Prevalence and phylogenetic analysis of protozoan in Haemaphysalis ticks from Chongming, Shanghai

doi:10.12140/j.issn.1000-7423.2026.01.011

Abstract

Abstract:

Objective To investigate the prevalence and genetic characteristics of common Apicomplexan protozoan in ticks collected from Chongming, Shanghai, so as to provide insights into management of tick-borne diseases in Chongming area. Methods Free-living ticks were collected using the flagging method in Chongming, Shanghai, from July to September 2023. Following morphological identification, ticks were pooled according to species and developmental stage (one adult per pool, five nymphs per pool, or ten larvae per pool), and genomic DNA was extracted from each pool. Tick species were molecularly identified by PCR amplification of the cytochrome c oxidase subunit 1 (cox1) gene and 16S rDNA gene. The short fragment of the Babesia/Theileria 18S rRNA gene (approximately 400 bp in length) was amplified using nested PCR assay, and positive samples were further subjected to amplification of the long fragment of the 18S rRNA gene (approximately 1 600 bp in length), and positive nested PCR products were sequenced, followed by sequence alignment with BLAST. Genetic distances were calculated among different samples using the software MEGA 11 and phylogenetic trees were created. Differences in the detection of protozoan were tested for statistical significance with chi-square test. Results A total of 622 free-living ticks were collected, including 255 Haemaphysalis longicornis and 367 H. flava. H. longicornis ticks were divided into 83 pools (55 adult pools, 16 nymph pools and 12 larval pools), and H. flava were divided into 43 pools (2 adult pools, 9 nymph pools and 32 larval pools). Nested PCR amplification of the short fragment of the 18S rRNA gene yielded 17 positive pools, including 16 H. longicornis pools (9 adult pools and 7 nymph pools) and one H. flava pool (adult pool). and the long fragment of the 18S rRNA gene was successfully amplified from all 17 positive pools, with sequences deposited under accession numbers of PX453257 to PX453273. BLAST alignments revealed that one gene sequence showed 99.52% identity with the Babesia 18S rRNA gene sequence (MK930513), 7 gene sequences showed 91.53% to 99.94% identity with the 18S rRNA gene sequences of the novel protozoan Colpodella, and 9 gene sequences showed 95.66% to 99.36% identity with the Colpoda 18S rRNA gene sequences. Genetic distance analysis indicated high intraspecific sequence conservation, with genetic distances ranging from 0.003 to 0.147 among 7 Colpodella gene sequences and from 0.009 to 0.060 among 9 Colpoda gene sequences, and the genetic distance between Colpodella and Colpoda ranged from 0.124 to 0.221, indicating substantial interspecific divergence. Phylogenetic analysis revealed that Colpoda and Colpodella were clustered into a large clade, and Babesia and Theileria were clustered into another large clade. The detection rates of Babesia, Colpodella, Colpoda, and protozoan were 1.20% (1/83), 8.43% (7/83), 9.64% (8/83), and 19.28% (16/83) in H. longicornis, and 0 (0/43), 0 (0/43), 2.33% (1/43), and 2.33% (1/43) in H. flava, respectively. The overall detection of protozoan was significantly higher in H. longicornis than in H. flava (χ² = 6.29, P < 0.05). Conclusion There were Babesia, Colpoda, and novel protozoan Colpodella infections in free-living ticks collected from Chongming area, indicating a potential risk of tick-borne diseases.

Key words: Haemaphysalis, Babesia, Colpoda, Colpodella, Novel protozoan, Chongming

CLC Number:

R382

SHEN Yong, LI Yuanyuan, WANG Ziyi, YANG Limin, HUANG Lirong, LI Zhongqiu, HAN Qingchi, ZHANG Yi, GUO Yunhai, LIU Qin. Prevalence and phylogenetic analysis of protozoan in Haemaphysalis ticks from Chongming, Shanghai[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(1): 72-78.

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引物名称 Primer name	引物序列（5′→3′） Primer sequence (5′→3′)	产物大小/bp Product size/bp
cox1	F：TAAACTTCAGGGTGACCAAAAAATCA	700
	R：GGTCAACAAATCATAAAGATATTGG
16S rDNA	F：CTGCTCAATGATTTTTTAAATTGCTGTGG	400
	R：CCGGTCTGAACTCAGATCAAGT
18S rRNA基因短片段 18S rRNA short fragment	F1：AATTACCCAATCCTGACACAGG	400
18S rRNA基因短片段 18S rRNA short fragment	R1：TTTCGCAGTAGTTCGTCTTTAACA
	F2：GACACAGGGAGGTAGTGACAAGA
	R2：CCCAACTGCTCCTATTAACCATTAC
18S rRNA基因长片段 18S rRNA long fragment	F1：GCCAGTAGTCATATGCTTGTGTTA	1 600
18S rRNA基因长片段 18S rRNA long fragment	R1：CTCCTTCCTYTAAGTGATAAGGTTCAC
	F2：CCATGCATGTCTWAGTAYAARCTTTTA
	R2：CCTYTAAGTGATAAGGTTCACAAAACTT

特征 Fature	采集蜱数 No. ticks collected
特征 Fature	长角血蜱 H. longicornis	褐黄血蜱 H. flava	合计 Total
采集环境 Sampling environment
居民区 Residential area	0	20	20
公园 Park	194	347	541
跑马场 Racecourse	61	0	61
生长阶段 Growth stage
幼蜱 Larva	120	320	440
若蜱 Nymph	80	45	125
成蜱 Adult	55	2	57
合计 Total	255	367	622

序列登录号 Sequence account numbers	PX453257	PX453263	PX453266	PX453267	PX453268	PX453270	PX453272	PX453258	PX453259	PX453261	PX453262	PX453264	PX453265	PX453269	PX453271	NJ-T-37	2016-T18	HLJ	C. grandis
PX453263	0.127
PX453266	0.027	0.125
PX453267	0.120	0.027	0.125
PX453268	0.110	0.144	0.118	0.139
PX453270	0.113	0.147	0.121	0.142	0.003
PX453272	0.123	0.136	0.125	0.132	0.138	0.141
PX453258	0.213	0.189	0.210	0.185	0.205	0.208	0.204
PX453259	0.221	0.194	0.218	0.189	0.204	0.208	0.205	0.040
PX453261	0.218	0.194	0.213	0.189	0.207	0.210	0.209	0.040	0.040
PX453262	0.206	0.180	0.201	0.177	0.197	0.201	0.193	0.010	0.038	0.032
PX453264	0.218	0.191	0.212	0.185	0.206	0.210	0.203	0.046	0.040	0.028	0.041
PX453265	0.212	0.187	0.208	0.184	0.201	0.204	0.201	0.033	0.031	0.036	0.032	0.026
PX453269	0.220	0.197	0.219	0.193	0.205	0.209	0.213	0.039	0.037	0.042	0.041	0.038	0.025
PX453271	0.209	0.188	0.207	0.183	0.196	0.201	0.203	0.051	0.053	0.054	0.050	0.060	0.055	0.058
NJ-T-37	0.127	0.002	0.125	0.026	0.146	0.149	0.136	0.190	0.195	0.195	0.181	0.191	0.188	0.198	0.189
2016-T18	0.109	0.127	0.115	0.124	0.120	0.123	0.043	0.195	0.200	0.200	0.185	0.198	0.192	0.205	0.197	0.129
HLJ	0.024	0.125	0.017	0.127	0.115	0.118	0.126	0.212	0.218	0.216	0.202	0.211	0.208	0.220	0.208	0.127	0.112
C. grandis	0.213	0.188	0.207	0.183	0.202	0.205	0.201	0.040	0.033	0.039	0.039	0.021	0.015	0.025	0.055	0.190	0.195	0.206
C. reniformis	0.213	0.186	0.207	0.180	0.200	0.203	0.201	0.039	0.032	0.037	0.038	0.022	0.016	0.026	0.053	0.187	0.193	0.206	0.004

蜱种 Tick species	检测样品数 No. detected samples	原虫检出率/%（阳性样品数/检测样品数） Protozoan positive rate/% (No. positive samples/No. detected samples)
蜱种 Tick species	检测样品数 No. detected samples	巴贝虫 Babesia	Colpodella	肾形虫 Colpoda	合计 Total
长角血蜱 H. longicornis
成蜱 Adult	55	1.82（1/55）	7.27（4/55）	7.27（4/55）	16.36（9/55）
若蜱 Nymph	16	0/16	3/16	4/16	7/16
幼蜱 Larva	12	0/12	0/12	0/12	0/12
小计 Subtotal	83	1.20（1/83）	8.43（7/83）	9.64（8/83）	19.28（16/83）
褐黄血蜱 H. flava
成蜱 Adult	2	0/2	0/2	1/2	1/2
若蜱 Nymph	9	0/9	0/9	0/9	0/9
幼蜱 Larva	32	0（0/32）	0（0/32）	0（0/32）	0（0/32）
小计 Subtotal	43	0（0/43）	0（0/43）	2.33（1/43）	2.33（1/43）