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Article

Characterization of “Candidatus Ehrlichia Pampeana” in Haemaphysalis juxtakochi Ticks and Gray Brocket Deer (Mazama gouazoubira) from Uruguay

1
Laboratorio de Vectores y Enfermedades Transmitidas, Departamento de Ciencias Biológicas, CENUR Litoral Norte—Salto, Universidad de la República, Rivera 1350, Salto 50000, Uruguay
2
Departamento de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad de Concepción, Av. Vicente Méndez 595, Casilla 537, Chillán 3780000, Chile
3
AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand
4
Laboratorio de Ecología de Vertebrados, CENUR Noreste, Universidad de la República, Ituzaingó 667, Rivera 40000, Uruguay
5
INVESAGA Group, Department of Animal Pathology, Faculty of Veterinary Sciences, Universidade de Santiago de Compostela, 27002 Lugo, Spain
*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(10), 2165; https://doi.org/10.3390/microorganisms9102165
Submission received: 2 September 2021 / Revised: 30 September 2021 / Accepted: 2 October 2021 / Published: 17 October 2021
(This article belongs to the Section Veterinary Microbiology)

Abstract

:
Human ehrlichiosis are scantily documented in Uruguay. The aim of this study was to investigate the presence of Ehrlichia spp. in Haemaphysalis juxtakochi and in a gray brocket deer (Mazama gouazoubira) from Uruguay. The presence of Ehrlichia DNA was investigated in free-living H. juxtakochi in five localities of southeast and northeast Uruguay, as well as blood, spleen, and ticks retrieved from a M. gouazoubira. Ehrlichia spp. DNA was detected in six out of 99 tick pools from vegetation, in the spleen of M. gouazoubira, and in one out of five pools of ticks feeding on this cervid. Bayesian inference analyses for three loci (16S rRNA, dsb, and groEL) revealed the presence of a new rickettsial organism, named herein as “Candidatus Ehrlichia pampeana”. This new detected Ehrlichia is phylogenetically related to those found in ticks from Asia, as well as Ehrlichia ewingii from USA and Cameroon. Although the potential pathogenicity of “Ca. E. pampeana” for humans is currently unknown, some eco-epidemiological factors may be relevant to its possible pathogenic role, namely: (i) the phylogenetic closeness with the zoonotic agent E. ewingii, (ii) the evidence of H. juxtakochi parasitizing humans, and (iii) the importance of cervids as reservoirs for zoonotic Ehrlichia spp. The molecular detection of “Ca. E. pampeana” represents the third Ehrlichia genotype described in Uruguay.

1. Introduction

Ehrlichiae are small Gram-negative tick-transmitted coccobacilli that obligately dwell inside cells. These microorganisms are classified as α-proteobacteria belonging to the family Anaplasmataceae included in the order Rickettsiales [1]. Wild mammals, and probably birds [2], constitute natural vertebrate hosts for Ehrlichia spp., which are horizontally transmitted through tick bites [3]. Ehrlichia species infect different cells in mammals and ticks. While monocytes, neutrophils, or endothelial cells have been detected as the mammalian target cells, salivary glands, intestinal epithelium, and hemolymph cells, are infected in the vectors [4]. Some Ehrlichia spp. exhibit tropism for mononuclear phagocyttablees and compose microcolonies within membrane-bound cytoplasmic vacuoles known as morulae [5]. These bacteria are the agents of ehrlichiosis, a complex of life-threatening emerging zoonoses and diseases of worldwide veterinary concern [6]. The genus Ehrlichia currently consists of six validly published species, namely Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia minasensis, Ehrlichia muris, and Ehrlichia ruminantium [7]. For all these species, sequences of the complete chromosome are already available in genetic databases [3]. Four species (E. chaffeensis, E. ewingii, E. canis, and E. muris) are known to infect humans and cause potentially severe to fatal ehrlichiosis [7]. Cervids (Cervidae) have been demonstrated to be reservoirs of human pathogenic Ehrlichiae, particularly E. chaffeensis, E. ewingii, and E. muris [8,9,10,11,12].
Recent molecular studies performed in South America have unveiled novel genotypes of Ehrlichia retrieved from domestic and wild vertebrates, as well as from their ticks [2,13]. For instance, in Uruguay there is only one characterization of Ehrlichia which corresponds to two novel genotypes currently found in Ixodes auritulus [14]. There are several studies reporting the evidence of deer as reservoirs of pathogenic Ehrlichia spp. in northern latitudes of the globe [9,10]. Despite the report of a case of autochthonous human ehrlichiosis in 2001 [15], this disease in Uruguay has unclear status. The fact that I. auritulus does not bite humans [16] prompted us to consider the presence of other tick vectors of Ehrlichia spp., and that the disease could be underdiagnosed in this country. Interestingly, Haemaphysalis juxtakochi is one of the five species of ticks that have been reported parasitizing humans in Uruguay [16], and cervids are common hosts mainly for its adult stages [17,18]. Therefore, the present study aimed to investigate the presence of Ehrlichia spp. in H. juxtakochi and its host, the gray brocket deer (Mazama gouazoubira) in Uruguay.

2. Materials and Methods

Between March 2014 and August 2017 field work was conducted in five localities of Uruguay: Gruta de los Cuervos (31°37′08″ S, 56°02′47″ W), Tacuarembó Department; Amarillo (31°39′49″ S, 55°03′02″ W) and Lunarejo (31°08′29″ S, 55°54′01″ W), Rivera Department; Reserva Natural Salus (34°25′16″ S, 55°18′54″ W), Lavalleja Department; and Laguna Negra (34°05′09″ S, 53°44′17″ W), Rocha Department.
Ticks were collected from vegetation using the flagging method as described previously [14] and stored in plastic containers with 95% ethanol. In addition, a juvenile female M. gouazoubira carcass killed by dogs in September 2017 at Gruta de los Cuervos, Tacuarembó Department was included in this study. Ticks and a sample from the spleen and blood were retrieved from the carcass and stored at −20 °C until use. Ticks were identified using a Nikon stereo microscope SMZ1000 following morphological keys for larval, nymph, and adult stages [17,19]. Since Ehrlichia species are not maintained by transovarial transmission [20], only nymphs and adult ticks were analyzed in this study. Ticks were pooled according to sex, developmental stage, site, collection date, and host. Ticks were rinsed with distilled water to remove ethanol, and the ticks were cut thoroughly with dissecting scissors. DNA was extracted using a GeneJET Genomic DNA Purification kit (Thermo Scientific, Vilnius, Lithuania), according to manufacturer’s instructions. DNA concentration was estimated using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
For Ehrlichia DNA detection, a molecular screening targeting a fragment of the 16S rRNA gene of the Anaplasmataceae family was carried out using primers and protocols as described previously [21]. Subsequently, positive samples were subjected to two additional PCR protocols to amplify a nearly full-length sequence (~1431 bp) of the 16S rRNA gene based on two overlapping fragments [22,23]. In addition, a nested and a heminested PCR targeting partial fragments of groEL (60 kDa chaperonin) and dsb (disulfide oxireductase) genes, respectively, were performed [24,25,26,27,28]. All primers used in this study and fragment sizes are listed in Table 1. Distilled water and DNA of E. canis were included as negative and positive controls, respectively. PCR products were analyzed by electrophoresis in 1.5% agarose gels. Amplicons were purified using a GeneJET PCR purification kit (Thermo Fisher Scientific, Vilnius, Lithuania) and sent for sequencing to Macrogen (Seoul, Korea). BLASTn analyses (www.ncbi.nlm.nih.gov/blast, accessed date: 4 October 2021) were performed in order to infer closest identities with microorganisms available in the GenBank database [29], and to include those sequences in a phylogenetic analysis.
Phylogenies for the genus Ehrlichia were constructed with sequences of each amplified gene and GenBank-retrieved homologues. The alignments for 16S rRNA, dsb, and groEL were implemented in CLUSTAL W [30]. We used Bayesian inferences to reconstruct evolutionary relationships in the genus with MrBayes 3.2.5 [31]. The general time reversible (GTR) model was selected to run all the phylogenies using 1,000,000 generations. Each tree was sampled every 100 generations, beginning with random seeds, and ran four times. The first 25% of the trees were considered burn-in, and the remaining trees used to calculate Bayesian posterior probabilities. Sequences of Neoehrlichia mikurensis (EU810406; AB213021) and E. ruminantium (AF308669) rooted the phylogenetic trees.

3. Results

A total of 5772 H. juxtakochi ticks (89 females, 107 males, 1681 nymphs, and 3895 larvae) were collected from vegetation in nineteen samplings carried out in the five selected localities (Table S1). Additionally, 18 H. juxtakochi (five females, four males, and nine nymphs) were obtained from the of M. gouazoubira carcass.
For Ehrlichia DNA detection, 1864 H. juxtakochi collected from vegetation (85 females, 98 males, and 1681 nymphs) were divided in 99 samples and analyzed (Table 2). The ticks were processed individually or grouped in pools containing 2 to 62 specimens collected upon the vegetation. The samples examined from M. gouazoubira were: five pools containing H. juxtakochi ticks (one containing five females, one with four males, and three with three nymphs each) as well as a blood and a spleen sample.
Six out of the 99 H. juxtakochi samples containing ticks from the vegetation were positive (6.1%) (4 out of 71 nymph pools, 1 out of 14 male pools, and 1 of 14 female pools) (Table 2). Partial sequences generated for 16S rRNA, dsb, and groEL loci of Ehrlichia sp. were deposited in GenBank with the accession numbers listed in Table 2. Moreover, one out of the three nymph pools containing H. juxtakochi specimens retrieved from M. gouazoubira was positive (GenBank accession numbers: MZ733621, MZ779087, and MZ779096 for 16S rRNA, dsb, and groEL, respectively). For the samples of blood and spleen of M. gouazoubira only a partial sequence for groEL of Ehrlichia sp. was obtained from the spleen (GenBank accession number MZ779099).
The comparison among the sequences obtained herein revealed a similarity percentage of 100 and between 99.38–100% and 99.69–100% for 16S rRNA, groEL, and dsb fragment genes, respectively. Analyses of the 16S rRNA sequences retrieved from H. juxtakochi (610 to 1234 bp) revealed 100% identity with Ehrlichia sp. clone HLAE331 obtained from Haemaphysalis longicornis from South Korea (GenBank accession number: GU075697) and 99.92% with two sequences named as Ehrlichia sp. TC249-2 and Ehrlichia sp. TC251-2 from Dermacentor nuttalli from China (KJ410252-KJ410253). Sequences of the 16S rRNA gene of other Ehrlichia spp. obtained from ticks from different parts of the world were <90% identical. Accordingly, partial sequences of groEL obtained from H. juxtakochi and spleen of M. gouazoubira (1140 and 1242 bp, respectively) also showed high identity (97.73–97.91%) with sequences from the Ehrlichia sp. detected in D. nuttalli from China (KJ410294-KJ410296). In contrast, the closest identity for the dsb gene (295 to 330 bp) was E. ewingii, detected in Amblyomma americanum from USA (AY428950: 90.25%) and from a dog from Cameroon (DQ151999: 90.10%).
The phylogenetic relationship of characterized Ehrlichia genes was assessed through Bayesian analyses. Phylogenetic trees constructed with partial sequences of 16S rRNA and groEL produced similar topologies (Figure 1a,c). Although with low support, the Ehrlichia 16S rRNA sequences obtained in this study formed a clade with sequences of Ehrlichia spp. characterized from H. longicornis (HQ697588, MT258398, MT258399) and Haemaphysalis flava (MT258401) from Japan, and D. nuttalli from China (KJ410251–KJ410253) (Figure 1a). Similarly to the results obtained for the 16S rRNA gene, the groEL sequences formed a clade with sequences of Ehrlichia spp. from Haemaphysalis, Dermacentor, and Hyalomma ticks from Asia, as well as E. ewingii from a human and A. americanum from USA (AF195273, KJ907744) (Figure 1c). In contrast, dsb sequences clustered with E. ewingii homologues retrieved from A. americanum and Amblyomma inornatum from USA (AY428950, KM458249), and Rhipicephalus sanguineus from Cameroon (DQ902688) (Figure 1b).

4. Discussion

In recent decades, molecular advances have favored the determination of novel species and strains of Ehrlichia in ticks from South America; for instance, E. minasensis and E. canis in Rhipicephalus microplus and R. sanguineus, respectively [32]. In addition, Ehrlichia cf. chaffeensis, and a series of strains (Ehrlichia sp. strain Córdoba, Ehrlichia sp. strain San Luis, Ehrlichia sp. strain Iberá, Ehrlichia sp. strain Jaguar, Ehrlichia sp. strain Delta, Ehrlichia sp. strain La Dormida, and a Ehrlichia sp.) were detected in ticks of the genus Amblyomma [13,33,34,35,36,37,38,39]. Recently, new Ehrlichia genotypes were described in Ixodes uriae from Chile and Ixodes auritulus from Uruguay [2,14]. Collectively, these findings suggest that different Ehrlichia species and genotypes are circulating in South American ecosystems.
The genetic distances and phylogenetic relationships for the 16S rRNA, groEL, and dsb genes of the Ehrlichia sp. characterized in this study clearly denote the finding of a novel species related to the Ehrlichia species harbored by Haemaphysalis spp., Hyalomma anatolicum, and D. nuttalli ticks from Asia [3,40,41,42,43,44,45]. We propose its denomination as “Candidatus Ehrlichia pampeana”. The species name is in allusion to the Pampa biome where positive ticks and deer were found. Remarkably, “Ca. E. pampeana” is also related to E. ewingii detected in Amblyomma spp. and dog blood from USA and R. sanguineus from Cameroon [46,47,48,49,50]. The topology of the phylogenetic trees also suggested that “Ca. E. pampeana” is closely related to E. ewingii. Although 16S rRNA and groEL phylogenies indicated that other Ehrlichia spp. detected in ticks from Asia clustered with “Ca. E. pampeana”, there are no dsb sequences available for the Asian Ehrlichiae genotypes, thus no phylogenetic relationship could be established with this locus (Figure 1b).
Candidatus E. pampeana” is associated with the gray brocket deer since a fragment of the groEL gene was retrieved from the spleen of this cervid. This Ehrlichia sp. deer association was previously reported for two human-pathogenic Ehrlichia species such as E. chaffeensis and E. ewingii that use Odocoileus virginianus (white-tailed deer) as their main animal reservoir in the USA [9]. This fact, added to the detection of “Ca. E. pampeana” DNA in H. juxtakochi ticks, suggests that M. gouazoubira could act as a reservoir for this bacterium, which could be transmitted by its associated tick species (H. juxtakochi).
The molecular characterization of “Ca. E. pampeana” represents the third genotype of Ehrlichia determined in Uruguay, and the first report of an Ehrlichia sp. in H. juxtakochi, as well as for Haemaphysalis spp. from South America.
Human ehrlichiosis was documented in Uruguay more than ten years ago [15], and further cases have not been reported. Notably, Amblyomma triste, a tick that commonly bites humans in South America, has been positive to Ehrlichia spp. detection in Brazil and Argentina [37,51]; however, bacteria of this genus have not been detected in A. triste Uruguayan populations to date [37,52]. While the pathogenicity of “Ca. E. pampeana” for humans is uncertain, our results highlight eco-epidemiological features that might be relevant to suggest this novel Ehrlichia as a putative human pathogen as follows: (i) “Ca. E. pampeana” is phylogenetically closely related to E. ewingii, a recognized zoonotic pathogen [7], (ii) although H. juxtakochi parasitizes cervids, it has been also recorded feeding on humans [16,17], and (iii) the role of cervids as reservoirs for pathogenic Ehrlichia species has been previously suggested in North America [9].
Ehrlichiae are transstadially transmitted bacteria that need vertebrate hosts in order to thrive in nature [1]. For this reason, more studies are needed to determine the presence of “Ca. E. pampeana” in different H. juxtakochi hosts along the distribution range of this tick species. Moreover, further studies will be necessary to understand the eco-epidemiology of this novel bacteria and to assess its pathogenicity.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/microorganisms9102165/s1, Table S1: Complete data of Haemaphysalis juxtakochi collected in vegetation for each site and detection of “Candidatus Ehrlichia pampeana”.

Author Contributions

Conceptualization, M.L.F., S.M.-L., M.T.A.-F., and J.M.V.; data curation, M.L.F., and J.M.V.; methodology, M.L.F., S.M.-L., L.A.C., D.Q., S.R., M.T.A.-F., and J.M.V.; validation, M.L.F., formal analysis, M.L.F., S.M.-L., M.T.A.-F., and J.M.V.; investigation, M.L.F., S.M.-L., L.A.C., D.Q., S.R., M.T.A.-F., and J.M.V.; resources, M.L.F., and J.M.V.; writing—original draft preparation, M.L.F., S.M.-L., M.T.A.-F., and J.M.V.; writing—review and editing, M.L.F., S.M.-L., L.A.C., D.Q., S.R., M.T.A.-F., and J.M.V.; validation, M.L.F.; supervision, J.M.V.; project administration, M.L.F. and J.M.V.; funding acquisition, M.L.F. and J.M.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partly supported by the Comisión Sectorial de Investigación Científica (Programa Iniciación a la Investigación 2017—Project ID 160) to M.L.F., and PEDECIBA (Programa de Desarrollo de las Ciencias Básicas)—Área Biología support 2020–2021 to J.M.V.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data presented in this study are available in the article.

Acknowledgments

We would like to thank Gustavo de Souza and Fernando Ramos (Colonia Don Bosco, Laguna Negra, Rocha); Eduardo Méndez, Alejandro Rodríguez, and Andrés de Mello (Reserva Natural Salus, Lavalleja); Hugo Pereda and Ricardo Palle (Gruta de los Cuervos, Tacuarembó); Daniel Casalás, Winderson Rodriguez da Cunha, and Lorena Ojeda (Amarillo, Rivera), for their collaboration during the field work.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Bayesian phylogenetic analyses inferred for partial fragments of the genes (a) 16S rRNA, (b) dsb, and (c) groEL. Bayesian posterior probabilities are indicated upon each branch. The positions of “Candidatus Ehrlichia pampeana” are highlighted with blue. Scale bar indicates the number of substitutions per nucleotide position. GenBank accession numbers are in brackets.
Figure 1. Bayesian phylogenetic analyses inferred for partial fragments of the genes (a) 16S rRNA, (b) dsb, and (c) groEL. Bayesian posterior probabilities are indicated upon each branch. The positions of “Candidatus Ehrlichia pampeana” are highlighted with blue. Scale bar indicates the number of substitutions per nucleotide position. GenBank accession numbers are in brackets.
Microorganisms 09 02165 g001aMicroorganisms 09 02165 g001bMicroorganisms 09 02165 g001c
Table 1. PCR primers used to amplify the partial 16S rRNA, groEL, and dsb genes of Ehrlichia spp.
Table 1. PCR primers used to amplify the partial 16S rRNA, groEL, and dsb genes of Ehrlichia spp.
Primer NameTargeted GeneSequence (5′–3′)Amplicon Size (bp)Reference
EHR16SD *
EHR16SR *
fD1
16S rRNAGGT ACC YAC AGA AGA AGT CC345[21]
TAG CAC TCA TCG TTT ACA GC[21]
AGA GTT TGA TCC TGG CTC AG~1431[22,23]
rP2ACG GCT ACC TTG TTA CGA CTT[22,23]
HS1agroELAIT GGG CTG GTA ITG AAA T~1400[24,26]
HS6aCCI CCI GGI ACI AIA CCT TC[24,26]
HS43ATW GCW AAR GAA GCA TAG TC1297[25]
HSVRCTC AAC AGC AGC TCT AGT AGC[25]
Dsb-330dsbGAT GAT GTT TGA AGA TAT SAA ACA AAT401[27,28]
Dsb-720 **
Dsb-380
CTA TTT TAC TTC TTA AAG TTG ATA WAT C349[27,28]
ATT TTT AGR GAT TTT CCA ATA CTT GG[28]
* Primers used in the initial PCR screening, ** primer used on first and second round.
Table 2. Data of Haemaphysalis juxtakochi collected in vegetation for each site and processed for detection of “Candidatus Ehrlichia pampeana”.
Table 2. Data of Haemaphysalis juxtakochi collected in vegetation for each site and processed for detection of “Candidatus Ehrlichia pampeana”.
Collection SiteStageN° of Ticks ProcessedPoolsPositive PoolsPositive Pools CodeGenBank Accession Numbers
16S rRNAdsbgroEL
Gruta de los Cuervos (T)Female6571S16HH13MZ733618MZ779092MZ779098
Male7781S12HM25-MZ779089-
Nymph969292S11HN18
S14HN39
MZ733620
MZ733619
MZ779088
MZ779091
MZ779097
MZ779095
Amarillo (Ri)Female000
Male000
Nymph1220
Lunarejo (Ri)Female1030
Male1540
Nymph401151S14HN30-MZ779090MZ779094
Reserva Natural Salus (L)Female320
Male410
Nymph12470
Laguna Negra (Ro)Female720
Male210
Nymph175181S8HN5MZ733617MZ779093-
Total 1864996
(T) Tacuarembó, (Ri) Rivera, (L) Lavalleja, (Ro) Rocha.
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Félix, M.L.; Muñoz-Leal, S.; Carvalho, L.A.; Queirolo, D.; Remesar, S.; Armúa-Fernández, M.T.; Venzal, J.M. Characterization of “Candidatus Ehrlichia Pampeana” in Haemaphysalis juxtakochi Ticks and Gray Brocket Deer (Mazama gouazoubira) from Uruguay. Microorganisms 2021, 9, 2165. https://doi.org/10.3390/microorganisms9102165

AMA Style

Félix ML, Muñoz-Leal S, Carvalho LA, Queirolo D, Remesar S, Armúa-Fernández MT, Venzal JM. Characterization of “Candidatus Ehrlichia Pampeana” in Haemaphysalis juxtakochi Ticks and Gray Brocket Deer (Mazama gouazoubira) from Uruguay. Microorganisms. 2021; 9(10):2165. https://doi.org/10.3390/microorganisms9102165

Chicago/Turabian Style

Félix, María Laura, Sebastián Muñoz-Leal, Luis Andrés Carvalho, Diego Queirolo, Susana Remesar, María Teresa Armúa-Fernández, and José Manuel Venzal. 2021. "Characterization of “Candidatus Ehrlichia Pampeana” in Haemaphysalis juxtakochi Ticks and Gray Brocket Deer (Mazama gouazoubira) from Uruguay" Microorganisms 9, no. 10: 2165. https://doi.org/10.3390/microorganisms9102165

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