Transovarial transmission of Anaplasma phagocytophilum in Haemaphysalis ticks under field conditions

Background: Anaplasmosis, a zoonotic tick-borne pathogen affecting both livestock, companion animals, and humans, exhibits 15 to 18% seropositivity among hunting dogs in Korea. The dominant tick species in Korea, Haemaphysalis longicornis can transmit this pathogen to both humans and animals. Given the limited understanding of transovarial transmission of Anaplasma spp., our study aimed to assess the prevalence of questing larval ticks containing Anaplasma DNA. Additionally, we aimed to gather data for establishing a nationwide forecasting and alert system on seasonal variation of tick developmental stages and tick-borne zoonotic pathogens. Methods: From March to October 2021 and again from March to October 2022, we collected a total of 36,912 unfed, questing ticks of Haemaphysalis spp. from 149 sites in Korea. Ticks were collected from herbaceous vegetation using the flagging method using a white flannel cloth. After species identification, one third of collected ticks underwent analysis for Anaplasma gene. Nymph ticks were pooled in groups of one to 10, larvae in groups of one to 50, while adults were examined individually. Nested PCR was performed to detect the genus Anaplasma by amplifying the 16S rRNA gene, followed by sequencing for species identification and phylogenetic analysis. Results: Of the 36,912 questing ticks collected, 13,082 (35.4%) were identified as nymphs and adults of H. longicornis and 3,850 (10.4%) as those of H. flava . The morphologically indistinguishable larval stage of Haemaphysalis predominated, with 19,980 (54.1%) collected primarily from July to October. From the 939 tick pools, 24 pools (2.6%) tested positive for Anaplasma , with the larval stage exhibiting the highest number of positive pools (16, 1.7%). Phylogenetic analysis revealed that 21 of the 24 Anaplasma -positive pools contained A. phagocytophilum -specific genes, while the remaining 1 was identified as Anaplasma sp. and 2 as A. bovis . Conclusions: Our study provides evidence of transovarial transmission of Anaplasma phagocytophilum in Haemaphysalis spp. larvae under field conditions, as A. phagocytophilum originates from their mother ticks in unengorged questing larval ticks. Additionally, our findings contribute significant data for establishing a nationwide forecasting and alert system on seasonal variation of tick developmental stages and tick-borne zoonotic pathogens.