Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus)

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Abstract

Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasma, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Paraná River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of São Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n = 99), MS02 (n = 18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n = 9; Peixe River, after flooding), and AGUA (n = 17; Aguapeí River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals.

Introduction

In the past few centuries, changes in land-use patterns have been causing several transformations in the environment, such as the alteration of habitats, and the diminishment of overland species size and its genetic flow. These modifications have drastically transformed disease ecology and impacted human and animal global health [1].

The Brazilian marsh deer (Blastocerus dichotomus), the largest of Brazilian deer [2], is a species that struggles for survival in an extremely impacted habitat, the floodplain. The retraction of the area where this species lives is due to complex factors, such as the effects of hydroelectric plant installation, the advancing agricultural and urban frontiers, disease transmission by close contact with livestock, changes in host–parasite relationships, and unregulated hunting [3], [4]. These factors likely contributed to the emergence of Anaplasmataceae agents (Rickettsiales: Anaplasmataceae), important gram negative obligate intracellular pathogens causing tick-borne diseases in humans and animals [5].

We have recently reported Ehrlichia chaffeensis and Anaplasma marginale in Brazilian wild marsh deer living in proximity to the newly installed Porto Primavera Hydroelectric Power Plant, on the Paraná River [6]. Also, A. marginale, E. chaffeensis, A. bovis and Anaplasma spp. have been detected in brocket deer (Mazama gouazoubira) and Brazilian marsh deer from Minas Gerais State [7]. A. marginale has also been detected in Pampas deer (Ozotoceros bezoarticus) inhabiting the Brazilian Pantanal wetlands [8]. These findings are alarming given that E. chaffeensis is the recognized zoonotic agent of human monocytic ehrlichiosis [9]. Furthermore, A. marginale is incriminated in large economic losses in cattle [10].

Additionally, antibodies against E. chaffeensis have been detected in dogs from Minas Gerais state in Brazil [10], a location in which suspected cases of human monocytic ehrlichiosis have also been recently reported [11], [12], [13], [14]. More recently, E. ewingii as well as A. phagocytophilum have been reported in dogs from Minas Gerais and Rio de Janeiro states, respectively [15], [16]. Regarding the occurrence of tick-borne agents in Brazilian deer, the protozoans Theileria sp. and Babesia sp. have also been detected in deer from Minas Gerais state [17].

Despite the detection of Anaplasmataceae agents in deer, the vectors are not yet certain in Brazil. Regarding the occurrence of ticks in marsh deer habitat, Szabó et al. (2007) found five free-living tick species, in the following order of abundance: Amblyomma cajennense, A. dubitatum, A. triste, A. coelebs, and A. nodosum. Also, A. cajennense, A. triste, Dermacentor nitens, and Rhipicephalus (Boophilus) microplus tick species have been found parasitizing marsh deer in Paraná River region; A. triste, especially, is a tick species highly related to the environment and also the floodplain marsh deer [18], [19].

Given the recognized zoonotic potential of Anaplasmataceae agents, these findings highlight the need for a better epidemiologic evaluation of vector-borne diseases among Brazilian wildlife. The identification of domestic and wild reservoirs for Anaplasmataceae agents could help in the identification of risk areas for human infection. Furthermore, better knowledge of the ecology of tick-borne diseases among free-ranging wild animals is essential for planning successful conservation strategies for endangered species, such as translocation and reintroduction programs, or the management of wild animals in captivity [19].

In light of the scarcity of published data on tick-borne disease prevalence among wildlife in Brazil, the present work aimed to evaluate the prevalence of Anaplasmataceae agents in Brazilian marsh deer using molecular and serologic techniques, before and after the impact caused by the installation of the Porto Primavera hydroelectric power plant. Phylogenetic analyses were then performed to properly classify the tick-borne agents found in these deer populations.

Section snippets

Physiographic region and sampled animals

The areas surveyed in this study were the extensive floodplains near the Porto Primavera hydroelectric power plant (UHE “Sérgio Motta”), installed in the Paraná River, between the states of São Paulo and Mato Grosso do Sul, Brazil. The region is characterized by tropical weather with rainy summers (December to February) and dry winters (June to August), temperatures ranging between a maximum of 40 °C and a minimum of 10 °C, and average annual rainfall between 1200 mm and 1400 mm [20]. Between 1998

Results

Out of 143 animals sampled, 116 (81.12%) and 24 (16.78%) were IFA positive for E. chaffeensis and A. phagocytophilum, respectively, while PCR amplification of the bacterial 16S rRNA gene identified 61 (42.65%) and 70 (48.95%) positive samples for E. chaffeensis (GenBank Accession number JQ085940) and Anaplasma spp. (GenBank Accession number JQ085939), respectively (Table 1). Forty-nine (80.33%) samples were positive to both 16S rRNA PCR and IFA for E. chaffeensis; on the other hand, 13 (18.57%)

Discussion

Here we showed the presence of E. chaffeensis and Anaplasma spp. in blood samples from Brazilian marsh deer populations living in floodplains along the Paraná River. Our results help to further characterize the disease ecology of rickettsial agents in the Brazilian Southwest, and corroborate previously reported data [6].

Wild cervids have been confirmed, via both serology and molecular tests, to be important reservoirs of the genera Ehrlichia and Anaplasma in Europe and in the United States [41]

Acknowledgments

The authors would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the scholarship (number #07/52181-8) and financial support (07/555500-7). The authors are also thankful to John Stephen Dumler (Johns Hopkins School of Medicine, Baltimore, MD, USA) for kindly supplying the Anaplasmataceae DNA positive controls.

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