Our original study design aimed to identify equine piroplasmosis in horse samples collected in close proximity to Ordos City, Inner Mongolia, located in northern China (longitude 106°42′40″–111°27′20″, latitude 37°35′24″–40°51′40″). Whole blood was collected using EDTA anticoagulant tubes, with collection location, time, and sample number recorded, prior to storage at –20^°C.

DNA was extracted from whole blood samples using Blood DNA Extraction Kits (Tiangen Biotechnique Inc., Beijing, China) following the manufacturer’s instructions. Obtained DNA were stored in a –20^°C refrigerator while awaiting further testing.

A universal primer PIRO 0F (5′-GCC AGT CAT ATG CTT GTG TTA-3′) and PIRO 6R (5′-CTC CTT CCT YTA AGT GAT AAG GTT CAC-3′) (Kawabuchi et al., 2005) targeting 1,600 bp segment sequences of 18S rRNA gene of Babesia spp. and Theileria spp. were used to test for piroplasma infection. The PCR mixture consisted of 10.5 μl ddH₂O, 12.5 μl 2 × Ex Taq Plus Master Mix, 0.5 μl of each primer, and 1 μl template DNA. An initial denaturation step at 95°C for 5 min was followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. A final extension was done at 72°C for 7 min, followed by a hold step at 4°C. PCR products were then detected by 1% agarose gel electrophoresis. To minimize risks of contamination, template isolation, PCR setup, and agarose gel electrophoresis occurred in separate rooms, in addition to including a negative control in each amplification.

Positive amplicons were sent to Comate Biosciences Co., Ltd. (Changchun, China) for sequencing on a 3730 DNA Sequencer (Applied Biosystems, Foster City, CA, United States) from both directions. The bidirectional determination of the sequences were used for sequence splicing with SeqMan software. The clear sequences obtained were then compared with previously published sequences deposited in GenBank using the BLAST program from the National Center for Biotechnology Information website. Nucleotide sequences reported in this study were then deposited into GenBank with accession numbers.

The alignments of novel sequences, together with reference sequences from GenBank, were constructed with ClustalW using MEGA software (6.0). Phylogenetic trees were reconstructed using neighbor-joining, maximum likelihood, and maximum parsimony methods, respectively. Three analysis methods were performed using MEGA (6.0), with best fit selected according to the topology of the phylogenetic trees. Phylograms were drawn using the Fig Tree (v. 1.4.2).

Statistical analysis was performed by SPSS 22.0 software, and tick-borne pathogen risk factors were analyzed by χ² test or Fisher’s exact test. Bilateral p < 0.05 means the difference is statistically significant.

Whole blood samples were collected from a total of 400 horses and screened for piroplasma infection. A total of 136 (34.0%) samples were positive using common primers targeting 18S rRNA for piroplasms (Kawabuchi et al., 2005), which showed ∼1,600-bp amplicons under electrophoretic diagram (Figure 1). Entire sequences were obtained from positive amplicons. After conducting BLAST search, 132 (33%) horses were confirmed infected with Theileria equi, two were infected with Babesia caballi, and another two (0.5%), numbered NM103 and NM115, were infected with Colpodella sp. (Table 1). The infection rates of these three pathogens are significantly different in horses (p < 0.001, χ² = 280.296). Analyses suggested that the phylogenetic position of Colpodella sequences fell within the sister group of the apicomplexan clade (Kuvardina et al., 2002), including Babesia spp., Theileria spp., Toxoplasma gondii, Cryptosporidium spp., and Plasmodium spp. Thus, it was not surprising to inadvertently amplify Colpodella with these common primers, similar to what was observed in a previous study (Jiang et al., 2018).

Two approximately 1,600-bp sequences (accession nos. MW261749 and MW261750) obtained from our sample NM103 and NM115 had three nucleotide bases difference, and were closely related to Colpodella sp. ATCC50594 strain (accession no. AY14207583) isolated from brown woodland soil at Gambrill State Park, located in Frederick, Maryland, with 99.18 and 98.73% similarity, respectively. We used the MegAlign component to perform multiple sequence alignments with the ClustalW algorithm to create the consensus phylogenetic tree based on nearly the entire 18S rRNA gene, along with other representative sequences from the GenBank database, respectively inferred by maximum likelihood, maximum parsimony, and the neighbor-joining method, with bootstrap values determined by 1,000 replicates in MEGA6.0 (Figure 2). Furthermore, the topology of all three trees were similar across methods. These two novel sequences fell within the same clade as Colpodella sp. ATCC50594, and represent the first evidence of Colpodella sp. detected in horses in China (Figures 2A–C). In addition, the consensus phylogenetic tree based on partial 18S rRNA gene, especially along with other representative sequences originated from Amur tiger deposited in GenBank database, inferred by maximum likelihood still confirmed the same topology and relationship (Figure 2D).

Colpodella spp. are uncommon in the natural environment according to the current research report; however, our present study detected two potential pathogenic agents, which were closely related to Colpodella sp. ATCC50594 isolated from soil and has possible links to the origin of intracellular parasitism (Getty et al., 2021), and close to Colpodella sp. HLJ (KT364261) and Colpodella sp. HEP (GQ411073) isolated from patients in China (Yuan et al., 2012; Jiang et al., 2018). Two Colpodella spp. were also found in positive Ixodes persulcatus ticks collected around the living area of the patients mentioned previously (Jiang et al., 2018). One of the Rhipicephalus microplus infesting cattle was also positive for Colpodella spp. (Matsimbe et al., 2017). In addition, the Colpodella sp. sequence under the accession number MH012044-46 was detected in Dermacentor nuttalli, which is commonly found in Inner Mongolia. Thus, it is likely that ticks might be important carriers of these microorganisms and serve as potential transmission vectors in both humans and animals. However, it remains unclear whether the ticks are mechanical or biological transmitters, which requires further study. On the basis of molecular biology, recent modified light microscopy using a new trichrome staining technique by Sam-Yellowe et al. (2020) will aid in diagnosis of infections of Colpodella sp. and provide another available avenue for future surveillance and research.

The two previous reports of human cases of Colpodella sp. infection presented with clinical symptoms similar to other erythrocyte parasitic infections, such as fever and hemolytic anemia (Yuan et al., 2012; Jiang et al., 2018). The Heilongjiang case presented with moderate nuchal rigidity neurological symptoms, with isolates obtained from the patient’s cerebral spinal fluid and from ticks collected in close proximity to the patient’s house.

In addition, five partial sequences of Colpodella sp. obtained from Amur tiger (Panthera tigris altaica) without other available detail information were also reported. The present study first showed that another vertebrate, an animal that has high contact with human and is often attached by ticks, is affected by Colpodella sp. with no obvious symptoms like human cases. However, it is certain that horses are an important risk factor or act as a reservoir for potential zoonotic pathogen Colpodella sp. infection in human. Extending investigation on them among vertebrates, vector ticks, and human is further needed.