Giardia Duodenalis and Cryptosporidium Sp

Yu et al. Parasites Vectors (2020) 13:403 https://doi.org/10.1186/s13071-020-04274-0 Parasites & Vectors

RESEARCH Open Access Molecular characterization and zoonotic potential of Enterocytozoon bieneusi, Giardia duodenalis and Cryptosporidium sp. in farmed masked palm civets (Paguma larvata) in southern China Zhengjie Yu1, Xi Wen1, Xitong Huang1, Ruohong Yang1, Yaqiong Guo1, Yaoyu Feng1,2, Lihua Xiao1,2 and Na Li1,2*

Abstract Background: Masked palm civets are known to play an important role in the transmission of some zoonotic pathogens. However, the distribution and zoonotic potential of Enterocytozoon bieneusi, Giardia duodenalis and Crypto- sporidium spp. in these animals remain unclear. Methods: A total of 889 fecal specimens were collected in this study from farmed masked palm civets in Hainan, Guangdong, Jiangxi and Chongqing, southern China, and analyzed for these pathogens by nested PCR and DNA sequencing. Results: Altogether, 474 (53.3%), 34 (3.8%) and 1 (0.1%) specimens were positive for E. bieneusi, G. duodenalis and Cryptosporidium sp., respectively. Sequence analysis revealed the presence of 11 novel E. bieneusi genotypes named as PL1–PL11 and two known genotypes Peru8 and J, with PL1 and PL2 accounting for 90% of E. bieneusi infections. Phylogenetically, PL4, PL5, PL9, PL10 and PL11 were clustered into Group 1, while PL1, PL2, PL3, PL6, PL7 and PL8 were clustered into Group 2. Assemblage B (n 33) and concurrence of B and D (n 1) were identifed among G. duodenalis-positive animals. Further multilocus genotyping= of assemblage B has revealed= that all 13 multilocus genotypes in civets formed a cluster related to those from humans. The Cryptosporidium isolate from one civet was identifed to be genetically related to the Cryptosporidium bamboo rat genotype II. Conclusions: To the best of our knowledge, this frst report of enteric protists in farmed masked palm civets suggests that these animals might be potential reservoirs of zoonotic E. bieneusi and G. duodenalis genotypes. Keywords: Enterocytozoon bieneusi, Giardia duodenalis, Cryptosporidium, Masked palm civet, Zoonotic potential

Background various wild and domestic animals, causing diarrhea Enterocytozoon bieneusi, Giardia duodenalis and Crypto- and other gastrointestinal symptoms. Humans can be sporidium spp. are enteric pathogens in humans and infected by these pathogens through contact of contaminated fomites or ingestion of contaminated food or water (food-borne or water-borne transmission) [1–3]. *Correspondence: [email protected] Te identifcation of genetic diversity in these patho- 1 Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, gens has accelerated in recent years. Tus far, nearly 500 Guangdong, China E. bieneusi genotypes have been identifed, belonging to Full list of author information is available at the end of the article 11 phylogenetic groups with diferent host preferences,

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including the zoonotic Group 1 and host-adapted Groups Specimens were stored at 4 °C in 2.5% potassium dichro- 2–11 [2]. Similarly, eight distinct G. duodenalis assem- mate before DNA extraction. blages of A–H with diferent host ranges have been identifed by genetic characterization [4]. Among them, DNA extraction assemblages A and B are the most common causes of Prior to genomic DNA extraction, potassium dichromate human giardiasis [5]. Tere are also nearly 40 recognized was removed by washing 500 μl of fecal suspension three Cryptosporidium species and at least as many genotypes times with distilled water by centrifugation at 2000×g for of unknown species status, most of which have host pref- 10 min. DNA was extracted from the sediment using the erence [3]. Recently, high-resolution multilocus geno- FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, typing (MLG) tools have been employed to elucidate the USA) as described [15]. Te extracted DNA was stored at genetic heterogeneity of these pathogens in humans and − 20 °C until being used in PCR analyses. animals [6–8]. Masked palm civets (Paguma larvata), belonging to the PCR amplifcation order Carnivora and family Viverridae, are wild mam- Enterocytozoon bieneusi was identifed by nested PCR mals distributed widely in Asia [9]. In southern China, amplifcation of the internal transcribed spacer (ITS) of they are raised as new farm animals, as their meat is con- the rRNA gene [16]. Giardia duodenalis was detected by sidered a culinary delicacy. In 2003, masked palm civets nested PCR targeting the triosephosphate isomerase (tpi), gained attention due to their potential involvement in the β-giardin (bg), and glutamate dehydrogenase (gdh) genes outbreak of severe acute respiratory syndrome (SARS), [17]. Cryptosporidium spp. were detected by PCR and which originated from southern China and spread to sequence analyses of the small subunit (SSU) rRNA and over 30 countries [10]. In addition, results of other stud- further characterized by sequence analysis of the 60-kDa ies have suggested that they may play a role in the trans- glycoprotein (gp60), 70-kDa heat-shock protein (hsp70) mission of other zoonotic pathogens such as Salmonella and actin genes [18–21]. Two replicates were used in enterica, Campylobacter spp., Bartonella henselae and PCR analysis of each target for each specimen. DNA of Toxoplasma gondii [11–14]. Tus far, the occurrence genotype PtEb IX from dogs, assemblage E from cattle, and zoonotic potential of E. bieneusi, G. duodenalis and and Cryptosporidium bovis from cattle were used as posi- Cryptosporidium spp. in masked palm civets remain tive controls in PCR analysis of E. bieneusi, G. duodenalis unclear. and Cryptosporidium spp., respectively, while reagent- Tis study was conducted to examine the prevalence, grade water was used as the negative control. genetic identity and zoonotic potential of E. bieneusi, G. duodenalis and Cryptosporidium spp. in farmed masked Sequence analysis palm civets in southern China. All positive secondary PCR products were sequenced bi- directionally on an ABI 3730 Genetic Analyzer (Applied Methods Biosystems, Foster City, CA, USA). To determine the Specimens genetic identity of E. bieneusi, G. duodenalis and Crypto- From April 2018 to March 2019, a total of 889 fecal sporidium spp., sequences obtained were assembled specimens were collected from masked palm civets on using ChromasPro 2.1.6. (http://technelysium.com.au/ four commercial farms in Hainan, Guangdong, Jiangxi ChromasPro.html), edited using BioEdit 7.1 (http:// and Chongqing, southern China (Fig. 1). Te manage- www.mbio.ncsu.edu/BioEdit/bioedit.html), and aligned ment of animals on all four farms was similar, with the with each other and reference sequences downloaded farm in Jiangxi being the largest in scale and having the from GenBank using ClustalX 2.0.11 (http://clustal.org). longest history of over 20 years. Te sanitary conditions Genotypes or subtypes of these pathogens were named of farms in Hainan and Chongqing were poor compared according to the established nomenclature system [22, with the other two farms. Masked palm civets were kept 23]. Maximum likelihood (ML) trees were constructed in groups of 2–6 animals per cage, with some interac- to infer the phylogenetic relationships among species or tions with those in neighboring cages. Fresh fecal drop- genotypes of these pathogens using MEGA 7.0.14 (http:// pings from civets were collected on the ground under www.megasoftware.net/). Te general time-reversible each cage, with one fecal specimen being collected from model was used in substitution rate calculations and 1000 each cage for this study. Te age of animals was divided replicates were used in bootstrapping analysis. into three groups: < 1 year (n = 469); 1–2 years (n = 129); Statistical analysis and > 2 years (n = 291). All animals sampled in this study were clinically normal without obvious signs of diarrhea. Te Chi-square test was used to compare diferences in the prevalence of pathogens among geographical Yu et al. Parasites Vectors (2020) 13:403 Page 3 of 10

locations or age groups. Diferences were considered sig- and 32.149, respectively; P < 0.0001) and Jiangxi (35.2%; 2 nifcant at P < 0.05. χ = 152.05 and 76.11, respectively; P < 0.0001) (Table 1). By age, E. bieneusi infection rates in animals of < 1 year 2 Nucleotide accession numbers (61.0%; χ = 29.110, P < 0.0001) and 1–2 years (53.5%; χ2 5.734, P 0.0166) were signifcantly higher than in Representative nucleotide sequences generated in this = = animals of > 2 years (40.9%) (Table 1). study were deposited in the GenBank database under the For G. duodenalis, 34 (3.8%) of the 889 specimens were accession numbers MT497888–MT497900 (E. bieneusi), positive based on the PCR positivity at any one of the MT487560–MT487588 (G. duodenalis), MT487589– three genetic loci. Te highest infection rate was 13.2% MT487591 and MT779807 (Cryptosporidium sp.). (14/106) on the farm in Guangdong, followed by Chong- qing (8.2%, 7/85), Hainan (3.8%, 8/209) and Jiangxi (1.0%, Results 5/489). Te infection rate on the farm in Guangdong was Occurrence of E. bieneusi, G. duodenalis 2 signifcantly higher than in Hainan (χ = 9.525, P = 0.002) and Cryptosporidium sp 2 and Jiangxi (χ = 37.993, P < 0.0001) (Table 1). Addition- Occurrence of the three pathogens was determined based ally, animals of 1–2 years (10.9%) had signifcantly higher on the PCR positivity of at least one PCR replicate. Of the prevalence of G. duodenalis than those of < 1 year (2.8%; 2 2 889 fecal specimens collected from masked palm civets, χ = 15.324, P < 0.0001) and > 2 years (2.4%; χ = 13.427, 474 (53.3%) were positive for E. bieneusi, with infection P = 0.0002) (Table 1). rates ranging from 35.2% (172/489) to 86.1% (180/209) Among the 889 specimens analyzed, only one (0.1%) among the four farms sampled. Infection rates on the from the farm in Hainan was positive for Cryptosporid- farms in Hainan (86.1%) and Chongqing (85.9%) were sig- ium sp. Tis civet was co-infected with E. bieneusi. In 2 addition, co-infection of E. bieneusi and G. duodenalis nifcantly higher than in Guangdong (46.2%; χ = 56.407

Chongqing

Jiangxi

Guangdong

Hainan Fig. 1 Locations (triangles) of four farms in southern China examined in the present study Yu et al. Parasites Vectors (2020) 13:403 Page 4 of 10

was detected in 31 other animals, with a signifcantly PL1 (53.0% or 242/457) and PL2 (37.0% or 169/457) higher occurrence of co-infection of the two pathogens were the two most prevalent genotypes (Table 1). PL1 on the farm in Guangdong (12.3%) than in Hainan (2.9%; was the dominant genotype on the three farms in Hainan, 2 2 χ = 10.949, P = 0.0009) and Jiangxi (1.0%; χ = 33.793, Chongqing and Jiangxi, while PL2 was the most com- P < 0.0001). By age, a signifcantly higher co-infection mon one on the farm in Guangdong. Te highest genetic rate was detected in civets aged 1–2 years (10.1%) than diversity was observed on the farm in Jiangxi, with 2 < 1 year (2.6%; χ2 14.278, P 0.0002) and > 2 years known and 8 novel genotypes. Several unique genotypes 2 = = (2.1%; χ = 13.296, P = 0.0003) (Table 1). were detected on some of the farms, including PL4, PL5, PL6, PL9, PL10, PL11 on the farm in Jiangxi, PL7 and Enterocytozoon bieneusi genotypes PL8 on the farm in Guangdong, and PL3 on the farm in Sequence analysis of the ITS PCR products was suc- Chongqing. cessful for 457 of 474 E. bieneusi-positive specimens. Te remaining 17 specimens generated sequences with Giardia duodenalis assemblages underlying signals of mixed nucleotides, probably due Among the 34 G. duodenalis-positive specimens, assem- to concurrence of more than one E. bieneusi genotype blage B was the dominant genotype, being detected in 33 in each specimen. Altogether, 13 E. bieneusi genotypes specimens. One specimen, however, had the concurrence were detected, including two known ones (Peru8 and J) of assemblages B and D. By genetic locus, 13 sequence and 11 novel genotypes (named as PL1–PL11) (Table 1). types were present among the 28 assemblage B-positive Te latter were identifed based on nucleotide sequence specimens at the tpi locus, including 3 known ones and diferences from known E. bieneusi genotypes. Te ITS 10 new ones. Te three known sequence types, i.e. MB8 sequences of Peru8 in Group 1 and genotype J in Group (n = 8), MB7 (n = 3) and B14 (n = 1), were identical to 2 were identical to the GenBank reference sequences GenBank reference sequences KF679746, KF679745 and AY371283 and MF592787, respectively. Among the 11 KF679737, respectively. In contrast, the new sequence novel E. bieneusi genotypes, PL4, PL5, PL9, PL10 and types B-PL01 to B-PL10 had 1 to 6 single nucleotide pol- PL11 were phylogenetically placed in Group 1, while ymorphisms (SNPs) compared with the GenBank refer- PL1, PL2, PL3, PL6, PL7 and PL8 formed a clade named ence sequence KF679746. as Group 2-like, which was mostly related to Group 2 At the bg locus, one known and two new sequence (Fig. 2). types were obtained among the 16 assemblage B-positive specimens. Bb-3 (n = 5) was identical to the GenBank

Table 1 Distribution of Enterocytozoon bieneusi and Giardia duodenalis genotypes in farmed masked palm civets in southern China by sampling location and age Specimen No. of E. bieneusi G. duodenalis Co-infection specimens No. positive (%) Genotype (n) No. positive (%) Genotype (n) No. positive (%)

Hainan 209 180 (86.1)a PL1 (95), PL2 (75) 8 (3.8) B (8) 6 (2.9) Chongqing 85 73 (85.9)a PL1 (57), PL3 (11), PL2 (5) 7 (8.2) B (6), B D (1) 7 (8.2) + Guangdong 106 49 (46.2) PL2 (30), PL1 (16), PL7 (1), PL8 (1) 14 (13.2)c B (14) 13 (12.3)e Jiangxi 489 172 (35.2) PL1 (74), PL2 (59), PL4 (15), PL9 (5), PL6 (4), PL5 (3), 5 (1.0) B (5) 5 (1.0) PL10 (2), PL11 (2), J (1), Peru8 (1) < 1 year 469 286 (61.0) PL1 (157), PL2 (87), PL3 (11), PL4 (10), PL9 (4), PL6 (4), 13 (2.8) B (12), B D (1) 12 (2.6) PL5 (3), PL11 (2), PL10 (1), Peru8 (1) + 1–2 years 129 69 (53.5) PL2 (35), PL1 (30), PL7 (1), PL8 (1) 14 (10.9)d B (14) 13 (10.1)f > 2 years 291 119 (40.9)b PL1 (55), PL2 (47), PL4 (5), PL9 (1), PL10 (1), J (1) 7 (2.4) B (7) 6 (2.1) Total 889 474 (53.3) PL1 (242), PL2 (169), PL4 (15), PL3 (11), PL9 (5), PL6 (4), 34 (3.8) B (33), B D (1) 31 (3.5) PL5 (3), PL10 (2), PL11 (2), PL7 (1), PL8 (1), J (1), Peru8 + (1) a P < 0.01, for Hainan and Chongqing in comparison with Guangdong and Jiangxi b P < 0.05, for above 2 years-old in comparison with 1–2 years-old c P < 0.01, for Guangdong in comparison with Hainan and Jiangxi d P < 0.01, for 1–2 years old in comparison with under 1 year and above 2 years-old e P < 0.01, for Guangdong in comparison with Hainan and Jiangxi f P < 0.01, for 1–2 years-old in comparison with under 1 year and above 2 years-old Yu et al. Parasites Vectors (2020) 13:403 Page 5 of 10

reference sequence KJ888976, while the new sequence palm civet (Paradoxurus hermaphroditus) for G. duode- types of B-PL11 and B-PL12 had one SNP compared with nalis but did not detect this pathogen [30]. the GenBank reference sequence KJ888976. Results of the present study have revealed some dif- At the gdh locus, 10 new sequence types ferences in infection rates of E. bieneusi and G. duode- (B-PL13 ~ B-PL22) were detected among the 22 assem- nalis among farms and age groups. Signifcantly higher blage B-positive specimens, with 1–4 SNPs compared infection rates of E. bieneusi were observed on farms in with the GenBank reference sequence LC430575. Hainan (86.1%) and Chongqing (85.9%) than in Guang- dong (46.2%) and Jiangxi (35.2%). Tis was likely due to Multilocus genotypes of G. duodenalis assemblage B the poor sanitary conditions of the two farms in Hainan Of the 33 assemblage B-positive specimens, 13 were pos- and Chongqing, as the housing and management of ani- itive by PCR at all three loci, forming 13 MLGs (Civet- mals were similar among the four farms. By age, a higher MLG-B1 to Civet-MLG-B13) based on the concatenated prevalence of E. bieneusi was obtained from young civets sequences of the gdh, bg and tpi loci. Phylogenetic analy- of < 1 year. Tis is in agreement with previous fndings sis of these MLGs was conducted together with those of age-associated occurrence of E. bieneusi infections in from previous studies of assemblage B in humans and other farmed wild animals such as macaques, boars and various animals [24–28]. It showed that all 13 MLGs squirrels [31–33]. In contrast, a signifcantly higher infec- from civets in this study formed a cluster, which was a tion rate of G. duodenalis was observed on the farm in sister group with MLGs from humans in Sweden (Fig. 3). Guangdong, and masked palm civets of 1–2 years had the highest infection rate of G. duodenalis, suggesting that E. Cryptosporidium genotype bieneusi and G. duodenalis are transmitted diferently in For the Cryptosporidium-positive specimen SCAU12558, these animals. DNA sequencing of PCR products of the SSU rRNA, Results of the sequence analysis have demonstrated a gp60 and hsp70 genes revealed that it was phylogeneti- high genetic diversity of E. bieneusi in masked palm civ- cally related to the Cryptosporidium bamboo rat geno- ets. Altogether, 13 E. bieneusi ITS genotypes were identi- type II (Fig. 4). Te SSU rRNA sequence generated had fed in this study, with two novel genotypes PL1 and PL2 one SNP and three nucleotide insertions compared with accounting for about 90% E. bieneusi infections in civets. the sequence from the bamboo rat genotype II (Gen- Tese two ITS genotypes together with several other Bank: MK731962). Te gp60 sequence generated had novel ones formed a clade related to Group 2, thus might a maximum nucleotide identity of 83.0% to the bam- be civet-adapted E. bieneusi genotypes. In comparison, boo rat genotype II (GenBank: MK731966), with exten- the remaining novel genotypes PL4, PL5, PL9, PL10 and sive sequence diferences in the non-repeat regions. Te PL11 were phylogenetically clustered into the zoonotic hsp70 sequence generated had 17 SNPs and 1 nucleo- Group 1. In addition, one civet was found positive for tide deletion with the bamboo rat genotype II (Gen- Peru8, which is a common zoonotic genotype in humans Bank: MK731969), mostly over the repeat region at the [2]. Tus, masked palm civets could carry human-patho- 3′-end of the gene. Due to the absence of the actin gene genic E. bieneusi. sequence from the bamboo rat genotype II in the Gen- High genetic heterogeneity of E. bieneusi was observed Bank database, the actin sequence of SCAU12558 was in masked palm civets on a large farm in Jiangxi. Tis found to be most similar to the Cryptosporidium ferret farm is the largest among the four study farms and has genotype (GenBank: MF411076) with 12 SNPs. been established for over 20 years. It has 10 of the 13 E. bieneusi genotypes identifed in the study, including all Discussion six ITS genotypes (Peru8, PL4, 5, 9, 10 and 11) of the In this study, to the best of our knowledge, we report zoonotic Group 1. One explanation for the high het- for the frst time the occurrence and genetic identity of erogeneity is that genetic exchange might have occurred E. bieneusi, G. duodenalis and Cryptosporidium sp. in among ancestral types on this farm during the past 20 masked palm civets, with infection rates of 53.3%, 3.8% years [34]. Te long history of the farm and high geno- and 0.1%, respectively. Within the family Viverridae, type diversity have probably facilitated the genetic several small carnivores genetically related to masked exchange among E. bieneusi isolates and emergence of palm civets have been examined for these pathogens novel genotypes in farmed masked palm civets. without much success. For example, a recent study in Te zoonotic assemblage B appears to be the domi- Spain reported the occurrence of an unknown Crypto- nant G. duodenalis genotype in masked palm civets. In sporidium genotype in one of six genets (Genetta genetta) this study, all G. duodenalis-positive civets harbored examined for Cryptosporidium spp. and G. duodenalis assemblage B, with only one having concurrent infec- [29]. Another study in the Philippines examined an Asian tion of assemblages B and D. Assemblage B is the most Yu et al. Parasites Vectors (2020) 13:403 Page 6 of 10

JF681177-Baboon-KB3 KR062130-Yellow necked mouse-WR4 60 AY237215 -Beaver-WL7 DQ683748-Human-CAF3 AY371285-Human-Peru10 AY371283-Human-Peru8 KT750158-Fox-NCF5 KU847362-Raccoon dog-NCR1 AY371278-Human-Peru3 56 KJ728789-Monkey-CM10 KF305589-Cynomolgus Monkey-CM3 KT984491-Cattle-BEB13 AF242478 -Human-type IV HM992511-Human-CHN4 50 DQ683747-Human-CAF2 JX994258-Human-SH2 AF242477-Human-type III AY371282-Human-Peru7 DQ683746-Human-CAF1 KP732485-Cattle-NECA5 Group 1 59 KM272395-Human-NEC4 KF607048-Pig-CS-2 66 JQ029727-Human-Henan-IV KM272392-Human-NEC1 MT497899-Masked palm civet-PL10 MT497894-Masked palm civet-PL5 78 MT497898-Masked palm civet-PL9 MT497893-Masked palm civet-PL4 MT497900-Masked palm civet-PL11 99 AF101199-Human-C AF267147-Human-Q AF076041-Pig-EbpB 69 JX994266-Human-SH10 JX994259-Human-SH3 AF267145-Human-O KP318001-Pig-EbpA GU198949-Human-CZ1 KP318016-Pig-PigEb15 JQ863269-Wastewater-WW1 MT497890-Masked palm civet-PL1 53 MT497896-Masked palm civet-PL7 MT497897-Masked palm civet-PL8 MT497895-Masked palm civet-PL6 Group 2-like MT497891-Masked palm civet-PL2 MT497892-Masked palm civet-PL3 AF135836-Cattle-I 56 KJ867481-white-tailed deer-DeerEb2 EF139195-Cattle -CEbA MF592787-Cattle-J KF675196-Cattle-BEB10 AY331007-Cattle-BEB3 89 KU604930-Golden snub-nosed monkey-CM20 79 MH899210-Cattle-BEB8 KP262389-Sheep-CHG3 98 KM591946-Monkey-BSH Group 2 MK573330-Sheep-CM7 KP732478-Sheep-NESH4 KP262391-Sheep-CHS6 MH822622-Sheep-OEB1 60 KJ668739-Goat-CC1 EU153584-Cattle-BEB6 67 KU245697-Dog-CD6 52 MH822620-Sheep-COS-II AY237212-Muskrat-WL4 KF591688-Raccoon-WL24 KY706126-Red Kangaroo-CHK1 89 JN997479-Human-Nig3 97 JF681179-Baboon-KB-5 Group 3-11 KJ728811-Lemur-CM18 95 DQ683749-Human-CAF4 AY237210-Raccoon-WL2 DQ885585-Dog-PtEbIX KY706128-Red Kangaroo-CSK2 Outgroup

0.1 Fig. 2 Phylogenetic relationship among Enterocytozoon bieneusi genotypes based on the maximum-likelihood analysis of the internal transcribed spacer of the rRNA gene. Bootstrap values greater than 50% from 1000 replicates are shown on the branches. Known and novel genotypes identifed in this study are indicated by blue and red triangles, respectively Yu et al. Parasites Vectors (2020) 13:403 Page 7 of 10

64 MLG3-Pig tailed macaque-China 52 MLG8-Golden monkey-China 56 36413-Macaques-China MLG15-Rhesus macaque-China ISSGdA711-Macaque-Italy 36395-Macaques-China 88 MLG4-Ring tailed lemur-China MLG7-Yellow baboon-China ISSGdA722-Macaque-Italy Bnew(a)-Human-England ZB6-Rabbit-China 95 ZC47-Rabbit-China 15-Pet chinchillas-China 92-Pet chinchillas-China MLG10-Ring tailed lemur-China VANC/90/UBC/50-Water-Canada VANC/90/UBC/46-Human-Canada Bnew(b)-Human-England 50 Bnew(c)-Human-England 83 Egyh14-Human-Egypt 93 Egyh35-Human-Egypt Q334-Racehorse-China 92 H929-Racehorse-China MLG6-Ring tailed lemur-China MLG5-Squirrel monkey-China MLG9-Ring tailed lemur-China Swegp138-Guinea pig-Sweden ECUST5367-Human-China TB4-Human-Malaysia CK20-Human-Malaysia SE12-Human-Malaysia TB7-Human-Malaysia ECUST6453-Human-China TB5-Human-Malaysia 19864-Cat-China DC01-Human-Brazil Swerabbit176-Rabbit-Sweden 99 AP15-Human-Czech Republic ECUST6534-Human-China ECUST1710-Human-China ECUST2729-Human-China 20277-Cat-China 65 19997-Cat-China 72 20268-Cat-China 99 Swemon200-Vervet monkey-Sweden 58 M7-Human-Belgium 91 DC39-Human-Brazil ECUST5414-Human-China 20-Human-Iran MLG-Crab-eating macaques-B24 100 MLG-Crab-eating macaques-B7 MLG-Crab-eating macaques-B40 71 Sweh107-Human-Sweden 100 Sweh136-Human-sweden 82 Sweh059-Human-Sweden 100 Sweh158-Human-Sweden Civet-MLG-B11 57 Civet-MLG-B12 Civet-MLG-B13 77 55 Civet-MLG-B8 Civet-MLG-B9 Civet-MLG-B4 50 Civet-MLG-B3 66 Civet-MLG-B5 Civet-MLG-B6 Civet-MLG-B10 Civet-MLG-B7 Civet-MLG-B1 56 Civet-MLG-B2

0.1 Fig. 3 Phylogeny of multilocus genotypes (MLGs) of Giardia duodenalis assemblage B from this and previous reports based on the maximum-likelihood analysis of concatenated sequences of the gdh, bg and tpi loci. Bootstrap values greater than 50% from 1000 replicates are shown on the branches. MLGs identifed in this study are indicated by red triangles Yu et al. Parasites Vectors (2020) 13:403 Page 8 of 10

a EF641051-Mink genotype b KJ802723-C. parvum-IIdA15G1 KM012045-C. parvum 84 KC885904-C. parvum-IIpA9 GQ214082-C. erinacei 99 FJ897787-C. parvum-IInA8 EF641026-Chipmunk genotype I AF262330-Deer mouse genotype KC885906-C. parvum-IIoA13G1 LC089977-Bat genotype V DQ648547-Horse genotype-VIa KT336622-C. cuniculus AM937006-C. parvum-IIlA18 94 KR296813-C. hominis 99 AY873780-C. parvum-IIgA9 HQ917077-C. meleagridis MK095339-C. parvum-IIaA15G2R1 MT779807-SCAU12558-Civet 90 FJ262730-C. cuniculus-VaA18 MK731962-Bamboo rat genotype II 56 63 AY737562-Vole genotype 74 EF208067-C. hominis-IgA24 MH187877-C. suis GQ121028-C. wrairi-VIIaA17T1 GQ121030-C. tyzzeri-IXaA6R3 MG699179-C. occultus 72 MK731960-Bamboo rat genotype I 61 AY700401-C. parvum-IImA7G1 50 KU608308-C. canis AY873782-C. parvum-IIiA10 MG516762-C. ubiquitum AY262031-C. hominis-IbA10G2 KJ000486-C. tyzzeri 99 AF440638-C. hominis-IfA19G1R5 JX644908-C. viatorum 99 56 MG516747-C. fayeri 75 AY873781-C. parvum-IIhA7G4 KT749819-C. felis AF164501-C. parvum-IIcA5G3b JX424840-C. scrofarum 100 MK731966-Bamboo rat genotype II 97 54 LC019004-Deer genotype 98 MT487589-SCAU12558-Civet JN400880-C. ryanae 52 99 GQ121029-Ferret genotype-VIIIaA5G2 EU408317.1-C. bovis 82 HM234174-Mink genotype-XaA5G1 GU553016-C. xiaoi FJ490078-C. fayeri-IVaA10G3T1R1 89 KU058876-C. avium 100 HM234181-Opossum genotype I-XIaA4G1T1 92 EU827297-C. baileyi AF093500-C. serpentis KP115936-C. viatorum-XVaA3a 99 99 94 AJ307669-C. muris KC204981-C. ubiquitum-XIIa JN400881-C. andersoni 0.01 0.1

c 95 KP314260-C. hominis d 100 GU327783-C. cuniculus 99 KJ941149-C. hominis 65 JQ413433-C. hominis 94 KC157562-C. cuniculus 98 KF059969-C. parvum KC885897-C. parvum 59100 JQ073414-C. tyzzeri KF612325-C. erinacei 53 JX471003-C. meleagridis 99 AF221536-C. wrairi LC106149-Bat genotype V AF221537-C. meleagridis 93 83 MT487590-SCAU12558-Civet 73 MK731969-Bamboo rat genotype II 99 68 MF411076-Ferret genotype MT487591-SCAU12558-Civet AF382348-C. wrairi JX978274-C. viatorum 85 JN846707-C. viatorum JX978276-Chipmunk genotype I MH145309-Vole genotype III MH145326-Vole genotype VI 65 97 JX485418-C. suis 51 MH145327-Vole genotype VII 59 GQ337960-C. ubiquitum MK731968-Bamboo rat genotype I 89 57 MN065774-Bamboo rat genotype I DQ898164-C. suis 100 55 AF382340-C. canis MG699175-C. occultus 84 JX485414-Rat genotype I EU754843-C. canis 100 AF382347-C. felis KP642069-C. felis EF502043-C. saurophilum 100 KF907826-C. xiaoi LT976832-C. scrofarum LC019013-Deer genotype 70 99 FJ463206-C. ryanae 100JQ798893-Environmental sequence 83 KU058886-C. avium FJ896042-C. xiaoi 99 EU741861-C. baileyi EU741853-C. baileyi 89 51 AF382353-C. serpentis AF221541-C. serpentis 100 100 AF382352-C. andersoni AF221543-C. muris 77 95 KR090628-C. muris 100 AF221542-C. muris 98 AB610481-C. andersoni KT731194-C. proliferans

0.02 0.05 Fig. 4 Phylogeny of Cryptosporidium spp. based on the maximum-likelihood analyses of the small subunit rRNA (a), 60-kDa glycoprotein (b), 70-kDa heat-shock protein (c) and actin (d) genes. Bootstrap values greater than 50% from 1000 replicates are shown on the branches. The Cryptosporidium-positive specimen detected in this study is indicated by red triangle common human-pathogenic genotype in both indus- donkeys, rabbits, chinchillas and bamboo rats, mostly trialized and developing countries [4, 35]. In animals, in China [24, 36–43]. Within assemblage B found in this assemblage B has been recently reported as the dominant study, a high genetic heterogeneity was observed at the G. duodenalis genotype in farmed macaques, horses, three genetic loci examined. Of the known subtypes, Yu et al. Parasites Vectors (2020) 13:403 Page 9 of 10

MB7 and MB8 at the tpi locus and Bb-3 at the bg locus Availability of data and materials Data supporting the conclusions of this article are included within the article. were reported in non-human primates [27, 44], while B14 Representative nucleotide sequences generated in this study were deposited at the tpi locus was detected in urban wastewater previ- in the GenBank database under the accession numbers MT487560-MT487591, ously [45]. In addition, phylogenetic analysis of the con- MT497888-MT497900 and MT779807. catenated sequences from the three loci showed that the Ethics approval and consent to participate MLGs of assemblage B in civets formed a separate clus- This research was approved by the Ethics Committee of the South China ter, which confrms the previous suggestion on the likely Agricultural University. Masked palm civets sampled were handled following the Animal Ethics Procedures and Guidelines of the People’s Republic of China. occurrence of host adaptation within assemblage B [24, Specimen collection was conducted with the permission of the owners or 27]. Nevertheless, the assemblage B MLGs from civets farm managers. were genetically related to several MLGs from humans, Consent for publication indicating that they could have zoonotic potential. Not applicable. Te Cryptosporidium isolate from masked palm civets was found to be genetically related to the Crypto- Competing interests The authors declare that they have no competing interests. sporidium bamboo rat genotype II from bamboo rats in south-central China based on sequence analysis of the Author details 1 SSU rRNA, gp60 and hsp70 genes [46]. As only one fecal Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, Guangdong, China. specimen was positive for Cryptosporidium, whether it is 2 Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, a native parasite of masked palm civets remains unclear. Guangdong 510642, China. Further studies are needed to understand its host range. Received: 1 June 2020 Accepted: 30 July 2020

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Deng L, Li W, Yu X, Gong C, Liu X, Zhong Z, et al. First report of the human-pathogenic Enterocytozoon bieneusi from red-bellied tree squirrels • gold Open Access which fosters wider collaboration and increased citations (Callosciurus erythraeus) in Sichuan, China. PLoS ONE. 2016;11:e0163605. • maximum visibility for your research: over 100M website views per year 34. Li W, Xiao L. Multilocus sequence typing and population genetic analysis of Enterocytozoon bieneusi: host specifcity and its impacts on public At BMC, research is always in progress. health. Front Genet. 2019;10:307. 35. Ryan U, Caccio SM. Zoonotic potential of Giardia. Int J Parasitol. Learn more biomedcentral.com/submissions 2013;43:943–56.