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Description and molecular characterisation of Babesia ailuropodae n. sp., a new piroplasmid species infecting giant pandas

Parasites & Vectors volume 17, Article number: 315 (2024) Cite this article

Abstract

Background

Babesia spp. are protozoan parasites that infect the red blood cells of domesticated animals, wildlife and humans. A few cases of giant pandas (a flagship species in terms of wildlife conservation) infected with a putative novel Babesia sp. have been reported. However, comprehensive research on the morphological and molecular taxonomic classification of this novel Babesia sp. is still lacking. This study was designed to close this gap and formally describe this new Babesia sp. infecting giant pandas.

Methods

Detailed morphological, molecular and phylogenetic analyses were conducted to characterise this Babesia sp. and to assess its systematic relationships with other Babesia spp. Blood samples from giant pandas infected with Babesia were subjected to microscopic examination. The 18S ribosomal RNA (18S rRNA), cytochrome b (cytb) and mitochondrial genome (mitogenome) of the new Babesia sp. were amplified, sequenced and assembled using DNA purified from blood samples taken from infected giant pandas. Based on the newly generated 18S rRNA, cytb and mitogenome sequences, phylogenetic trees were constructed.

Results

Morphologically, the Babesia sp. from giant pandas exhibited various forms, including round to oval ring-shaped morphologies, resembling those found in other small canine Babesia spp. and displaying typical tetrads. Phylogenetic analyses with the 18S rRNA, cytb and mitogenome sequences revealed that the new Babesia sp. forms a monophyletic group, with a close phylogenetic relationship with the Babesia spp. that infect bears (Ursidae), raccoons (Procyonidae) and canids (Canidae). Notably, the mitogenome structure consisted of six ribosomal large subunit-coding genes (LSU1-6) and three protein-coding genes (cytb, cox3 and cox1) arranged linearly.

Conclusions

Based on coupled morphological and genetic analyses, we describe a novel species of the genus Babesia, namely, Babesia ailuropodae n. sp., which infects giant pandas.

Graphical Abstract

Background

A flagship species in terms of wildlife protection, giant pandas (Ailuropoda melanoleuca) are primarily distributed across the Daxiangling, Xiaoxiangling, Qinling, Qionglai and Minshan mountain ranges in the Sichuan, Shaanxi and Gansu provinces of China. Habitat reduction, low reproductive rates and outbreaks of various diseases continue to threaten the survival of giant pandas [1, 2]. Among the threats to giant pandas, diseases caused by parasites are considered one of the significant factors affecting the health of giant pandas [3, 4].

The genus Babesia comprises protozoan parasites that are transmitted by ticks; these parasites parasitise red blood cells in livestock, wild animals and humans [5, 6]. There are more than 120 Babesia species globally; these species can infect various hosts and induce babesiosis. Haemolysis, fever, jaundice, anaemia and haemoglobinuria are typical clinical signs of babesiosis [7]. Babesiosis primarily affects mammals, including, but not limited to, members of the Canidae, Felidae, Ursidae, Procyonidae, Bovidae, Equidae and Cervidae families. The most common agents of babesiosis include Babesia canis, Babesia rossi, Babesia vogeli, Babesia conradae, Babesia gibsoni, Babesia vulpes, Babesia felis, Babesia cati, Babesia lengau, Babesia leo, Babesia microti, Babesia bovis, Babesia bigemina, Babesia caballi and a series of newly identified Babesia spp. [8,9,10,11,12,13,14,15]. Notably, giant pandas, classified as carnivores within the Ursidae family [16], have exhibited cases of Babesia infection, especially among those that have been released into the wild in recent years. These cases have exhibited symptoms, including anaemia, jaundice and haemoglobinuria. Additionally, two cases of naturally occurring Babesia infection in wild giant pandas have been documented [7]. Blood protozoa testing was subsequently conducted on all captive giant pandas at the giant panda base in Sichuan Province, as well as on wild giant pandas rescued from the wilderness. The results showed that 14 giant pandas tested positive for Babesia, with two wild giant pandas exhibiting typical clinical symptoms of babesiosis, and up to ten parasites were detected within a single erythrocyte, while the remaining positive giant pandas showed no clinical symptoms.

Considering the scarcity of previous research, this study explores the morphological features, phylogenetic relationships and taxonomic status of Babesia in giant pandas. This study aims to gain in-depth insights into the characterisation of this parasite within the Babesia genus and to offer guidance for future disease prevention and control efforts related to babesiosis.

Methods

Samples

This study investigated a total of 14 Babesia-positive giant pandas—eight from wild giant pandas across China (three from the Qionglai mountain range, three from the Xiaoxiangling mountain range, one from the Liangshan mountain range and one from the Minshan mountain range) and six from captive giant pandas in Sichuan Province (Fig. 1).

Fig. 1
figure 1

Map showing the distribution of Babesia-positive samples collected from giant pandas. XXL Xiaoxiangling, DXL Daxiangling, LS Liangshan, QLS Qionglaishan, MS Minshan

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Morphological characterisation of the parasites

Blood smears were made using fresh whole blood collected from giant pandas and were subsequently stained with a Diff-Quick stain kit (Diff-Quick Stain Kit, Solarbio, Beijing, China). The stained blood smears were observed, and photographs were taken using a Zeiss Axio Imager M2 optical microscope (Carl Zeiss, Oberkochen, Germany) with an oil immersion magnification of 1000×; these images were processed using the Zeiss ZEN 2.3 lite software package (Carl Zeiss, Oberkochen, Germany).

Cloning and sequencing analysis of the mitogenome and 18S rRNA

Following the instructions provided in the DNeasy® Blood and Tissue Kit manual (QIAGEN, Hilden, Germany), DNA extraction was carried out on 200 µl of EDTA-anticoagulated whole blood samples from giant pandas. A NanoDrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) was used to measure the DNA concentrations after the extracted DNA samples were eluted using 200 µl of elution buffer. The isolated DNA was then preserved at –80 °C for use in subsequent molecular biology investigations. Both the 18S ribosomal RNA (18S rRNA) and the mitogenome were amplified using primers designed for the genus Babesia (Table 1).

Table 1 Sequences of the PCR primers used to amplify the 18S rRNA and mitogenome from giant panda DNA samples
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The polymerase chain reaction (PCR) amplification conditions were as follows: initial denaturation at 98 °C for 3 min, 20 s of denaturation at 98 °C, 20 s of annealing at 50–60 °C (depending on the primers used), 10 s of extension at 72 °C, 38 cycles of repetition and a final 5–8 min extension at 72 °C (depending on amplicon size, 1 min/kb). The amplification of the 18S rRNA was accomplished using the primers 18SF/R and BP18SF/R as previously described [7]. The cytb gene and the mitogenome were amplified using the primers Bab-Forc1/Rev1, F5F/R, MTF1-F/R, MTF2-F/R, MTF3-F/R, MCF1/R1, MGF1/R1, cox1-F/R, cox3-F/R and cytb-F/R as previously described [17,18,19,20].

The PCR amplicons were cloned into the pMD-19T vector (Takara, China) and transformed into Escherichia coli DH5α cells. Plasmid DNA was isolated and sent for sequencing by Sangon Biotech (Shanghai, China). Thereafter, the sequencing results were confirmed through sequence alignment using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/BLAST).

Gene annotation and sequence analysis

Referring to previously published data on B. canis (GenBank KC207822), B. rossi (KC207823), B. vogeli (KC207825) and B. gibsoni (KP666169), the mitogenome sequences obtained from the giant panda samples examined in this study were assembled and annotated using the online websites GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq) and Artemis (https://www.sanger.ac.uk/resources/software/artemis/). Previous annotations of B. canis, B. rossi, B. vogeli and B. gibsoni were used to infer the protein-coding genes of the new Babesia sp. Sequence data alignment and analyses were conducted using the online websites ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) and MAFFT 7.0 (https://mafft.cbrc.jp/alignment/server/). The rRNA genes were further confirmed by referencing the rRNA sequences of B. gibsoni (KP666169), B. rossi (KC207823) and B. canis (KC207822). Moreover, the prediction of transfer RNA (tRNA) genes was performed using tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/index.html).

Phylogenetic analysis

Molecular Evolutionary Genetics Analysis (MEGA 5.0) was used to compare the amplified sequences of 18S rRNA, cytochrome b (cytb) and the complete mitogenome. The amplified sequences were aligned with those of other similar species listed in GenBank to construct a phylogenetic tree. During the sequence alignment stage, the ClustalW tool was used. The construction of the phylogenetic tree involved various analysis methods, including the maximum likelihood (ML) and neighbour-joining (NJ) methods. The ML tree was constructed using the general time reversible model (G + I) and calculated using the Akaike information criterion (AIC). The NJ tree was constructed with the Tamura-Nei model (G + I) according to the AIC [21, 22]. In the process of constructing the phylogenetic tree, 1000 bootstrap replicates were performed to assess the tree’s reliability [23]. DNAStar was utilised for pairwise genetic analysis of the 18S rRNA and cytb datasets, aimed at identifying possible evolutionary differences at the nucleotide level [22]. The first evolutionary tree was constructed based on 61 (almost) full-length sequences of the giant panda Babesia 18S rRNA and sequences of protozoa collected from other related species. Additionally, Toxoplasma gondii 18S rRNA sequences were used as outgroups. For the analysis of the cytb gene, 36 sequences from related protozoan species were used, with the cytb gene sequences of Hepatozoon canis being used as outgroups. Moreover, in constructing the mitogenome tree, 17 related Babesia spp. sequences were used, with Cytauxzoon felis sequences serving as outgroups.

Results

Family Babesiidae Poche, 1913

Genus Babesia Starcovici, 1893

Babesia ailuropodae n. sp.

Type host: giant panda Ailuropoda melanoleuca (Mammalia: Ursidae).

Type locality: the mountains of Minshan (31°04′18′′–33°58′28″N, 103°08′24″–105°35′22″E), Qionglaishan (29°38′24″–31°30′36″N, 102°10′48″–103°32′24″E), Daxiangling (29°22′48″–29°48′00″N, 102°36′00″–103°11′24″E), Xiaoxiangling (28°24′36″–29°20′24″N, 101°51′00″–102°33′00″E) and Liangshan (28°12′00″–29°11′24″N, 102°37′12″–103°45′00″E), Sichuan Province, China.

Other localities: unknown.

Type material: the study samples, including whole blood, DNA (wild positive sample accession number: GYYXL 2301–8; captive positive sample accession number: GYYXL 2309–14) and dyed thin blood smears (additional file 1: Fig. S1, accession number: GYYXL 230501) from Babesia-infected giant pandas containing the holotype (Fig. 2H) were deposited at the Department of Parasitology at Sichuan Agricultural University in Sichuan, China.

Fig. 2
figure 2

Various morphologies of giant panda Babesia merozoites inside erythrocytes, including round-to-oval ring-shaped (A, B), paired pyriform (C, D), irregularly shaped (E) and tetrad-shaped (F) merozoites. Babesia ailuropodae n. sp., type material in blood smears from a giant panda (G, H). The holotype is marked with an arrow in H. Diff-Quick stains (modified Wright’s and quick Romanowsky staining). Scale bar, 10 μm

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Vector: unknown. Haemaphysalis flava Neumann, 1897 is suspected [24, 25].

Representative DNA sequences: GenBank accession numbers PP117080-PP117093 for 18S rRNA, PP215402-PP215412 for cytb and PP236906 for the mitogenome.

ZooBank registration: details of the new Babesia species were submitted to ZooBank in accordance with the guidelines mentioned in Article 8.5 of the 2012 amendment to the International Code of Zoological Nomenclature (ICZN) [26]. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:5BD943D8-989D-4DB2-8373-86F7673E3686. The LSID for the new Babesia ailuropodae n. sp. is urn:lsid:zoobank.org:act:55427CD6-EF60-4981-B679-886FEAC7A5B4.

Etymology: the species name was derived from the genus name of the type host.

Morphological description

In the stained blood smears, round to oval ring-shaped, paired pyriform, irregularly shaped and tetrad-shaped forms were observed (Fig. 2). There were varying numbers of parasites infecting erythrocytes, with individual ring-shaped parasites constituting the majority. Round-to-oval-shaped merozoites exist as single or double parasites within infected erythrocytes. The range of parasites infecting a single erythrocyte was between 1 and 10. The following were the size ranges of the different parasitic forms: round-to-oval ring-shaped merozoites measuring 0.926–2.505 µm (mean ± standard deviation: 1.574 ± 0.439) in length and 0.665–2.29 µm (1.365 ± 0.395) in width (n = 30); paired pyriform structures measuring 1.987–2.686 µm (2.350 ± 0.219) in length and 1.072–1.763 µm (1.399 ± 0.236) in width (n = 11); irregularly shaped structures measuring 1.756–3.917 µm (2.381 ± 0.849) in length and 1.204–3.331 µm (1.982 ± 0.705) in width (n = 8); and tetrads measuring 2.815–4.074 µm (3.519 ± 0.467) in length and 2.022–3.198 µm (2.605 ± 0.358) in width (n = 12). Moreover, the average blood parasitemia in the erythrocytes of the affected animals was 44.60 ± 5.36% (positive cells, 70.143 ± 8.030; total cells, 160.429 ± 14.932).

Sequence analysis of the 18S rRNA and cytb

We successfully cloned nearly complete (1679 bp) 18S rRNA sequences from all 14 positive giant panda blood samples. Compared with the previously reported sequence of the Babesia sp. EBP strain in giant pandas (MT256300), the new Babesia ailuropodae n. sp. cloned sequence exhibited an additional 75 base pairs while maintaining a high similarity (99.75%). Furthermore, the 18S rRNA consensus sequence (PP117082) of Babesia ailuropodae n. sp. showed significant similarities with those of Japanese black bears (99.51%, AB586027) and brown bears (99.49%, AB566229). Simultaneously, the similarity to Babesia spp. from canids (AY190123, KR017880) and raccoons (OK524313) exceeded 99%. Compared with B. felis, which infects felids (AY452707) in other regions, the similarity reached only 86.52% (Table 2).

Table 2 Comparison of the identities between the complete 18S rRNA sequence (1679 bp) of the giant panda Babesia and other published Babesia sequences
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Eleven cytb gene sequences (1092 bp) from positive panda blood samples were successfully cloned. BLASTn analysis demonstrated that the cytb gene consensus sequence (PP215403) from Babesia ailuropodae n. sp. shared 82.6% similarity with that of B. vogeli (KC207825). Its similarity with the cytb gene sequences of B. bigemina (AB499085), B. bovis (EU075182), B. ovate (LC146481) and B. naoakii (LC684769) was less than 80% (Table 3).

Table 3 Comparison of the identities between the complete cytb sequence (1092 bp) of the giant panda Babesia and other published Babesia sequences
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We found that Babesia gene sequences from giant pandas collected from different geographical sources exhibited relatively conserved intraspecific genetic relationships, with 14 high-quality 18S rRNA sequences showing 99.4–100% similarity and nine high-quality cytb sequences displaying 97.4–100% nucleotide identity.

Phylogenetic analysis

Phylogenetic analysis of Babesia, encompassing 61 sequences, revealed four distinct clades: Babesia (senso stricto), Babesia (sensu lato), Theileriidae, and Hepatozoidae. The 18S rRNA sequences from the 14 positive giant panda blood samples in this study clustered with a published Babesia sequence from giant pandas (MT256300) within the Babesia (s.s.) clade. Specifically, the giant panda Babesia 18S rRNA sequence showed highest similarity to those of Japanese black bears (AB586027) and brown bears (AB566229), with decreasing similarity to that of the Babesia sequences from Japanese domestic dogs (AY190123), Chinese red pandas (OK524313) and Japanese wild raccoons (AB251608). These sequences also exhibited similarity to Babesia strains infecting other canids and procyonids. The remaining branches included the Babesia (s.l.) clade (three sequences), Theileriidae clade (four sequences) and Hepatozoidae clade (four sequences), positioned further apart from the giant panda Babesia sequences, which included species such as B. leo (AF244911), B. felis (Y452707), T. equi (KY111761), T. parva (HQ895985), H. canis (AY150067), and H. felis (KX017290) (Fig. 3).

Fig. 3
figure 3

Phylogenetic analysis of the nearly complete giant panda Babesia 18S rRNA sequences was conducted using the maximum likelihood method. The 18S rRNA sequence of T. gondii served as the outgroup. With a sequence length of 1679 bp, the general time reversible (G + I) model was used to construct the ML tree. The analysis did not include gaps or missing data. For each sequence, information on the host, the country of origin and the GenBank accession number are provided. Only bootstraps > 50% are shown, and the bootstrap values are based on 1000 repetitions

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Furthermore, the cytb gene sequences of 11 positive giant panda blood samples were aligned with 25 protozoan cytb gene sequences obtained from the GenBank database to construct an evolutionary tree, with the H. canis cytb sequence serving as the outgroup. The evolutionary tree revealed three main clades; one clade represented the Babesia (s.s.) group, where all 11 cytb sequences of the giant panda Babesia formed a distinct clade and were situated within this group. They shared a close phylogenetic relationship with other Babesia cytb sequences, primarily including B. sp. pudui, B. sp. Dunhuang, B. sp. Coco, B. rossi, B. gibsoni, B. canis and some Babesia subspecies. Additionally, the other two main clades included Babesia (s.l.) and Theileriidae, which were relatively distantly related to the cytb clade of the giant panda Babesia and were situated closer to the outgroup (Fig. 4).

Fig. 4
figure 4

Phylogenetic analysis of the giant panda Babesia cytb gene sequences was conducted using the maximum likelihood method. The cytb sequence of H. canis served as the outgroup. With a sequence length of 1092 bp, the general time reversible (G + I) model was used to construct the ML tree. The analysis did not include gaps or missing data. For each sequence, information on the host, the country of origin and the GenBank accession number are provided. Only bootstraps > 50% are shown, and the bootstrap values are based on 1000 repetitions

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The mitogenome tree showed two primary clades: one representing the Babesia (s.s.) group, which was distinctively separate from the outgroup C. felis. Within Babesia (s.s.), the mitogenome sequence of the giant panda Babesia was closely related to that of B. gibsoni, B. rossi and B. canis. The remaining clades belonged to the Babesia (s.l.) group, positioned nearer to the outgroup C. felis, and primarily included B. conradae, B. microti, B. rodhaini and B. duncani (Fig. 5).

Fig. 5
figure 5

Phylogenetic analysis of the giant panda Babesia mitogenome was conducted using the maximum likelihood method. The sequence of C. felis served as the outgroup. With a sequence length of 5609 bp, the TN93 + G model was used to construct the NJ tree. The analysis did not include gaps or missing data. Only bootstraps > 50% are shown, and the bootstrap values are based on 1000 repetitions

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Mitogenome

The giant panda Babesia mitogenome, designated with GenBank accession number PP236906, spans 5608 bp and includes six ribosomal large subunit genes (LSU) and three protein-coding genes (cox1, cox3 and cytb), with no amplification of TIR at either end. The sizes of the cox1, cox3 and cytb genes were 1431 bp, 642 bp and 1092 bp, respectively. The sizes of LSU1, LSU2, LSU3, LSU4, LSU5 and LSU6 were 321 bp, 35 bp, 111 bp, 48 bp, 72 bp and 43 bp, respectively. Unlike other Apicomplexan parasites, it lacks tRNA and exhibits a linear structure typical of Babesia mitogenomes, resembling those of B. gibsoni (AB499087), B. bovis (AB499088), B. bigemina (AB499085), B. caballi (AB499086) and B. orientalis (KF218819) [27,28,29,30]. The mitogenome structure and gene arrangement are consistent with this lineage, which typically does not exceed 6000 bp (Fig. 6).

Fig. 6
figure 6

Comparison results of the mitogenome map of the giant panda Babesia and other published Babesia linear mitogenome maps, including B. gibsoni (AB499087), B. bovis (AB499088), B. bigemina (AB499085), B. caballi (AB499086) and B. orientalis (KF218819). White boxes represent protein-encoding genes (cox1, cox3 and cytb). The grey boxes represent large subunits (LSU1-6), and the arrows represent terminal inverted repeats (TIRs)

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Discussion

To date, Babesia infections have been detected in animals from the Canidae, Felidae, Ursidae, and Procyonidae families worldwide, all of which are closely related to giant pandas. Among canids [31,32,33,34,35,36,37,38,39,40,41,42], including domestic dogs, wild dogs, foxes, raccoon dogs, black-backed jackals, grey wolves, coyotes and maned wolves, as well as felids such as domestic cats, wild cats, bobcats, caracals, ocelots, cheetahs, leopards, cougars, jaguars and lions, Babesia infections have been identified [43,44,45,46,47,48]. Additionally, Babesia infections have been diagnosed in Ursidae animals, including black bears in Japan and the USA, brown bears in Japan, polar bears and sun bears [49,50,51,52,53,54,55]. Moreover, reports of Babesia infection in Procyonidae animals have also been increasing steadily [56,57,58,59,60]. Recently, confirmed cases of Babesia infection have been detected in wild giant pandas undergoing training [7]. This study systematically classifies the Babesia species found in giant pandas, identifying it as a new taxonomic unit meeting the criteria for a new species according to the ICZN guidelines [26]. According to ICZN standards, naming a newly discovered parasite involves several steps: providing a valid species description encompassing the morphological, biological and potential taxonomic characteristics; designating a type specimen; and assigning a Latinised name that conforms to international naming conventions to ensure uniqueness. This study meticulously described the shape, size, vector, type locality, type host, type material, ZooBank registration, designation and taxonomic status of Babesia in giant pandas. Consequently, the study named this species Babesia ailuropodae n. sp. The species name reflects the giant panda (Ailuropoda melanoleuca) as the primary natural host of this Apicomplexan parasite [7].

The morphological analysis in this study reveals that Babesia in giant pandas exhibits pleomorphism within erythrocytes, typically appearing as individual ring-shaped parasites. This parasite predominantly parasitises the periphery of erythrocytes and shares parasitic characteristics similar to small Babesia species found in canids and felids [33, 61,62,63]. Individual parasites of Babesia in giant pandas exhibit a size range of 1.365 µm × 1.574 µm in their various morphological forms. Additionally, B. ailuropodae n. sp. also appears in irregular forms, differing somewhat from Babesia species found in canids and felids. Furthermore, they share similarities in tetrad morphology with B. canis, B. microti, B. duncani, B. conradae, B. negevi and B. panickeri [64,65,66]. Compared with the larger Babesia species reported in canids, such as B. canis (2 µm × 5 μm), B. vogeli (2.5 µm × 4.5 μm) and B. rossi (2 µm × 5 μm), B. ailuropodae n. sp. (1.365 ± 0.395 µm × 1.574 ± 0.439 μm) is relatively smaller. In contrast, compared with smaller Babesia species in felids such as B. lengau (0.6 µm × 2.3 µm) and B. leo (0.62 µm × 1.73 µm), B. ailuropodae n. sp. tends to be larger [46, 67]. The size of B. ailuropodae n. sp. overlaps significantly with that of B. gibsoni (1 µm × 3 μm), placing it in the category of medium-sized parasites [65, 68]. These distinct morphological characteristics indicate that the Babesia species found in giant pandas is unique. This finding presents an exciting prospect, as research on Babesia in Ursidae and Procyonidae has thus far been limited to epidemiological investigations, with detailed studies on morphology and systematic classification yet to be conducted.

Although a single-gene evolutionary tree may not fully capture the parasite’s position in species evolution, combining multiple gene features with potentially diverse evolutionary histories can construct a more accurate phylogenetic tree that reflects the true relationships among related species [69]. This study conducted a genetic analysis of the 18S rRNA and cytb genes in the giant panda Babesia to elucidate the evolutionary relationships of the newly identified species B. ailuropodae n. sp. The results indicated that Babesia isolates from giant pandas form a distinct evolutionary branch in different phylogenetic trees (Figs. 3 and 4), clearly distinguishing them from other Babesia species. This finding supports the idea that B. ailuropodae n. sp. could be unique to giant pandas because of its distinctive molecular evolutionary history.

In this study, four branches – Babesia (s.s.), Babesia (s.l.), Theileria and Hepatozoon – were visible in the phylogenetic tree built using the 18S rRNA gene sequence. Within Babesia (s.s.), B. ailuropodae n. sp. formed a sister clade along with the Babesia gathered from black bears and brown bears in Japan. It shares a high degree of similarity with the Babesia collected from domestic dogs in Japan and from the red panda in China [51, 70]. A geographical analysis revealed that the distributions of black bears, brown bears and domestic dogs in Japan do not overlap with those of giant pandas in China, which largely impedes inter-species disease transmission. However, this study revealed significant genetic similarities in the Babesia strains infecting these animals. The strong evolutionary kinship among giant pandas, black bears and brown bears, which are all members of the order Carnivora, likely explains this phenomenon. Despite their geographical separation, their genetic similarity suggests the potential for disease transmission among these species [71]. H. flava, primarily found in East Asia, including China, Japan and Korea, is suspected to be a vector for giant pandas and has been detected in bear species in Japan, indicating its potential role in disease transmission [24, 25]. Therefore, we speculate that H. flava may serve as a common vector for Babesia infection. In this study, within the Babesia (s.s.) group, B. ailuropodae n. sp. and Babesia from the red panda were closely related, showing 99.23% sequence similarity in their 18S rRNA (OK524313), despite significant differences in their hosts’ evolutionary lineages. Notably, the red panda, a member of the Ailuridae family within the superfamily Musteloidea, is evolutionarily distinct from the giant panda, which shares closer kinship with members of the bear family (Ursidae) [16, 72, 73]. Despite their differences, giant pandas and red pandas have both inhabited the same geographical region and have undergone convergent evolution over time, thereby adapting similarly to environmental pressures and acquiring morphological and physiological similarities [74]. Instances of Babesia infection in Procyonidae animals are well documented worldwide, and the similarity in genetic sequences of the Babesia derived from these animals corresponds to the phylogenetic relationships among their hosts [57,58,59, 75, 76]. Simultaneously, the 18S rRNA sequences of Babesia in Japanese domestic dogs and those in giant pandas showed high similarity in this study, possibly due to ticks incidentally infecting Japanese domestic dogs, which may have originated from Japanese wild raccoons. This hypothesis was supported by the evolutionary tree analysis comparing Babesia sequences from Japanese domestic dogs with the 18S rRNA sequences from raccoons in Japan and the United States of America [14, 59, 70]. These findings further support that Babesia infections in domestic dogs across various regions – Japan (AB118032, AF271082), China (HG328235, KP666168), Brazil (AY371196), India (L19079), Nigeria (JN982353), the USA (EU109717), and Europe (AY072926) – exhibit distinct evolutionary branches compared with B. ailuropodae n. sp. within Babesia (s.s.). Therefore, based on the 18S rRNA sequence classification analysis, the giant panda Babesia demonstrated a close phylogenetic relationship with Babesia species found in the bear (Ursidae), panda (Ailuridae), raccoon (Procyonidae) and dog (Canidae) families.

The cytb sequences of B. ailuropodae n. sp. differed by 1 to 53 base pairs from those of other Babesia species. Alignments of 82.6% with canine Babesia species and 77.92% with B. naoakii were obtained. This variability indicates its genetic independence in evolutionary development. Overall, the cytb gene sequences of 11 B. ailuropodae n. sp. formed a distinct branch, mirroring the branching pattern observed in the 18S rRNA analysis. This underscores the unique position of the giant panda Babesia within Babesia (s.s.). The cytb sequence of B. ailuropodae n. sp. showed close phylogenetic relationships with those of B. sp. Pudui (ON995403), B. sp. Dunhuang (MK962314), B. sp. Coco (KC207824), B. caballi (AB499086), B. gibsoni (OM933649) and B. rossi (KC207823). This genetic evolutionary analysis was consistent with the morphological characteristics of this species, supporting the classification of the giant panda Babesia as an independent taxonomic unit. Additionally, B. conradae (KC207826) and B. duncani (NC039721) within Babesia (s.l.) were found to be more distantly related to the outgroup, indicating a less close phylogenetic relationship with B. ailuropodae n. sp. This divergence may be attributed to environmental selection pressures between the parasites and their hosts, as well as inherent evolutionary differences among various parasitic species [62].

To date, the mitogenomes of most piroplasmids, including B. canis, B. vogeli, B. rossi, B. gibsoni, B. bovis, B. orientalis, B. caballi, B. bigemina, B. conradae, B. microti, B. rodhaini, B. duncani, T. equi, T. parva, T. orientalis, H. canis and C. felis,, have been extensively studied and analysed [17, 18, 29, 77,78,79]. The study of mitogenomes has contributed significantly to the provision of crucial information about the biological characteristics, inheritance and species categorisation of pathogens [27]. In this study, the mitogenome of the giant panda Babesia was amplified, sequenced, assembled and then compared with those of other piroplasms from the GenBank database. The findings revealed that the mitogenome of the giant panda Babesia shares substantial similarity in size, structure and content with those of other Babesia species. It contains three protein-coding genes (cox1, cox3 and cytb) and six large subunit rRNA genes (LSU1-6), which are arranged linearly. However, attempts to amplify the two TIRs of the mitogenome using various methods, including high-throughput deep sequencing, inverted PCR, designing specific primers and optimising the PCR conditions, were unsuccessful in this study. This failure may be due to the extensive diversity and variability of TIRs among Babesia species within the phylum Apicomplexa, compounded by potential host-specific factors influencing the availability of TIRs in the mitogenomes of the studied Babesia species [17, 30, 80]. The genetic analysis in this study revealed that the mitogenome of the giant panda Babesia is closely related to those of B. gibsoni, B. rossi, B. vogeli and B. canis, which form a sister branch. In contrast, it exhibits a more distant branching relationship with B. duncani, B. rodhaini, B. microti and B. conradae.

Conclusions

Based on morphological and molecular analyses (including mitogenome), we formally described a new species of piroplasmid infecting giant pandas in China, namely, Babesia ailuropodae n. sp. Further research is required to confirm the full host range and geographical distribution of the Babesia ailuropodae n. sp. vector.

Availability of data and materials

The molecular data have been deposited in GenBank under the following accession numbers: 18S rRNA, PP117080-PP117093; cytb, PP215402-PP215412; and mitogenome, PP236906.

Abbreviations

B. ailuropodae n. sp.:

Babesia ailuropodae n. sp.

T. gondii :

Toxoplasma gondii

H. canis :

Hepatozoon canis

C. felis :

Cytauxzoon felis

18S rRNA:

18 Svedberg ribosomal RNA

cox :

Cytochrome c oxidase

cytb :

Cytochrome b

Mitochondrial genome:

Mitogenome

s.s.:

Sensu stricto

s.l.:

Sensu lato

PCR:

Polymerase chain reaction

LSU:

Large subunit

ICZN:

International code of zoological nomenclature

References

  1. Li J, Karim MR, Li J, Zhang L, Zhang L. Review on parasites of wild and captive giant pandas (Ailuropoda melanoleuca): diversity, disease and conservation impact. Int J Parasit Parasites Wildl. 2020;13:38–45.

    Article  Google Scholar 

  2. Wang T, Xie Y, Zheng Y, Wang C, Li D, Koehler AV, et al. Parasites of the giant panda: a risk factor in the conservation of a species. Adv Parasit. 2018;99:1–33.

    Article  Google Scholar 

  3. Ma H, Wang Z, Wang C, Li C, Wei F, Liu Q. Fatal Toxoplasma gondii infection in the giant panda. Parasite. 2015;22:30.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Qin Z, Liu S, Bai M, Geng Y, Miller DL, Zhao R, et al. First report of fatal baylisascariasis-induced acute pancreatitis in a giant panda. Parasit Int. 2021;84:102380.

    Article  Google Scholar 

  5. Jalovecka M, Sojka D, Ascencio M, Schnittger L. Babesia life cycle—when phylogeny meets biology. Trends Parasit. 2019;35:356–68.

    Article  Google Scholar 

  6. Wilhelmsson P, Pawełczyk O, Jaenson TGT, Waldenström J, Olsen B, Forsberg P, et al. Three Babesia species in Ixodes ricinus ticks from migratory birds in Sweden. Parasit Vectors. 2021;14:183.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Yue C, Deng Z, Qi D, Li Y, Bi W, Ma R, et al. First detection and molecular identification of Babesia sp. from the giant panda, Ailuropoda melanoleuca, in China. Parasit Vectors. 2020;13:537.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Penzhorn BL. Don’t let sleeping dogs lie: unravelling the identity and taxonomy of Babesia canis, Babesia rossi and Babesia vogeli. Parasit Vectors. 2020;13:184.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Zygner W, Gójska-Zygner O, Bartosik J, Górski P, Karabowicz J, Kotomski G, et al. Canine babesiosis caused by large Babesia species: global prevalence and risk factors-a review. Animals. 2023;13:2612.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Panti-May JA, Rodríguez-Vivas RI. Canine babesiosis: a literature review of prevalence, distribution, and diagnosis in Latin America and the Caribbean. Vet Parasit Reg Stud Rep. 2020;21:100417.

    Google Scholar 

  11. Kelly P, Marabini L, Dutlow K, Zhang J, Loftis A, Wang C. Molecular detection of tick-borne pathogens in captive wild felids. Zimbabwe Parasit Vectors. 2014;7:514.

    Article  PubMed  Google Scholar 

  12. André MR, Adania CH, Teixeira RH, Allegretti SM, Machado RZ. Molecular and serological detection of Babesia spp. in neotropical and exotic carnivores in Brazilian zoos. J Zoo Wildl Med. 2011;42:139–43.

    Article  PubMed  Google Scholar 

  13. Melissa S, Nikolai K, Jane E, et al. Babesia spp. in Ursus americanus (Black bear) in New Jersey. Northeast Nat. 2015;22:451–8.

    Article  Google Scholar 

  14. Birkenheuer AJ, Whittington J, Neel J, Large E, Barger A, Levy MG, et al. Molecular characterization of a Babesia species identified in a North American raccoon. J Wildl Dis. 2006;42:375–80.

    Article  CAS  PubMed  Google Scholar 

  15. AbouLaila M, El-Sayed SAE, Omar MA, Al-Aboody MS, Aziz ARA, Abdel-Daim MM, et al. Myrrh oil in vitro inhibitory growth on bovine and equine piroplasm parasites and Babesia microti of mice. Pathogens. 2020;9:173.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yu L, Luan PT, Jin W, Ryder OA, Chemnick LG, Davis HA, et al. Phylogenetic utility of nuclear introns in interfamilial relationships of Caniformia (order Carnivora). Syst Biol. 2011;60:175–87.

    Article  CAS  PubMed  Google Scholar 

  17. Guo J, Miao X, He P, Li M, Wang S, Cui J, et al. Babesia gibsoni endemic to Wuhan, China: mitochondrial genome sequencing, annotation, and comparison with apicomplexan parasites. Parasit Res. 2019;118:235–43.

    Article  Google Scholar 

  18. Schreeg ME, Marr HS, Tarigo JL, Cohn LA, Bird DM, Scholl EH, et al. Mitochondrial genome sequences and structures aid in the resolution of piroplasmida phylogeny. PLoS ONE. 2016;11:e0165702.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Corduneanu A, Hrazdilová K, Sándor AD, Matei IA, Ionică AM, Barti L, et al. Babesia vesperuginis, a neglected piroplasmid: new host and geographical records, and phylogenetic relations. Parasit Vectors. 2017;10:598.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Wickramasekara Rajapakshage BK, Yamasaki M, Hwang SJ, Sasaki N, Murakami M, Tamura Y, et al. Involvement of mitochondrial genes of Babesia gibsoni in resistance to diminazene aceturate. J Vet Med Sci. 2012;74:1139–48.

    Article  PubMed  Google Scholar 

  21. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35:4453–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–91.

    Article  PubMed  Google Scholar 

  24. Cheng WY, Zhao GH, Jia YQ, Bian QQ, Du SZ, Fang YQ, et al. Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling subspecies of giant panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by morphology and molecular markers. PLoS ONE. 2013;8:e69793.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang C, Wang L, Liu Y, Deng L, Wei M, Wu K, et al. The mitochondrial genome of the giant panda tick Haemaphysalis flava (Acari, Ixodidae) from Southwest China. Mitochondrial DNA B Resour. 2020;5:1188–90.

    Article  PubMed  PubMed Central  Google Scholar 

  26. ICZN. International commission on zoological nomenclature: amendment of articles 8, 9, 10, 21 and 78 of the international code of zoological nomenclature to expand and refine methods of publication. Bull Zool Nomenc. 2012;692012:161–9.

    Google Scholar 

  27. Wang X, Wang J, Liu J, Liu A, He X, Xiang Q, et al. Insights into the phylogenetic relationships and drug targets of Babesia isolates infective to small ruminants from the mitochondrial genomes. Parasit Vectors. 2020;13:378.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wang T, Guan G, Korhonen PK, Koehler AV, Young ND, Hall RS, et al. Mitochondrial genomes of two Babesia taxa from sheep in China as a foundation for population genetic and epidemiological investigations. Infect Genet Evol. 2017;47:51–5.

    Article  CAS  PubMed  Google Scholar 

  29. He L, Zhang Y, Zhang QL, Zhang WJ, Feng HH, Khan MK, et al. Mitochondrial genome of Babesia orientalis, apicomplexan parasite of water buffalo (Bubalus babalis, Linnaeus, 1758) endemic in China. Parasit Vectors. 2014;7:82.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Hikosaka K, Watanabe Y, Tsuji N, Kita K, Kishine H, Arisue N, et al. Divergence of the mitochondrial genome structure in the apicomplexan parasites Babesia and Theileria. Mol Biol Evol. 2010;27:1107–16.

    Article  CAS  PubMed  Google Scholar 

  31. Weingart C, Helm CS, Müller E, Schäfer I, Skrodzki M, von Samson-Himmelstjerna G, et al. Autochthonous Babesia canis infections in 49 dogs in Germany. J Vet Intern Med. 2023;37:140–9.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Shabangu N, Penzhorn BL, Oosthuizen MC, Vorster I, van Schalkwyk OL, Harrison-White RF, et al. A shared pathogen: Babesia rossi in domestic dogs, black-backed jackals (Canis mesomelas) and African wild dogs (Lycaon pictus) in South Africa. Vet Parasitol. 2021;291:109381.

    Article  CAS  PubMed  Google Scholar 

  33. Baneth G, Cardoso L, Brilhante-Simões P, Schnittger L. Establishment of Babesia vulpes n. sp. (Apicomplexa: Babesiidae), a piroplasmid species pathogenic for domestic dogs. Parasit Vectors. 2019;12:129.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Baneth G, Florin-Christensen M, Cardoso L, Schnittger L. Reclassification of Theileria annae as Babesia vulpes sp. nov. Parasit Vectors. 2015;8:207.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Stayton E, Lineberry M, Thomas J, Bass T, Allen K, Chandrashekar R, et al. Emergence of Babesia conradae infection in coyote-hunting Greyhounds in Oklahoma, USA. Parasit Vectors. 2021;14:402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Phair KA, Carpenter JW, Smee N, Myers CB, Pohlman LM. Severe anemia caused by babesiosis in a maned wolf (Chrysocyon brachyurus). J Zoo Wildl Med. 2012;43:162–7.

    Article  PubMed  Google Scholar 

  37. Han JI, Lee SJ, Jang HJ, Na KJ. Asymptomatic Babesia microti-like parasite infection in wild raccoon dogs (Nyctereutes procyonoides) in South Korea. J Wildl Dis. 2010;46:632–5.

    Article  PubMed  Google Scholar 

  38. Matjila PT, Leisewitz AL, Jongejan F, Bertschinger HJ, Penzhorn BL. Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Africa. Vet Parasit. 2008;157:123–7.

    Article  CAS  Google Scholar 

  39. Williams BM, Berentsen A, Shock BC, Teixiera M, Dunbar MR, Becker MS, et al. Prevalence and diversity of Babesia, Hepatozoon, Ehrlichia, and Bartonella in wild and domestic carnivores from Zambia. Africa Parasit Res. 2014;113:911–8.

    Article  Google Scholar 

  40. Erdélyi K, Mezősi L, Vladov S, Földvári G. Fatal acute babesiosis in captive grey wolves (Canis lupus) due to Babesia canis. Ticks Tick Borne Dis. 2014;5:281–3.

    Article  PubMed  Google Scholar 

  41. Javeed NN, Shultz L, Barnum S, Foley JE, Hodzic E, Pascoe EL, et al. Prevalence and geographic distribution of Babesia conradae and detection of Babesia vogeli in free-ranging California coyotes (Canis latrans). Int J Parasit Parasit Wildl. 2022;19:294–300.

    Article  Google Scholar 

  42. Furman H, Scimeca RC. Detection of Babesia conradae in coyotes (Canis latrans) and coyote-hunting greyhound dogs (Canis familiaris). Pathogens. 2023;12:528.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bosman AM, Penzhorn BL, Brayton KA, Schoeman T, Oosthuizen MC. A novel Babesia sp. associated with clinical signs of babesiosis in domestic cats in South Africa. Parasit Vectors. 2019;12:138.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Penzhorn BL, Oosthuizen MC. Babesia species of domestic cats: Molecular characterization has opened pandora’s box. Front Vet Sci. 2020;7:134.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Bosman AM, Venter EH, Penzhorn BL. Occurrence of Babesia felis and Babesia leo in various wild felid species and domestic cats in Southern Africa, based on reverse line blot analysis. Vet Parasit. 2007;144:33–8.

    Article  Google Scholar 

  46. Bosman AM, Oosthuizen MC, Peirce MA, Venter EH, Penzhorn BL. Babesia lengau sp. nov., a novel Babesia species in cheetah (Acinonyx jubatus, Schreber, 1775) populations in South Africa. J Clin Microbiol. 2010;48:2703–8.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Githaka N, Konnai S, Kariuki E, Kanduma E, Murata S, Ohashi K. Molecular detection and characterization of potentially new Babesia and Theileria species/variants in wild felids from Kenya. Acta Trop. 2012;124:71–8.

    Article  CAS  PubMed  Google Scholar 

  48. Yabsley MJ, Murphy SM, Cunningham MW. Molecular detection and characterization of Cytauxzoon felis and a Babesia species in cougars from Florida. J Wildl Dis. 2006;42:366–74.

    Article  CAS  PubMed  Google Scholar 

  49. DiVincenti L, Garner M, Thomas B, Birkenheuer A. Babesia sp. infection in a zoo-housed polar bear (Ursus maritimus). Vet Parasit Reg Stud Rep. 2019;18:100350.

    Google Scholar 

  50. Chua TH, Yeoh BN, Manin BO, Wong ST. First detection of Babesia sp. in Bornean sun bear (Helarctos malayanus euryspilus Horsfield) in Sabah Malaysia. Trop Biomed. 2022;39:179–84.

    Article  CAS  PubMed  Google Scholar 

  51. Moustafa MAM, Sasaki A, Shimozuru M, Nakao R, Sashika M, Yamazaki K, et al. Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus). Parasitol Res. 2020;119:3739–53.

    Article  PubMed  Google Scholar 

  52. Jinnai M, Kawabuchi-Kurata T, Tsuji M, Nakajima R, Hirata H, Fujisawa K, et al. Molecular evidence of the multiple genotype infection of a wild Hokkaido brown bear (Ursus arctos yesoensis) by Babesia sp. UR1. Vet Parasit. 2010;173:128–33.

    Article  CAS  Google Scholar 

  53. Ikawa K, Aoki M, Ichikawa M, Itagaki T. The first detection of Babesia species DNA from Japanese black bears (Ursus thibetanus japonicus) in Japan. Parasitol Int. 2011;60:220–2.

    Article  CAS  PubMed  Google Scholar 

  54. Westmoreland LSH, Stoskopf MK, Sheppard E, DePerno CS, Gould NP, Olfenbuttel C, et al. Detection and prevalence of Babesia spp. in American black bears (Ursus americanus) from eastern and western north Carolina, USA. J Wildl Dis. 2019;55:678–81.

    Article  CAS  PubMed  Google Scholar 

  55. Skinner D, Mitcham JR, Starkey LA, Noden BH, Fairbanks WS, Little SE. Prevalence of Babesia spp., Ehrlichia spp., and tick infestations in Oklahoma black bears (ursus americanus). J Wildl Dis. 2017;53:781–7.

    Article  CAS  PubMed  Google Scholar 

  56. Birkenheuer AJ, Marr HS, Hladio N, Acton AE. Molecular evidence of prevalent dual piroplasma infections in North American raccoons (Procyon lotor). Parasitology. 2008;135:33–7.

    Article  CAS  PubMed  Google Scholar 

  57. Clark K, Savick K, Butler J. Babesia microti in rodents and raccoons from northeast Florida. J Parasit. 2012;98:1117–21.

    Article  PubMed  Google Scholar 

  58. Kawabuchi T, Tsuji M, Sado A, Matoba Y, Asakawa M, Ishihara C. Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido. Japan J Vet Med Sci. 2005;67:825–7.

    Article  PubMed  Google Scholar 

  59. Jinnai M, Kawabuchi-Kurata T, Tsuji M, Nakajima R, Fujisawa K, Nagata S, et al. Molecular evidence for the presence of new Babesia species in feral raccoons (Procyon lotor) in Hokkaido. Japan Vet Parasit. 2009;162:241–7.

    Article  CAS  Google Scholar 

  60. Mehrkens LR, Shender LA, Yabsley MJ, Shock BC, Chinchilla FA, Suarez J, et al. White-nosed coatis (Nasua narica) are a potential reservoir of Trypanosoma cruzi and other potentially zoonotic pathogens in Monteverde. Costa Rica J Wildl Dis. 2013;49:1014–8.

    Article  PubMed  Google Scholar 

  61. Kjemtrup AM, Wainwright K, Miller M, Penzhorn BL, Carreno RA. Babesia conradae, sp. Nov., a small canine Babesia identified in California. Vet Parasit. 2006;138:103–11.

    Article  Google Scholar 

  62. Orkun Ö. Description of a novel Babesia sp. genotype from a naturally infected Eurasian lynx (Lynx lynx) in Anatolia, Turkey, with remarks on its morphology and phylogenetic relation to other piroplasmid species. Ticks Tick Borne Dis. 2022;13:102026.

    Article  PubMed  Google Scholar 

  63. He L, Miao X, Hu J, Huang Y, He P, He J, et al. First molecular detection of Babesia gibsoni in dogs from Wuhan. China Front Microbiol. 2017;8:1577.

    Article  PubMed  Google Scholar 

  64. Yabsley MJ, Shock BC. Natural history of zoonotic Babesia: Role of wildlife reservoirs. Int J Parasitol Parasites Wildl. 2013;2:18–31.

    Article  PubMed  Google Scholar 

  65. Solano-Gallego L, Baneth G. Babesiosis in dogs and cats–expanding parasitological and clinical spectra. Vet Parasitol. 2011;181:48–60.

    Article  PubMed  Google Scholar 

  66. Panicker VP, Sreedharannair AK, Narayanan A, George S, Hameed SV. Molecular identification of a novel species, Babesia panickeri sp. nov., from a naturally infected domestic cat of india and its comparison with canine Babesia isolates. Acta Parasit. 2020;65:913–8.

    Article  Google Scholar 

  67. Penzhorn BL, Kjemtrup AM, López-Rebollar LM, Conrad PA. Babesia leo n. sp. from lions in the Kruger National Park, South Africa, and its relation to other small piroplasms. J Parasit. 2001;87:681–5.

    Article  CAS  PubMed  Google Scholar 

  68. Solano-Gallego L, Sainz Á, Roura X, Estrada-Peña A, Miró G. A review of canine babesiosis: the European perspective. Parasit Vectors. 2016;9:336.

    Article  PubMed  PubMed Central  Google Scholar 

  69. Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA. Babesia: a world emerging. Infect Genet Evol. 2012;12:1788–809.

    Article  PubMed  Google Scholar 

  70. Inokuma H, Yoshizaki Y, Shimada Y, Sakata Y, Okuda M, Onishi T. Epidemiological survey of Babesia species in Japan performed with specimens from ticks collected from dogs and detection of new Babesia DNA closely related to Babesia odocoilei and Babesia divergens DNA. J Clin Microbiol. 2003;41:3494–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Wei F, Hu Y, Zhu L, Bruford MW, Zhan X, Zhang L. Black and white and read all over: the past, present and future of giant panda genetics. Mol Ecol. 2012;21:5660–74.

    Article  PubMed  Google Scholar 

  72. Eizirik E, Murphy WJ, Koepfli KP, Johnson WE, Dragoo JW, Wayne RK, et al. Pattern and timing of diversification of the mammalian order Carnivora inferred from multiple nuclear gene sequences. Mol Phylogenet Evol. 2010;56:49–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. McKenney EA, Maslanka M, Rodrigo A, Yoder AD. Bamboo specialists from two mammalian orders (Primates, Carnivora) share a high number of low-abundance gut microbes. Microb Ecol. 2018;76:272–84.

    Article  PubMed  Google Scholar 

  74. Hu Y, Wu Q, Ma S, Ma T, Shan L, Wang X, et al. Comparative genomics reveals convergent evolution between the bamboo-eating giant and red pandas. Proc Natl Acad Sci USA. 2017;114:1081–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Myśliwy I, Perec-Matysiak A, Hildebrand J. Invasive raccoon (Procyon lotor) and raccoon dog (Nyctereutes procyonoides) as potential reservoirs of tick-borne pathogens: data review from native and introduced areas. Parasit Vectors. 2022;15:126.

    Article  PubMed  PubMed Central  Google Scholar 

  76. Modarelli JJ, Westrich BJ, Milholland M, Tietjen M, Castro-Arellano I, Medina RF, et al. Prevalence of protozoan parasites in small and medium mammals in Texas, USA. Int J Parasitol Parasites Wildl. 2020;11:229–34.

    Article  PubMed  PubMed Central  Google Scholar 

  77. Léveillé AN, Baneth G, Barta JR. Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences. Int J Parasitol. 2019;49:375–87.

    Article  PubMed  Google Scholar 

  78. Virji AZ, Thekkiniath J, Ma W, Lawres L, Knight J, Swei A, et al. Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes. Int J Parasitol. 2019;49:105–13.

    Article  CAS  PubMed  Google Scholar 

  79. Hikosaka K, Tsuji N, Watanabe Y, Kishine H, Horii T, Igarashi I, et al. Novel type of linear mitochondrial genomes with dual flip-flop inversion system in apicomplexan parasites, Babesia microti and Babesia rodhaini. BMC Genomics. 2012;13:622.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Lau AO. An overview of the Babesia, Plasmodium and Theileria genomes: a comparative perspective. Mol Biochem Parasit. 2009;164:1–8.

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to the staff at the Chengdu Research Base of Giant Panda Breeding and the China Conservation and Research Center for the Giant Panda for their invaluable assistance in gathering the blood samples.

Funding

This study was financially supported by grants from the Chengdu Research Base of Giant Panda Breeding [Grant number CPF2017-24].

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Authors and Affiliations

  1. Sichuan Agricultural University, Sichuan, China

    Lang Xiong & Guangyou Yang

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  1. Lang Xiong
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  2. Guangyou Yang
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Contributions

L.X. participated in the conception and design of the study, conducted the experiments, performed the data analysis, prepared the figures and wrote the main manuscript. The manuscript was conceived, designed, reviewed and edited collaboratively by L.X. and G.Y.Y. All the authors reviewed the manuscript.

Corresponding author

Correspondence to Guangyou Yang.

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Ethics approval and consent to participate

The blood samples from routine health screenings of giant pandas were obtained noninvasively, ensuring no harm to the animals during the sampling process. The Institutional Animal Care and Use Committee of the China Conservation and Research Center for the Giant Panda and Chengdu Research Base of Giant Panda Breeding approved the use of materials and all animal-based experimental procedures.

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Not applicable.

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All the authors declare that they have no competing interests.

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Xiong, L., Yang, G. Description and molecular characterisation of Babesia ailuropodae n. sp., a new piroplasmid species infecting giant pandas. Parasites Vectors 17, 315 (2024). https://doi.org/10.1186/s13071-024-06402-6

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Keywords

Parasites & Vectors

ISSN: 1756-3305

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