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

Comparative Immunology, Microbiology and Infectious Diseases 35 (2012) 325–334

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Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid

Presented at the 6th International Meeting on Rickettsia and Rickettsial Diseases at Heraklion, Crete, Greece on June 5-7, 2011

Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus) A.B.V. Sacchi, J.M.B. Duarte, M.R. André, R.Z. Machado ∗ Universidade Estadual Paulista, UNESP, Jaboticabal, SP, Brazil

a r t i c l e

i n f o

Article history: Received 16 August 2011 Received in revised form 30 January 2012 Accepted 2 February 2012 Keywords: Marsh deer (Blastocerus dichotomus) Ehrlichia chaffeensis Anaplasma spp. Brazil

a b s t r a c t Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tickborne 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. © 2012 Elsevier Ltd. All rights reserved.

1. 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.

∗ Corresponding author at: Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias Júlio de Mesquita Filho (UNESP), Campus de Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane, s/n, Zona Rural, CEP: 14884-900, Jaboticabal, São Paulo, Brazil. Tel.: +55 16 3202 2663; fax: +55 16 3202 4275. E-mail address: [email protected] (R.Z. Machado). 0147-9571/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2012.02.001

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].

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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 [11–14]. 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. 2. Materials and methods 2.1. 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 and 2001, 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). Blood samples were collected from the jugular veins of 143 anesthetized deer as previously described [21]. Serum and buffy coat samples were transferred to microtubes and stored at −196 ◦ C. Samples were divided in four groups, according to the location and time of deer capture, as described below (Fig. 1): MS01 and MS02 (n = 117): deer blood samples were collected in the state of Mato Grosso do Sul, in the region between the villages of Anaurilandia and Bataguassu (21◦ 58 29.61 S 52◦ 27 56.53 W). This region comprises the marginal floodplains of the Paraná River, well-preserved, where 90% of the lowland areas were inundated [22]. Blood samples MS01 (n = 99) were obtained from animals captured in the months of June, July and October 1998, prior to the first occurrence of flooding (November, 1998); on the other hand, blood samples MS02 (n = 18) were obtained from deer captured after the flooding in February, 2001. PX (n = 9): blood samples were collected in the region of Rio do Peixe, one of the main tributaries on the left bank of the Paraná River (21◦ 35 06.18 S 51◦ 49 31.42 W), where deer population was less affluent because the topographic characteristics of the region consists primarily of banks, resulting in fewer wetlands. In this area, animals were captured after the initial filling of the reservoir, which caused moderate loss of the deer’s natural habitat (September 1998) [23]. AGUA (n = 17): blood samples were collected in the region of Rio Aguapeí (21◦ 05 22.20 S 51◦ 42 22.26 W), a tributary on the left bank of the River Paraná. In this area, animals were captured two years after the initial flooding, which had also low impact on the deer’s habitat (April 2001) [22]. 2.2. Serological assay for the detection of IgG antibodies to E. chaffeensis and A. phagocytophilum The presence of antibodies to E. chafeensis and A. phagocytophilum in the serum of each animal was detected by Indirect Immunofluorescence Assay (IFA). Slides coated with E. chaffeensis and A. phagocytophilum antigens (Focus Diagnostics, Cypress, CA, USA) were removed from storage and allowed to thaw at room temperature (25 ◦ C) for 30 min. Ten microliters of diluted sera at 1:64 (cut-off) were placed in wells on antigen slides [24]. Slides were incubated at 37 ◦ C in a moist chamber for 30 min, washed 3 times in PBS (pH 7.2) for 5 min, and air dried at room temperature. Anti-deer conjugate was diluted according to the manufacturer (1:10 dilution, KPL; Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) and then added to each well. Slides were incubated again at 37 ◦ C, washed 3 times in PBS, and air dried at room temperature. Next, slides were overlaid with buffered glycerine (pH 9.5), covered with glass

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Fig. 1. Schematic map of the Porto Primavera physiographic region showing the capture location of the four different marsh deer subpopulations (MS01, MS02, PX01 and AGUA) surveyed between 1998 and 2001.

coverslips, and examined under an epifluorescence microscope using a 40× objective (Olympus BX60; Olympus America Inc., FL, USA). Samples were considered positive if fluorescent morulae were observed inside the cells. Positive and negative human serum controls for A. phagocytophilum and E. chaffeensis (included in the diagnostic kit) were used in the first reactions. Deer sera confirmed positive or negative for each agent subsequently replaced the human serum controls. 2.3. Polymerase chain reaction (PCR) for Anaplasmataceae agents DNA was extracted from 200 ␮l aliquots of deer whole blood samples using the QIAamp DNA Blood Mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Each sample of extracted DNA was used as a template in 50 ␮l reaction mixtures containing

10× PCR buffer, 1.5 mM MgCl2 , 10 mM deoxynucleotide triphosphate (dNTPs) mixture, and Taq DNA polymerase (Invitrogen, Carlsbad, California, USA) with genus- and species-specific primers for the 16S rRNA gene of E. canis [25], E. chaffeensis [26], E. ewingii [27], A. phagocytophilum [28], A. platys [5], and Neorickettsia risticci [29]. Additionally, PCR assays for the dsb [30] and the groESL [31–33] genes of Ehrlichia/Anaplasma spp. were performed for further molecular characterization. Positive samples for 16S rRNA Anaplasma spp. were submitted to both conventional [34] and real-time [35] specific A. phagocytophilum msp2 gene PCR. Anaplasma platys DNA positive control was obtained from a naturally infected dog from Governador Laudo Natel Veterinary Hospital-UNESP, Jaboticabal, São Paulo [36]. The positive controls for A. phagocytophilum, E. chaffeensis, and N. risticii were kindly supplied by J. Stephen Dumler, Department of Pathology, Johns Hopkins Hospital, Baltimore, MD, USA. Ultra-purified water was used

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Table 1 IFA and 16S rRNA PCR results for E. chaffeensis, IFA results for A. phagocytophilum and 16S rRNA PCR results for Anaplasma spp. in different subpopulations of Brazilian marsh deer. Group (n)

MS01 (99) PX (9) MS02 (18) AGUA (17) Total

E. chaffeensis

A. phagocytophilum

Anaplasma spp.

IFA+

PCR+

IFA+

PCR+

n (%)

n (%)

n (%)

n (%)

38 (38.38a) 04 (44.44a) 12 (66.66a) 07 (41.18a)

20 (20.20a) 02 (22.22a) 01 (05.55a) 01 (05.88a)

51 (51.51a) 0 (0.0b) 12 (66.66a) 07 (41.18a)

61 (42.65)

24 (16.78)

70 (48.95)

76 (76.76a) 08 (88.88a) 16 (88.88a) 16 (94.12a) 116 (81.12)

Different letters in each column indicate statistical significance (P > 0.05).

as negative control. PCR and nested amplifications were performed in a Gradient Cycler (Perkin-ElmerTM model PT-200). 2.4. Phylogenetic analysis Amplified DNA fragments from positive samples were sequenced in an automatic sequencer (ABI Prism 310 Genetic Analyser; Applied Byosystem/Perkin Elmer) for confirmation, and used for subsequent phylogenetic analysis. Phylogenetic reconstructions were based upon deoxyribonucleic acid. Consensus sequences were obtained through the analysis of the sense and antisense sequences using the CAP3 program [http://mobyle. pasteurfr/cqi-bin/MobylePortal/portal.py]. Comparisons with sequences deposited in GenBank were done using the basic local alignment search tool (BLAST). The BioEdit 7.0.9 [http://www.mbio.ncsu.edu/bioedit/bioedit. html], CLUSTAL X [37] and TreeView [38] programs were used for alignment and phylogenetic analysis, respectively. The neighbor-joining method was used to build phylogenetic trees [39]. The Bootstrap test with 1000 replications was applied to estimate the confidence of branching patterns of the neighbor-joining tree [40]. 2.5. Statistical analysis The statistical analysis was performed using the Minitab Release 14 Statistical Software (2003). The statistical method was the Q-square, used to compare the prevalence of agents in different areas, and in the moments before and after flooding (MS01, MS02, PX and AGUA). Results with a P value was less than or equal to 0.05 were considered statistically significant.

3. 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%) samples were positive to 16S rRNA Anaplasma spp. PCR and A. phagocytophilum IFA (Table 2). Samples tested positive to 16S rRNA PCR for E. chaffeensis were also positive to PCR assays targeting dsb (GenBank Accession number JQ085942) and groESL genes (GenBank Accession number JQ085941). On the other hand, positive samples to 16S rRNA PCR for Anaplasma spp., showed negative results at amplification reactions targeting groESL and msp-2 genes. A BLAST analysis of 16S rRNA amplicons revealed 100% identity with E. chaffeensis entries from Argentina and from the United States (Genbank Accession numbers EU826516 and AF416764, respectively). BLAST comparisons of the dsb and groESL amplicons from the same samples also revealed 96.0% (GenBank Accession number AF403711) and 98.0% (GenBank Accession number L10917) identity with E. chaffeensis, respectively. Interestingly, gene products amplified with primers for the 16S rRNA A. phagocytophilum genogroup revealed the highest similarity (99.0%) with a sample of A. platys detected in Italy (EU439943.1). However, these samples were subsequently tested with specific primers for A. platys and yielded no amplification products. The same samples were also PCR negative for A. marginale, E. canis, E. ewingii, and N. risticii.

Table 2 Association between 16S rRNA PCR and IFA results for E. chaffeensis and between 16S rRNA PCR results for Anaplasma spp. and IFA results for A. phagocytophilum in Brazilian marsh deer blood samples. IFA+

IFA−

Total

Agent

PCR+

49 (80.33%) 13 (18.57%)

12 (19.67%) 57 (81.43%)

61 70

E. chaffeensis Anaplasma spp.

PCR−

67 (81.71%) 11 (15.07%)

15 (18.29%) 62 (84.93%)

82 73

E. chaffeensis Anaplasma spp.

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Deer 201

95

Deer 207

66 80 49

329

Ehrlichia chaffeensis AF416764.1 Deers D1D4 Ehrlichia ewingii M73227.1

97

Ehrlichia ruminantium DQ482915.1 Ehrlichia canis EF195134.1

99

Anaplasma phagocy tophilum AF486636.1 Anaplasma platys EF139459.1

52

93

Anaplasma bovis AB211163.1 67

Anaplasma marginale AF414877.1 89

Anaplasma centrale AB211164.1

Wolbachia pipientis U23709.1 Neorickett sia risticii AF037210.1 0.02

Fig. 2. Phylogenetic dendogram calculated with partial sequences of the 16S rRNA gene (5 –3 ) amplified from blood of marsh deer naturally infected with Ehrlichia chaffeensis (Deer 201, Deer 207 and Deer D1D4) using the “Neighbor-Joining” algorithm with 1000 bootstrap repetitions. Sequences used for comparison are labeled with the Genbank accession numbers.

Phylogenetic analysis of sequences obtained from 16S rRNA, dsb and groESL, placed the Ehrlichia spp. samples amplified from the study populations within the same clade as E. chaffeensis (Figs. 2–4), whereas samples positive for Anaplasma spp. fell in the same clade as A. platys only when amplified with primers for the 16S rRNA gene (Fig. 5).

genera Ehrlichia and Anaplasma in Europe and in the United States [41–50]. In Brazil, the detection of E. chaffeensis DNA in marsh deer blood samples points toward a similarly important role for these cervids in disease ecology in areas where human cases of ehrlichiosis have been clinically suspected and serologically detected [11–14]. The detection of E. ewingii and A. phagocytophilum in dogs living in the Southeastern region of Brazil, in the same geographic area [15,16], further highlights the need for a thorough survey of tick-borne agents in this region. Dogs, as well as other domestic animals such as horses and cattle, are not only susceptible to the same tick species as those infesting wild mammals such as deer and capibaras, but also share the same habitat, which leads to increased risk of inter-species transmission of potentially zoonotic diseases [51,52]. In this context, knowledge of the ecology of diseases is fundamental. It is known that in North America, a number of surveys of American white-tailed deer populations

4. 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

DSB Deers 92 88. 729 Ehrlichia chaffeensis CP000236.1 93

Ehrlichia chaffeensis EF375887.1 Ehrlichia chaffeensis EF375885.1

93

Ehrlichia chaffeensis EF375886.1 Ehrlichia chaffeensis DQ902686.1 Ehrlichia ruminantium NC005295.2 Ehrlichia ruminantium CR925677.1 Ehrlichia ewingii AY428950.1 Ehrlichia muris AY236484.1

90

Ehrlichia canis DQ460716.1 83

Ehrlichia canis DQ460715.1

Fig. 3. Phylogenetic dendogram calculated with partial sequences of the dsb gene (5 –3 ) amplified from blood of marsh deer naturally infected with Ehrlichia chaffeensis (Deer 92 88. 729) using the “Neighbor-Joining” algorithm with 1000 bootstrap repetitions. Sequences used for comparison are labeled with the Genbank accession numbers.

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Fig. 4. Phylogenetic dendogram calculated with partial sequences of the groESL gene (5 –3 ) amplified from blood of marsh deer naturally infected with Ehrlichia chaffeensis (Deer #205, Deer #218, Deer #201) using the “Neighbor-Joining” algorithm with 1000 bootstrap repetitions. Sequences used for comparison are labeled with the Genbank accession numbers.

show seroprevalence to Anaplasmataceae agents ranging from 2.0 to 92.0% [41–47]. While in the U.S.A. and Europe, Amblyomma americanum and Ixodes spp. (I. scapularis, I. ricinus and I. pacificus) are the main vectors of E. chaffeensis and A. phagocytophilum, respectively [53,54], the vectors have not been identified yet in Brazil. In a previous study evolving capture of free-living ticks in floodplain areas, natural habitat of sampled deer in the present study, A. triste was the only species to be collected in significantly higher numbers in the marsh than in surrounding drier areas such as forest patches. Among domestic animals living close to the marsh areas, horses were infested by Dermacentor (Anocentor) nitens, A. cajennense, and Rhipicephalus (Boophilus) microplus; bovines, on the other hand, were infested solely by B. microplus, the same tick species found parasitizing the marsh deer sampled in our survey. Marsh deer, however, are considered the most important hosts for the adult stages of A. triste, in that area [18,19].

However, further studies are needed not only to define the vector, but for a better understanding of the epidemiology of these diseases. Recently, Machado et al. [74] have identified E. chaffeensis and A. phagocytophilum DNA in carnivorous birds’ blood samples in Brazil, highlighting the possibility of new patterns of transmission and spread of these zoonotic agents. Herein, epidemiological studies represent an evaluation of the impact of installing a hydroelectric plant in the area of occurrence of deer, significantly affecting the habitat of these animals. Comparison of results obtained from MS01 and MS02 showed a trend (however not statistically significant [p > 0.05]) toward increased positivity (in both serology and PCR) for E. chaffeensis in deer populations that had endured flooding of its habitat. Robust statistical analysis was precluded by the large variation in sample size, given that capture conditions and population density varied from region to region. The MS01 region

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331

Anaplasma platys EF139459.1 62 Anaplasma platys EU439943.1

Deer 1F 89

Deer Cont3 Deer Cont2

66

82

68

Deer 5F Deer Cont4 Anaplasma phagocytophila AF486636.1

98 Anaplasma phagocytophilum M73224.1 99

Anaplasma bovis AB211163.1 Anaplasma centrale AB211164.1

99

99

Anaplasma marginale AF414877.1 81 Anaplasma marginale AF309867.1

Ehrlichia ruminantium DQ482915.1 Ehrlichia canis EF195134.1

100

Ehrlichia chaffeensis AF416764.1

48 23

Ehrlichia ewingii M73227.1 Wolbachia pipientis U23709.1 Neorickettsia risticii AF037210.1

0.08

0.06

0.04

0.02

0.00

Fig. 5. Phylogenetic dendogram calculated with partial sequences of the 16S rRNA gene (5 –3 ) amplified from blood of marsh deer naturally infected with Anaplasma spp. (Deer 1F, Deer Cont2, Deer Cont3, Deer Cont4, Deer 5F) using the “Neighbor-Joining” algorithm with 2000 bootstrap repetitions. Sequences used for comparison are labeled with the Genbank accession numbers.

was characterized by relatively well-preserved floodplains where deer had little contact with neighboring farm pastures, and were infested mostly by R. (Boophilus) microplus and A. triste [19]. After flooding, deer in MS02 continued to harbor these same tick species but were also infested by D. (Anocentor) nitens and A. cajennense. Interestingly, ticks belonging to the genus Amblyomma are considered the primary vectors for E. chaffeensis in the United States [26,53,55]. The increased seroprevalence for E. chaffeensis detected in MS02 may have been, therefore, linked to the infestation by Amblyomma spp. Moreover, inundation narrows the floodplain and results in higher cervid population density, which in turn may facilitate dissemination of the tick vector and of the rickettsial agent among these deer populations [22]. A higher number of ticks found parasitizing deer in MS02 area could also be explained by the time of the year when deer were sampled in that region (summer), when compared to time of sampling in MS01 (late winter and early spring). In PX and AGUA regions, flooding had already impacted the habitat, narrowed the floodplain, and consequently, stressed the local deer populations. Animals in both these regions were in a similar situation to those in MS02 region, once they had to compete for food and space, and were in proximity to neighboring farmland. On the other hand, adult stages of A. triste were more found in fall (April) [18] and in AGUA animals were sampled during early dry season. These factors could help to explain the high seroprevalence (PX 88.88% and AGUA 94.12%) and PCR-positive animals (PX 44.44% and AGUA 41.18%) for E. chaffeensis in both these regions.

As previously described, marsh deer (B. dichotomus) is considered to be the most important host for A. triste in the regions surveyed in this study [19]. In Uruguay, where B. dichotomus is currently extinct [56], adult A. triste ticks now parasitize dogs and humans [57,58], and immature stages of A. triste can be found in aberrant hosts such as marsupials and rodents [59]. Furthermore, in Uruguay, A. triste is thought to be the vector of cutaneous-ganglionar rickettsiosis to humans, a disease caused by Rickettsia coronii [56–58]. In light of the above data, our results implicate A. triste as the biological vector of E. chaffeensis. This is an important consideration, given that the dwindling of the marsh deer’s natural habitat could have two possible outcomes: if the deer population is forced to retract to drier areas then it becomes more heavily infested with A. cajennense, a tick with a much broader range of hosts. Conversely, when the deer population decreases in a certain region, A. triste may adapt to parasitize other species, as observed in Uruguay. In both situations there may be an increased risk of infection of human beings and other animal species by this agent. Hence, ecological changes and loss of natural habitat result in altered host–parasite relationships, interfere with wildlife conservation strategies, and expose humans and other animals to infectious agents that may not be well-characterized yet. Molecular analyses of the 16S rRNA, dsb, and groESL partial genes reveal high prevalence of E. chaffeensis in Brazilian marsh deer and confirm that the ehrlichial DNA in our study samples match those previously reported. These molecular findings associated to a high presence of

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antibodies to E. chaffeensis among sampled deer, suggest that deer can play an important role in the enzootyc cycle of this agent in rural areas in Brazil. In the U.S.A, whitetailed deer (O. virginianus), when experimentally infected with E. chaffeensis, remains bacteremic for at least 2 weeks after infection, whereas antibodies can only be detected around 10 days post-infection, but before the onset of clinical signs [24]. A more recent study demonstrated that white-tailed deer, when inoculated intravenously with E. chaffeensis, were bacteremic for 24 days post-inoculation and developed antibodies to the agent (≥1:64) 17 days post-inoculation, at which time none showed clinical signs of infection [60]. Similarly, the high prevalence of E. chaffeensis-positive deer suggest that deer act as carriers of this agent and not necessarily show clinical signs of infection; also, the presence seropositive animals show a previous established contact (Table 2). Future studies need to be done aiming at verifying the kinetics of an experimental infection with E. chaffeensis in Brazilian marsh deer, regarding parasitemia, DNA load, antibody levels, and appearance of clinical signs during the course of infection. Regarding the molecular approaches used in the present study, it is important to mention that the conventional PCR techniques used here could not have been sufficiently sensitive to detect low levels of bacteremia. It is worth mentioning that real-time PCR increases significantly the specificity and sensitivity of the molecular detection for rickettsial agents [8,61]. Additionally, serologic tests are not specific, and cross reactivity is known to occur among different species of Ehrlichia, among different species of Anaplasma, and between Ehrlichia spp. and Bartonella spp. [62,63]. Cross reactivity between A. marginale and A. phagocytophilum [63], and E. canis and E. chaffeensis [59] have also been detected in ELISA and IFA techniques. Regarding the occurrence of Anaplasma spp., we noted an increase in PCR-positive animals in MS02 compared to MS01, but this increase was not accompanied by a rise in seroprevalence, which remained higher in MS01. Considering that sequencing pointed toward an organism more closely related to A. platys, the above results are expected given that the PCR primers used in this survey were designed to detect all organisms within the Anaplasma genus, whereas antigen used for IFA aimed at detecting antibodies to A. phagocytophilum. Our results suggest the occurrence of cross-reactivity between different species of Anaplasma sp. in serology, since the sequencing showed greater similarity to A. platys than with A. phagocytophilum. Similarly, the so-called “WTD agent,” a species of Anaplasma sp. reported in white-tailed deer from the United States, is phylogenetically closer to A. platys than to A. phagocytophilum [64]. It is worth noting that several studies show that the antigenic variation of Anaplasma species is subject to evade the host immune response, which could have influence on the specific diagnosis [65–67]. Evading the immune response, bacteremia can be maintained in cycles, allowing the transmission by vectors [68], which helps explain the high prevalence of PCR positive for deer Anaplasma spp. Although our results show a greater proximity of the detected agent to A. platys, more studies are needed.

Recently, at least three different A. phagocytophilum genotypes have been identified circulating in lizards, woodrats, sciurids, dogs and horses in the U.S.A., suggesting tropism for certain hosts [65]. In this study, deer were not positive for E. canis PCR, supporting previously reported data in white-tailed deer [24,50]. Experimental infection of white-tailed deer with E. canis does not cause bacteremia or seroconversion in deer [24]. In a previous study, free-ranging white-tailed deer, captured during the hunting season in the USA, were negative to E. canis DNA amplification [50]. However, cross reactivity between E. canis and E. chaffeensis in serology is commonly reported [59], and was also observed in our study (data not shown). Similarly, all samples examined in this study were negative for E. ewingii by PCR. These data differ from those reported in the United States for white-tailed deer, which is considered a natural host of E. ewingii, with a PCR-positive prevalence of 5.5% (6/110 animals) [47]. Importantly, E. ewingii has been previously detected in dogs in Brazil, a finding that warrants further survey of this agent in other animal species [15]. All animals examined in our cohorts were negative (via both serology and PCR) for N. risticii, presumably due to the lack of intermediate hosts. Marsh deer should, however, be further surveyed for this agent given that this species inhabits wetlands that are also conducive to propagation of trematodes necessary to complete the bacteria’s life cycle [9,69]. In Brazil, fresh water snails of the genus Heleobia harboring cercaria of Parapleurolophocercous cercarie, the trematode that carries N. risticii, were identified in the Southern state of Rio Grande do Sul [70]. A few years later Potomac horse fever was reported in horses from the same state, confirming that N. risticii is present in Brazil [71]. However, there are currently no published reports of N. risticii infections in Brazilian deer. In this study, negative PCR results for the msp-5 gene of A. marginale warrant further investigation because the agent has been previously detected both in marsh deer via conventional PCR [6,7], and in Pampas deer via real-time PCR. Additionally, Pampas deer share the habitat with cattle, the main host for A. marginale, and infectious agent transmission across closely related species, such as cervids and bovines, is relatively common [8]. It is important to identify Anaplasmataceae bacteria, and to determine their geographic distribution, both because of their zoonotic potential and the implications of disease ecology in directing conservation efforts [19,72,73]. Survey of other Brazilian cervid species, mainly via realtime PCR, becomes essential to confirm the diagnosis of rickettsial diseases in deer that inhabit a variety of geographical regions and that are susceptible to distinct tick species. Subsequent molecular characterization of isolates will help elucidate antigenic variation among these bacteria, and aid in the implementation of appropriate diagnostic techniques. Together these efforts will advance our knowledge about tick-borne disease ecology, not only in cervids but globally, ultimately informing decisions that will impact wildlife conservation, land use, and human and animal health.

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