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Detection of Toxoplasma gondii in a free-ranging giant anteater/Deteccao de Toxoplasma gondii em tamandua-bandeira de vida livre.

Toxoplasmosis is a worldwide distribution disease caused by Toxoplasma gondii, an obligatory intracellular protozoan. This parasite has felids as final hosts, causing acute and chronic infections in a wide variety of intermediate hosts, including humans (DUBEY, 2010).

Identification of T. gondii by molecular techniques in new hosts is essential to both clarify its interaction with hosts and indicate enviromnental contamination (RICHINI-PEREIRA et al., 2016). Toxoplasmosis can be diagnosed by biological, serological, and histological methods ora combination of them (DUBEY, 2010). Despite the serological evidence of infection with T. gondii in free-ranging wild animals, their role in the epidemiological chain and species susceptibility to protozoan are not yet well described (VITALIANO, 2014).

The giant anteater (Myrmecophaga tridactyla) population is decreasing, and this species has been classified as "vulnerable" (MIRANDA & MEDRI, 2010). Therefore, the objective of this study was to describe the first positive case of T. gondii detected by PCR in the peripheral blood of a young free-ranging giant anteater.

A female and orphan specimen of a giant anteater (M. tridactyla: approximate age: 10 days; weight: 1.26kg) was attended at the Veterinary Hospital (HOVET), Universidade Federal do Mato Grosso (UFMT), receiving veterinary medical care and breastfeeding. The pup had no contact with other species of wild or domestic animals during the period when it was in the hospital. Although, it was clinically healthy, at three months of age it was chemically contained with Ketamine (8.0mg/kg) and Midazolam (0.2mg/kg) for routine exams, such as hematology, clinical biochemistry, and search for infectious agents in the epidemiological survey of wild animals received at the HOVET-UFMT. Blood collected by venipuncture of the cephalic vein, and the sample so obtained was divided into three aliquots: i) 10% EDTA tube (lmL), ii) tube without anticoagulant (2mL), and Hi) microtube (0.5mL), depending on the type of analysis. The values for the hematological and biochemical parameters were within normality as expected for the species.

The serum sample was submitted to the modified agglutination test (MAT) for T. gondii, being performed according to DUBEY & DESMONTS (1987). Reagent samples were considered positive at dilutions equal to or greater than 1:25. Antigen obtained from mice peritoneal fluid, previously inoculated with RH strain tachyzoites, was used. The whole blood sample was submitted to DNA extraction using the phenol-chloroform method (SAMBROOK & RUSSELL, 2001). Then a PCR was performed, using the universal oligonucleotides TOXO 1 (5'-GGAACTGCATCCGTTCATGAG-3') and TOXO 2 (5'-TCTTTAAAGCGTTCGTGGTC-3') as described by BURG et al. (1989), seeking to amplify a 194 base pair (bp) fragment of the B1 gene species specific for T. gondii. The following reagents were used for amplification (final volume: 25(il): 8pmol of each primer: 2.7mM Mg[Cl.sub.2]; lx PCR buffer (20mM Tris-HCl; 50mM KC1; pH 8.4); 0.2mM DNTPs; 1U Taq DNA polymerase; and lOng DNA. The reaction was carried out in a thennocycler (BIO-RAD My CyclerThermalCycler) with initial denaturation (95[degrees]C, 5min), followed by 30 cycles (95[degrees]C, 30sec), annealing (56[degrees]C, 45sec) and extension (72[degrees]C, 2min), with a final extension cycle (72[degrees]C, 5min). Ultrapure water was used for the negative control and genomic DNA (lOng) from the protozoan cell culture was used for the positive control. The control DNA was extracted using the method described above. The amplification products were analyzed in 2.0% agarose gel electrophoresis, stained with Gel Red[TM] (Biotium[R]; 10V/cm), and observed in ChemiDoc[TM] XRS using the ImageLab[TM] software. Ladder (Ludwig[R]; 0.1([micro]g/([micro]l; 100bp) DNA was used as base pair marker.

The sample was positive in PCR. The amplicon thus obtained was purified on the GFX PCR DNA and Gel Band purification kit, sequenced on the ABI 3500 (Applied Biosvstems[R]) Genetic Analyzer using specific oligonucleotides, and the sequence was finally deposited in the GenBank (No. KU288755). The sequence was also analyzed by the BLAST (NCBI) program, obtaining 100% identity with the AF179871 sequence of T. gondii. In the MAT, the serum sample was positive for anti-T: gondii antibodies (title: 1:800).

It should be considered that PCR positivity for T. gondii will only be observed in peripheral venous blood samples in cases of parasitemia, i.e., when protozoan dissemination or passage occurs in the blood. This may occur in the acute phase of infection or, alternatively, when there is recrudescence of encysted bradyzoites, with possible appearance of clinical signs, such as in situations in which the immune system is affected (DUBEY, 2010).

The T. gondii parasite causes an asymptomatic infection in most hosts due to a rapid induction of the cellular immune response, which results in the control of tachyzoite multiplication and establishment of a chronic infection (DUBEY, 2010). Non-regenerative anemia, neutrophilic leukocytosis, lymphocytosis, monocytosis, and eosinophilia are most commonly observed (LAPPIN et al., 1996). However, the hematological and biochemical parameters remained within the normal range in this case, even during parasitemia.

In this giant anteater, no clinical sign was observed during parasitemia. In this case, stating that parasitemia occurred by toxoplasmosis reactivation due to either recrudescence of the encysted bradyzoites or the acute phase of an infection is uncertain. The absence of clinical signs in this specimen can be explained by strain pathogenicity and virulence, in addition to host immunity. Little is known about the susceptibility of the giant anteater to this infection.

For the first time, Vitaliano (2014) conducted a serological survey, and described anti-T. gondii antibodies detected in two free-ranging anteaters by the same MAT technique, and their titles (1:100 and 1:800) were similar to that obtained in the present study. Moreover, RICHINIPEREIRA et al. (2016) performed the genotyping of T. gondii in wild mammals killed in traffic accidents on a road (Central-West Region, State of Sao Paulo, Brazil), and they reported T. gondii DNA in samples of tissues from two M. tridactyla.

The diagnostic sensitivity of serological tests may be low when they are used in cases of acute toxoplasmosis (GYIMESI et al., 2016). In these cases, the clinical signs may appear before the development of an immune response, and failure can occur in the detection of class G (IgG) or M (IgM) immunoglobulins at the beginning of parasitemia (CASTILHO-MORALES et al., 2012).

Thus, diagnosis of T. gondii by PCR using peripheral blood to detect DNA is imperative in cases in which confirmation of parasitemia in the host is necessary, especially in those with immunological deficiency (ROBERT-GANGNEUX, 2015). In addition, PCR also allows diagnosis in animals with high sensitivity to protozoan infection (which can develop fatal toxoplasmosis) and early detection; although, in the acute phase, prior to antibody production. Therefore, early detection enables treatment in highly susceptible species, such as neotropical primates and giant panda (Ailuropoda melanoleuca) among others (CASAGRANDE et al., 2013; MA et al., 2015).

The positive results obtained from serological tests do not always reflect that the host is infected with T. gondii. They only indicate contact with the protozoan. The PCR confirms the presence of parasite DNA (KORNACKA et al., 2016). GYIMESI et al. (2006) conducted a study using different toxoplasmosis diagnostic techniques in a colony of woolly monkeys (Lagothrix lagotricha). Histopathological and biochemical findings showed that death of monkeys was due to fatal toxoplasmosis. Later, these authors were able to perform PCR of DNA extracted from stored serum samples, thus confirming that they were positive for T. gondii. These results showed effective diagnosis through the use of PCR.

Giant anteaters are historically hunted for food in all their territorial distribution, and such practice is part of the culture of some indigenous peoples (MIRANDA & MEDRI, 2010). Since large felines are natural predators of this species, the sexual phase of T. gondii continues in these wild felines as definitive hosts, thus completing the protozoan cycle in the wild environment (DUBEY, 2010).

Thus, the giant anteatercanbe considered as an intermediate host, being a probable source of infection for humans and wild carnivores through the consumption of raw or undercooked meat from giant anteater. In this case, pathogenicity is related to the T. gondii sample virulence of and species susceptibility. Although, parasitemia was confirmed, by amplification of T. gondii DNA (GenBank; No. KU288755) and a high antibody title (1:800), we emphasize the fact that this specimen of M. tridactyla showed no clinical sign of infection. Therefore, it is impossible to draw a conclusion based only on serological data or DNA detection in tissues from dead animals. Therefore, further studies are suggested on genotyping and pathogenicity of strains of T. gondii reported in M. tridactyla.

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

Approved by the Ethics Committee for Animal Use (CEUA-UFMT; protocol N[degrees] 23108.017917/12-0).

http://dx.doi.org/ 10.1590/0103-8478cr20161127

Received 12.27.16

Approved 04.26.17

Returned by the author 06.15.17

CR-2016-1127.R1

ACKNOWLEDGEMENTS

To Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES); to Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for the scholarships of V. Dutra and R.C. Pacheco.

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Thais Oliveira Morgado (1) * Francielle Cristina Kagueyama (2) Janaina Marcela Assuncao Rosa (2) Melissa Dehesa Belizario (2) Richard de Campos Pacheco (2) Valeria Dutra (2) Sandra Helena Ramiro Correa (1, 2) Regina Celia Rodrigues da Paz (2)

(1) Universidade Federal de Mato Grosso (UFMT), Av. Femando Correa da Costa, 2367, Boa Esperanca, 78060-900, Cuiaba, MT, Brasil. E-mail: thaismorgadoMgmail.com. * Corresponding author.

(2) Faculdade de Medicina Veterinaria, Universidade Federal de Mato Grosso (UFMT), Cuiaba, MT, Brasil.
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Title Annotation:PARASITOLOGY
Author:Morgado, Thais Oliveira; Kagueyama, Francielle Cristina; Rosa, Janaina Marcela Assuncao; Belizario,
Publication:Ciencia Rural
Date:Aug 1, 2017
Words:2088
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