Potential zoonotic role of the tick Amblyomma cf. oblongoguttatum (Ixodida: Ixodidae) in the bacterial transmission of Ehrlichia chaffeensis (Rickettsiales: Anaplasmataceae) in a deciduous tropical forest in Mexico (2024)

Emily McDermott, Subject Editor

Abstract

Ehrlichia chaffeensis is a bacterium belonging to the Anaplasmataceae family. In Mexico, only 2 species have been recorded in association with tick species and humans. The objective of the present study was to detect the presence of bacteria of the genus Ehrlichia in ticks collected from the Chamela-Cuixmala Biosphere Reserve, Jalisco, Mexico. The collected ticks were identified and analyzed individually by polymerase chain reaction to amplify a fragment of the Anaplasmataceae 16S rRNA gene and the Ehrlichia-specific dsb gene. A total of 204 ticks, corresponding to 5 species of Ixodidae and 1 of Argasidae, were collected from 147 mammals of 6 species and 4 orders; 57 ticks collected from vegetation were also included. Among the total ticks collected, 1.47% (3/204) was positive for Ehrlichia sp. DNA was obtained using the primers EHR 16SD and EHR 16SR for 16S rRNA and DSB-330 and DSB-728 for dsb. The positive samples corresponded to a larva (Amblyomma sp.) associated with Didelphis virginiana and 2 nymphs (Amblyomma cf. oblongoguttatum) infesting Nasua narica. None of the ticks collected from the vegetation tested positive for Ehrlichia sp. DNA on the basis of the 16S rRNA and dsb genes. The sequences from the larvae of Amblyomma sp. and the nymphs of A. cf. oblongoguttatum were similar to those of E. chaffeensis. The phylogenetic analysis inferred with maximum likelihood corroborated the identity as E. chaffeensis. Although the role of these tick species as vectors of E. chaffeensis is still undetermined, the presence of infected ticks in the area indicates a potential zoonotic risk.

Keywords: bacteria, mammal, reserve, tick, disease

Introduction

Ehrlichioses were considered a group of diseases of exclusively veterinary interest until 1986, when Ehrlichia chaffeensis was detected as a zoonotic pathogen (Maeda et al. 1987). Currently, E. chaffeensis is recognized as an emerging etiological agent that causes human monocytic ehrlichiosis (HME), one of the most prevalent tick-borne diseases in North America (Paddock and Childs 2003).

Ehrlichia chaffeensis is an intracellular gram-negative bacterial species in the Anaplasmataceae family that is transmitted to humans, cattle, dogs, and other mammals by ticks (Dumler et al. 2001). These authors, in the most recent revision of Ehrlichia, recognized 5 valid species, and Cabezas-Cruz et al. (2016) described an additional species distributed mainly in the Americas but with records elsewhere.

Little is known about Ehrlichia infections in Mexico, and only 2 species (i.e., Ehrlichia canis and E. chaffeensis) have been recorded in association with tick species of the genera Amblyomma and Rhipicephalus (Ojeda-Chi et al. 2019, Alcántara-Rodríguez et al. 2020). Nonetheless, 2 of the 4 human cases caused by E. chaffeensis reported to date have been fatal, 1 in Mexico City (Alcántara-Rodríguez et al. 2020) and the other in Estado de México (Sosa-Gutiérrez et al. 2013). A unique human case of E. canis was detected on the coast of the state of Oaxaca (Beatriz-Silva et al. 2014). Guzmán-Cornejo et al. (2022) detected the presence of Rickettsia amblyommatis, R. parkeri, R. bellii, and Rickettsia sp. in mammalian ectoparasites of the Reserva de la Biosfera Chamela-Cuixmala, state of Jalisco, Mexican Pacific. These findings have clear implications for the spread of zoonotic diseases given the number of visitors to the Estación de Biología Chamela and the tourism development in the surrounding areas (Martínez and Corgo 2014, Magallón-Puebla 2019). In this context, we continued our investigations on the richness of pathogenic bacteria transmitted by ectoparasites, and the objective of the present study was to test for the presence of species of the genus Ehrlichia in ticks collected in the same area.

Materials and Methods

In a previous work, bacteria of the genus Rickettsia associated with ticks were detected in the Chamela-Cuixmala Biosphere Reserve (Guzmán-Cornejo et al. 2022). During April and November of 2018 and 2019, 4 follow-up surveys were carried out at the Estación de Biología Chamela (EBCh) (19°30’00’’ N; 105°02’00’ W), Jalisco, Mexico. This locality is a tropical deciduous forest (dry forest). The samples were collected under the permits SGPA/DGVS/12771/16 and SGPA/DGVS/011184/18, which were issued to C. G. C. by the Secretaría de Medio Ambiente y Recursos Naturales, Mexico. Mammal sampling and tick collection procedures were conducted as described by Guzmán-Cornejo et al. (2020). Briefly, Tomahawk (baited with sardine) and Sherman (baited with a mix of oat grains and vanilla) traps were used to capture mammals, whereas beat nets and flannel cloth drags were used to collect ticks from vegetation. The procedures for trapping and handling carnivores were performed according to the guidelines of the American Society of Mammalogists (Sikes and Gannon 2011). All ticks were fixed and preserved in 96% ethanol.

Ticks were identified morphologically based on specialized taxonomical keys: those of Keirans and Jones (1972) and Martins et al. (2010) for nymphs of Ixodidae and Guzmán-Cornejo et al. (2011) for adult specimens. Argasid larvae were identified using the keys of Kohls et al. (1965) and Guzmán-Cornejo et al. (2019).

Total DNA was extracted from ticks from each specimen using 250 µl of 10% (w/v) Chelex (Bio-Rad, CA, USA) and 10 mg/ml proteinase K. Each tick was digested at 56 °C for 2 h, followed by heat inactivation of proteinase K at 95 °C for 15 min. The samples were vortexed, and Chelex was pelleted by centrifugation at 14,000 rpm for 1 min.

All ticks were tested individually for Anaplasmataceae by conventional polymerase chain reaction (PCR) using the primers EHR16SD (5ʹ-GGT ACC YAC AGA AGA AGT CC-3ʹ) and EHR16SR (5ʹ-TGC ACT CAT CGT TTA CAG-3ʹ), which amplify an ~344 bp fragment of the 16S rRNA gene of Anaplasmataceae bacterial agents (Inokuma et al. 2000). Positive samples were further tested by a PCR assay with primers DSB-330 (5ʹ-GAT GAT GTC TGA AGA TAT GAA ACA AAT-3ʹ) and DSB-728 (5ʹ-CTG CTC GTC TAT TTT ACT TCT TAA AGT-3ʹ), which amplify an ~409 bp fragment of the Ehrlichia genus-specific disulfide bond formation protein-encoding gene (dsb) (Doyle et al. 2005).

PCRs were performed in a total volume of 15 µl, including 13 µl of master mix (0.5 µl of 50 mM MgCl₂, 3µl of 5X buffer, 0.2 µl of each primer at 10 mM, 0.1 µl of Taq DNA polymerase [MyTaq], and 9 µl of nuclease-free water) and 2 µl of genomic DNA. Negative (water) and positive (donated to Dr. Jorge Zavala Castro by the University of Texas Medical Branch) control samples were included in each PCR assay. Amplicons were visualized on 1.5% agarose gels, and positive PCR products were sequenced at the Laboratorio de Biología Molecular y de la Salud (LANABIO), Instituto de Biología, Universidad Nacional Autónoma de México (UNAM).

Sequences were edited with the program Geneious (Kearse et al. 2012) and compared with sequences available in GenBank using the basic local alignment search tool (BLASTn) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Based on the BLASTn results, our newly generated sequences and sequences obtained from GenBank were aligned with the online version of MAFFT v7 software (Katoh et al. 2019, https://mafft.cbrc.jp/alignment/server/).

The final dataset of the dsb partition included 25 terminals and 423 aligned characters. A phylogenetic analysis was run under the maximum likelihood (ML) criterion, employing a TN93 + I model. ML analysis was implemented with RAxML-NG v.1.1 (Kozlov et al. 2019), with bootstrap support values obtained through 1,000 pseudoreplicates. The phylogenetic tree was visualized and edited with FigTree version 1.4.2 software (Rambaut 2012).

Results

Among the total ticks (204: 147 infesting mammals and 57 collected directly from vegetation) tested for Ehrlichia (Table 1), only 3 (1.47%) were positive: 1 larva of Amblyomma associated with Didelphis virginiana and 2 nymphs of Amblyomma cf. oblongoguttatum associated with Nasua narica (Table 1). None of the ticks collected from the vegetation tested positive for Ehrlichia.

The 16S rDNA sequences obtained from A. cf. oblongoguttatum, showed a similarity of 99.67% (298/299; E-value 2e-150) with that of Ehrlichia sp. (MT514732). The dsb bacterial sequences obtained from the Amblyomma larva of the opossum was 99.7% (406/407; E-value: 0.0) similar to that of E. chaffeensis (GenBank CP007480), while the bacterial sequences generated from the nymphs of A. cf. oblongoguttatum were 99% (403/407; E-value: 0.0) and 97.3% (285/293; E-value: 2e-136) similar to those of E. chaffeensis (GenBank CP007480.1 and KM458247.1, respectively). On the other hand, phylogenetic analysis based on the dsb gene, inferred through ML, grouped the sequences generated in the present study with those of E. chaffeensis (Fig. 1). 16S rDNA is widely used for broad phylogenetic studies in bacteria; however, in the present article, phylogenetic relationships within Ehrlichia were inferred based on dsb alone, given its greater variability.

Discussion

Ehrlichia chaffeensis is known to be the etiological agent of HME, a disease characterized by high fever, headaches, malaise, myalgia, nausea, anorexia, and, in some cases, rash. Severe infections may result in death (CDC 2019).

Ehrlichia chaffeensis is transmitted to mammals, including humans, by hard ticks. In nature, the life cycle of this bacterial species includes mammals that serve as reservoirs; the white-tailed deer Odocoileus virginianus has been recorded as the main natural reservoir for E. chaffeensis, with Amblyomma americanum being the tick species implicated in its life cycle. Furthermore, additional mammals have been implicated as potential reservoirs, including the domestic dog Canis lupus familiaris, the coyote Canis latrans, the raccoon Procyon lotor, and the opossum Didelphis virginiana (Paddock and Childs 2003), all of which are distributed in the EBch (Ceballos and Miranda 2000). It is expected that these mammals [and others among the 68 species distributed in the reserve according to Ceballos et al. (2010)] play an important role in the maintenance and dispersion of E. chaffeensis through infected ticks in the region, as has been suggested by the presence of this bacterium in O. virginianus and in nymphs of Amblyomma spp. in Yucatán, Mexico (Ojeda-Chi et al. 2019). Currently, the role of A. cf. oblongoguttatum as a vector of this pathogen is unclear; however, it is known that the adult specimens of this tick are closely associated with cervids, while the nymphs are associated with didelphids. If A. cf. oblongoguttatum is confirmed as a vector, opossums may represent important agents in the potential spread of E. chaffeensis to humans, given the synanthropic relationship with these marsupials.

During the life cycle of E. chaffeensis, non-infected tick larvae feed on a bacteremic vertebrate reservoir, and subsequently, bacteria undergo transstadial transmission to the nymph. The Amblyomma larva positive for E. chaffeensis found on the opossum in the present study may have acquired the infection by feeding on the opossum. Opossums are synanthropic marsupials that can inhabit wild, peri-urban, and urban environments interchangeably, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of public health interest (Braga et al. 2023).

Lockhart et al. (1997) reported 8% seropositivity for E. chaffeensis in D. virginiana in Georgia, USA. Braga et al. (2023) demonstrated for the first time the occurrence of different vector-borne agents in D. marsupialis in northeastern Brazil, with the description of potential novel genotypes of Ehrlichia. Similarly, the Ehrlichia sp. strain Natal has been detected in other marsupials, such as the white-eared opossum Didelphis albiventris in Argentina and Brazil and the agile gracile opossum Gracilinanus agilis in Brazil (Tarragona et al. 2022).

Both opossums and coatis are known to be highly synanthropic, and this behavior warrants caution (Bermúdez et al. 2017). In addition to the opossum and coati, 4 other mammals were also found to be positive for ticks but negative for Ehrlichia. These mammals included the ocelot Leopardus pardalis, the Mexican cottontail Silvilagus cunicularius, the painted spiny pocket mouse Heteromys pictus, and the Magdalena rat Xenomys nelsoni. Negative results should be interpreted with caution, given the low number of ticks screened.

In Mexico, ehrlichiosis caused by E. canis has been widely studied in C. lupus familiaris (Nuñez 2003). To date, this bacterium has been found to be responsible for 1 human infection, whose clinical resolution was reached after 3 weeks of treatment with antibiotics (Beatriz-Silva et al. 2014). An additional agent reported to be involved in human infections in Yucatán, Estado de México, and Mexico City is E. chaffeensis, whose vectors remain unknown (Góngora-Sosa-Gutiérrez et al. 2013, Góngora-Bianchi et al. 1999, Alcántara-Rodríguez et al. 2020). Recently, a new case of ehrlichiosis (Ehrlichia sp.) in a 15-year-old female was reported in an urban area in northeastern Mexico, representing the first pediatric case in the country (Cisneros-Saldaña et al. 2023). Even though it is unknown whether A. cf. oblongoguttatum is the vector for E. chaffeensis, the fact that this tick species can parasitize a wide variety of mammals, including species of the families Cervidae, Tayassuidae, and Canidae, and birds of the families Cracidae, Cathartidae, and Phasianidae, as well as humans (Guglielmone et al. 2014), is of major concern. To date, records of this tick parasitizing humans have been reported in Brazil, Colombia, French Guiana, Guyana, Panama, Suriname, and Venezuela, but most likely, they are only a subset of the real number of cases (Guglielmone and Robbins 2018).

The finding of E. chaffeensis in wild animals represents a potential health risk not only for the staff of the EBCh; according to prepandemic data published by Magallón-Puebla (2019), 754 visitors to this station belonged to 17 Mexican and 4 international institutions; likewise, the touristic developments near the reserve have monopolized large extents of land (Martínez and Corgo 2014), which may result in increased exposure of people to these bacteria.

The record of E. chaffeensis reported here, together with the Rickettsia species reported in a previous study in the same area (Guzmán-Cornejo et al. 2022), gives rise to new research questions that should be addressed to better understand the dynamics of bacterial infections, both in nature and in humans. For example, it is necessary to prove if negative results may be due to the presence of PCR inhibitors in the chitin of ticks. Furthermore, is of particular interest the characterization of seasonality, host spectrum, prevalence in wildlife, and exposure to humans of these bacteria. In addition, it should be determined whether A. cf. oblongoguttatum is the vector of E. chaffeensis or whether other invertebrates are involved. However, an important limitation to human studies is the collection of human blood samples, which should also be analyzed to detect these potential zoonoses in early infections and propose mechanisms to control the problem.

Acknowledgments

We thank Dr. Katherine Renton and the staff of the Chamela Biological Station, Instituto de Biología, UNAM for allowing us to use the facilities for this project. Laura Del Castillo Martínez and Ana Ugalde Medina assisted in the collection and cataloging of ectoparasites, while Jonathan López, Gerardo Contreras, Martín Cabrera-Garrido, and Belem Isaak helped with trapping hosts. Andrea Jiménez and Andrea Rebollo for the PCR procedures. Laura Márquez and Nelly López of LANABIO assisted in the sequencing procedures.

Contributor Information

Carmen Guzmán-Cornejo, Laboratorio de Acarología, Departamento de Biología Comparada, Facultad de Ciencias, Universidad Nacional Autónoma de México, 04510, Ciudad de México, México.

Angel Herrera-Mares, Laboratorio de Ecología de Enfermedades y Una Salud, Departamento de Etología, Fauna Silvestre y Animales de Laboratorio, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad de México, México.

Luis García-Prieto, Laboratorio de Helmintología, Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México, 04510, Ciudad de México, México.

Alejandro Oceguera-Figueroa, Laboratorio de Helmintología, Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México, 04510, Ciudad de México, México.

Andrés M López-Pérez, Red de Biología y Conservación de Vertebrados, Instituto de Ecología (INECOL A.C.), Veracruz, México.

Karla Dzul-Rosado, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi,” Universidad Autónoma de Yucatán, Mérida, Yucatán, México.

Funding

Financial support for this study was obtained from the Dirección General de Asuntos del Personal Académico-Universidad Nacional Autónoma de México through the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT, Project IN214918).

Author Contributions

Carmen Guzmán-Cornejo (Conceptualization [lead], Data curation [equal], Formal analysis [equal], Funding acquisition [lead], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Supervision [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Angel Herrera-Mares (Formal analysis [equal], Investigation [equal], Methodology [equal], Software [equal], Validation [equal], Writing—original draft [equal], Writing—review & editing [equal]), Luis García-Prieto (Conceptualization [equal], Supervision [equal], Writing—original draft [equal], Writing—review & editing [equal]), Alejandro Oceguera-Figueroa (Data curation [equal], Resources [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Andrés M. López-Pérez (Investigation [equal], Methodology [equal], Writing—review & editing [equal]), and Karla Dzul-Rosado (Validation [equal], Visualization [equal], Writing—review & editing [equal])

References