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Seroprevalence and spatial distribution of Toxoplasma gondii infection in cats, dogs, pigs and equines of the Fernando de Noronha Island, Brazil
Seroprevalence and spatial distribution of Toxoplasma gondii infection in cats, dogs, pigs and equines of the Fernando de Noronha Island, Brazil Fernando J.R. Magalh˜aes, M¨uller Ribeiro-Andrade, F´atima M. Souza, Carlos D.F. Lima Filho, Alexander Welker Biondo, Odilon Vidotto, Italmar Teodorico Navarro, Rinaldo A. Mota PII: DOI: Reference:
S1383-5769(16)30178-7 doi:10.1016/j.parint.2016.11.014 PARINT 1607
To appear in:
Parasitology International
Received date: Revised date: Accepted date:
6 June 2016 4 November 2016 23 November 2016
Please cite this article as: Magalh˜aes Fernando J.R., Ribeiro-Andrade M¨ uller, Souza F´ atima M., Lima Filho Carlos D.F., Biondo Alexander Welker, Vidotto Odilon, Navarro Italmar Teodorico, Mota Rinaldo A., Seroprevalence and spatial distribution of Toxoplasma gondii infection in cats, dogs, pigs and equines of the Fernando de Noronha Island, Brazil, Parasitology International (2016), doi:10.1016/j.parint.2016.11.014
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Seroprevalence and spatial distribution of Toxoplasma gondii infection in cats, dogs, pigs and equines of the Fernando de Noronha Island, Brazil
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Fernando J. R. Magalhães1, Müller Ribeiro-Andrade2, Fátima M. Souza1, Carlos D. F.
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Lima Filho1, Alexander Welker Biondo3, Odilon Vidotto4, Italmar Teodorico Navarro4,
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Rinaldo A. Mota2*
Unidade de Vigilância em Saúde do Distrito de Fernando de Noronha, Pernambuco,
Brazil.
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco,
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2
Recife, Pernambuco, Brazil.
Departamento de Medicina Veterinária, Universidade Federal do Paraná, Curitiba,
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3
Paraná, Brazil. 4
Departamento de Medicina Veterinária, Universidade Estadual de Londrina, Londrina,
Correspondence:
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Paraná, Brazil.
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Rinaldo Aparecido Mota
Rua Dom Manoel de Medeiros S/N, Dois Irmãos, CEP: 52171-900. Departamento de Medicina Veterinária – Universidade Federal Rural de Pernambuco, Recife-PE, Brazil.
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Telephone: +55 81 33206425; Fax: +55 81 3320 6400. email: [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Little is known about toxoplasmosis in animals of the Fernando de Noronha Island, Brazil. Therefore, we investigated the prevalence of Toxoplasma gondii
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infection in the total population of pet cats (n = 348), dogs (n = 320), pigs (n = 27),
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equines (n = 101), as well as a significant portion of the population of feral cats (n = 247) of the Island by Indirect Fluorescent Antibody Test. Anti-T. gondii IgG antibodies
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were found in 71.26%, 54.74%, 48.75%, 51.85% and 22.7%, of the pet and feral cats, dogs, pigs and equines, respectively, demonstrating a high prevalence of T. gondii infection in the wild and domestic animals of the Island. The Kernel intensity estimator
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showed a correlation between areas with high prevalence of infection in cats and occurrence of infection in the other studied species. We suggest that the island’s health authorities should develop initiatives to reduce the population of cats and alert the
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island’s population about the risk of T. gondii infection.
1. INTRODUCTION
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KEYSWORDS: animal toxoplasmosis, Fernando de Noronha Island, serology
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The Fernando de Noronha Archipelago belongs to the state of Pernambuco, northeastern Brazil, and consists of 21 islands and islets. The main island, named Fernando de Noronha, measures approximately 18.4 km2, being the largest Brazilian oceanic island, and an important region for conservation of biological diversity. It has a
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National Marine Park (Parque Nacional Marinho - PARNAMAR) and two Environmental Protection Areas (Áreas de Proteção Ambiental - APA), focusing on wildlife preservation. Toxoplasmosis is a cosmopolitan zoonosis of medical and veterinary importance. It is caused by coccidia Toxoplasma gondii, an obligate intracellular parasite that can be found in different tissues of infected animals [1,2,3]. Felines, especially cats, are the definitive hosts; they shed the infective forms (oocyst) in their feces contributing to environmental contamination. Studies have shown that the presence of cats in animal production areas is a major risk factor for T. gondii infection [4,5]. The main routes of transmission of T. gondii are through consumption of food contaminated with sporulated oocysts, the transplacental route, and through ingestion of tissue cysts (bradyzoites) in undercooked or uncooked meat [6].
ACCEPTED MANUSCRIPT The clinical manifestations in humans include eye abnormalities, reproductive disorders and neuro-muscular alterations [7]. In animals can cause reproductive disorders mainly in sheep, goats and pigs [2,8,9]. In Brazil, seroepidemiological surveys
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showed that 90% of domestic and wild animals have antibodies against this protozoan
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[10].
Data on animal toxoplasmosis or dispersal of its etiological agent in oceanic islands
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are scarce [11]. Therefore, the present study assessed the prevalence of anti-Toxoplasma gondii antibodies in cats, dogs, pigs and equines of the Fernando de Noronha Island,
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2. MATERIAL AND METHODS
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Brazil, as well as the spatial distribution of infection.
2.1 Sampling
The sample size was determined based on the records of companion (dogs and pet
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cats) and production animals (pigs and equines) provided by the Island’s Administration
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office. No official record was available for feral cats; thus, their sampling sites were chosen considering data concerning the monitoring of their predation activity. Blood
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samples were collected from 348 pet cats, 247 feral cats, 320 dogs, 27 pigs and 101 equines, what represents their total population in the Island, except for the feral cats. The dogs and pet cats are domiciled animals in the houses of habitants of island. While, pigs and cattle are raised in semi-intensive system in small paddocks in the
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properties.
2.2 Biological samples The procedures adopted in the present study were approved by the Animal Ethics Committee of the Federal Rural University of Pernambuco (Comitê de Ética no Uso de animais da Universidade Federal Rural de Pernambuco - CEUA-UFRPE - License n. 116/2015) and are in accordance with the current legislation of the Brazilian College of Animal Experimentation (Colégio Brasileiro de Experimentação Animal - COBEA). The blood samples of pet cats and dogs were collected during the third census of dogs and cats population of the Fernando de Noronha Island. Veterinarians of the Animal Health Surveillance (Núcleo de Vigilância Animal) captured the feral cats with the aid of traps and fishes as bait in predetermined locations. Blood samples were
ACCEPTED MANUSCRIPT collected by puncture of the cranial vena cava in pigs and puncture of the jugular vein in cats, dogs and equines. The blood samples were stored in test tubes, placed in insulated boxes containing
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reusable ice packs and sent to the Animal Health Surveillance of the Health
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Surveillance Unit of the Island Administration where they were centrifuged (2000g for 5 mi). Subsequently, the sera was transferred to Eppendorf® tubes properly identified,
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stored at -20°C and sent to the Laboratory of Infectious Diseases of Domestic Animals of UFRPE where the serological analysis were performed.
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2.3 Serological tests
Anti-T. gondii IgG antibodies were detected using the Indirect Fluorescent
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Antibody Test (IFAT) with a cut-off of 1:16 for cats and dogs, and 1:64 for pigs and equines. Tachyzoites of the RH strain were used as antigens and positive and negative controls (to each specie) were included in all reactions. The second antibodies used
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were anti-IgG antibody specie-specific conjugated with fluorescein isothiocyanate
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(FITC). The slides were examined using an epifluorescent microscope and reactions in which at least 50% of the tachyzoites of each well, presented total peripheral
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fluorescence were deemed positive.
2.4 Spatial distribution
Plane coordinates obtained by Global Positioning System (GPS) were used for the
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development of the spatial map of the Fernando de Noronha Island. The GPS was set to provide the plane coordinates in the UTM (Universal Transverse Mercator) projection in the SAD-69 (South American Datum of 1969), which is the coordinate system of the cartographic base in the Fernando de Noronha Island. The geo-referenced data were plotted in the ArcGIS 10.1 software, using the Kernel intensity estimator, a non-parametric technique that enables filtration of the variability of a data set, retaining the essential characteristics of local data. The color gradient represents the density of cases per property, ranging from green (lowest prevalence) to red (highest prevalence). Localities with high prevalence of T. gondii infection in pigs and equines were also included in the map.
3. RESULTS
ACCEPTED MANUSCRIPT The prevalence of anti-T. gondii IgG antibodies in pets and feral cats, dogs, pigs, equines, were 71.26% (248/348), 54.74% (150/247), 48.75% (156/320) (Table 1), 51.85% (14/27) (Table 2), 22.7% (23/101) (Table 3). The spatial distribution of cats
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positive for T. gondii and its relationship with positivity in dogs, pigs and equines are
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shown in Figure 1.
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4. DISCUSSION
We collected blood samples from the entire population of pet cats, dogs, pigs and equines as well as from a significant portion of the population of feral cats of the
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Fernando de Noronha Island, and registered all the infection foci in these species through spatial distribution. The data presented here can contribute to the development
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of measures to control toxoplasmosis in the Island by the health authorities. The results of the present study show an increase in the prevalence of T. gondii infection in cats, dogs, pigs and equines compared to those reported by Costa et al [12].
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The high prevalence of anti-T. gondii IgG antibodies in pet (71.26%) and feral cats
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(54.74%) have a strong impact on the disease prevalence in other production animals and in humans, because felines, especially feral cats, roam widely over the Island and
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prey on birds and wild rodents, perpetuating the life cycle of T. gondii. Oocysts shed in cats’ feces contaminate pastures, water sources and food, increasing the chances of transmission of toxoplasmosis [3]. In the present study, the highest prevalence of T. gondii infection in dogs, pigs and equines were observed in areas with high prevalence
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of seropositive pet cats, such as the Vila dos Três Paus (Figure 1), which recorded the highest prevalence of T. gondii infection in pigs, equines and dogs. The prevalence of anti-T. gondii IgG antibodies in the pet cats of the Island is similar to those reported by Cavalcante et al [13], Garcia et al [14] and Dubey et al [15] who reported 87.3% seroprevalence in the state of Amazonas, 73% and 84.4% in the state of Paraná, Brazil, respectively. Other studies showed lower prevalence rates in the states of Rio de Janeiro (17.7%) and São Paulo (19%) [16,17]. The prevalence of T. gondii infection in cats varies depending on the animal’s lifestyle, and feral cats, which hunt for their food, usually have higher prevalence than pet cats [18]. In our study, we observed a higher seroprevalence in pet cats than in feral cats what is likely related to the fact that in the Fernando de Noronha Island, pet cats roam freely, prey on rodents and birds and consume viscera of slaughtered animals, increasing their chances of becoming infected.
ACCEPTED MANUSCRIPT Costa et al [12] detected a higher prevalence of T. gondii infection in feral cats (66.6%) than in pet cats (54.2%) of the Fernando de Noronha Archipelago, showing that feral cats become infected through ingestion of tissue cysts from intermediate hosts or
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oocysts from the contaminated environment. The population of feral cats of the
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Fernando de Noronha Island has increased in recent years and given the fact that the environment is highly contaminated with oocysts; a higher prevalence of T. gondii
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infection is expected in these animals.
In the Pacific Islands [19], Australia [20] and United States [21] no evidence of T. gondii infection was observed in regions with no cats, highlighting the role of these
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animals in the transmission cycle of this protozoan. On the other hand, high prevalence of T. gondii infection in pet and feral cats were observed in the Caribbean Islands [22].
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The occurrence of T. gondii infection in companion animals shows the parasite dissemination in the habitat shared with humans [14,23]. As reviewed by Fialho et al [24], the prevalence of anti-T. gondii antibodies in dogs ranges from 3.1% to 91% in
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Brazil. Costa et al [12] found 39.6% (36/91) prevalence of T. gondii infection in dogs of
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the Fernando de Noronha Archipelago, which is lower than that found in the present study.
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T. gondii only completes the enteroepithelial cycle, which results in the development and elimination of oocysts in the environment, in cats [3]. The high prevalence of anti-T. gondii IgG antibodies in pet and feral cats of the Island, demonstrates the ability of these animals to eliminate oocysts after primary infection,
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contributing to the dissemination and maintenance of the protozoan. Although in the present study feral cats had a lower seroprevalence than pet cats, their role in the transmission of T. gondii should not be underestimated because they roam over a wider area than pet cats, and consequently have a significant dispersal capability. The prevalence of T. gondii infection detected in pigs (51.85 %) is above those observed in other studies conducted in continental Brazil [14,25,26,27]. The relationship between the breeding system and exposure of pigs to T. gondii is already known. The pig farming industry is highly mechanized; thus, the possibility of infection by T. gondii or other pathogens is low [3]. However, in the Fernando de Noronha Island, pigs are raised for subsistence in rudimentary facilities, with varied diet (from corn bran to food leftovers) and poor sanitary management increasing their chances to become infected [14].
ACCEPTED MANUSCRIPT According to Dubey [3], in Brazil, the prevalence of anti - T. gondii antibodies in equines ranges from 4.4% to 16%, being close to that reported by Oliveira et al [28] for donkeys and mules (23.8%) in four states of northeastern, Brazil. Although equines
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meat are not commonly consumed in Brazil, the study of T. gondii infection in this
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species is important because they can contribute to the transmission of this protozoa to the definitive host, which may feed on equines viscera. Equines are herbivorous and
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they become infected through ingestion of oocysts in contaminated pastures and water [29].
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5. CONCLUSION
T. gondii infection is highly prevalent in animals of the Fernando de Noronha
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Island. The Kernel intensity estimator showed a correlation between the areas with high prevalence of infection in cats and occurrence of infection in the other studied species. We suggest that the island’s health authorities should develop initiatives to reduce the
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Acknowledgments
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population of cats and alert the island’s population about the risk of T. gondii infection.
To the Pernambuco Research and Technology Foundation (Fundação de Amparo à Ciência e Tecnologia de Pernambuco - FACEPE) for financial support [grant numbers
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APQ-0531-5.05/14].
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[13] G. T. Cavalcante, D. M. Aguiar, D. Chiebao, Seroprevalence of Toxoplasma gondii antibodies in cats and pigs from rural western Amazon, Brazil. J. Parasitol. 92 (2006) 863–864. [14] J. L. Garcia, I. T. Navarro, L. Ogawa, R. C. de Oliveira, Seroepidemiology of toxoplasmosis in cats and dogs from rural properties of Jaguapitã county, Paraná state, Brazil. Ciencia. Rural. 29 (1999) 99–104. [15] J. P. Dubey, I. T. Navarro, C. Sreekumar, Toxoplasma gondii infections in cats from Parana, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates. J. Parasitol. 90 (2004) 721–726. [16] S. R. R. Lucas, M. K. Hagiwara, V. S. Loureiro, J. Y. H. Ikesaki, E. H. Birgel, Toxoplasma gondii Infection in Brazilian Domestic Outpatient Cats. Rev. Inst. Med. Trop. São Paulo 41 (1999) 221-224. [17] A. V. Silva, A. A. Cutolo, and H. Langoni, Comparison of indirect fluorescent antibody test and modified agglutination test to detect antibodies to Toxoplasma gondii in sera from sheep, goats, dogs and cats. Arq. Inst. Biol. 69 (2002) 7–11.
ACCEPTED MANUSCRIPT [18] Y. Sukthana, , J. Kaewkungwal, C. Jantanavivat, A. Lekkla, R. Chiabchalard, W. Aumarm, Toxoplasma gondii antibody in Thai cats and their owners. Southeast. Asian. J. Trop. Med. Pub. Health. 34 (2003) 733–738.
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[19] G.D. Wallace, Serologic and epidemiologic observations on toxoplasmosis on three pacific attols. Am. J. Epidemiol. 90 (1969) 103-111.
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[21] J.P. Dubey, E. A. Rollor, K. Smith, O. C. H. Kwok, P. Thulliez, Low seroprevalence of Toxoplasma gondii in feral pigs from a remote island lacking cats. J. Parasitol. 83 (1997) 839-841.
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[22] J.P. Dubey, M. R. Lappin, O. C. H. Kwok, S. Mofya, A. Chikweto, A. Baffa, D. Doherty, J. Shakeri, C. N. L. Macpherson, R. N. Sharma, Seroprevalence of Toxoplasma gondii and concurrent Bartonella spp., feline immunodeficiency virus, and feline leukemia virus infections in cats from Grenada, West Indies. J. Parasitol. 95 (2009) 1129–1133.
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[23] N.P. Varandas, P. A. A. Rached, G. H. N. Costa, L. M. Souza, K. C. Castagnoli, A. J. Costa, Frequence of antibodies for Neospora caninum and Toxoplasma gondii in dogs in northest of São Paulo State. Semina: Ciências Agrárias. 22 (2001) 105-111.
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[24] C. G. Fialho, M. C. Teixeira, F. A. P. Araujo, Animal Toxoplasmosis in Brazil. Acta. Vet. 37 (2009) 1-23.
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[25] E.H.G. Caporali, A.V. Silva, A.O. Mendonca, H. Langoni, Methodologies comparison for prevalence of anti-Toxoplasma gondii antibodies determination in swine from São Paulo and Pernambuco states - Brazil. Arq. Cien. Vet. Zool. 8 (2005) 19–24. [26] A. B. Moura, S. C. Osaki, D. L. Zulpo, R. M. Marana, Occurrence of antiToxoplasma gondii antibodies in swine and ovine slaughtered at Municipality of Guarapuava in the State of Paraná, Brazil. Rev. Brasil. Parasitol. Vet. 16 (2007) 54–56. [27] E. F. T. S. Fernandes, M. F. T. S. Fernandes, P. C. P. Kim, P. P. F. Albuquerque, O. L. S. Neto, A. S. Santos, E. P. B. X. Moraes, E. G. F. Morais, R. A. Mota, Prevalence of Toxoplasma gondii in slaughtered pigs in the state of Pernambuco, Brazil. J. Parasitol. 98 (2012) 690–691. [28] E. Oliveira, P. P. F. Albuquerque, O. L. Souza Neto, E. B. Faria, J. W. P. Júnior, R. A. Mota, Occurrence of antibodies to Toxoplasma gondii in mules and donkeys in the northeast of Brazil, J. Parasitol. 99 (2013) 343-345. [29] P. Tassi, Toxoplasma gondii infection in horses. A review, Parasitol. 49 (2007) 7– 15.
ACCEPTED MANUSCRIPT Figure 1 - Kernel density map for T. gondii infection in pet cats of the Fernando de
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Noronha Island. Brazil.
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Graphical Abstract
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ACCEPTED MANUSCRIPT Table 1 - Absolute and relative frequency of dogs positive and negative for antiToxoplasma gondii antibodies according to IFAT, Fernando de Noronha, Brazil. Number of Number of Positives Properties
samples
Negatives
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Localities
RF (%)
AF
43
Floresta Nova
40
45
Vila dos Remédios
35
38
Vila dos Três Paus
5
7
Vila da Basinha
10
Vila da Coréia
8
RF (%)
21
48.83
22
51.17
23
51.11
22
48.89
22
57.89
16
42.11
5
71.42
2
28.58
14
9
64.28
5
35.72
8
2
25.0
6
75.0
17
7
41.17
10
58.83
38
14
36.84
24
63.16
Santo 5
6
2
33.33
4
66.67
Vila da Vacaria
6
9
2
22.22
7
77.78
Vila do Boldró
30
38
26
68.42
12
31.58
Vila da Quixaba
8
9
4
44.44
5
55.56
Vila do DPV
22
28
13
46.42
15
53.58
Vila do Sueste
9
9
2
22.22
7
77.78
Vila da Conceição
8
11
4
36.36
7
63.64
do 17
Velha
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Sueste
30
de
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Vila do Trinta Porto
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Estrada
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39
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Floresta Velha
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AF
AF: Absolute Frequency; RF: Relative Frequency
ACCEPTED MANUSCRIPT Table 2 - Absolute and relative frequency of pigs positive and negative for antiToxoplasma gondii antibodies according to IFAT, Fernando de Noronha, Brazil. Number of Number of Positives Properties
samples
Negatives
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Localities
RF (%)
AF
09
Vila dos Remédios
1
04
Vila dos Três Paus
1
07
Vila do Trinta
1
03
Estrada
Velha
do 1
04
Sueste
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AF: Absolute Frequency; RF: Relative Frequency
RF (%)
4
44.44
5
55.56
2
50.00
2
50.00
5
71.43
2
28.57
01
33.33
2
66.67
02
50.00
2
50.00
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2
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Floresta Nova
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AF
ACCEPTED MANUSCRIPT Table 3 - Absolute and relative frequency of equines positive and negative for antiToxoplasma gondii antibodies according to IFAT, Fernando de Noronha, Brazil. Number of Number of Positives Properties
samples
Negatives
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Localities
RF (%)
07
Floresta Nova
10
10
Vila dos Remédios
13
13
Vila dos Três Paus
2
5
Vila da Basinha
4
Vila da Coréia
2
AF
RF (%)
0
00.00
7
100.00
3
30.00
7
70.00
4
30.77
9
69.23
3
60.00
2
40.0
7
2
28.6
5
71.4
4
0
00.00
4
100.00
6
0
00.00
6
100.00
15
5
33.33
10
66.67
Santo 5
5
1
20.00
4
80.00
Vila da Vacaria
3
3
0
00.00
3
100.00
Vila do Boldró
6
6
3
50.00
3
50.00
Vila da Quixaba
6
9
2
22.22
7
77.78
Vila do DPV
6
6
0
00.00
7
100.00
Vila do Sueste
3
3
0
00.00
3
100.00
Vila da Conceição
1
2
0
00.00
2
100.00
do 5
Velha
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Sueste
10
de
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Antônio
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Vila do Trinta Porto
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Estrada
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7
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Floresta Velha
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IP
AF
AF: Absolute Frequency; RF: Relative Frequency
ACCEPTED MANUSCRIPT Highlights - We investigated T. gondii infection in cats, dogs, pigs, equines from Fernando de Noronha Island;
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- There are high prevalence of T. gondii infection in cats from island;
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- Areas with high infection of cats has correlation with infection in other species.
S1383-5769(16)30178-7 doi:10.1016/j.parint.2016.11.014 PARINT 1607
To appear in:
Parasitology International
Received date: Revised date: Accepted date:
6 June 2016 4 November 2016 23 November 2016
Please cite this article as: Magalh˜aes Fernando J.R., Ribeiro-Andrade M¨ uller, Souza F´ atima M., Lima Filho Carlos D.F., Biondo Alexander Welker, Vidotto Odilon, Navarro Italmar Teodorico, Mota Rinaldo A., Seroprevalence and spatial distribution of Toxoplasma gondii infection in cats, dogs, pigs and equines of the Fernando de Noronha Island, Brazil, Parasitology International (2016), doi:10.1016/j.parint.2016.11.014
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Seroprevalence and spatial distribution of Toxoplasma gondii infection in cats, dogs, pigs and equines of the Fernando de Noronha Island, Brazil
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Fernando J. R. Magalhães1, Müller Ribeiro-Andrade2, Fátima M. Souza1, Carlos D. F.
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Lima Filho1, Alexander Welker Biondo3, Odilon Vidotto4, Italmar Teodorico Navarro4,
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Rinaldo A. Mota2*
Unidade de Vigilância em Saúde do Distrito de Fernando de Noronha, Pernambuco,
Brazil.
Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco,
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2
Recife, Pernambuco, Brazil.
Departamento de Medicina Veterinária, Universidade Federal do Paraná, Curitiba,
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3
Paraná, Brazil. 4
Departamento de Medicina Veterinária, Universidade Estadual de Londrina, Londrina,
Correspondence:
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Paraná, Brazil.
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Rinaldo Aparecido Mota
Rua Dom Manoel de Medeiros S/N, Dois Irmãos, CEP: 52171-900. Departamento de Medicina Veterinária – Universidade Federal Rural de Pernambuco, Recife-PE, Brazil.
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Telephone: +55 81 33206425; Fax: +55 81 3320 6400. email: [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Little is known about toxoplasmosis in animals of the Fernando de Noronha Island, Brazil. Therefore, we investigated the prevalence of Toxoplasma gondii
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infection in the total population of pet cats (n = 348), dogs (n = 320), pigs (n = 27),
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equines (n = 101), as well as a significant portion of the population of feral cats (n = 247) of the Island by Indirect Fluorescent Antibody Test. Anti-T. gondii IgG antibodies
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were found in 71.26%, 54.74%, 48.75%, 51.85% and 22.7%, of the pet and feral cats, dogs, pigs and equines, respectively, demonstrating a high prevalence of T. gondii infection in the wild and domestic animals of the Island. The Kernel intensity estimator
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showed a correlation between areas with high prevalence of infection in cats and occurrence of infection in the other studied species. We suggest that the island’s health authorities should develop initiatives to reduce the population of cats and alert the
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island’s population about the risk of T. gondii infection.
1. INTRODUCTION
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KEYSWORDS: animal toxoplasmosis, Fernando de Noronha Island, serology
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The Fernando de Noronha Archipelago belongs to the state of Pernambuco, northeastern Brazil, and consists of 21 islands and islets. The main island, named Fernando de Noronha, measures approximately 18.4 km2, being the largest Brazilian oceanic island, and an important region for conservation of biological diversity. It has a
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National Marine Park (Parque Nacional Marinho - PARNAMAR) and two Environmental Protection Areas (Áreas de Proteção Ambiental - APA), focusing on wildlife preservation. Toxoplasmosis is a cosmopolitan zoonosis of medical and veterinary importance. It is caused by coccidia Toxoplasma gondii, an obligate intracellular parasite that can be found in different tissues of infected animals [1,2,3]. Felines, especially cats, are the definitive hosts; they shed the infective forms (oocyst) in their feces contributing to environmental contamination. Studies have shown that the presence of cats in animal production areas is a major risk factor for T. gondii infection [4,5]. The main routes of transmission of T. gondii are through consumption of food contaminated with sporulated oocysts, the transplacental route, and through ingestion of tissue cysts (bradyzoites) in undercooked or uncooked meat [6].
ACCEPTED MANUSCRIPT The clinical manifestations in humans include eye abnormalities, reproductive disorders and neuro-muscular alterations [7]. In animals can cause reproductive disorders mainly in sheep, goats and pigs [2,8,9]. In Brazil, seroepidemiological surveys
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showed that 90% of domestic and wild animals have antibodies against this protozoan
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[10].
Data on animal toxoplasmosis or dispersal of its etiological agent in oceanic islands
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are scarce [11]. Therefore, the present study assessed the prevalence of anti-Toxoplasma gondii antibodies in cats, dogs, pigs and equines of the Fernando de Noronha Island,
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2. MATERIAL AND METHODS
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Brazil, as well as the spatial distribution of infection.
2.1 Sampling
The sample size was determined based on the records of companion (dogs and pet
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cats) and production animals (pigs and equines) provided by the Island’s Administration
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office. No official record was available for feral cats; thus, their sampling sites were chosen considering data concerning the monitoring of their predation activity. Blood
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samples were collected from 348 pet cats, 247 feral cats, 320 dogs, 27 pigs and 101 equines, what represents their total population in the Island, except for the feral cats. The dogs and pet cats are domiciled animals in the houses of habitants of island. While, pigs and cattle are raised in semi-intensive system in small paddocks in the
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properties.
2.2 Biological samples The procedures adopted in the present study were approved by the Animal Ethics Committee of the Federal Rural University of Pernambuco (Comitê de Ética no Uso de animais da Universidade Federal Rural de Pernambuco - CEUA-UFRPE - License n. 116/2015) and are in accordance with the current legislation of the Brazilian College of Animal Experimentation (Colégio Brasileiro de Experimentação Animal - COBEA). The blood samples of pet cats and dogs were collected during the third census of dogs and cats population of the Fernando de Noronha Island. Veterinarians of the Animal Health Surveillance (Núcleo de Vigilância Animal) captured the feral cats with the aid of traps and fishes as bait in predetermined locations. Blood samples were
ACCEPTED MANUSCRIPT collected by puncture of the cranial vena cava in pigs and puncture of the jugular vein in cats, dogs and equines. The blood samples were stored in test tubes, placed in insulated boxes containing
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reusable ice packs and sent to the Animal Health Surveillance of the Health
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Surveillance Unit of the Island Administration where they were centrifuged (2000g for 5 mi). Subsequently, the sera was transferred to Eppendorf® tubes properly identified,
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stored at -20°C and sent to the Laboratory of Infectious Diseases of Domestic Animals of UFRPE where the serological analysis were performed.
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2.3 Serological tests
Anti-T. gondii IgG antibodies were detected using the Indirect Fluorescent
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Antibody Test (IFAT) with a cut-off of 1:16 for cats and dogs, and 1:64 for pigs and equines. Tachyzoites of the RH strain were used as antigens and positive and negative controls (to each specie) were included in all reactions. The second antibodies used
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were anti-IgG antibody specie-specific conjugated with fluorescein isothiocyanate
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(FITC). The slides were examined using an epifluorescent microscope and reactions in which at least 50% of the tachyzoites of each well, presented total peripheral
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fluorescence were deemed positive.
2.4 Spatial distribution
Plane coordinates obtained by Global Positioning System (GPS) were used for the
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development of the spatial map of the Fernando de Noronha Island. The GPS was set to provide the plane coordinates in the UTM (Universal Transverse Mercator) projection in the SAD-69 (South American Datum of 1969), which is the coordinate system of the cartographic base in the Fernando de Noronha Island. The geo-referenced data were plotted in the ArcGIS 10.1 software, using the Kernel intensity estimator, a non-parametric technique that enables filtration of the variability of a data set, retaining the essential characteristics of local data. The color gradient represents the density of cases per property, ranging from green (lowest prevalence) to red (highest prevalence). Localities with high prevalence of T. gondii infection in pigs and equines were also included in the map.
3. RESULTS
ACCEPTED MANUSCRIPT The prevalence of anti-T. gondii IgG antibodies in pets and feral cats, dogs, pigs, equines, were 71.26% (248/348), 54.74% (150/247), 48.75% (156/320) (Table 1), 51.85% (14/27) (Table 2), 22.7% (23/101) (Table 3). The spatial distribution of cats
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positive for T. gondii and its relationship with positivity in dogs, pigs and equines are
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shown in Figure 1.
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4. DISCUSSION
We collected blood samples from the entire population of pet cats, dogs, pigs and equines as well as from a significant portion of the population of feral cats of the
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Fernando de Noronha Island, and registered all the infection foci in these species through spatial distribution. The data presented here can contribute to the development
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of measures to control toxoplasmosis in the Island by the health authorities. The results of the present study show an increase in the prevalence of T. gondii infection in cats, dogs, pigs and equines compared to those reported by Costa et al [12].
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The high prevalence of anti-T. gondii IgG antibodies in pet (71.26%) and feral cats
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(54.74%) have a strong impact on the disease prevalence in other production animals and in humans, because felines, especially feral cats, roam widely over the Island and
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prey on birds and wild rodents, perpetuating the life cycle of T. gondii. Oocysts shed in cats’ feces contaminate pastures, water sources and food, increasing the chances of transmission of toxoplasmosis [3]. In the present study, the highest prevalence of T. gondii infection in dogs, pigs and equines were observed in areas with high prevalence
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of seropositive pet cats, such as the Vila dos Três Paus (Figure 1), which recorded the highest prevalence of T. gondii infection in pigs, equines and dogs. The prevalence of anti-T. gondii IgG antibodies in the pet cats of the Island is similar to those reported by Cavalcante et al [13], Garcia et al [14] and Dubey et al [15] who reported 87.3% seroprevalence in the state of Amazonas, 73% and 84.4% in the state of Paraná, Brazil, respectively. Other studies showed lower prevalence rates in the states of Rio de Janeiro (17.7%) and São Paulo (19%) [16,17]. The prevalence of T. gondii infection in cats varies depending on the animal’s lifestyle, and feral cats, which hunt for their food, usually have higher prevalence than pet cats [18]. In our study, we observed a higher seroprevalence in pet cats than in feral cats what is likely related to the fact that in the Fernando de Noronha Island, pet cats roam freely, prey on rodents and birds and consume viscera of slaughtered animals, increasing their chances of becoming infected.
ACCEPTED MANUSCRIPT Costa et al [12] detected a higher prevalence of T. gondii infection in feral cats (66.6%) than in pet cats (54.2%) of the Fernando de Noronha Archipelago, showing that feral cats become infected through ingestion of tissue cysts from intermediate hosts or
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oocysts from the contaminated environment. The population of feral cats of the
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Fernando de Noronha Island has increased in recent years and given the fact that the environment is highly contaminated with oocysts; a higher prevalence of T. gondii
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infection is expected in these animals.
In the Pacific Islands [19], Australia [20] and United States [21] no evidence of T. gondii infection was observed in regions with no cats, highlighting the role of these
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animals in the transmission cycle of this protozoan. On the other hand, high prevalence of T. gondii infection in pet and feral cats were observed in the Caribbean Islands [22].
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The occurrence of T. gondii infection in companion animals shows the parasite dissemination in the habitat shared with humans [14,23]. As reviewed by Fialho et al [24], the prevalence of anti-T. gondii antibodies in dogs ranges from 3.1% to 91% in
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Brazil. Costa et al [12] found 39.6% (36/91) prevalence of T. gondii infection in dogs of
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the Fernando de Noronha Archipelago, which is lower than that found in the present study.
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T. gondii only completes the enteroepithelial cycle, which results in the development and elimination of oocysts in the environment, in cats [3]. The high prevalence of anti-T. gondii IgG antibodies in pet and feral cats of the Island, demonstrates the ability of these animals to eliminate oocysts after primary infection,
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contributing to the dissemination and maintenance of the protozoan. Although in the present study feral cats had a lower seroprevalence than pet cats, their role in the transmission of T. gondii should not be underestimated because they roam over a wider area than pet cats, and consequently have a significant dispersal capability. The prevalence of T. gondii infection detected in pigs (51.85 %) is above those observed in other studies conducted in continental Brazil [14,25,26,27]. The relationship between the breeding system and exposure of pigs to T. gondii is already known. The pig farming industry is highly mechanized; thus, the possibility of infection by T. gondii or other pathogens is low [3]. However, in the Fernando de Noronha Island, pigs are raised for subsistence in rudimentary facilities, with varied diet (from corn bran to food leftovers) and poor sanitary management increasing their chances to become infected [14].
ACCEPTED MANUSCRIPT According to Dubey [3], in Brazil, the prevalence of anti - T. gondii antibodies in equines ranges from 4.4% to 16%, being close to that reported by Oliveira et al [28] for donkeys and mules (23.8%) in four states of northeastern, Brazil. Although equines
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meat are not commonly consumed in Brazil, the study of T. gondii infection in this
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species is important because they can contribute to the transmission of this protozoa to the definitive host, which may feed on equines viscera. Equines are herbivorous and
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they become infected through ingestion of oocysts in contaminated pastures and water [29].
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5. CONCLUSION
T. gondii infection is highly prevalent in animals of the Fernando de Noronha
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Island. The Kernel intensity estimator showed a correlation between the areas with high prevalence of infection in cats and occurrence of infection in the other studied species. We suggest that the island’s health authorities should develop initiatives to reduce the
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Acknowledgments
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population of cats and alert the island’s population about the risk of T. gondii infection.
To the Pernambuco Research and Technology Foundation (Fundação de Amparo à Ciência e Tecnologia de Pernambuco - FACEPE) for financial support [grant numbers
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APQ-0531-5.05/14].
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[23] N.P. Varandas, P. A. A. Rached, G. H. N. Costa, L. M. Souza, K. C. Castagnoli, A. J. Costa, Frequence of antibodies for Neospora caninum and Toxoplasma gondii in dogs in northest of São Paulo State. Semina: Ciências Agrárias. 22 (2001) 105-111.
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[24] C. G. Fialho, M. C. Teixeira, F. A. P. Araujo, Animal Toxoplasmosis in Brazil. Acta. Vet. 37 (2009) 1-23.
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[25] E.H.G. Caporali, A.V. Silva, A.O. Mendonca, H. Langoni, Methodologies comparison for prevalence of anti-Toxoplasma gondii antibodies determination in swine from São Paulo and Pernambuco states - Brazil. Arq. Cien. Vet. Zool. 8 (2005) 19–24. [26] A. B. Moura, S. C. Osaki, D. L. Zulpo, R. M. Marana, Occurrence of antiToxoplasma gondii antibodies in swine and ovine slaughtered at Municipality of Guarapuava in the State of Paraná, Brazil. Rev. Brasil. Parasitol. Vet. 16 (2007) 54–56. [27] E. F. T. S. Fernandes, M. F. T. S. Fernandes, P. C. P. Kim, P. P. F. Albuquerque, O. L. S. Neto, A. S. Santos, E. P. B. X. Moraes, E. G. F. Morais, R. A. Mota, Prevalence of Toxoplasma gondii in slaughtered pigs in the state of Pernambuco, Brazil. J. Parasitol. 98 (2012) 690–691. [28] E. Oliveira, P. P. F. Albuquerque, O. L. Souza Neto, E. B. Faria, J. W. P. Júnior, R. A. Mota, Occurrence of antibodies to Toxoplasma gondii in mules and donkeys in the northeast of Brazil, J. Parasitol. 99 (2013) 343-345. [29] P. Tassi, Toxoplasma gondii infection in horses. A review, Parasitol. 49 (2007) 7– 15.
ACCEPTED MANUSCRIPT Figure 1 - Kernel density map for T. gondii infection in pet cats of the Fernando de
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Noronha Island. Brazil.
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Graphical Abstract
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ACCEPTED MANUSCRIPT Table 1 - Absolute and relative frequency of dogs positive and negative for antiToxoplasma gondii antibodies according to IFAT, Fernando de Noronha, Brazil. Number of Number of Positives Properties
samples
Negatives
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Localities
RF (%)
AF
43
Floresta Nova
40
45
Vila dos Remédios
35
38
Vila dos Três Paus
5
7
Vila da Basinha
10
Vila da Coréia
8
RF (%)
21
48.83
22
51.17
23
51.11
22
48.89
22
57.89
16
42.11
5
71.42
2
28.58
14
9
64.28
5
35.72
8
2
25.0
6
75.0
17
7
41.17
10
58.83
38
14
36.84
24
63.16
Santo 5
6
2
33.33
4
66.67
Vila da Vacaria
6
9
2
22.22
7
77.78
Vila do Boldró
30
38
26
68.42
12
31.58
Vila da Quixaba
8
9
4
44.44
5
55.56
Vila do DPV
22
28
13
46.42
15
53.58
Vila do Sueste
9
9
2
22.22
7
77.78
Vila da Conceição
8
11
4
36.36
7
63.64
do 17
Velha
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Sueste
30
de
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Vila do Trinta Porto
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Estrada
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39
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Floresta Velha
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AF
AF: Absolute Frequency; RF: Relative Frequency
ACCEPTED MANUSCRIPT Table 2 - Absolute and relative frequency of pigs positive and negative for antiToxoplasma gondii antibodies according to IFAT, Fernando de Noronha, Brazil. Number of Number of Positives Properties
samples
Negatives
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Localities
RF (%)
AF
09
Vila dos Remédios
1
04
Vila dos Três Paus
1
07
Vila do Trinta
1
03
Estrada
Velha
do 1
04
Sueste
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AF: Absolute Frequency; RF: Relative Frequency
RF (%)
4
44.44
5
55.56
2
50.00
2
50.00
5
71.43
2
28.57
01
33.33
2
66.67
02
50.00
2
50.00
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2
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Floresta Nova
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AF
ACCEPTED MANUSCRIPT Table 3 - Absolute and relative frequency of equines positive and negative for antiToxoplasma gondii antibodies according to IFAT, Fernando de Noronha, Brazil. Number of Number of Positives Properties
samples
Negatives
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Localities
RF (%)
07
Floresta Nova
10
10
Vila dos Remédios
13
13
Vila dos Três Paus
2
5
Vila da Basinha
4
Vila da Coréia
2
AF
RF (%)
0
00.00
7
100.00
3
30.00
7
70.00
4
30.77
9
69.23
3
60.00
2
40.0
7
2
28.6
5
71.4
4
0
00.00
4
100.00
6
0
00.00
6
100.00
15
5
33.33
10
66.67
Santo 5
5
1
20.00
4
80.00
Vila da Vacaria
3
3
0
00.00
3
100.00
Vila do Boldró
6
6
3
50.00
3
50.00
Vila da Quixaba
6
9
2
22.22
7
77.78
Vila do DPV
6
6
0
00.00
7
100.00
Vila do Sueste
3
3
0
00.00
3
100.00
Vila da Conceição
1
2
0
00.00
2
100.00
do 5
Velha
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Sueste
10
de
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Antônio
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Vila do Trinta Porto
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Estrada
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7
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Floresta Velha
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AF
AF: Absolute Frequency; RF: Relative Frequency
ACCEPTED MANUSCRIPT Highlights - We investigated T. gondii infection in cats, dogs, pigs, equines from Fernando de Noronha Island;
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- There are high prevalence of T. gondii infection in cats from island;
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- Areas with high infection of cats has correlation with infection in other species.