Molecular investigation of ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from Eurasian lynx (Lynx lynx) in northwestern China: first identification of Cytauxzoon manul in this host species

doi:10.21203/rs.3.rs-4197979/v1

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Molecular investigation of ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from Eurasian lynx (Lynx lynx) in northwestern China: first identification of Cytauxzoon manul in this host species

https://doi.org/10.21203/rs.3.rs-4197979/v1

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Background: Multiple species of the genera Cytauxzoon and Hepatozoon can infect wild felines, but the diversity of these and other apicomplexan parasites in Eurasian lynx is scarelyknown. The aim of this study was to detect Cytauxzoon and Hepatozoon species with molecular methods in Eurasian lynxes and their ticks in northwestern China.

Methods: DNA was extracted from the heart, liver, spleen, lung and kidney samples of three Eurasian lynxes as well as from their five ixodid ticks. These DNA samples were screened with PCRs for Cytauxzoonwith the partial cytochrome b gene (CytB), cytochrome c oxidase subunit I gene (COI) and small subunit ribosomal RNA gene (18S rRNA), and Hepatozoon with three different fragments of small subunit ribosomal RNA gene (18S rRNA). PCR products were sequenced, aligned and phylogenetically analyzed.

Results: One adult female of Eurasian lynx (#1, adult female) was co-infected with Cytauxzoon manul and Hepatozoon felis genotype I, while an adult male lynx (#2) was infected with C. manul. Interestingly, H. felis genotype I was both detected in a male cub (#3) and two out of five infesting Hyalomma asiaticum ticks.

Conclusion: For the first time, Cytauxzoon manul is reported here from Eurasian lynx. In addition, H. felis has not been known to occur in this host species in China and Central Asia. Thus, findings of this study extend our knowledge on the geographical distribution and host range of these haemoprotozoan parasites. Moreover, this is also the first evidence of C. manul and H. felisco-infection in Eurasian lynx.

Eurasian lynx

Cytauxzoon manul

Hepatozoon felis

northwestern China

Vector-borne pathogens are the causative agents of several emerging or re-emerging infectious diseases among felines [1–2]. Nowadays, their epidemiological patterns undergo changes, their geographical ranges tend to expand, and their global incidence rates also increase owing to climatic change and various environmental, demographic and human-related factors [3].

Cytauxzoonosis is an emerging tick-borne disease of domestic cats and wild felids, caused by members of the genus Cytauxzoon (Apicomplexa: Aconoidasida: Piroplasmida: Theileriidae) [4–5]. The clinic-pathologically most important species, Cytauxzoon felis was described from domestic cat [5], and was thought to be endemic only to North America, specifically in the southern, southeastern, and mid-Atlantic regions of the United States [3, 6]. In North America, the bobcat (Lynx rufus) is the most common natural host for C. felis. Bobcats usually experience a brief and mild illness upon infection, followed by a full recovery [7–9]. More recently, Cytauxzoon manul and further Cytauxzoon spp. infecting felids in Eurasia were discovered [10].

Hepatozoonosis is a parasitic disease caused by members of the genus Hepatozoon (Apicomplexa: Conoidasida: Coccidia: Eucoccidiorida: Adeleorina: Hepatozoidae). These protozoa infect various mammals, birds, reptiles, and amphibians [11]. Among felids, Hepatozoon felis and other species can cause anorexia, pale mucous membranes, weight loss, pain, diarrhea, vomiting, gait abnormalities, fever, polyuria, polydipsia and even death in severe cases [12–14]. The life cycle of Hepatozoon spp. involves a vertebrate host, which gets infected by ingesting arthropod vectors (e.g., ticks) [13, 15–16]. The infection can also occur by predation [17–18].

In this study, Cytauxzoon and Hepatozoon spp. were molecularly screened from Eurasian lynx (Lynx lynx) and their ixodid ticks in northwestern China.

Sample collection

Three Eurasian lynxes were involved in this study. Two of them, an adult female (#1, 4–5 years old) and an adult male (#2, 3–4 years old), were found dead due to natural causes during our field investigation in China-Kazakhstan border, the West Junggar Mountain in 2018 and 2019, respectively. The third one, a road-killed male cub (#3, 4–6 months old), was also collected in this region in 2019 as already reported in Liu et al., 2021 [19]. The sex of the lynx was determined, and their ages were estimated on the basis of body mass, date of collection and tooth wear [20]. Five ticks were collected from male cub the latter and molecularly identified as Hyalomma asiaticum [21].

DNA extraction

Genomic DNA was individually extracted from the heart, liver, spleen, lung and kidney samples of three lynxes. The sampled ticks were carefully surface-sterilized, prior to processing, the exterior of all ticks was disinfected using 3% sodium hypochlorite for 1min, 70% ethanol for 1min and phosphate-buffered saline (PBS) for 1min. DNA was extracted from whole ticks, and was extracted using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China), with an overnight following the manufacturer’s instructions. DNA extracts were eluted in 60 µL of Tris-EDTA buffer and stored at − 80°C under sterile conditions to prevent contamination until polymerase chain reaction (PCR) analysis.

Polymerase chain reaction amplification

DNA extracts (all from lynxes and five from ticks) were individually screened for the presence of Cytauxzoon and Hepatozoon spp. with PCR and sequencing. For genotyping Hepatozoon spp, 325-, 620-, and 1700-bp-long fragments of the small subunit 18S ribosomal RNA gene (18S rRNA) were chosen [22]. PCR was also performed using primer set of Cytauxzoon targeting the partial 18S rRNA gene fragment (900bp), 1150 bp fragment of the CytB gene and 1320 bp fragment of the COI gene [23]. The primers and PCR cycling conditions are shown in Additional file 1. A negative control (distilled water) was included in each run to validate primer-specific amplification. The PCR products were subjected to electrophoresis in 1.5% agarose gel and visualized under UV light by staining the gel with Goldview (Biotopped, Beijing, China). All PCR products were purified using the TIANgel Midi Purification Kit (TIANGEN, Beijing, China) and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) using the same primers.

Sequencing and data analyses

Sequencing data were subjected to Basic Local Alignment Search Tool (BLAST) searches (http://www.ncbi.nlm.nih.gov/blast/) and then aligned and analyzed with reference sequences downloaded from GenBank. Phylogenetic trees were constructed based on the sequence distance method using the maximum likelihood algorithms implemented in the Molecular Evolutionary Genetics Analysis MEGA 7.0 software [24]. All sequences from this study were deposited in the GenBank (http://www.ncbi.nlm.nih.gov) database (C. manul 18S rRNA: PP033938; C. manul CytB: PP442054; C. manul COI: PP503316; H. felis 18S rRNA: PP033238, PP528680-PP528683, OR497518 and OR497519).

The results of molecular analysis and sequencing indicated that i) Eurasian lynx #1 was found to be coinfected with Cytauxzoon manul and Hepatozoon felis; ii) Eurasian lynx #2 and #3 was infected with a H. felis genotype I that was significantly same from that in Hyalomma asiaticum infesting Eurasian lynx cub #3. Phylogenetic trees and BLAST analyses showed that C. manul 18S rRNA gene sequences from this study were grouped with those from Pallas’ cat (Otocolobus manul) in Mongolia (shown in Additional file 2: Fig. S1), and shared 99.89% (871/872) identities with sequences available in GenBank (AY485690 and AY485691). To assess the genetic variability of Cytauxzoon spp., sequence analyses of mitochondrial COI gene were performed. The results of our phylogenetic analyses indicated a sister group relationship among C. manul and Cytauxzoon spp. from felines (Fig. 1), which was similar to the result based on CytB gene (shown in Additional file 3: Fig. S2). The phylogenetic tree of Hepatozoon spp. based on the partial 18S rRNA gene fragment showed that this genotype was clustered into the clade of “genogroup I”, separately from other isolates of which corresponding sequences available in GenBank (Fig. 2), and shared 99.94% (1684/1685 bp) identities with those from Asiatic lion in India (ON075470).

This study provides the first evidence for the occurrence of C. manul and H. felis in Eurasian lynx, and this is the first time that infection with H. felis genotype I has been reported in Eurasian lynx and Hyalomma asiaticum ticks in China. To the best of our knowledge, this is also the first report of C. manul and H. felis co-infection in Felid.

Cytauxzoonosis, caused by C. felis, C. manul, and three recently described new Cytauxzoon European species, is an emerging infectious disease that affects wild felids as well as the domestic cat [25–27]. Cytauxzoon manul is endemic in free-ranging Pallas’s cats (Otocolobus manul) in Mongolia [28], and it was also reported from lions (Panthera leo) in Zimbabwe [29]. More recently, Cytauxzoon spp. different from C. manul were detected in Romania in four Eurasian lynxes (Lynx lynx) and 12 wild cats (Felis silvestris) [10]. This study reports the first detection of C. manul in Eurasian lynx. There are currently very few studies on C. manul, in the future, more research is needed to prove the C. manul species.

Three Hepatozoon species are known to infect felines, including Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris [30–32]. Hepatozoon felis was previously detected in wild cat in the Republic of Cape Verde, in jaguar (Panthera onca) and in jaguarundi (Puma yagouaroundi) in Brazil, in leopard cat (Prionailurus bengalensis) in Korea, in Eurasian lynx (Lynx lynx) in Turkey, as well as in Asiatic lion (Panthera leopersica), Indian tiger (Panthera tigris tigris) and Indian leopard (Panthera pardus fusca) in India [33–38]. H. felis was reported in Haemaphysalis longicornis (Acari: Ixodidae) ticks from free-ranging domestic sheep in Hebei Province, China [39]. In the present study, H. felis genotype I was detected both in a lynx cub and its infecting Hyalomma asiaticum ticks.

In this study, although H. felis was detected in ticks, it is still impossible determine whether these ticks are truly infected with H. felis. Given that these ticks were collected from the Eurasian lynx, so they were engorged by lynx blood was possible. Therefore, the question of whether ticks are infected with H. felis still needs further research and confirmation.

Protozoan co-infections are relatively frequent in carnivores [40–45]. Although this phenomenon, as also observed in this study, is seldom reported in lynxes, Babesia sp. and H. felis were detected simultaneously in Eurasian lynx in Turkey [46]. Eurasian lynx is included in the Red List of Threatened Species by IUCN and is also listed on the second level of National Key Protected Wildlife in China [47]. To better understand the impact of these parasites on the health and conservation status of the Eurasian lynx, future studies should identify its complete pathogen profile by metagenomic next-generation sequencing.

In this study, C. manul and H. felis genotype I were molecularly identified in Eurasian lynx. These two haemoprotozoan parasites caused co-infection in a lynx. Hepatozoon felis was detected both in a lynx cub and its Hyalomma asiaticum ticks. These finding extends our knowledge on the geographical distribution and host range of C. manul and H. felis.

18S rRNA: 18S Ribosomal RNA

COI: Cytochrome c oxidase subunit I

CytB: Cytochrome B

Acknowledgements

The author would like to thank all the veterinarians who participated in the study as well as all the colleagues who contributed to sample collection and sample preparation.

Funding

This work was supported in part by the Natural Science Foundation of China (82260399 and 82260414), Natural Science Key Project of Xinjiang Uygur Autonomous Region (2022B03014), Key Scientific and Technological Projects in Key Areas of XPCC (2022AB014).

Availability of data and materials

The sequences obtained and analyzed during the present study are deposited in the GenBank database under the accession numbers (C. manul 18S rRNA: PP033938; C. manul CytB: PP442054; C. manul COI: PP503316; H. felis 18S rRNA: PP033238, PP528680-PP528683, OR497518 and OR497519).

Authors’ contributions

NC, LS, ZW, HS and YW conceived and designed the study, and wrote the manuscript. NC, YZ, GZ, LT and MY performed the experiments and analyzed the data. NC, HS and YW contributed to study design and edited the manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

This study was reviewed and approved by the ethics committee of School of Medicine, Shihezi University in accordance with the medical regulations of China (Approval numbers 2015-063-01 and A2018-144-01).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

Additionalfile1.pdf
Additional file 1: Table S1. Characteristics of PCRs used in this study: target genes, primer sequences and cycling conditions.
Additionalfile2.pdf
Additional file 2: Fig. S1. Phylogenetic tree based on 18S rRNA gene sequences of Cytauxzoon manul (▲) from Eurasian lynx, constructed with the maximum likelihood method and using the Tamura 3-parameter substitution model with discrete Gamma distributed with Invariant sites (bootstrap replicates: 1000). The GenBank accession number, strain name, host, and area of origin were listed. Plasmodium falciparumwas used as an outgroup.
Additionalfile3.pdf
Additional file 3: Fig. S2. Phylogenetic tree based on CytB gene sequences of Cytauxzoon manul(▲) from Eurasian lynx, constructed with the maximum likelihood method and using the Hasegawa-Kishino-Yano model with discrete Gamma distributed with Invariant sites (bootstrap replicates: 1000). The GenBank accession number, strain name, host, and area of origin were listed. Plasmodium falciparum was used as an outgroup.
Graphicalabstracts.tiff

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You are reading this latest preprint version

Molecular investigation of ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from Eurasian lynx (Lynx lynx) in northwestern China: first identification of Cytauxzoon manul in this host species

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Abstract

Figures

Background

Methods

Sample collection

DNA extraction

Polymerase chain reaction amplification

Sequencing and data analyses

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1