the genus Turdus (Turdus albicollis, Turdus amaurochalinus, Turdus leucomelas and Turdus rufiventris) has a partially migratory habit; and the two other genus, Myodynastes maculatus and Lathrotriccus euleri, both of the Tyrannidae family, have migratory habit. Regarding bird feeding habits, 24 species are insectivorous and 13 species are omnivorous. The birds live in different strata: forest floor, understory, middle layer, and canopy, and most of them are able to live in more than one stratum. Antibodies were found in 73 (36.1%) of 202 birds with titers of 1:5 in 16 samples, 1:10 in 26 samples, 1:20 in 17 samples, 1:40 in 10 samples, 1:80 in three samples and 1:160 in one sample. No association was observed between the different occurrence of T. gondii antibodies and the place where the birds were collected (p = 0.666; df = 6), as presented in Table 1. Table 2 presents the occurrence of T. gondii antibodies per bird species, habits and antibody titers. Table 3 presents the occurrence of T. gondii antibodies in wild birds based on their habitat and feeding behavior. There was a strong association between higher (77.7%, 21/27) seropositivity of birds and the forest floor ecosystem (p < 0.001; U = 1230.0). No association between bird seropositivity and living above the forest floor was observed. There was association (p = 0.007; U = 3939.0) of
S.M. Gennari et al. / Veterinary Parasitology 200 (2014) 193–197
195
Table 2 Occurrence of anti-T. gondii antibodies, antibody titers and habits of the birds per bird species. Bird order/family
Species
Number of samples
Tested Galbuliformes Bucconidae Piciformes Picidae Passeriformes Conopophagidae Dendrocolaptidae
Emberizidae
Furnariidae
Parulidae
Pipridae Scleruridae Thamnophilidae
Thraupidae
Tityridae Turdidae
Habits
Titer (no. positives per titer)
Positive
%
Migratory
Feed
Strat
IUCNa
Crescent-chested Puffbird (Malacoptila striata)
3
2
66.7
5(2)
N
IN
U/M
LC
White-spotted Woodpecker (Veniliornis spilogaster)
1
1
100.0
40(1)
N
IN
U/C
LC
15
6
40.0
10(3), 20(2), 160(1)
N
IN
U
LC
7
0
0
<5
N
IN
M/C
LC
2
0
0
<5
N
IN
M
LC
2
1
50.0
10(1)
N
IN
M
LC
2
1
50.0
5(1)
N
IN
M
LC
1 1
0 0
0 0
<5 <5
N N
OM IN
M/C M
LC LC
2
0
0
<5
N
IN
M
NT
14
3
21.4
5(1), 10(1), 20(1)
N
IN
U
LC
1
0
0
<5
N
IN
T
LC
2
0
0
<5
N
IN
U/M
VU
7
0
0
<5
N
IN
U/M
LC
7
4
57.1
5(3), 40(1)
N
IN
U
LC
12
2
16.7
5(1), 20(1)
N
IN
U/M
LC
5
1
20.0
20(1)
N
IN
U
LC
4
0
0
<5
N
OM
U/M
LC
1
1
100.0
10(1)
N
IN
T
LC
11
5
45.4
5(2), 10(2), 20(1)
N
IN
U/M
LC
4
3
75.0
5(1), 10(2)
N
IN
T
LC
11
4
36.4
5(1), 20(3)
N
IN
U
LC
4
0
0
<5
N
IN
U/M
LC
13
8
61.5
5(1), 10(3), 20(3), 40(1)
N
OM
U/M
LC
6
1
16.7
10(1)
N
OM
M/C
LC
15
5
33.3
5(1), 10(3), 40(1)
N
OM
U/M
LC
8
0
0
<5
N
OM
U
LC
5
5
100.0
20(1), 40(3), 80(1)
P
OM
U/M
LC
2
1
50.0
10(1)
P
OM
T/C
LC
1
1
100.0
10(1)
P
OM
T/C
LC
18
15
83.3
10(6), 20(4), 40(3), 80(2)
P
OM
T/C
LC
Rufous Gnateater (Conopophaga lineata) Scaled Woodcreeper (Lepidocolaptes squamatus) Olivaceous Woodcreeper (Sittasomus griseicapillus) White-throated Woodcreeper (Xiphocolaptes albicollis) Lesser Woodcreeper (Xiphorhynchus fuscus) Uniform Finch (Haplospiza unicolor) Chestnut-bellied Seed-Finch (Oryzoborus (Sporophila) angolensis) White-browed Foliage-gleaner (Anabacerthia amaurotis) White-eyed Foliage-gleaner (Automolus leucophthalmus) Sharp-tailed Streamcreeper (Lochmias nematura) Bahia Spinetail (Synallaxis whitneyi) Rufous-capped Spinetail (Synallaxis ruficapilla) Buff-browed Foliage-gleaner (Syndactyla rufosuperciliata) Golden-crowned Warbler (Basileuterus culicivorus) White-browed Warbler (Basileuterus leucoblepharus) Swallow-tailed Manakin (Chiroxiphia caudata) Rufous-breasted Leaftosser (Sclerurus scansor) Plain Antvireo (Dysithamnus mentalis) Squamate Antbird (Myrmeciza squamosa) White-shouldered Fire-eye (Pyriglena leucoptera) Variable Antshrike (Thamnophilus caerulescens) Red-crowned Ant-Tanager (Habia rubica) Ruby-crowned Tanager (Tachyphonus coronatus) Black-goggled Tanager (Lanio melanops) Greenish Schiffornis (Schiffornis virescens) White-necked Thrush (Turdus albicollis) Creamy-bellied Thrush (Turdus amaurochalinus) Pale-breasted Thrush (Turdus leucomelas) Rufous-bellied Thrush (Turdus rufiventris)
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Table 2 (Continued) Bird order/family
Species
Number of samples
Tested Tyrannidae
Rhynchocyclidae
Incertae sedis Vireonidae
Streaked Flycatcher (Myiodynastes maculatus) Euler’s Flycatcher (Lathrotriccus euleri) Sepia-capped Flycatcher (Leptopogon amaurocephalus) Gray-hooded Flycatcher (Mionectes rufiventris) White-throated Spadebill (Platyrinchus mystaceus) Rufous-browed Peppershrike (Cyclarhis gujanensis)
Positive
Titer (no. positives per titer)
Habits
Migratory
Feed
Strat
IUCNa
10(1)
M
OM
M/C
LC
%
1
1
100.0
2
0
0
<5
M
IN
U/M
LC
3
0
0
<5
N
IN
U/M
LC
4
1
25.0
5(1)
N
OM
U/M
LC
3
0
0
<5
N
IN
U/M
LC
2
1
50.0
5(1)
N
OM
M/C
LC
M, migratory; P, partially migratory; N, no migratory; IN, insectivorous; OM, omnivorous; T, forest floor; U, understory; M, middle layer; C, canopy; LC, least concern; VU, vulnerable; NT, near threatened. a IUCN-RED LIST (2010).
feeding behavior and seropositivity with T. gondii antibodies detected in 48.7% (39/80) of omnivorous birds and in 27.8% (34/122) of insectivores. There are no data on the Felidae and T. gondii seropositivity in cats in this part of the sampled forests; however, the seropositivity in omnivorous birds and higher infection rate in ground feeding birds indicate possible transmission by oocysts. In the present study antibodies to T. gondii were found in 23 species of birds for the first time, especially in 22 of 26 birds of the genus Turdus. The only other previous records that we are aware were in other species of Turdus, from Europe and Central America. In these studies viable T. gondii was isolated from 2 of 54 and 1 of 4 Black Bird (Turdus merula) in Czech Republic (Literák et al., 1992) and Slovakia ˇ 1974), respectively. Frenkel et al. (1995) reported (Catár, T. gondii antibodies (MAT ≥ 5) in Clay-colored Robin (Turdus grayi) from Panama. Remarkably, the collected species of the genus Turdus are know as partial migratory birds (Table 2) what may not be associated with the chance of be infected, but may address a potential important role for T. gondii spreading, since those birds were highly infected and they are able to easily move among forest patches.
The biome Atlantic Forest is a set of forest types and different ecosystems. Originally occupying 1,300,000 km2 in Brazilian territory, however currently only 7% of this area is well preserved in fragments of more than 100 ha. Even reduced in the Atlantic Forest there are about 20,000 species of plants (about 35% of all Brazilian species) and in relation to fauna, the region is home to 849 species of birds, 370 species of amphibians, 200 species of reptiles, 270 species of mammals and 350 species of fish (MMA, 2013). Birds are part of the alimentary chain of many of these animal species, and neotropical felids are also present in this biome, keeping the sylvatic cycle of T. gondii in this important ecosystem. Conflict of interest None declared. Acknowledgments We would like to thanks State of São Paulo Research Foundation (FAPESP) for the grant to M.O., H.S.S and D.G.S, and the National Council of Scientific and Technologic Development (CNPq) for the grant to M.B.L. and S.M.G. References
Table 3 Risk assessment and T. gondii seropositivity among birds by forest strata preference, feeding and migratory behavior. Bird groups by behavior
Toxoplasma gondii
Proportion
Positive
Examined
(%)
Forest strata preferenceA Forest floor Understory Middle layer Canopy
21 64 50 20
27 170 135 38
77.7a 37.6b 37.0b 51.6b
Feeding behavior Omnivorous Insectivores
39 34
80 122
48.7c 27.8d
Same letters in the proportion column means there is no significant difference (p > 0.05). A Total number of birds in this group is larger than the total sampled birds because some bird species fit in more than one categories.
ˇ ´ podmienkach na slovensku. Catár, G., 1974. Toxoplazmóza v ekologickych Biol. Práce (Bratislava) 20, 1–138. Dubey, J.P., 2002. A review of toxoplasmosis in wild birds. Vet. Parasitol. 106, 121–153. Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans. CRC Press, Boca Raton, FL, pp. 313. Dubey, J.P., Desmonts, G., 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Vet. J. 19, 337–339. Frenkel, J.K., Hassanein, K.M., Hassanein, R.S., Brown, E., Thulliez, P., Quintero-Nunez, R., 1995. Transmission of Toxoplasma gondii in Panama City, Panama: a five-year prospective cohort study of children, cats, rodents, birds, and soil. Am. J. Trop. Med. Hyg. 53, 458–468. IUCN, 2010. IUCN Red List of Threatened Species, Version 2010.1, Available at: http://www.iucnredlist.org Leite, A.S., Alves, L.C., Faustino, M.A.G., 2007. Serological survey of toxoplasmosis in birds from Cracidae family in a wild bird center facility at Pernambuco State, Northeast of Brazil. Med. Vet. 1, 55–57. Literák, I., Hejlicek, K., Nezval, J., Folk, C., 1992. Incidence of Toxoplasma gondii in populations of wild birds in the Czech Republic. Avian Pathol. 21, 659–665.
S.M. Gennari et al. / Veterinary Parasitology 200 (2014) 193–197 Loss, S.R., Will, T., Marra, P.P., 2013. The impact of free-ranging domestic cats on wildlife of the United States. Nat. Commun. 4, 1396. MMA – Ministério do Meio Ambiente, 2013. Mata Atlântica. Ministry of Environment, Atlantic Forest, Available at: http://http://www.mma.gov.br/biomas/mata-atlantica Ogrzewalska, M., Saraiva, D.G., Moraes-Filho, J., Martins, T.F., Costa, F., Pinter, A., Labruna, M.B., 2012. Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of Sa˜o Paulo, Brazil. Parasitology 139, 1283–1300. Ruiz, A., Frenkel, J.K., 1980. Intermediate and transport hosts of Toxoplasma gondii in Costa Rica. Am. J. Trop. Med. Hyg. 29, 1161–1166.
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Soares, H.S., Alves, N.D., Pereira, R.H.M.A., Matos, S.M., Pena, H.F.J., Gennari, S.M., Feijó, F.M.C., Amóra, S.S.A., Peixoto, G.C.X., 2010. Occurrence of anti-Toxoplasma gondii antibodies in Greater Rheas (Rhea americana) at the Reproduction Centre for Wild Animals in Mossoró, Rio Grande do Norte, Brazil. Arq. Bras. Med. Vet. Zootec. 62, 489–491. Sigrist, T., 2007. Aves do Brasil Oriental – Birds of Eastern Brazil. Avis Brasilis, São Paulo, Brazil, pp. 448. Vitaliano, S.N., Mineo, T.W.P., Andréa, M.R., Machado, R.Z., Mineo, J.R., Werther, K., 2010. Experimental infection of Crested Caracara (Caracara plancus) with Toxoplasma gondii simulating natural conditions. Vet. Parasitol. 172, 71–75.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Occurrence of Toxoplasma gondii antibodies in birds from the Atlantic Forest, state of São Paulo, Brazil夽 S.M. Gennari a,∗ , M. Ogrzewalska a , H.S. Soares a , D.G. Saraiva a , A. Pinter b , M.B. Labruna a , J.P. Dubey c a Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP CEP 05508-270, Brazil b Superintendência de Controle de Endemias (Sucen), São Paulo, Brazil c United States Department of Agriculture, Agricultural Research Service, Beltsville Agriculture Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 4 October 2013 Accepted 7 October 2013 Keywords: Toxoplasma gondii Birds Atlantic Forest Brazil
a b s t r a c t Avian are considered important intermediate hosts for Toxoplasma gondii because they serve as source of infection for Felidae, which shed environmentally resistant oocysts after ingesting infected tissues. Little is known of epidemiology of toxoplasmosis in wild birds. In the present study, antibodies to T. gondii were determined in 202 wild birds of 37 species captured in seven small areas of the Atlantic Forest, in the state of São Paulo, Brazil, and provided information on possible associated risk factors. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 73 (36.1%) of 202 birds with titers of 1:5 in 16 samples, 1:10 in 26 samples, 1:20 in 17 samples, 1:40 in 10 samples, 1:80 in three samples, and 1:160 in one sample. No association was observed between T. gondii seropositivity and the local where the birds were collected. Seropositivity was higher in birds that lived on the forest floor (p < 0.001; U = 1230.0), and in omnivorous birds (p = 0.007; U = 3939.0). T. gondii antibodies were reported for the first time in 23 species of birds enlarging the host range of this parasite. Notably, T. gondii antibodies were found in 83.3% (15/18) of the Rufous-bellied Thrush (Turdus rufiventris). © 2013 Elsevier B.V. All rights reserved.
1. Introduction Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including birds (Dubey, 2010). Avian are considered important intermediate hosts for T. gondii because they serve as source of infection for Felidae, which shed environmentally resistant oocysts after ingesting infected tissues. It has been
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel: +55 11 30917654; fax: +55 11 30917928. E-mail address: [email protected] (S.M. Gennari). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.10.003
estimated that cats prey billions of birds annually (Loss et al., 2013). Additionally, ground-feeding birds are considered sentinels for soil contamination with T. gondii oocysts (Ruiz and Frenkel, 1980); additionally, toxoplasmosis is also cause of mortality in certain avian species (Dubey, 2002, 2010). In many countries, including Brazil, most surveys for toxoplasmosis have been focused on domestic birds and little information is available on wild birds (Leite et al., 2007; Dubey, 2010; Soares et al., 2010; Vitaliano et al., 2010). Aims of the present study were to investigate the occurrence of T. gondii antibodies in wild birds captured in seven areas of the Atlantic Forest, in the state of São Paulo, Brazil, and provide information on possible associated risk factors.
194
S.M. Gennari et al. / Veterinary Parasitology 200 (2014) 193–197
2. Materials and methods 2.1. Ethics
Table 1 Number of birds analyzed and positive (%) for the presence of T. gondii antibody per examined locality. Municipality
Protocol that agrees with Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation was followed. The use of the wild animals was approved by the Brazilian Institute of Natural Resources (IBAMA number 22212-2). 2.2. Study areas In all seven localities, wild birds were sampled inside the forest fragment: three small forest areas in the southern part of the São Paulo metropolitan area (SPMA), within the municipalities of São Bernardo do Campo, Diadema and Santo André; three small forest areas in the northern part of the SPMA (municipalities of Arujá, Mairiporã and Nazaré Paulista) and one large forest area (Cantareira State Park) in the northern part of the SPMA, within the São Paulo municipality. The detailed characteristics of the study areas are described elsewhere (Ogrzewalska et al., 2012). Sampled areas are located between 765 and 1000 m above sea level and have a subtropical climate. Summer is warm and rainy, while winter is mild. The average annual temperature is around 18 ◦ C. The coldest month is July (average 14 ◦ C) and the warmest is February (average 22 ◦ C). The annual rainfall index is approximately 1400 mm.
Number of birds Sampled
S. Bernardo do Campo Diadema Santo André Arujá Mairiporã Nazaré Paulista São Paulo Total
Positive
Occurrence (%)
22 13 21 37 40 44 25
8 6 10 13 11 14 11
36.4a 46.1a 47.6a 35.1a 27.5a 31.8a 44.0a
202
73
36.1
a
Same letters in the occurrence column means the proportion has no significant difference (p > 0.05).
naturally infected chickens (Gallus domesticus) with this level of seropositivity (Dubey, 2010). There is no easy way to establish sensitivity and specificity for rare wild species (as sampled here), therefore we state all titers. 2.5. Statistical analysis Association among bird preferential forest strata and different feeding behavior with the presence of antibodies anti-T. gondii were analyzed by non-parametric tests. All associations were analyzed by Mann–Whitney test; furthermore the association of infection with the sampled place were previously analyzed by Kruskal–Wallis test.
2.3. Capture evenness 3. Results and discussion From each of the seven study areas samples were collected four times, at 3-month intervals. The first visit was in autumn (April–May) 2010, followed by collections in winter (July–August), spring (September–October) and summer (November–January 2011). Birds were caught using 10 mist nets (12 m long × 2 m wild, 36 mm mesh) displayed along animal trails (two work days per area) inside the forest. In each area, mist nets were left open from 6:00 am to 6:00 pm on the first day, and from 6:00 am to 12:00 pm on the second day, resulting in 5040 net-hours for the whole study. Mist nets were checked every 40 min. Captured birds were identified to species following Sigrist (2007). Blood collection were obtained from a jugular vein and placed in a serum collection tube until clotted and then centrifuged. Sera were stored at −20 ◦ C until analysis. The birds were released after sample collection. 2.4. Serological test Sera were tested for the presence of antibodies to T. gondii by modified agglutination test (MAT) as previously described (Dubey and Desmonts, 1987). Sera were first screened at 1:5 dilution and the positive samples were tested at two-fold serial dilutions. Positive and negative control sera were included in each test. The MAT is considered as one of the most sensitive test for the detection of T. gondii antibodies in human or animal sera (Dubey, 2010). The low serum dilution (1:5) was used to screen sera because viable T. gondii was isolated occasionally from
Serum samples were obtained from 202 birds of 37 species, belonging to the orders: Galbuliformes (1 family, 3 samples), Piciformes (1 family, 1 sample) and Passeriformes (15 families, 198 samples). From those, the genus Turdus (Turdus albicollis, Turdus amaurochalinus, Turdus leucomelas and Turdus rufiventris) has a partially migratory habit; and the two other genus, Myodynastes maculatus and Lathrotriccus euleri, both of the Tyrannidae family, have migratory habit. Regarding bird feeding habits, 24 species are insectivorous and 13 species are omnivorous. The birds live in different strata: forest floor, understory, middle layer, and canopy, and most of them are able to live in more than one stratum. Antibodies were found in 73 (36.1%) of 202 birds with titers of 1:5 in 16 samples, 1:10 in 26 samples, 1:20 in 17 samples, 1:40 in 10 samples, 1:80 in three samples and 1:160 in one sample. No association was observed between the different occurrence of T. gondii antibodies and the place where the birds were collected (p = 0.666; df = 6), as presented in Table 1. Table 2 presents the occurrence of T. gondii antibodies per bird species, habits and antibody titers. Table 3 presents the occurrence of T. gondii antibodies in wild birds based on their habitat and feeding behavior. There was a strong association between higher (77.7%, 21/27) seropositivity of birds and the forest floor ecosystem (p < 0.001; U = 1230.0). No association between bird seropositivity and living above the forest floor was observed. There was association (p = 0.007; U = 3939.0) of
S.M. Gennari et al. / Veterinary Parasitology 200 (2014) 193–197
195
Table 2 Occurrence of anti-T. gondii antibodies, antibody titers and habits of the birds per bird species. Bird order/family
Species
Number of samples
Tested Galbuliformes Bucconidae Piciformes Picidae Passeriformes Conopophagidae Dendrocolaptidae
Emberizidae
Furnariidae
Parulidae
Pipridae Scleruridae Thamnophilidae
Thraupidae
Tityridae Turdidae
Habits
Titer (no. positives per titer)
Positive
%
Migratory
Feed
Strat
IUCNa
Crescent-chested Puffbird (Malacoptila striata)
3
2
66.7
5(2)
N
IN
U/M
LC
White-spotted Woodpecker (Veniliornis spilogaster)
1
1
100.0
40(1)
N
IN
U/C
LC
15
6
40.0
10(3), 20(2), 160(1)
N
IN
U
LC
7
0
0
<5
N
IN
M/C
LC
2
0
0
<5
N
IN
M
LC
2
1
50.0
10(1)
N
IN
M
LC
2
1
50.0
5(1)
N
IN
M
LC
1 1
0 0
0 0
<5 <5
N N
OM IN
M/C M
LC LC
2
0
0
<5
N
IN
M
NT
14
3
21.4
5(1), 10(1), 20(1)
N
IN
U
LC
1
0
0
<5
N
IN
T
LC
2
0
0
<5
N
IN
U/M
VU
7
0
0
<5
N
IN
U/M
LC
7
4
57.1
5(3), 40(1)
N
IN
U
LC
12
2
16.7
5(1), 20(1)
N
IN
U/M
LC
5
1
20.0
20(1)
N
IN
U
LC
4
0
0
<5
N
OM
U/M
LC
1
1
100.0
10(1)
N
IN
T
LC
11
5
45.4
5(2), 10(2), 20(1)
N
IN
U/M
LC
4
3
75.0
5(1), 10(2)
N
IN
T
LC
11
4
36.4
5(1), 20(3)
N
IN
U
LC
4
0
0
<5
N
IN
U/M
LC
13
8
61.5
5(1), 10(3), 20(3), 40(1)
N
OM
U/M
LC
6
1
16.7
10(1)
N
OM
M/C
LC
15
5
33.3
5(1), 10(3), 40(1)
N
OM
U/M
LC
8
0
0
<5
N
OM
U
LC
5
5
100.0
20(1), 40(3), 80(1)
P
OM
U/M
LC
2
1
50.0
10(1)
P
OM
T/C
LC
1
1
100.0
10(1)
P
OM
T/C
LC
18
15
83.3
10(6), 20(4), 40(3), 80(2)
P
OM
T/C
LC
Rufous Gnateater (Conopophaga lineata) Scaled Woodcreeper (Lepidocolaptes squamatus) Olivaceous Woodcreeper (Sittasomus griseicapillus) White-throated Woodcreeper (Xiphocolaptes albicollis) Lesser Woodcreeper (Xiphorhynchus fuscus) Uniform Finch (Haplospiza unicolor) Chestnut-bellied Seed-Finch (Oryzoborus (Sporophila) angolensis) White-browed Foliage-gleaner (Anabacerthia amaurotis) White-eyed Foliage-gleaner (Automolus leucophthalmus) Sharp-tailed Streamcreeper (Lochmias nematura) Bahia Spinetail (Synallaxis whitneyi) Rufous-capped Spinetail (Synallaxis ruficapilla) Buff-browed Foliage-gleaner (Syndactyla rufosuperciliata) Golden-crowned Warbler (Basileuterus culicivorus) White-browed Warbler (Basileuterus leucoblepharus) Swallow-tailed Manakin (Chiroxiphia caudata) Rufous-breasted Leaftosser (Sclerurus scansor) Plain Antvireo (Dysithamnus mentalis) Squamate Antbird (Myrmeciza squamosa) White-shouldered Fire-eye (Pyriglena leucoptera) Variable Antshrike (Thamnophilus caerulescens) Red-crowned Ant-Tanager (Habia rubica) Ruby-crowned Tanager (Tachyphonus coronatus) Black-goggled Tanager (Lanio melanops) Greenish Schiffornis (Schiffornis virescens) White-necked Thrush (Turdus albicollis) Creamy-bellied Thrush (Turdus amaurochalinus) Pale-breasted Thrush (Turdus leucomelas) Rufous-bellied Thrush (Turdus rufiventris)
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Table 2 (Continued) Bird order/family
Species
Number of samples
Tested Tyrannidae
Rhynchocyclidae
Incertae sedis Vireonidae
Streaked Flycatcher (Myiodynastes maculatus) Euler’s Flycatcher (Lathrotriccus euleri) Sepia-capped Flycatcher (Leptopogon amaurocephalus) Gray-hooded Flycatcher (Mionectes rufiventris) White-throated Spadebill (Platyrinchus mystaceus) Rufous-browed Peppershrike (Cyclarhis gujanensis)
Positive
Titer (no. positives per titer)
Habits
Migratory
Feed
Strat
IUCNa
10(1)
M
OM
M/C
LC
%
1
1
100.0
2
0
0
<5
M
IN
U/M
LC
3
0
0
<5
N
IN
U/M
LC
4
1
25.0
5(1)
N
OM
U/M
LC
3
0
0
<5
N
IN
U/M
LC
2
1
50.0
5(1)
N
OM
M/C
LC
M, migratory; P, partially migratory; N, no migratory; IN, insectivorous; OM, omnivorous; T, forest floor; U, understory; M, middle layer; C, canopy; LC, least concern; VU, vulnerable; NT, near threatened. a IUCN-RED LIST (2010).
feeding behavior and seropositivity with T. gondii antibodies detected in 48.7% (39/80) of omnivorous birds and in 27.8% (34/122) of insectivores. There are no data on the Felidae and T. gondii seropositivity in cats in this part of the sampled forests; however, the seropositivity in omnivorous birds and higher infection rate in ground feeding birds indicate possible transmission by oocysts. In the present study antibodies to T. gondii were found in 23 species of birds for the first time, especially in 22 of 26 birds of the genus Turdus. The only other previous records that we are aware were in other species of Turdus, from Europe and Central America. In these studies viable T. gondii was isolated from 2 of 54 and 1 of 4 Black Bird (Turdus merula) in Czech Republic (Literák et al., 1992) and Slovakia ˇ 1974), respectively. Frenkel et al. (1995) reported (Catár, T. gondii antibodies (MAT ≥ 5) in Clay-colored Robin (Turdus grayi) from Panama. Remarkably, the collected species of the genus Turdus are know as partial migratory birds (Table 2) what may not be associated with the chance of be infected, but may address a potential important role for T. gondii spreading, since those birds were highly infected and they are able to easily move among forest patches.
The biome Atlantic Forest is a set of forest types and different ecosystems. Originally occupying 1,300,000 km2 in Brazilian territory, however currently only 7% of this area is well preserved in fragments of more than 100 ha. Even reduced in the Atlantic Forest there are about 20,000 species of plants (about 35% of all Brazilian species) and in relation to fauna, the region is home to 849 species of birds, 370 species of amphibians, 200 species of reptiles, 270 species of mammals and 350 species of fish (MMA, 2013). Birds are part of the alimentary chain of many of these animal species, and neotropical felids are also present in this biome, keeping the sylvatic cycle of T. gondii in this important ecosystem. Conflict of interest None declared. Acknowledgments We would like to thanks State of São Paulo Research Foundation (FAPESP) for the grant to M.O., H.S.S and D.G.S, and the National Council of Scientific and Technologic Development (CNPq) for the grant to M.B.L. and S.M.G. References
Table 3 Risk assessment and T. gondii seropositivity among birds by forest strata preference, feeding and migratory behavior. Bird groups by behavior
Toxoplasma gondii
Proportion
Positive
Examined
(%)
Forest strata preferenceA Forest floor Understory Middle layer Canopy
21 64 50 20
27 170 135 38
77.7a 37.6b 37.0b 51.6b
Feeding behavior Omnivorous Insectivores
39 34
80 122
48.7c 27.8d
Same letters in the proportion column means there is no significant difference (p > 0.05). A Total number of birds in this group is larger than the total sampled birds because some bird species fit in more than one categories.
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