Investigation of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in captive giant anteaters ( Myrmecophaga tridactyla )

This research aimed to investigate the occurrence of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in captive giant anteaters. Blood and fecal samples were taken from 16 animals in institutions from the states of Minas Gerais, Bahia and Distrito Federal, which had been in captivity for at least a year. A commercial rapid chromatographic immunoassay test was used for detecting coronavirus and parvovirus antigens, in addition to antibodies against leishmaniasis, all results being negative. In the case of the test for antibodies against distemper, four (4/16; 25%) anteaters had an average titration, two (2/16; 12.5%) a low titration and ten (10/16; 62.5%) were non-reactive. Using the DOT-ELISA (dot blotting) method for detection of immunoglobulin G, only one specimen obtained a 1 : 40 titration. For the polymerase chain reaction tests for Leishmania and Chlamydia , all samples were negative.


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In Brazil, the giant anteater (Myrmecophaga tridactyla Linnaeus, 1758) is part of the group of 110 taxa of endangered mammals (Young et al., 2003), with a conservation status classified as "vulnerable" (Miranda et al., 2014).The main threats to the species include being run over, Arq.Bras.Med. Vet. Zootec., v.74, n.5, p.833-840, 2022 Furthermore, many of the emerging zoonoses arose from changes in the interaction of fauna promoted by humans, which resulted in the contact of pathogens with new hosts (Chang, 2020).
There is little research on the effects of proximity between domestic animals and wild specimens, especially with the species M. tridactyla (Miranda, 2008).In the case of free-living mammals, Vilela and Guedes (2014) highlighted the impact of the presence of domestic dogs in protected areas, as in addition to preying on and competing with native species for territory and food, they can transmit infectious and contagious diseases.
When it comes to xenarthras, distemper was reported in seven two-toed sloths (Choloepus didactylus) by Sheldon et al. (2017), with diagnosis made by polymerase chain reaction (PCR) test and gene sequencing.A positive result was also found in an anteater (Tamandua tetradactyla) by Lunardi et al. (2018), from postmortem PCR tests, immunohistochemical assay and gene sequencing.In M. tridactyla, Granjeiro et al. (2020) reported the presence of Lentz inclusions in an animal from Mato Grosso state, that died with positive results also in the rapid test for canine distemper antigen, PCR of gene N and phylogenetic analysis of gene H.
Additionally, for giant anteaters, there are reports of some infectious agents occurring in domestic animals, such as Toxoplasma gondii (Ferrari, 2016) and bacteria of the genera Brucella and Leptospira in free-living animals (Miranda, 2008), in addition to influenza virus A in captive specimens (Nofs et al., 2009).Cases of interspecific transmission of infectious agents highlight the importance of biosecurity measures and monitoring of wild animals under human care (Granjeiro et al., 2020).Determining the health status of captive individuals is essential in the case of threatened species, since it is possible to include them in reintroduction, translocation, or population invigoration programs.
Due to the scarcity of information about the occurrence of pathogens in anteaters, this study aimed to investigate the occurrence of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in captive M. tridactyla.

MATERIAL AND METHODS
The methodology of this study was approved by the Animal Use Ethics Committee of the University of Brasília (CEUA-UnB), protocol no.55/2019, and the Biodiversity Information and Authorization System (SISBIO), no.68635-4.Blood and feces samples from 16 giant anteaters (M.tridactyla) were used, seven being males and nine females.One of the specimens was born in captivity, while the others were received as puppies by the institutions and kept under human care for at least a year.
The samples were collected from seven animals from the TamanduAsas rehabilitation project and two from the Parque do Sabiá Municipal Zoo, both in the municipality of Uberlândia, MG.Four animals were under the care of the Fundação Jardim Zoológico de Brasília, DF, and three were from Parque Vida Cerrado, in Luís Eduardo Magalhães, BA (Table 1).
The anteaters were captured and immobilized with the aid of a catching net for the application of drugs for chemical restraint, with variation of the protocols according to what was established by each institution.The physical examination included inspection, palpation, and auscultation of the anteaters, which had a body score between 2.5 and 3 (scale 1 to 5), mean rectal temperature of 33 °C, normohydrated, well-formed stools, with no change in coat or lesions to the skin.Blood collection for blood count and serum biochemistry was also performed.
Blood was punctured from the cephalic (Figure 1A), lateral saphenous or femoral vein, using a 5 mL syringe and 25 × 0.70 mm disposable needle.The samples were placed in tubes containing ethylenediamine tetraacetic acid (EDTA) and a tube without anticoagulant.In the case of feces, samples were collected fresh directly in the enclosure when defecation was witnessed, and by rectal swab (Figure 1B), with storage in a universal collector flask and Eppendorf tubes containing phosphate-buffered saline (PBS), respectively.tubes.For that, a commercial kit was used (Illustra Blood genomicPrep Mini Spin Kit ® , GE Healthcare, Piscataway, NJ, USA) and the procedures followed the manufacturer's recommendations.The material obtained was stored in Eppendorf tubes in a freezer at −20 °C until the time of performing the PCR tests.

All
samples were submitted to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) test to verify the extraction quality, the integrity of the obtained DNA and the absence of PCR inhibitors.The mixture for this first step consisted of 16.55 µL of water; 2.5 µL of PCR buffer; 1.5µL of magnesium chloride (50mM MgCl 2 /µL); 1µL of each oligonucleotide (Table 2) (10pmol/µL GAPDH F and GAPDH R); 0.2µL of phosphated deoxyribonucleotides (25mM/µL DNTP); 0.25µL of Taq DNA polymerase (5U/µL; Invitrogen ® , Vila Guarani, SP, Brazil); and 2µL of the sample (DNA), with a final volume of 25µL.After preparation, the compound was amplified by denaturation steps.For this, initially a temperature of 95°C for 5 min was used, followed by 40 cycles of amplification at 94°C for 30s, 52°C for 1 min, 72°C for 1 min and final extraction at 72°C for 5 min, as described by Birkenheyer et al. (2003).Amplification was performed in three steps, starting with denaturation at 94°C for 5 min, followed by 40 cycles of amplification, with denaturation at 94°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 5 min, as indicated by Disch et al. (2003).
The mix for Chlamydia was composed of 17.05µL of water; 2.5µL of PCR buffer; 1.0µL of MgCl 2 (50mM/µL); 1.0µL of Chls A and Chls B primers; 0.2µL of 25µM DNTP (25mM/µL); 0.2 µL of Taq polymerase (Invitrogen ® , Vila Guarani, SP, Brazil); and 2µL of the sample (DNA) in a final volume of 25µL.Then, the mixture was submitted to the amplification protocol, starting from initial denaturation at 94°C for 10 min, followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1 min.Finally, the mixture underwent final extension at 72°C for 4 min and cooling at 4°C, according to the methodology of Silva (2013).
All PCR tests were performed in a thermocycler device (Bio-Rad ® C1000TM Thermal Cycler, Hercules, CA, USA) and their products were visualized on a 2% agarose gel by electrophoresis.To assess the size of the bands of the amplified compound, the gel was stained with ethidium bromide (Vetec, Sigma-Aldrich ® , St. Louis, MO, USA) and photographed in a transilluminator (UV Transilluminator UVP ® , Upland, CA, USA) under ultraviolet (UV) light.In all tests, a molecular marker (Invitrogen ® , Vila Guarani, São Paulo, Brazil) was used, with a molecular weight range of 100bp.The negative control was carried out with sterile water devoid of DNA (autoclaved Milli-Q), and the positive control with samples from dogs diagnosed and confirmed by the Laboratory of Microbiology and Molecular Pathology of the Veterinary Hospital of the University of Brasília and vaccine for detection of Chlamydia.
The samples were analyzed for IGG detection against distemper and parvovirus using the DOT-ELISA (dot blotting) technique.Results were interpreted as follows: score zero = negative; score 1 and 2 = low positive, score 3 = medium positive, and score 4 or greater = high positive.
Subsequently, using the indirect immunofluorescence reaction (IFAT) method, the results were obtained in the table of conversion of titers to 1 : 40; 1 : 80; 1 : 160 or 1 : 320.The laboratory analysis of ELISA was performed by the laboratories TECSA ® (Animal Health Technology Laboratory, Belo Horizonte, MG, Brazil) and CIVET (Uberlândia, MG, Brazil) with blood serum.

RESULTS
For the investigated infectious agents, all 16 animals showed non-reactive results for coronavirus and parvovirus antigens, as well as for antibodies against Leishmania.All animals tested were positives for GAPDH and negative for Leishmania 18S, Leishmania kinetoplast and Chlamydia sp. in PCR.None of the samples evaluated were reactive for parvovirus virus in the DOT-ELISA test.
In the rapid test for detection of antibodies against distemper, four (4/16; 25%) animals had a medium titer marking, two (2/16; 12.5%) had a low titer and ten (10/16; 62.5%) were nonreactive (Table 3).When comparing the rapid test with the laboratory test, the agreement was 68.75% (11/16), with 31.25% (5/16) of the samples showing false-positive results by the rapid test.Only sample VC2 was positive by DOT-ELISA (1/16, 6.25%), with a score of 1 and a 1 : 40 titer for canine distemper virus.The VC2 animal, which showed medium titration results in the rapid immunochromatographic test, was received by the Brasília Zoo as a puppy, without clinical signs of disease, and at 1 year of age was transferred to the Parque Vida Cerrado, in Bahia.
The three animals of the institution are housed in neighboring rooms separated by a wire mesh and showed positive results in the rapid test.Animals VC1 and VC2 were mated in 2018, resulting in the birth of VC3.

DISCUSSION
The scarcity of data about the sanitary conditions of wildlife species represents one of the challenges for conservation strategies, as this information can be used to estimate the risk of extinction (Radchuk et al., 2016).Surveys about the occurrence of agents causing diseases such as distemper, parvovirus, leishmaniasis and chlamydiasis in specimens of M. tridactyla kept under human care are rare or non-existent, this being the first study in which rapid and comparative laboratory tests were carried out to investigate antibodies against the morbillivirus in the species.
Of the six anteaters positive in the rapid antibody detection test for distemper, only one confirmed a reaction in the DOT-ELISA IGG laboratory test.This specific anteater did not show clinical signs of disease and there are reports that stray dogs sporadically invade the park.Studies in this regard are important since Lunardi et al. (2018) followed the occurrence of death of giant anteaters by distemper with signs similar to those presented by canids, but there is still a lack of further information about the morbidity and mortality of the disease for the species and this is the first investigation of this disease in xenarthras from the Distrito Federal, and Bahia and Minas Gerais states.
The M. tridactyla positive for antibodies against distemper in the laboratory test was rescued as a puppy and has been in captivity for 10 years, so it is not possible to determine when and where its contact with the morbillivirus occurred.
According to Monti (2004), dogs that were experimentally exposed to canine distemper virus presented antibody titers on the ninth day after exposure and a study showed that the duration of immunity against the disease can extend for a period greater than 9 years (Angelico and Pereira, 2012).(δCoV).Species of the first genus mainly infect domestic dogs and cats, but there are species that infect pigs and humans, and a study has already detected the circulation of Alphacoronavirus in wild bats, mice, and rabbits in France (Monchatre-Leroy et al., 2017;Stout et al., 2020).Due to the high mutation potential of coronaviruses, including as zoonotic agents, and the previous reports of infectious diseases from dogs in anteaters (Sheldon et al., 2017;Lunardi et al., 2018;Granjeiro et al., 2020), investigations such as the one in the present study are important for their health.
Chlamydia is a bacterium that causes reproductive disturbance in several species of domestic mammals and stands out for its zoonotic potential and for causing economic damage (Lima et al., 2019).The Institute for Research on the Conservation of Anteaters in Brazil (IPCTB) described an individual of the species M. tridactyla from captivity in a zoo in the state of São Paulo, with a positive diagnosis for the disease after miscarriage (Miranda, 2008).All animals sampled in this study were negative for Chlamydia, similarly to the research carried out by Miranda (2008) with free-living specimens.
Understanding how diseases are transmitted, how they progress and how to prevent their spread is important to ensure the sanitary health of wild animals under human care.Some anteaters in this research are part of rehabilitation projects; therefore, the performance of sanitary tests is essential to assess the health of animals and reduce the chances of transmission of infectious agents to free-living populations.The data obtained in this study can support decisions to be implemented in the National Action Plan (PAN) for conservation of the species M. tridactyla.

CONCLUSION
The specimens of M. tridactyla from captivity showed negative results for the investigation of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in the tests performed.Six anteaters obtained a positive reaction in the rapid immunochromatographic test for detection of antibodies against Morbillivirus, but only one was confirmed by the DOT-ELISA technique, with an occurrence of 31.25% of false-positive results.

Figure 1 .
Figure 1.Collection of biological samples in Myrmecophaga tridactyla.A: blood collection in the right cephalic vein.B: rectal swab sample collection Immediately after the collection of biological samples, rapid immunochromatographic tests (Alere S.A. -Bionote, Belo Horizonte, MG,Brazil)  were performed to detect antibodies against distemper and Leishmania infantum (rK9, rK39 and rK26 antigens), using whole blood sampling.Additionally, the same types of tests were also used to investigate the presence of parvovirus and canine coronavirus antigens (Test

Table 1 .
Information of sex, age (approximate) and institution responsible for the specimens of *Animal born in captivity.The other specimens were born in the wild and were rescued.

Table 2 .
Sequence of oligonucleotides, gene of origin, size of amplification products and references of polymerase chain reactions with deoxyribonucleic acid samples used in the research