Infection, Genetics and Evolution 34 (2015) 32–35
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China Ge-Ru Tian a,1, Guang-Hui Zhao a,⇑,1, Shuai-Zhi Du a, Xiong-Feng Hu a, Hui-Bao Wang a, Long-Xian Zhang b, San-Ke Yu a,⇑ a b
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province 712100, China College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province 450002, China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 11 June 2015 Accepted 12 June 2015 Available online 12 June 2015 Keywords: Ailuropoda melanoleuca Ailurus fulgens Enterocytozoon bieneusi Prevalence Genotype
a b s t r a c t Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n = 4) and genotype EbpC (n = 5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens), two precious and endangered animals for wildlife conservation (Yonzon and Hunter, 1991; Peng et al., 2001), are respectively classified as Category I and II protected species in China. Habitats destruction, infective factors, intrinsic (poor reproduction and low densities) and accidental mortality are the major threats of their extinction (Zhang et al., 2008). Of them, pathogen infections have been identified as the most serious threat for survival of wild pandas in China currently (He et al., 2012). Enterocytozoon bieneusi, a unicellular enteric pathogen from the phylum Microspora, can inhabit in small intestines of a wide range of invertebrate and vertebrate hosts including humans, domestic and wild animals, and even birds, causing chronic diarrhea and wasting syndrome (Didier and Weiss, 2006.). E. bieneusi has been ⇑ Corresponding authors. E-mail addresses: [email protected] (G.-H. Zhao), [email protected] (S.-K. Yu). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.meegid.2015.06.015 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
detected in immune-competent individuals, resulting in self-limiting diarrhea (Nkinin et al., 2007). More importantly, it is considered as an opportunistic pathogen which can cause life-threatening diarrhea in immune-compromised individuals especially in AIDS patients raising public health concerns (Li et al., 2015). Therefore, E. bieneusi has been classified as the Category B potentially dangerous pathogen by National Institutes of Health (NIH) (Didier et al., 2004). Complicate population structure of E. bieneusi increased the difficulty to control the infection. Because of a high diversity in the internal transcribed spacer region (ITS) of the ribosomal RNA (rRNA) gene within E. bieneusi isolates, it is widely used for genotyping E. bieneusi (Matos et al., 2012). To date, more than 200 genotypes have been reported in humans and animals based on ITS region (Henriques-Gil et al., 2010). Most of these genotypes represented host-adapted groups (groups 2–8) associated with specific hosts and wastewater, and probably have no significant public health importance, while almost all genotypes in group 1 are frequently found in humans and animals and are considered to be zoonotic (Thellier and Breton, 2008). However, the single ITS marker may be inadequate in identifying its genotypes due to the
G.-R. Tian et al. / Infection, Genetics and Evolution 34 (2015) 32–35
uncertainty about a sexual phase in E. bieneusi lifecycle (Widmer and Akiyoshi, 2010). Recently, a high-resolution multilocus sequencing typing tool (MLST) using three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) markers has been developed (Feng et al., 2011), and successfully used to investigate the genetic diversity and host specificity of E. bieneusi (Widmer and Akiyoshi, 2010). Here, we, for the first time, explored the occurrence and genetic diversity of E. bieneusi in giant and red pandas, and elucidated the zoonotic potential of E. bieneusi with the ITS-based sequence typing and the MLST tool. 2. Materials and methods 2.1. Ethics statement Faecal samples of this study were collected under the permission of the relevant zoos. During sample collection, the animal welfare was taken into consideration. All procedures were reviewed and approved by the Animal Welfare Committee of Northwest A&F University. 2.2. Sampling Sampling was carried out from September 2013 to June 2014 in Rare Wildlife Rescue Breeding Research Center and Xi’an Qinling Wildlife Park in Shaanxi province, northwestern China. A total of 82 fresh faecal samples were collected from 46 giant pandas and 36 red pandas. Each sample was collected from each panda on the ground immediately after defecation and was then put into a plastic container labeled with the time and number. All faecal specimens were stored at 4 °C in 2.5% potassium dichromate prior to DNA extraction. 2.3. DNA extraction Faecal specimens were washed with distilled water until supernatant became clear. Genomic DNA was extracted from approximately 0.2 g of washed faecal specimens using the E.Z.N.AÒ Stool DNA kit (Omega Bio-tek Inc., GA, USA) according to the manufacturer-recommended procedures. The extracted DNA was stored at 20 °C until PCR analysis. 2.4. PCR amplification A nested PCR protocol was used to amplify of 390 bp fragment of the partial ribosomal ITS region of E. bieneusi (Karim et al., 2014). The PCR amplification primers and amplification conditions of ITS gene were referred to previous study (Sulaiman et al., 2003). Positive specimens were further characterized by MLST analyses using MS1, MS3, MS4 and MS7 according to Feng et al. (2011). The TaKaRa Ex Taq DNA polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all PCR amplifications. A negative control without DNA was included in all PCR tests. All the secondary PCR products were subjected to electrophoresis in a 1% agarose gel containing ethidium bromide. 2.5. Sequence analysis The secondary PCR products and inner set of the primers of all PCR-positive samples were sent to the Sangon Company (Shanghai, China) using ABI 3730xl automated DNA sequencer (Big Dye Terminator Chemistry) for DNA sequencing in both directions. Obtained sequences were aligned using the Clustal X 1.81 (Thompson et al., 1997) and compared with reference sequences from GenBank. Aligned sequences were used to determine
33
genotypes/subtypes by using the Basic Local Alignment Search Tool (BLAST) in National Center for Biotechnology Information (NCBI). 2.6. Phylogenetic analysis To assess the genetic relationship of ITS genotypes of E. bieneusi obtained in the present study and those published in the previous studies, a phylogenetic analysis was performed by constructing a neighboring-joining tree (Saitou and Nei, 1987) using the program Mega 5.0 (Tamura et al., 2011) based on the evolutionary distances calculated by Kimura-2-parameter model (Kimura, 1980). The reliability of these trees was assessed using the bootstrap analysis with 1000 replicates. 3. Results 3.1. Prevalence and genotypes of E. bieneusi in pandas Of 82 faecal specimens examined by nested PCR amplifying the ITS gene of E. bieneusi, nine (10.98%) were positive for infection of E. bieneusi, with four (8.70%/46) in giant pandas and five (13.89%/36) in red pandas. Sequence analysis showed two genotypes in this study, including one known genotype EbpC (n = 5) in red pandas and one novel genotype I-like (n = 4) in giant pandas. The novel genotype I-like had five nucleotide polymorphisms (transversions: T/A; C/G; T/G; C/G; transition: A/G) in comparison with the genotype I (KJ668738), with 99% of homology. 3.2. Phylogenetic relationships of E. bieneusi based on sequences of ITS regions A phylogenetic analysis using NJ method based on the ITS gene sequences of E. bieneusi indicated that all positive samples in the present study belonged to two groups. Five positive specimens of red panda (QYLP1, QYLP2, QYLP3, LGLP1 and LGLP2) were clustered into 1d of zoonotic group 1, and four positive samples of giant panda (GP1–GP4) located into group 2 (Fig. 1). 3.3. Multilocus sequence typing of E. bieneusi ITS-positive specimens were further characterized using three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4). Nine, six, six and nine positive amplicons were amplified successfully in loci MS1, MS3, MS4, and MS7, respectively. Sequencing analysis showed two haplotypes in all four loci and formed two distinct MLGs (Table 1). Of all haplotypes in four loci, BLAST search in GenBank showed that only the Type I of MS1 locus was a new one, which had two nucleotide polymorphism (transversion: T/A; transition: G/A) with E. bieneusi isolate 147 clone 5 from Homo sapiens in Uganda (KF261756), with 99% of homology. 4. Discussion Recently, the epidemiological study of E. bieneusi has been employed (Zhao et al., 2014). E. bieneusi has been reported in humans and many animals (cattle, pigs, dogs, cats, horses, goats, birds, etc) worldwide (Rinder et al., 2000; Santín and Fayer, 2011). Some parasitic protozoa have been reported in pandas. Cryptosporidium giant panda genotype, Cryptosporidium parvum mouse genotype, and Cryptosporidium andersoni have been detected in giant and red pandas (Karanis et al., 2007; Liu et al., 2013; Wang et al., 2015). The present study reported the presence of E. bieneusi infection in giant and red pandas for the first time. Infective rate (13.89%) of E. bieneusi for red pandas in the present
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G.-R. Tian et al. / Infection, Genetics and Evolution 34 (2015) 32–35
Fig. 1. Phylogenetic relationships of ITS nucleotide sequences of E. bieneusi genotypes in this study and known E. bieneusi genotypes was inferred by a neighbor-joining analysis based on genetic distance by the Kimura two-parameter model. The numbers on the branches are percent bootstrapping values from 1000 replicates, with more than 50% shown in tree. The genotypes found in giant and red pandas in this study were marked with black triangle and circle, respectively.
study was higher than that (6.36%) of C. andersoni in captive red pandas (Wang et al., 2015). Previous studies have indicated that humans could be infected mainly through E. bieneusi in human and animal feces (Sulaiman et al., 2003). Therefore, zoonotic potential of giant and red pandas to transmit E. bieneusi to humans should be further evaluated by more samples investigation and animal adaptation tests. Two genotypes were identified in this study by analyzing ITS gene sequences of E. bieneusi, namely novel genotypes I-like (n = 4) and EbpC (n = 5). The homology was 99% between genotype I and genotype I-like. The genotype I was previously thought to be cattle-specific (Del Coco et al., 2014), but it has also been reported in humans (Zhang et al., 2011), suggesting zoonotic potential of this genotype. The genotype EbpC has an extensive host range and was previously found in humans, monkeys, pigs, dogs, and other wildlife animals (Del Coco et al., 2014). The present study firstly reported it in red pandas, expanding host range of this
genotype. In the genetic tree re-constructed based on ITS sequences, five specimens of red pandas belonged to 1d of zoonotic group 1 (subgroups 1a–1h), clustered with the genotype EbpC in human and the genotype HenanIII in humans (Fig. 1), suggesting zoonotic potential of the genotype EbpC. Four specimens of giant pandas were classified into the group 2 with genotype I and PtEBXI in cattle (Fig. 1). Most genotypes of this group are host-adapted and probably have no significant public health risk, but some genotypes were also found in humans, such as genotype I and BEB6 (Zhang et al., 2011; Wang et al., 2013). Thus, it remains unknown whether the novel genotype I-like has a potential of zoonotic transmission currently. Therefore, more studies are needed to assess zoonotic potential of these genotypes and the role of pandas as a source of zoonotic genotypes of E. bieneusi to humans. In order to better substantiate public health significance of animal-derived E. bieneusi isolates and to trace sources of human E. bieneusi infections, subsequently, multilocus sequence typing
G.-R. Tian et al. / Infection, Genetics and Evolution 34 (2015) 32–35 Table 1 Multi-locus sequence typing of Enterocytozoon bieneusi in giant and red pandas in China. Species
Isolate
ITS genotype
Multi-locus sequence type MS1
MS3
MS4
MS7
MLGs
Giant panda
GP1
I-like I-like
GP3
I-like
Type I Type I NA
GP4
I-like
Type I Type I Type I NA
Type I Type I Type I Type I
MLG1
GP2
Type I Type I Type I Type I
QYLP1
EbpC
QYLP3
EbpC
LGLP1
EbpC
Type II NA
LGLP2
EbpC
Type II Type II Type II
Type II Type II Type II Type II Type II
–
EbpC
Type II NA
NA
QYLP2
Type II Type II Type II Type II Type II
Red panda
Type II
Type I
NA
MLG1 – –
– MLG2 – MLG2
Note: NA = not amplified.
(MLST) tool containing more genetic information has been developed (Karim et al., 2014). In the present study, nine, six, six and nine positive amplicons were obtained successfully in loci MS1, MS3, MS4, and MS7, respectively. Sequencing analysis showed two haplotypes in all four loci, with one new type in locus MS1. Combining these haplotypes formed two distinct MLGs. These results showed genetic diversity of E. bieneusi in giant and red pandas. 5. Conclusion This study reported the prevalence and genotypes/subtypes of E. bieneusi in giant and red pandas by ITS combining with microsatellite and minisatellite markers for the first time. The overall infection rate was 10.98%, with 8.70% in giant pandas and 13.89% in red pandas. Two genotypes were detected in pandas based on ITS sequence analysis, with the genotype EbpC in red pandas and the novel genotype I-like in giant pandas. Genetic diversity was observed by MLST tool, and two MLGs were found in pandas. These results suggested that proper control strategies should be taken carefully to protect giant and red pandas from E. bieneusi infection and to avoid them transmit this parasite to human and other animals. Acknowledgments This study was supported by grants from the Program for New Century Excellent Talents in University (Grant No. NCET-13-0489), and the Fund for Basic Scientific Research (Grant No. ZD2012010). References Del Coco, V.F., Córdoba, M.A., Bilbao, G., de Almeida Castro, P., Basualdo, J.A., Santín, M., 2014. First report of Enterocytozoon bieneusi from dairy cattle in Argentina. Vet. Parasitol. 199, 112–115. Didier, E.S., Stovall, M.E., Green, L.C., Brindley, P.J., Sestak, K., Didier, P.J., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Vet. Parasitol. 126, 145–166.
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Didier, E.S., Weiss, L.M., 2006. Microsporidiosis: current status. Curr. Opin. Infect. Dis. 19, 485–492. Feng, Y., Li, N., Dearen, T., Lobo, M.L., Matos, O., Cama, V., Xiao, L., 2011. Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi. Appl. Environ. Microbiol. 77, 4822–4828. He, G., Chen, S., Wang, T., Yan, Y., Zhang, Z., Li, D., Yu, H., Xie, Y., Wang, C., Gu, X., Wang, S., Peng, X., Yang, G., 2012. Sequence analysis of the Bs-Ag1 gene of Baylisascaris schroederi from the giant panda and an evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag1 antigen in mice. DNA Cell Biol. 31, 1174–1181. Henriques-Gil, N., Haro, M., Izquierdo, F., Fenoy, S., del Aguila, C., 2010. Phylogenetic approach to the variability of the microsporidian Enterocytozoon bieneusi and its implications for inter- and intrahost transmission. Appl. Environ. Microbiol. 76, 3333–3342. Karanis, P., Plutzer, J., Halim, N.A., Igori, K., Nagasawa, H., Ongerth, J., Liqing, M., 2007. Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China. Parasitol. Res. 101, 1575–1580. Karim, M.R., Wang, R., Dong, H., Zhang, L., Li, J., Zhang, S., Rume, F.I., Qi, M., Jian, F., Sun, M., Yang, G., Zou, F., Ning, C., Xiao, L., 2014. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Appl. Environ. Microbiol. 80, 1893–1898. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Liu, X., He, T., Zhong, Z., Zhang, H., Wang, R., Dong, H., Wang, C., Li, D., Deng, J., Peng, G., Zhang, L., 2013. A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China. Parasitol. Int. 62, 454–458. Li, W., Li, Y., Song, M., Lu, Y., Yang, J., Tao, W., Jiang, Y., Wan, Q., Zhang, S., Xiao, L., 2015. Prevalence and genetic characteristics of Cryptosporidium, Enterocytozoon bieneusi and Giardia duodenalis in cats and dogs in Heilongjiang province, China. Vet. Parasitol. 208, 125–134. Matos, O., Lobo, M.L., Xiao, L., 2012. Epidemiology of Enterocytozoon bieneusi infection in humans. J. Parasitol. Res. 2012, 981424. Nkinin, S.W., Asonganyi, T., Didier, E.S., Kaneshiro, E.S., 2007. Microsporidian infection is prevalent in healthy people in Cameroon. J. Clin. Microbiol. 45, 2841–2846. Peng, J.J., Jiang, Z.G., Hu, J.C., 2001. Status and conservation of giant panda (Ailuropoda melanoleuca): a review. Folia Zool. 50, 81–88. Rinder, H., Thomschke, A., Dengje, B., Gothe, R., Löscher, T., Zahler, M., 2000. Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle. J. Parasitol. 86, 185–188. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Santín, M., Fayer, R., 2011. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Res. Vet. Sci. 90, 363–371. Sulaiman, I.M., Fayer, R., Lal, A.A., Trout, J.M., Schaefer, F.W., Xiao, L., 2003. Molecular characterization of microsporidia indicates that wild mammals Harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi. Appl. Environ. Microbiol. 69, 4495–4501. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Thellier, M., Breton, J., 2008. Enterocytozoon bieneusi in human and animals, focus on laboratory identification and molecular epidemiology. Parasite 15, 349–358. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Wang, L., Xiao, L., Duan, L., Ye, J., Guo, Y., Guo, M., Liu, L., Feng, Y., 2013. Concurrent infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS Negl. Trop. Dis. 7, e2437. Wang, T., Chen, Z., Yu, H., Xie, Y., Gu, X., Lai, W., Peng, X., Yang, G., 2015. Prevalence of Cryptosporidium infection in captive lesser panda (Ailurus fulgens) in China. Parasitol. Res. 114, 773–776. Widmer, G., Akiyoshi, D.E., 2010. Host-specific segregation of ribosomal nucleotide sequence diversity in the microsporidian Enterocytozoon bieneusi. Infect. Genet. Evol. 10, 122–128. Yonzon, P.B., Hunter, M.L., 1991. Conservation of the red panda Ailurus fulgens. Biol. Conserv. 57, 1–11. Zhang, J.S., Daszak, P., Huang, H.L., Yang, G.Y., Kilpatrick, A.M., Zhang, S., 2008. Parasite threat to panda conservation. Ecohealth 5, 6–9. Zhang, X., Wang, Z., Su, Y., Liang, X., Sun, X., Peng, S., Lu, H., Jiang, N., Yin, J., Xiang, M., Chen, Q., 2011. Identification and genotyping of Enterocytozoon bieneusi in China. J. Clin. Microbiol. 49, 2006–2008. Zhao, W., Zhang, W., Yang, F., Cao, J., Liu, H., Yang, D., Shen, Y., Liu, A., 2014. High prevalence of Enterocytozoon bieneusi in asymptomatic pigs and assessment of zoonotic risk at the genotype level. Appl. Environ. Microbiol. 80, 3699–3707.
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China Ge-Ru Tian a,1, Guang-Hui Zhao a,⇑,1, Shuai-Zhi Du a, Xiong-Feng Hu a, Hui-Bao Wang a, Long-Xian Zhang b, San-Ke Yu a,⇑ a b
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province 712100, China College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province 450002, China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 11 June 2015 Accepted 12 June 2015 Available online 12 June 2015 Keywords: Ailuropoda melanoleuca Ailurus fulgens Enterocytozoon bieneusi Prevalence Genotype
a b s t r a c t Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n = 4) and genotype EbpC (n = 5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens), two precious and endangered animals for wildlife conservation (Yonzon and Hunter, 1991; Peng et al., 2001), are respectively classified as Category I and II protected species in China. Habitats destruction, infective factors, intrinsic (poor reproduction and low densities) and accidental mortality are the major threats of their extinction (Zhang et al., 2008). Of them, pathogen infections have been identified as the most serious threat for survival of wild pandas in China currently (He et al., 2012). Enterocytozoon bieneusi, a unicellular enteric pathogen from the phylum Microspora, can inhabit in small intestines of a wide range of invertebrate and vertebrate hosts including humans, domestic and wild animals, and even birds, causing chronic diarrhea and wasting syndrome (Didier and Weiss, 2006.). E. bieneusi has been ⇑ Corresponding authors. E-mail addresses: [email protected] (G.-H. Zhao), [email protected] (S.-K. Yu). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.meegid.2015.06.015 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
detected in immune-competent individuals, resulting in self-limiting diarrhea (Nkinin et al., 2007). More importantly, it is considered as an opportunistic pathogen which can cause life-threatening diarrhea in immune-compromised individuals especially in AIDS patients raising public health concerns (Li et al., 2015). Therefore, E. bieneusi has been classified as the Category B potentially dangerous pathogen by National Institutes of Health (NIH) (Didier et al., 2004). Complicate population structure of E. bieneusi increased the difficulty to control the infection. Because of a high diversity in the internal transcribed spacer region (ITS) of the ribosomal RNA (rRNA) gene within E. bieneusi isolates, it is widely used for genotyping E. bieneusi (Matos et al., 2012). To date, more than 200 genotypes have been reported in humans and animals based on ITS region (Henriques-Gil et al., 2010). Most of these genotypes represented host-adapted groups (groups 2–8) associated with specific hosts and wastewater, and probably have no significant public health importance, while almost all genotypes in group 1 are frequently found in humans and animals and are considered to be zoonotic (Thellier and Breton, 2008). However, the single ITS marker may be inadequate in identifying its genotypes due to the
G.-R. Tian et al. / Infection, Genetics and Evolution 34 (2015) 32–35
uncertainty about a sexual phase in E. bieneusi lifecycle (Widmer and Akiyoshi, 2010). Recently, a high-resolution multilocus sequencing typing tool (MLST) using three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) markers has been developed (Feng et al., 2011), and successfully used to investigate the genetic diversity and host specificity of E. bieneusi (Widmer and Akiyoshi, 2010). Here, we, for the first time, explored the occurrence and genetic diversity of E. bieneusi in giant and red pandas, and elucidated the zoonotic potential of E. bieneusi with the ITS-based sequence typing and the MLST tool. 2. Materials and methods 2.1. Ethics statement Faecal samples of this study were collected under the permission of the relevant zoos. During sample collection, the animal welfare was taken into consideration. All procedures were reviewed and approved by the Animal Welfare Committee of Northwest A&F University. 2.2. Sampling Sampling was carried out from September 2013 to June 2014 in Rare Wildlife Rescue Breeding Research Center and Xi’an Qinling Wildlife Park in Shaanxi province, northwestern China. A total of 82 fresh faecal samples were collected from 46 giant pandas and 36 red pandas. Each sample was collected from each panda on the ground immediately after defecation and was then put into a plastic container labeled with the time and number. All faecal specimens were stored at 4 °C in 2.5% potassium dichromate prior to DNA extraction. 2.3. DNA extraction Faecal specimens were washed with distilled water until supernatant became clear. Genomic DNA was extracted from approximately 0.2 g of washed faecal specimens using the E.Z.N.AÒ Stool DNA kit (Omega Bio-tek Inc., GA, USA) according to the manufacturer-recommended procedures. The extracted DNA was stored at 20 °C until PCR analysis. 2.4. PCR amplification A nested PCR protocol was used to amplify of 390 bp fragment of the partial ribosomal ITS region of E. bieneusi (Karim et al., 2014). The PCR amplification primers and amplification conditions of ITS gene were referred to previous study (Sulaiman et al., 2003). Positive specimens were further characterized by MLST analyses using MS1, MS3, MS4 and MS7 according to Feng et al. (2011). The TaKaRa Ex Taq DNA polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all PCR amplifications. A negative control without DNA was included in all PCR tests. All the secondary PCR products were subjected to electrophoresis in a 1% agarose gel containing ethidium bromide. 2.5. Sequence analysis The secondary PCR products and inner set of the primers of all PCR-positive samples were sent to the Sangon Company (Shanghai, China) using ABI 3730xl automated DNA sequencer (Big Dye Terminator Chemistry) for DNA sequencing in both directions. Obtained sequences were aligned using the Clustal X 1.81 (Thompson et al., 1997) and compared with reference sequences from GenBank. Aligned sequences were used to determine
33
genotypes/subtypes by using the Basic Local Alignment Search Tool (BLAST) in National Center for Biotechnology Information (NCBI). 2.6. Phylogenetic analysis To assess the genetic relationship of ITS genotypes of E. bieneusi obtained in the present study and those published in the previous studies, a phylogenetic analysis was performed by constructing a neighboring-joining tree (Saitou and Nei, 1987) using the program Mega 5.0 (Tamura et al., 2011) based on the evolutionary distances calculated by Kimura-2-parameter model (Kimura, 1980). The reliability of these trees was assessed using the bootstrap analysis with 1000 replicates. 3. Results 3.1. Prevalence and genotypes of E. bieneusi in pandas Of 82 faecal specimens examined by nested PCR amplifying the ITS gene of E. bieneusi, nine (10.98%) were positive for infection of E. bieneusi, with four (8.70%/46) in giant pandas and five (13.89%/36) in red pandas. Sequence analysis showed two genotypes in this study, including one known genotype EbpC (n = 5) in red pandas and one novel genotype I-like (n = 4) in giant pandas. The novel genotype I-like had five nucleotide polymorphisms (transversions: T/A; C/G; T/G; C/G; transition: A/G) in comparison with the genotype I (KJ668738), with 99% of homology. 3.2. Phylogenetic relationships of E. bieneusi based on sequences of ITS regions A phylogenetic analysis using NJ method based on the ITS gene sequences of E. bieneusi indicated that all positive samples in the present study belonged to two groups. Five positive specimens of red panda (QYLP1, QYLP2, QYLP3, LGLP1 and LGLP2) were clustered into 1d of zoonotic group 1, and four positive samples of giant panda (GP1–GP4) located into group 2 (Fig. 1). 3.3. Multilocus sequence typing of E. bieneusi ITS-positive specimens were further characterized using three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4). Nine, six, six and nine positive amplicons were amplified successfully in loci MS1, MS3, MS4, and MS7, respectively. Sequencing analysis showed two haplotypes in all four loci and formed two distinct MLGs (Table 1). Of all haplotypes in four loci, BLAST search in GenBank showed that only the Type I of MS1 locus was a new one, which had two nucleotide polymorphism (transversion: T/A; transition: G/A) with E. bieneusi isolate 147 clone 5 from Homo sapiens in Uganda (KF261756), with 99% of homology. 4. Discussion Recently, the epidemiological study of E. bieneusi has been employed (Zhao et al., 2014). E. bieneusi has been reported in humans and many animals (cattle, pigs, dogs, cats, horses, goats, birds, etc) worldwide (Rinder et al., 2000; Santín and Fayer, 2011). Some parasitic protozoa have been reported in pandas. Cryptosporidium giant panda genotype, Cryptosporidium parvum mouse genotype, and Cryptosporidium andersoni have been detected in giant and red pandas (Karanis et al., 2007; Liu et al., 2013; Wang et al., 2015). The present study reported the presence of E. bieneusi infection in giant and red pandas for the first time. Infective rate (13.89%) of E. bieneusi for red pandas in the present
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G.-R. Tian et al. / Infection, Genetics and Evolution 34 (2015) 32–35
Fig. 1. Phylogenetic relationships of ITS nucleotide sequences of E. bieneusi genotypes in this study and known E. bieneusi genotypes was inferred by a neighbor-joining analysis based on genetic distance by the Kimura two-parameter model. The numbers on the branches are percent bootstrapping values from 1000 replicates, with more than 50% shown in tree. The genotypes found in giant and red pandas in this study were marked with black triangle and circle, respectively.
study was higher than that (6.36%) of C. andersoni in captive red pandas (Wang et al., 2015). Previous studies have indicated that humans could be infected mainly through E. bieneusi in human and animal feces (Sulaiman et al., 2003). Therefore, zoonotic potential of giant and red pandas to transmit E. bieneusi to humans should be further evaluated by more samples investigation and animal adaptation tests. Two genotypes were identified in this study by analyzing ITS gene sequences of E. bieneusi, namely novel genotypes I-like (n = 4) and EbpC (n = 5). The homology was 99% between genotype I and genotype I-like. The genotype I was previously thought to be cattle-specific (Del Coco et al., 2014), but it has also been reported in humans (Zhang et al., 2011), suggesting zoonotic potential of this genotype. The genotype EbpC has an extensive host range and was previously found in humans, monkeys, pigs, dogs, and other wildlife animals (Del Coco et al., 2014). The present study firstly reported it in red pandas, expanding host range of this
genotype. In the genetic tree re-constructed based on ITS sequences, five specimens of red pandas belonged to 1d of zoonotic group 1 (subgroups 1a–1h), clustered with the genotype EbpC in human and the genotype HenanIII in humans (Fig. 1), suggesting zoonotic potential of the genotype EbpC. Four specimens of giant pandas were classified into the group 2 with genotype I and PtEBXI in cattle (Fig. 1). Most genotypes of this group are host-adapted and probably have no significant public health risk, but some genotypes were also found in humans, such as genotype I and BEB6 (Zhang et al., 2011; Wang et al., 2013). Thus, it remains unknown whether the novel genotype I-like has a potential of zoonotic transmission currently. Therefore, more studies are needed to assess zoonotic potential of these genotypes and the role of pandas as a source of zoonotic genotypes of E. bieneusi to humans. In order to better substantiate public health significance of animal-derived E. bieneusi isolates and to trace sources of human E. bieneusi infections, subsequently, multilocus sequence typing
G.-R. Tian et al. / Infection, Genetics and Evolution 34 (2015) 32–35 Table 1 Multi-locus sequence typing of Enterocytozoon bieneusi in giant and red pandas in China. Species
Isolate
ITS genotype
Multi-locus sequence type MS1
MS3
MS4
MS7
MLGs
Giant panda
GP1
I-like I-like
GP3
I-like
Type I Type I NA
GP4
I-like
Type I Type I Type I NA
Type I Type I Type I Type I
MLG1
GP2
Type I Type I Type I Type I
QYLP1
EbpC
QYLP3
EbpC
LGLP1
EbpC
Type II NA
LGLP2
EbpC
Type II Type II Type II
Type II Type II Type II Type II Type II
–
EbpC
Type II NA
NA
QYLP2
Type II Type II Type II Type II Type II
Red panda
Type II
Type I
NA
MLG1 – –
– MLG2 – MLG2
Note: NA = not amplified.
(MLST) tool containing more genetic information has been developed (Karim et al., 2014). In the present study, nine, six, six and nine positive amplicons were obtained successfully in loci MS1, MS3, MS4, and MS7, respectively. Sequencing analysis showed two haplotypes in all four loci, with one new type in locus MS1. Combining these haplotypes formed two distinct MLGs. These results showed genetic diversity of E. bieneusi in giant and red pandas. 5. Conclusion This study reported the prevalence and genotypes/subtypes of E. bieneusi in giant and red pandas by ITS combining with microsatellite and minisatellite markers for the first time. The overall infection rate was 10.98%, with 8.70% in giant pandas and 13.89% in red pandas. Two genotypes were detected in pandas based on ITS sequence analysis, with the genotype EbpC in red pandas and the novel genotype I-like in giant pandas. Genetic diversity was observed by MLST tool, and two MLGs were found in pandas. These results suggested that proper control strategies should be taken carefully to protect giant and red pandas from E. bieneusi infection and to avoid them transmit this parasite to human and other animals. Acknowledgments This study was supported by grants from the Program for New Century Excellent Talents in University (Grant No. NCET-13-0489), and the Fund for Basic Scientific Research (Grant No. ZD2012010). References Del Coco, V.F., Córdoba, M.A., Bilbao, G., de Almeida Castro, P., Basualdo, J.A., Santín, M., 2014. First report of Enterocytozoon bieneusi from dairy cattle in Argentina. Vet. Parasitol. 199, 112–115. Didier, E.S., Stovall, M.E., Green, L.C., Brindley, P.J., Sestak, K., Didier, P.J., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Vet. Parasitol. 126, 145–166.
35
Didier, E.S., Weiss, L.M., 2006. Microsporidiosis: current status. Curr. Opin. Infect. Dis. 19, 485–492. Feng, Y., Li, N., Dearen, T., Lobo, M.L., Matos, O., Cama, V., Xiao, L., 2011. Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi. Appl. Environ. Microbiol. 77, 4822–4828. He, G., Chen, S., Wang, T., Yan, Y., Zhang, Z., Li, D., Yu, H., Xie, Y., Wang, C., Gu, X., Wang, S., Peng, X., Yang, G., 2012. Sequence analysis of the Bs-Ag1 gene of Baylisascaris schroederi from the giant panda and an evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag1 antigen in mice. DNA Cell Biol. 31, 1174–1181. Henriques-Gil, N., Haro, M., Izquierdo, F., Fenoy, S., del Aguila, C., 2010. Phylogenetic approach to the variability of the microsporidian Enterocytozoon bieneusi and its implications for inter- and intrahost transmission. Appl. Environ. Microbiol. 76, 3333–3342. Karanis, P., Plutzer, J., Halim, N.A., Igori, K., Nagasawa, H., Ongerth, J., Liqing, M., 2007. Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China. Parasitol. Res. 101, 1575–1580. Karim, M.R., Wang, R., Dong, H., Zhang, L., Li, J., Zhang, S., Rume, F.I., Qi, M., Jian, F., Sun, M., Yang, G., Zou, F., Ning, C., Xiao, L., 2014. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Appl. Environ. Microbiol. 80, 1893–1898. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Liu, X., He, T., Zhong, Z., Zhang, H., Wang, R., Dong, H., Wang, C., Li, D., Deng, J., Peng, G., Zhang, L., 2013. A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China. Parasitol. Int. 62, 454–458. Li, W., Li, Y., Song, M., Lu, Y., Yang, J., Tao, W., Jiang, Y., Wan, Q., Zhang, S., Xiao, L., 2015. Prevalence and genetic characteristics of Cryptosporidium, Enterocytozoon bieneusi and Giardia duodenalis in cats and dogs in Heilongjiang province, China. Vet. Parasitol. 208, 125–134. Matos, O., Lobo, M.L., Xiao, L., 2012. Epidemiology of Enterocytozoon bieneusi infection in humans. J. Parasitol. Res. 2012, 981424. Nkinin, S.W., Asonganyi, T., Didier, E.S., Kaneshiro, E.S., 2007. Microsporidian infection is prevalent in healthy people in Cameroon. J. Clin. Microbiol. 45, 2841–2846. Peng, J.J., Jiang, Z.G., Hu, J.C., 2001. Status and conservation of giant panda (Ailuropoda melanoleuca): a review. Folia Zool. 50, 81–88. Rinder, H., Thomschke, A., Dengje, B., Gothe, R., Löscher, T., Zahler, M., 2000. Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle. J. Parasitol. 86, 185–188. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Santín, M., Fayer, R., 2011. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Res. Vet. Sci. 90, 363–371. Sulaiman, I.M., Fayer, R., Lal, A.A., Trout, J.M., Schaefer, F.W., Xiao, L., 2003. Molecular characterization of microsporidia indicates that wild mammals Harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi. Appl. Environ. Microbiol. 69, 4495–4501. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Thellier, M., Breton, J., 2008. Enterocytozoon bieneusi in human and animals, focus on laboratory identification and molecular epidemiology. Parasite 15, 349–358. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Wang, L., Xiao, L., Duan, L., Ye, J., Guo, Y., Guo, M., Liu, L., Feng, Y., 2013. Concurrent infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS Negl. Trop. Dis. 7, e2437. Wang, T., Chen, Z., Yu, H., Xie, Y., Gu, X., Lai, W., Peng, X., Yang, G., 2015. Prevalence of Cryptosporidium infection in captive lesser panda (Ailurus fulgens) in China. Parasitol. Res. 114, 773–776. Widmer, G., Akiyoshi, D.E., 2010. Host-specific segregation of ribosomal nucleotide sequence diversity in the microsporidian Enterocytozoon bieneusi. Infect. Genet. Evol. 10, 122–128. Yonzon, P.B., Hunter, M.L., 1991. Conservation of the red panda Ailurus fulgens. Biol. Conserv. 57, 1–11. Zhang, J.S., Daszak, P., Huang, H.L., Yang, G.Y., Kilpatrick, A.M., Zhang, S., 2008. Parasite threat to panda conservation. Ecohealth 5, 6–9. Zhang, X., Wang, Z., Su, Y., Liang, X., Sun, X., Peng, S., Lu, H., Jiang, N., Yin, J., Xiang, M., Chen, Q., 2011. Identification and genotyping of Enterocytozoon bieneusi in China. J. Clin. Microbiol. 49, 2006–2008. Zhao, W., Zhang, W., Yang, F., Cao, J., Liu, H., Yang, D., Shen, Y., Liu, A., 2014. High prevalence of Enterocytozoon bieneusi in asymptomatic pigs and assessment of zoonotic risk at the genotype level. Appl. Environ. Microbiol. 80, 3699–3707.
