Pathogenic Rickettsia, Anaplasma, and Ehrlichia in Rhipicephalus microplus ticks collected from cattle and laboratory hatched tick larvae

Abstract

Background: The order Rickettsiales contains a group of vector-borne gram-negative obligate intracellular bacteria, which often cause human emerging infectious diseases and economic losses for dairy and meat industries. The purpose of this study is to investigate the distribution of the pathogens including Rickettsia spp., Anaplasma spp., and Ehrlichia spp. in the order Rickettsiales in ticks from Yueyang, a prefecture-level city of Hunan Province in Sothern China, and assess the potentiality of transovarial transmission of these rickettsial organisms.

Methods: Ticks were collected from cattle in a farm in Yueyang City and the tick DNA was used as template to amplify the htrA, rrs, gltA, ompA and ompB genes of Rickettsia as well as rrs and groEL genes of Anaplasma and Ehrlichia.

Results: All ticks (465) collected were the cattle tick, Rhipicephalus microplus. PCR showed the minimum infection rate (MIR) was 1.5% (7/465) for Candidatus Rickettsia xinyangensis, 1.9% (9/465) for C. Anaplasma boleense, 1.3% (6/465) for Anaplasma platys, 0.6% (3/465) for A. marginale, and 1.17% (2/465) for each of A. bovis, Ehrlichia minasensis, and a non-classified Ehrlichia sp. A human pathogen, C. Rickettsia xinyangensis and A. platys were detected in 100% (3/3) and 33.3% (2/6) laboratory-hatched larval pools from infected females respectively.

Conclusion: Our study revealed a diversity of pathogenic rickettsial species in R. microplus ticks from Hunan Province suggesting a threat to people and animals in China. This study also provided the first molecular evidence for the potential transovarial transmission of C. Rickettsia xinyangensis and A. platys in R. microplus, indicating that R. microplus may act as the host of these two pathogens.

Fig 1. Map of sampling site.

Ticks were collected from cattle in a farm in Xi Tang Town, Yueyang City, Hunan Province in Southern China from August 5 to August 22, 2022. The map was constructed using ArcGIS 10.6 software. The basemap shapefiles were downloaded from national platform for common geospatial information services (tianditu.gov.cn).

Fig 2. The oviposition of a female Rhipicephalus microplus.

(A) Photographs taken on the 3rd and 7th day of oviposition, respectively. (B) Micrographs of tick eggs (top) and laboratory hatched larvae (bottom).

Fig 3. Photos of the dorsal (left) and ventral (right) sides of Rhipicephalus microplus under a dissecting microscope.

(A) An adult female tick. (B) An adult male tick.

Fig 4. Sample pooling information.

Fig 5. Phylogenetic tree of Rhipicephalus microplus ticks.

The tree was constructed using the partial 16S rRNA gene (229 bp) of ticks based on the Maximum Likelihood (ML) method with the Kimura 2-parameter model in MEGA 7.0. Rhipicephalus sanguineus sequence was used for outgroup in the tree. The R. microplus sequences obtained in this study were marked with dots and have been submitted to the GenBank with accession numbers: OQ975295–OQ975297.

Fig 6. Phylogenetic tree of Rickettsia based on the concatenated sequences of htrA (365 bp), rrs (1241 bp), ompA (551 bp), gltA (604 bp), and ompB (344 bp) genes.

Phylogenetic tree was constructed based on the Maximum Likelihood (ML) method with the Kimura 2-parameter model in MEGA 7.0. Bootstrap values (inferred from 1,000 replicates) >60% were indicated. The Rickettsia sequences obtained in this study were marked with dots. For the Rickettsia species without complete genome sequences, the GenBank accession nos. in the order of htrA, rrs, ompA, gltA and ompB are KY617773, KY617772, KU853021, KU853023, and KY617776 for C. Rickettsia xinyangensis; MG906673, MT747412, MN026548, MN026549, and MT747415 for C. Rickettsia longicornii; KT187396, MT062904, KT326194, KT187394, and JF758826 for Rickettsia vini.

Fig 7. Phylogenetic trees of Anaplasma based on the partial rrs (470 bp) gene and groEL (205 bp) genes.

Phylogenetic trees were constructed based on the Maximum Likelihood (ML) method with the Kimura 2-parameter model in MEGA 7.0. Bootstrap values (inferred from 1,000 replicates) more than 60% were indicated. Wolbachia pipientis sequences were used for outgroup in the trees. The Anaplasma sequences obtained in this study were marked with dots.

Fig 8. Phylogenetic trees of Ehrlichia with the partial rrs (430 bp) and groEL (461 bp) genes.

Phylogenetic trees were constructed using the Maximum Likelihood (ML) method with the Kimura 2-parameter model in MEGA 7.0. Bootstrap values (inferred from 1,000 replicates) more than 60% were indicated. Anaplasma marginale sequences were used for outgroup in the trees. The Ehrlichia sequences obtained in this study were marked with dots.