Ehrlichia species are emerging infectious diseases affecting both animals and humans worldwide. These obligate intracellular bacteria are transmitted by ticks and can infect a range of hosts. While several Ehrlichia species are known, such as E. canis, E. chaffeensis, E. ewingii, E. muris, and E. ruminantium, recent discoveries have added Ehrlichia sp. AvBat and E. mineirensis to the list. Studies in Brazil and Oklahoma have shown evidence of Ehrlichia spp. reactivity in horses, but direct isolation or PCR detection had not been previously documented globally. In North America, cervids act as reservoir hosts for E. chaffeensis, which can cause monocytic ehrlichiosis in humans. Although horses are not typically known hosts for E. chaffeensis, bacterial DNA has been found in ticks collected from horses in Panama. Furthermore, Anaplasma phagocytophilum and Borrelia burgdorferi, responsible for granulocytic anaplasmosis and Lyme disease respectively, are tick-borne and can infect cats, dogs, horses, and humans.
In Mérida, Nicaragua, the prevalence of tick-borne pathogens in horses is a significant concern, exacerbated by poor horse body condition and heavy tick infestations. In 2013, a study was conducted to assess the exposure of equids to various tick-borne organisms. This investigation aimed to shed light on potential infections within the equine population of Nicaragua. The findings from this study suggest the presence of a potentially new Ehrlichia species, highlighting the ongoing need for research into tick-borne diseases in animal populations.
The Study and its Findings
The study, conducted with approval from the Oregon State University Animal Care and Use Committee, involved collecting blood samples from 92 horses in Mérida, Nicaragua, between August 28 and September 4, 2013. These horses were being evaluated for various medical conditions or were awaiting elective surgery. Blood samples were analyzed for antibodies against Anaplasma spp., B. burgdorferi sensu stricto, and Ehrlichia spp. using the SNAP 4DxPlus ELISA assay.
The results revealed that 51% of the horse serum samples tested were seroreactive to Ehrlichia spp. One sample showed reactivity to B. burgdorferi, while no samples reacted to Anaplasma spp. These seroreactive samples were then transported to North Carolina State University for PCR testing. DNA was extracted from the blood samples, and PCR was used to amplify specific gene fragments: the 16S rRNA gene, the groEL gene, and the sodB gene, which are commonly used for Ehrlichia identification.
Genetic Analysis Points to a Novel Species
Analysis of the amplified DNA sequences provided compelling evidence for a potentially new Ehrlichia species. Identical sequences of the 16S rDNA amplicon were found in 4 of the tested horses. Further comparisons of the amplified products with sequences in the GenBank database, including those from Ehrlichia spp., Anaplasma spp., and Neorickettsia risticii, are summarized in a table. This table illustrates the base pair similarities and differences, indicating that while the sequences shared similarities with known Ehrlichia species, they were not identical to any previously documented strains.
The sequencing of the groEL and sodB genes from three of the four positive horses further supported this conclusion. These protein-coding genes are considered crucial for accurate bacterial classification. Although the sequences showed homology to known Ehrlichia spp. in GenBank, they did not match existing entries completely. Current scientific recommendations suggest using multiple genes, including protein-coding genes, for the identification and classification of novel rickettsial species. Phylogenetic trees constructed from the 16S rRNA, groEL, and sodB gene sequences, when compared with reference sequences from representative members of the genera Anaplasma, Ehrlichia, and Neorickettsia, visually supported the classification of the organism found in Nicaraguan horses as a novel Ehrlichia species.
Table: DNA Sequence Similarities of Ehrlichia sp. from Nicaraguan Horses
| Bacteria | GenBank accession nos. | Gene, no. positive/no. tested (%) |
|---|---|---|
| 16S rRNA | GroEl | |
| E. ruminantium | CR925678 | 361/374 (96.5) |
| E. canis | CP000107 | 356/374 (95.2) |
| E. chaffeensis | CP000236 | 360/374 (96.2) |
| E. ewingii | NR_044747, AF195273, KC778986 | 352/374 (94.1) |
| A. marginale | CP006847 | 339/374 (90.6) |
| A. phagocytophilum | CP006618 | 348/374 (93.0) |
| N. risticii | CP001431 | 306/374 (81.8) |
Sequences for partial 16S rRNA, GroEl, and SodB genes are compared with GenBank database sequences from E. ruminantium, E. canis, E. chaffeensis, E. ewingii, Anaplasma marginale, A. phagocytophilum, and Neorickettsia risticii. GenBank sequence accession numbers for 16S rRNA, GroEl, and SodB for Ehrlichia sp. from horses in Nicaragua are KJ434178, KJ434179, and KJ434180, respectively.
Implications and Future Directions
The study’s findings indicate that exposure to Ehrlichia species is common among horses in Nicaragua, with over half showing serological evidence of exposure. While Anaplasma phagocytophilum was previously considered a possibility due to equine exposure in nearby Guatemala, the absence of its primary tick vectors (I. scapularis and I. pacificus) in Nicaragua makes this less likely. Future research will focus on collecting and identifying ticks in Nicaragua to pinpoint the vector for this novel Ehrlichia species.
The identification of a potentially novel Ehrlichia species in horses raises important questions about its pathogenicity and host range. Given that at least four Ehrlichia species are known to be pathogenic to canids and humans, further investigation is warranted to determine if dogs, other animals, and humans in Nicaragua are also exposed to and infected by this newly identified organism. Confirming this novel species will require further efforts, including attempts to isolate the organism through cell culture. The potential for this novel Ehrlichia species to impact both animal and human health in the region underscores the importance of continued research in vector-borne diseases.
The authors acknowledge the support from Oregon State University College of Veterinary Medicine, the veterinary students who assisted in sample collection, and Dana Riggins for technical assistance. Funding was also provided in part by the State of North Carolina and donations to the North Carolina State University College of Medicine Foundation for Vector-Borne Disease Research. B.A.Q. receives funding from IDEXX Laboratories Inc.
Suggested citation: O’Nion VL, Montilla HJ, Qurollo BA, Maggi RG, Hegarty BC, Tornquist J, et al. Potentially novel Ehrlichia species in horses, Nicaragua. Emerg Infect Dis. 2015 Feb [date cited]. http://dx.doi.org/10.3201/eid2102.140290
