First Molecular Screening of Plasmodium Species in Ungulates
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dos Santos et al. BMC Res Notes (2018) 11:536 https://doi.org/10.1186/s13104-018-3638-5 BMC Research Notes RESEARCH NOTE Open Access First molecular screening of Plasmodium species in ungulates from Southern Brazil Leonilda Correia dos Santos1,2†, Lilian de Oliveira Guimarães3†, Ana Laura Grazziotin4, Wanderlei de Morais5, Zalmir Silvino Cubas5, Marcos José de Oliveira5, Rafael Felipe da Costa Vieira1, Alexander Welker Biondo1 and Karin Kirchgatter3* Abstract Objective: Despite malaria epidemiology has been extensively studied in primates, few studies were conducted in ungulates. After half a century without descriptions of Plasmodium spp. in deer since its frst identifcation, recent research has rediscovered Plasmodium on ungulates in Africa, Asia, North America and South America, including Central Brazil. Here, a captive herd was evaluated in southern Brazil using light microscopy and PCR. DNA samples were tested for fragment amplifcation of two Plasmodium spp. genes: mitochondrial cytochrome b and small subunit ribosomal RNA. Results: All analyses were negative. However, the tests were performed on samples that were collected at a single time point, and parasitemia may fuctuate over the parasite’s life cycle. Thus, the possibility of occult infection cannot be ruled out. Despite the negative results of all of the methods applied, it cannot be categorically stated that these animals are free from Plasmodium sp. infection. Further monitoring and/or multiple sequential sampling may improve the success rate of detecting parasites. Moreover, although this survey of Plasmodium represents the frst molecular study on ungulate malaria parasites from Southern Brazil, further analysis of samples from diferent ungulate species is important for characterizing the epidemiology of Plasmodium of these mammals in this region. Keywords: Malaria, PCR, Deer, Zoo animals, Brazil Introduction deer (Odocoileus virginianus) in Texas, United States of Malaria parasites have been described in a wide range America (USA) [6, 9]. of hosts in the Americas, including humans [1], mon- Surprisingly, recent reports have “rediscovered” keys [2], free-living birds [3], reptiles [4] and rodents [5]. malaria parasites in cervids and other ungulates through- Among non-primate mammals, Plasmodium species had out the world [10–14]. Tese studies have raised ques- been thought to be limited to the Old World. In particu- tions regarding the evolution of Plasmodium parasites, lar, cervids had not been considered to be a vertebrate the cross-continental dispersion of these parasites and host due to the absence of parasites in blood smear inves- the role of ungulates as malaria reservoirs. Specifcally, tigations [6–8]. Tis idea was proved wrong in 1967 with the recent identifcation of Plasmodium-positive deer in the identifcation of a Plasmodium parasite (P. odocoilei) Central Brazil [14] led us to ask whether the presence of in a blood smear from a splenectomized white-tailed malaria reservoirs might extend to cervids from South- ern Brazil. A large herd of captive Brazilian dwarf brocket deer *Correspondence: [email protected] (Mazama nana) is protected in the Bela Vista Biologi- †Leonilda Correia dos Santos and Lilian de Oliveira Guimarães contributed cal Sanctuary (BVBS), Foz do Iguaçu, Brazil. Red brocket equally to this work deer (M. americana) and marsh deer (Blastocerus dichot- 3 Malaria Research Center, Superintendence for Endemic Disease Control, ′ ″ ′ ″ Institute of Tropical Medicine, University of São Paulo, São Paulo, SP, Brazil omous) are also present. BVBS (25° 26 57 S; 54° 33 18 Full list of author information is available at the end of the article W) is a zoo and animal rehabilitation center located in © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. dos Santos et al. BMC Res Notes (2018) 11:536 Page 2 of 5 a national protected area in southern Brazil and shares purposes, given that internationally, Brazilian dwarf borders with Argentina and Paraguay. Cervids are kept in brocket deer, marsh deer and red brocket deer have been fenced areas that are covered and surrounded by native considered vulnerable [18–20]. Tus, considering the vegetation, making contact with free-living wild animals recent reports about malaria parasites in cervids and possible. Capybaras (Hydrochoerus hydrochaeris) are other ungulates throughout the world, including Central often seen moving freely through zoo areas. Our research Brazil [10–12, 14], we conducted a re-examination, using group frst reported Plasmodium sp. infection in capyba- microscopic and molecular tools, of these 32 deer sam- ras that were captive animals at the BVBS, with fndings ples, searching for Plasmodium species. of infection in 1/11 via microscopy and 3/11 via molecu- lar testing [5]. In addition, the mosquito Anopheles sp., which is the Main text vector implicated in Plasmodium spp. transmission, Methods has been shown to have > 10% prevalence in this region, Animals and blood collection favored by the warm and humid climate [15]. Altogether, Blood samples from 32 cervids (22 M. nana, dwarf this region may provide conditions that are sufcient for brocket deer; 4 M. americana, red brocket deer and 6 a complete transmission cycle extending to wild mam- B. dichotomus, marsh deer) that were previously sur- mals, particularly cervids. veyed for other pathogens [16, 17] were included in this In Brazil, the Brazilian dwarf brocket deer, red brocket study. Te animals were maintained in captivity at Bela deer and marsh deer have been screened for pathogens, Vista Biological Sanctuary, Itaipu Binational Dam, in Foz with 27/31 (87%) infected by Mycoplasma ovis [16], 5/32 do Iguaçú, Paraná State, Brazil (Fig. 1). Since all animals (15.6%) seropositive for Toxoplasma gondii, 2/32 (6.2%) were clinically healthy at the time of constraining and positive for Neospora caninum and 1/32 (3.1%) posi- sampling, no preventive or curative treatment had been tive for Leptospira interrogans. Additionally, all animals given to any of the captive deer surveyed herein. EDTA tested negative for Brucella abortus, bovine viral diarrhea blood samples were collected by jugular venipuncture virus, foot-and-mouth disease virus, infectious bovine from the deer and were stored at − 20 °C. rhinotracheitis, Eastern equine encephalitis, Western Bela Vista Biological Sanctuary currently holds the big- equine encephalitis and Venezuelan equine encephalitis gest captive collection of Brazilian native deer worldwide. [17]. Tis monitoring has provided important informa- Death rates of Brazilian native deer due to constraint and tion for animal and public health and for conservation sedation have varied from 10 to 100% [21]. Although no Fig. 1 Origin of samples used in the study. A Location of Brazil in South America. B Location of Bela Vista Biological Sanctuary, Itaipu Binational Hydroelectric Power Plant, Foz do Iguaçu, Parana State Source: Modifed from Google Earth dos Santos et al. BMC Res Notes (2018) 11:536 Page 3 of 5 animal was lost as a result of the present survey, no fur- Results and discussion ther sampling has been performed. No Plasmodium-like parasites were observed through Tis study was approved by Bela Vista Biological direct examination of deer blood smears, and no DNA Sanctuary and the federal regulatory agency IBAMA amplifcation was detected by means of PCR for either (Brazilian Institute for the Environment and Renew- the SSU rRNA or the cytb gene (Fig. 2). However, nega- able Resources) under the Protocol Number 22.158-2 tive fndings obtained in this study may depend on the (November 12, 2009) and was conducted in accordance low parasitemia at the time of blood collection. Indeed, with IBAMA’s ethical rules. low parasitemia has been previously described in infected ungulates [6, 11, 12], and the incidence of infection has Microscopic examination been as low as 0.003% [12] in molecular tests, with no Peripheral blood smears were stained with May-Grün- detection of parasites in blood smears. Moreover, the life wald-Giemsa stain and were examined using optical cycle of Plasmodium in vertebrate hosts may include a microscopy at ×1000 (BX51, Olympus, Tokyo, Japan) long-lived dormant stage in the liver, with sequestration [22]. of the parasite from the general circulation, causing very low parasitemia in the absence of an immunosuppressed DNA extraction and molecular analyses state, as previously reported in water bufalos (Bubalus DNA was extracted using a commercial kit (DNeasy bubalis) [26]. Terefore, further studies are needed to Blood & Tissue Kit, Qiagen, Valencia, California, USA), confrm that examined deer are free from Plasmodium in accordance with the manufacturer’s instructions. DNA infection, despite the negative results obtained in the pre- quality was assessed through amplifcation of the mito- sent study. chondrial cytochrome b (cytb) gene [23], using the prim- Nested PCR-based screening of white-tailed deer in ers L14841 (5′-AAA AAG CTT CCA TCC AAC ATC 45 counties in the USA (all in the eastern region of the TCA GCA TGA TGA AA-3′) and H15149 (5′-AAA CTG country) found that 41/308 (13.3%) of the animals were CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3′). infected by Plasmodium sp. Conversely, previous studies PCR for Plasmodium spp. was performed using two have failed to detect Plasmodium in other ungulate spe- previously described protocols: one targeting the small cies, including elk (Cervus canadensis), pronghorn (Anti- subunit RNA gene (SSU rRNA) [5, 24] and the other tar- locapra americana) and mule deer (Odocoileus hemionus) geting the mitochondrial cytochrome b gene (cytb) of the [11].