Tick-Borne Bacteria in Free-Living Jaguars ( Panthera onca ) in Pantanal,Brazil

Introduction

W ild large carnivores must be regarded as useful sentinels for tick-borne diseases because they have large home ranges (exposing them to different host-seeking ticks) and are often in contact with different prey species (Azevedo 2008), exposing these predators to a wide variety of tick species in different habitat types (Labruna et al. 2005). The jaguar Panthera onca (Carnivora: Felidae), a near-threatened species, is the largest carnivore species in the New World (IUCN 2010). In this study, we investigated tick-borne bacteria in free-living jaguars in the Pantanal biome, central-west Brazil. To our knowledge, surveys on tick-borne zoonoses have been never conducted in the Pantanal biome. Because livestock activities and tourism related to fishing and safari are among the most important economic activities in the Brazilian Pantanal, the present study has important veterinary and public health implications.

Materials and Methods

The study site was a working 100-km² cattle ranch/wildlife reserve in the southern part of the Pantanal region of the Mato Grosso do Sul state, Brazil (19^o28′40′′ S, 57°00′49′′ W). From June to August 2008, 10 jaguars (Panthera onca; 3 males, 7 females) were captured using trained hounds and anesthetized with the association of tiletamine hydrochloride and zolazepam hydrocloride. Once immobilized, all animals were sexed and examined by a veterinarian for clinical conditions. Blood samples were collected from the cephalic or saphenous vein of each jaguar. All captured jaguars had a good overall health condition. Age was estimated based on tooth wear, ranging from 6 months to 9 years old. Jaguars were searched for attached/feeding ticks during a standardized period of 3 min of inspection per animal. The 10 captured jaguars were fitted with GPS radio-collars and monitored for at least 3 months, showing that all adult females produced offspring and that, based on each individual home range, this population was using an area of ≈850 km². Handling procedures were approved by the Brazilian Environment Institute (Permit No. 10608-1) and by the Animal Use Ethic Commission of the University of São Paulo (Protocol No. 1531/2008).

Blood sera were individually tested by indirect fluorescent antibody assays for immunoglobulin G, using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens, as previously described (Horta et al. 2007, Saito et al. 2008), using a goat anti-cat immunoglobulin G flurescein-labeled conjugate (KPL, Gaithersburg, MD) diluted at 1:400. Domestic cat sera were used as positive and negative controls (Horta et al. 2007). Serum was considered to contain antibodies against each of the bacterial agents if it displayed a reaction at 1:64 dilution. Endpoint titers against each bacterium species were determined by testing serial twofold serum dilutions.

DNA was isolated from jaguar blood clots using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). DNA from each adult tick or pools with no more than three larvae or nymphs collected from the same jaguar were extracted using the guanidine isothiocyanate phenol technique (Sangioni et al. 2005, Labruna et al. 2008). All DNA samples were tested by a battery of PCR protocols, using the following primer pairs: primers CS-78 (forward) and CS-323 (reverse), which amplify a 401-bp fragment of the citrate synthase gene (gltA) of possibly all Rickettsia species (Labruna et al. 2004); primers Rr190.70p and Rr190.602n, which amplify a 532-bp fragment of the 190-kDa outer membrane protein (ompA) of most of the spotted fever group Rickettsia species (Regnery et al. 1991); primers Dsb-330 (forward) and Dsb-728 (reverse), which amplify a 409-bp fragment of the Ehrlichia genus-specific disulfide bond formation protein gene (dsb) (Doyle et al. 2005); primers GE2′F2′(forward) and HE3 (reverse), which amplify a 360-bp fragment of the 16S rRNA gene of Anaplasmataceae species (Aguiar et al. 2008); and primers FlaLL (forward) and FlaRL (reverse) in a first reaction, and FlaLS (forward) and FlaRS (reverse) in a nested reaction, targeting a near 330-bp fragment of the Borrelia flagellin gene ( flaB) (Stromdahl et al. 2003). PCR products, when visualized in agarose gel stained with ethidium bromide, were purified and sequenced as described elsewhere (Horta et al. 2007). Partial sequences were subjected to BLAST analysis (Altschul et al. 1990) to determine similarities to other species.

A partial DNA sequence obtained from the dsb gene PCR was aligned with the corresponding sequences of Ehrlichia species available in GenBank using the CLUSTAL algorithm of the program MEGA version 4 (Tamura et al. 2007). The same procedure was done with a partial sequence obtained from the 16S rRNA PCR. Phylogenetic distances between homologous sequences were calculated using the Kimura's two-parameter model. A phylogram was constructed by the neighbor-joining method for each of the two ehrlichial genes, with bootstrap values at 1000 replicates.

Results

The number of collected ticks varied from none to 9 ticks per jaguar, totalizing 39 ticks encompassing three species: Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus (Table 1). Some Amblyomma larvae and nymphs could not be identified to the species level at the time they were analyzed, and they were regarded as Amblyomma sp. The results of these serological analyses are presented in Table 1. All 10 jaguar sera reacted (titer ≥64) to at least one Rickettsia species; 4 and 3 jaguar sera reacted with E. canis and C. burnetii, respectively.

No Rickettsia DNA was detected in jaguar blood samples, but one A. triste female collected on jaguar 2 was positive for both gltA and ompA rickettsial genes; a partial sequence of the latter gene was sequenced and showed to be 100% identical (430/430) to R. parkeri (EF102238). The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA. Partial sequences obtained from these samples resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. By Blast analysis, the 16S rRNA partial sequence of this strain showed to be closest (99.0%; 302/305) to uncultured strains of Ehrlichia sp. (AY309970, FJ966350) from Japan and Russia and 98.7% (301/305) identical to different strains of Ehrlichia ruminantium (CR925678, X62432, DQ482915). The ehrlichial dsb partial sequence (346-bp) of strain Jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank.

Through phylogenetic analysis for either dsb or 16S rRNA gene, Ehrlichia sp. strain Jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium (Figs. 1 and 2). Partial sequences (16S rRNA, dsb) from Ehrlichia sp. strain Jaguar generated in this study were deposited into GenBank and assigned nucleotide accession nos. HQ388286–HQ388287, respectively. No Borrelia DNA was detected in the jaguar blood or ticks.

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FIG. 1.

Phylogenetic analysis of Ehrlichia sp. strain Jaguar from Brazil. A total of 346 unambiguously aligned nucleotide sites of the ehrlichial gene dsb were subjected to analysis with closest available sequences in GenBank (accession numbers in brackets) using the neighbor-joining method with Kimura's two-parameter model. Bootstrap values with 1000 replicates are shown at the nodes. Scale bars indicate nucleotide substitutions per site.

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FIG. 2.

Phylogenetic analysis of Ehrlichia sp. strain Jaguar from Brazil. A total of 305 unambiguously aligned nucleotide sites of the ehrlichial gene 16S rRNA were subjected to analysis with closest available sequences in GenBank (accession numbers in brackets) using the neighbor-joining method with Kimura's two-parameter model. Bootstrap values with 1000 replicates are shown at the nodes. Scale bars indicate nucleotide substitutions per site.

Discussion

Carnivores such as jaguars have the potential to act as important sentinel hosts for emerging livestock and human diseases, providing a valuable tool for surveillance and for determining spatial and temporal patterns of infection (Cleaveland et al. 2006). This study reports serological evidence of rickettsial infection in jaguars. All jaguars, but animal 5 (Table 1), were seroreactive to R. parkeri, an emerging human pathogen that was only recently reported in Brazil (Silveira et al. 2007, Spolidorio et al. 2010). In addition, jaguar 6 presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri, as determined in previous studies (Saito et al. 2008, Horta et al. 2010). This result is corroborated by our finding of a jaguar that was parasitized by a R. parkeri-infected A. triste, which is a natural vector of this rickettsia in South America (Labruna 2009). Jaguar 10 showed serological evidence of exposure to either R. rhipicephalii or R. amblyommii or a close-related agent, whereas jaguar 5 presented a relatively high titer solely to R. bellii, also suggesting exposure to this agent in the Pantanal. Although no previous study investigated these agents in Pantanal, previous studies in other Brazilian biomes reported serological evidence of vertebrate infection by these Rickettsia species (Labruna et al. 2007, Pacheco et al. 2007), which have been isolated from ticks in Brazil (Labruna 2009). However, their pathogenicity to humans remains undetermined, despite a recent study provided serological evidence for R. amblyommii as the etiological agent of spotted fever in the United States (Apperson et al. 2008).

Ehrlichia sp. strain Jaguar was detected in the blood of jaguars 6 and 8 and in adult (A. cajennense and A. triste) and nymphal (Amblyomma sp.) ticks. This result suggests that the seropositivity of four jaguars to E. canis (Table 1) is a result of a cross-reaction with antibodies elicited during infection by Ehrlichia sp. strain Jaguar. Animal 8, which was PCR positive for this ehrlichia, had a threefold increase in serum transaminases (data not shown) and died at 3 months after capture, suggesting a potential pathogenicity of this novel ehrlichial strain.

Serological evidence of jaguar exposure to C. burnetii demands further investigations to identify the agent and to evaluate possible risks for human and animal health, considering that cattle ranching and ecotourism are the main economic activities in this Pantanal region. In this regard, our results indicate that both employees and tourists in this area are under the risk of acquiring spotted fever due to R. parkeri and highlight the presence of a potential novel ehrlichial pathogen in South America. As this novel ehrlichial agent was shown to be closest to E. ruminantium, a very important cattle pathogen in Africa (Allsopp 2010), it will be vital to know about the pathogenicity of strain Jaguar to cattle, as the state of Mato Grosso do Sul is within the most important region for the Brazilian beef industry (IBGE 2008).