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First report of Blastocystis infections in cattle in China
Veterinary Parasitology 246 (2017) 38–42
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Short communication
First report of Blastocystis infections in cattle in China Weining Zhu ⁎ Wei Lia, a b
a,1
a,1
, Wei Tao
b
a
a
MARK a
a
, Binbin Gong , Hang Yang , Yijing Li , Mingxin Song , Yixin Lu ,
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, China School of Chemical Engineering & Biotechnology, Xingtai University, Xingtai, Hebei 054001, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Blastocystis Prevalence Dairy cattle Subtyping Zoonotic potential
Blastocystis is one of the most common intestinal protists of humans and can also infect a variety of other mammals and birds. Blastocystis infections and subtype distribution in cattle have been documented, while nothing is known about those in China. Herein, a total of 526 dairy cattle from northeast China were sampled and investigated for the prevalence and genetic characteristics of Blastocystis and the potential role of bovine animals in zoonotic transmission of Blastocystis. The parasite was identified in 54 (10.3%) fecal specimens by nested PCR and DNA sequencing of the small subunit ribosomal RNA gene. Sequence analysis enabled identification of four Blastocystis subtypes (STs). Among those, subtype ST10 (75.9%, 41/54) has the highest frequency, followed by ST14 (18.5%, 10/54), ST4 (3.7%, 2/54), and ST5 (1.9%, 1/54). High prevalence and widespread distribution of ST10 and ST14 in cattle observed herein, together with analysis of their host distribution patterns in earlier studies, indicated some host-adapted potential in the two subtypes. The identification of human-pathogenic subtypes ST4 and ST5 might imply a potential zoonotic risk of cattle origin. This is the first study exploring the prevalence and genetic characteristics of Blastocystis in cattle in China. The host range of subtype ST4 was extended. The findings of this study should be helpful for a better understanding of the epidemiology and public health potential of Blastocystis.
1. Introduction Blastocystis, one of the most ubiquitous parasites of mammalian intestinal tracts, was reported in a variety of vertebrate hosts including humans (Fayer et al., 2012; Parkar et al., 2010; Roberts et al., 2013). The pathogenicity of Blastocystis is related to such factors including subtype variation and host immune status, while the exact mechanism remains controversial (Cirioni et al., 1999; Elwakil and Hewedi, 2010). Blastocystis is commonly transmitted through sewage-contaminated water and food that contains cysts and the fecal-oral route is a major mode of transmission (Leelayoova et al., 2004; Leelayoova et al., 2008; Yoshikawa et al., 2004b). Close contacts with infected animals may constitute risks of zoonotic transmission of Blastocystis (Osman et al., 2015; Wang et al., 2014). Genetic polymorphisms of the small subunit ribosomal RNA (SSU rRNA) gene enabled identification of 17 distinct Blastocystis subtypes (STs) in different human and animal species (Alfellani et al., 2013b; Stensvold et al., 2009; Wang et al., 2014). Nine subtypes (ST1–ST9) have been reported in human Blastocystis infections, with ST3 and ST1 most frequently examined. The variation in human Blastocystis subtype
⁎
1
by geographical region has been analyzed, with predominance of ST1 (followed by ST2 and ST3) in America, ST3 (ST4 and ST1) in Europe, ST3 (ST1 and ST6) in Africa, ST3 (ST1 and ST4) in Australia, and ST3 (ST1 and ST2) in Asia (Alfellani et al., 2013a). The host specificity and pathogenic potential among the various Blastocystis subtypes differ considerably as well, which might represent contributors to the symptom variability in patients with Blastocystis (Dominguez-Marquez et al., 2009; Khademvatan et al., 2017; Mohamed et al., 2017; Souppart et al., 2010). The rest eight subtypes such as ST10 and ST14 usually circulate in specific animal hosts and have never appeared in human infections. Most of the animal species surveyed can harbor potentially zoonotic Blastocystis subtypes and thus can be potential reservoirs of human infections (Khademvatan et al., 2017; Stensvold and Clark, 2016; Wang et al., 2014). In recent decades, Blastocystis infections have been repeatedly reported in a wide range of domestic animals and some factors associated with zoonotic transmission have been evaluated as well (Cian et al., 2017; Osman et al., 2015). Several reports have indicated the prevalence and distribution of Blastocystis subtypes ST1, ST3, ST5, ST6, ST10, and ST14 in cattle from Iran, USA, UK, Libya, Denmark, Japan,
Corresponding author. E-mail address: [email protected] (W. Li). These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.vetpar.2017.09.001 Received 26 July 2017; Received in revised form 31 August 2017; Accepted 1 September 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.
Veterinary Parasitology 246 (2017) 38–42
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screen positive PCRs.
and Colombia (Alfellani et al., 2013b; Badparva et al., 2015; Ramirez et al., 2014; Santin et al., 2011; Stensvold et al., 2009; Yoshikawa et al., 2004a). This study was conducted to investigate 526 dairy cattle of various ages in northeast China for the prevalence and genetic characteristics of Blastocystis as well as to assess the potential role of bovine animals in zoonotic transmission of Blastocystis.
2.4. Sequence analysis and phylogeny PCR amplicons of expected size were sequenced in both directions at the Beijing Genomics Institute. Raw sequences were proofread and edited manually with Chromas Pro 1.33 (Technelysium, Queensland, Australia) and BioEdit 7.0 (http://www.mbio.ncsu.edu/BioEdit/ bioedit.html). To determine Blastocystis subtypes, each of the corrected sequences was compared with GenBank sequences by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/). All the sequences obtained in this study were aligned with the reference sequences of known Blastocystis subtypes using MAFFT 7 (http://mafft.cbrc.jp/ alignment/software/). Their phylogenetic relationships were analyzed by the neighbor-joining (NJ) method under the Kimura 2-parameter model and the maximum parsimony (MP) method with bootstrap values out of 1000 replicates, using the MEGA 5.0 (http://www. megasoftware.net/). Unique SSU rRNA sequences gained herein were deposited in GenBank under accession numbers of MF573940 to MF573945.
2. Materials and methods 2.1. Ethics statement The protocol of the current study was reviewed and approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (no. SRM-08). Before the experiment, we contacted the farm owners for their permissions. No specific permits were required for the described field studies. And the locations where we sampled are not privately-owned or protected in any way. 2.2. Specimen collection A total of 526 fecal specimens were collected from cattle in cities Harbin (n = 196) in October 2013, Qiqihar (n = 190) in April 2014, and Daqing (n = 140) in July 2014. The three cities are closely linked and located in northeast China. Three age categories of animals were seen in Harbin, with preweaned animals aged < 3 months (n = 69), weaned animals aged 3–12 months (n = 61), and yearlings and adults aged > 12 months (n = 66) (Table 1). Specimens from Harbin were transferred individually into 50-ml bottles filled with 2.5% (m/v) potassium dichromate and stored at 4 °C. Only preweaned calves were kept on the farm in Qiqihar, and weaned cattle in Daqing likewise (Table 1). Specimens from the two locations were stored frozen in disposable plastic bags at −20 °C. All the specimens were collected as quickly as possible after defecation. One specimen per animal was used in this study.
2.5. Statistical analysis We used chi-square test in SPSS 17.0 (SPSS Inc., Chicago, IL, USA) to analyze the prevalence difference between animal groups. P values of < 0.05 were considered statistically significant. 3. Results 3.1. Prevalence of Blastocystis in cattle Nested PCRs and sequence analysis of the SSU rRNA gene identified Blastocystis in 54 of 526 (10.3%) cattle fecal specimens, with a prevalence of 23.0% (45/196) in Harbin and 6.4% (9/140) in Daqing (Table 1). The prevalence difference between the two cities was significant (p < 0.01, χ2 = 16.5) (Table 1). In addition, weaned cattle (36.1%, 22/61) from Harbin had a significantly higher prevalence than those (6.4%, 9/140) within the same age range from Daqing (p < 0.01, χ2 = 28.6) (Table 1). There were no Blastocystis-positive isolates found in preweaned cattle from Qiqihar (Table 1). In Harbin, the prevalence of Blastocystis in weaned cattle (36.1%, 22/61) was significantly higher than that in preweaned calves (18.8%, 13/69; p < 0.05, χ2 = 4.9) and that in yearlings and adults (15.2%, 10/66; p < 0.01, χ2 = 7.4) (Table 1). When the other two study areas were considered, the differences in the overall prevalence between weaned and preweaned cattle (15.4%, 31/201 versus 5.0%, 13/259) and between adult and preweaned cattle (15.2%, 10/66 versus 5.0%, 13/259) were significant (p < 0.01, χ2 = 14.2 and 6.7, respectively) (Table 1).
2.3. DNA extraction and PCR Prior to DNA extraction, fecal specimens stored in potassium dichromate were washed twice in double-distilled H2O by centrifugation at 12,000 × g for 5 min at room temperature. Genomic DNA was extracted from 0.2 g of feces using Stool DNA rapid extraction kits (TIANGEN, China) as instructed by the manufacturer. Nested PCR based on the SSU rRNA gene was performed using the primers RD3 (5′GGGATCCTGATCCTTCCGCAGGTTCACCTAC-3′) and RD5 (5′GGAAGCTTATCTGGTTGATCCTGCCAGTA-3′) that amplified a fragment of about 1780 bp in length in the first round PCR (Parkar et al., 2010) and the primers Blasto 2F (5′-TCTGGTTGATCCTGCCAGT-3′) and Blasto 2R (5′-AGCTTTTTAACTGCAACAACG-3′) that amplified a fragment of about 600 bp in length in the second round PCR (Souppart et al., 2009). PCR reaction systems used herein were exactly the same as those described (Souppart et al., 2009). Two parallel PCRs were conducted for each specimen using 2 μl DNA extract per reaction. Suitable positive and negative controls were placed with each plate. All PCRs were conducted in a GeneAmp PCR System 9700 (Applied Biosystems) thermal cycler. Agarose gel electrophoresis (1.5%) was performed to
3.2. Subtype distribution Sequencing is available for all 54 Blastocystis-positive isolates. Four Blastocystis subtypes were identified by sequence polymorphisms of the SSU rRNA gene (Table 1). Among those, subtype ST10 (75.9%, 41/54) has the highest frequency, followed by ST14 (18.5%, 10/54), ST4
Table 1 Prevalence and subtype distribution of Blastocystis in dairy cattle from northeast China. City
Harbin Qiqihar Daqing Total
No. of cattle
196 190 140 526
No. of subtype
Prevalence in age groups (% [positive no./total no.])
ST4
ST5
ST10
ST14
< 3 months
3–12 months
> 12 months
2 0 0 2
1 0 0 1
38 0 3 41
4 0 6 10
18.8 (13/69) 0.0 (0/190)
36.1 (22/61)
15.2 (10/66)
6.4 (9/140) 15.4 (31/201)
15.2 (10/66)
5.0 (13/259)
39
Total prevalence (% [positive no./total no.])
23.0 (45/196) 0.0 (0/190) 6.4 (9/140) 10.3 (54/526)
Veterinary Parasitology 246 (2017) 38–42
W. Zhu et al.
Table 2 Blastocystis subtypes and their frequency in cattle around the world. Country
China Colombia Denmark Iran Japan Libya UK USA Total
No. of subtype (frequency [%]) ST1
ST3
12 (60.0)
8 (40.0)
2 (12.5)
2 (10.5) 3 (12.5)
ST4
ST5
2 (3.7)
1 (1.9) 3 (12.0) 9 (47.4)
13 (8.0)
2 (1.2)
16 (9.8)
ST10
ST14
41 (75.9)
10 (18.5)
Reference
10.3 (54/526) 80.0 (20/25)
This study (Ramirez et al., 2014) (Stensvold et al., 2009) (Badparva et al., 2015) (Yoshikawa et al., 2004a) (Alfellani et al., 2013b) (Alfellani et al., 2013b) (Fayer et al., 2012; Santin et al., 2011)
Mixed
22 (88.0) 2 (10.5) 6 (75.0)
2 (13.3) 1 (14.3)
1 (14.3)
15 (9.2)
ST6
Total prevalence (% [positive no./total no.])
8 (4.9)
2 (10.5)
9.7 (19/196) 41.7 (15/36) 22.6 (7/31) 19.0 (16/84)
6 (40.0) 3 (42.9) 13 (81.3)
2 (13.3) 1 (6.3)
5 (33.3) 2 (28.6) 2 (12.5)
85 (52.1)
13 (8.0)
11 (6.7)
worldwide and population characteristics of the parasite varied by host types and geographical locations (Navarro et al., 2008; Ramirez et al., 2014; Wang et al., 2014). However, the relevant studies were rarely seen in China. This study surveyed Blastocystis infections in cattle and presented an overall prevalence of 10.3%. The prevalence is similar with that (9.7%) reported in cattle from Iran (Badparva et al., 2015), but lower than those from USA (19%), UK (22.6%), Libya (41.7%), and Colombia (80%) (Alfellani et al., 2013b; Fayer et al., 2012; Ramirez et al., 2014; Santin et al., 2011). Likewise, we found significant difference in Blastocystis prevalence among animal groups from different cities in this study. The prevalence difference may result from variations of animal ages, seasonality, and ecological environments. A significantly lower prevalence observed in preweaned calves than that in weaned, yearling, and adult cattle may be attributed to immune protection accomplished by maternal antibodies (Stenzel and Boreham, 1996; Zierdt, 1991). This would also be responsible for the absence of detection of Blastocystis in preweaned cattle from Qiqihar. Thus far, subtyping on the basis of the SSU rRNA gene has facilitated definition of 17 valid Blastocystis subtypes (ST1–ST17) (Alfellani et al., 2013b; Stensvold et al., 2009; Wang et al., 2014), four (ST10, ST14,
(3.7%, 2/54), and ST5 (1.9%, 1/54) (Table 2). Forty-five cattle in Harbin were affected with subtypes ST4 (n = 2), ST5 (n = 1), ST10 (n = 38), and ST14 (n = 4), and nine cattle in Daqing with subtypes ST10 (n = 3) and ST14 (n = 6) (Table 1). Subtypes ST4 (n = 2) and ST5 (n = 1) with zoonotic potential parasitized three preweaned calves in Harbin (Table 1). There were no mixed infections observed. 3.3. Phylogenetic analysis BLAST comparison to known sequence databases in GenBank recognized two novel partial SSU rRNA sequences from isolates HC104 and HC158 (Fig. 1). Sequences of isolates HC158 and HC104 differed from a GenBank sequence under accession number of KC148207 by one single nucleotide polymorphism (SNP) and four SNPs, respectively. They were both grouped with the known isolates that belong to subtype ST10 in phylogenetic analysis using NJ and MP methods (Fig. 1). 4. Discussion Blastocystis infections occurred in lots of human and animal species
Fig. 1. Phylogenetic relationships among nucleotide sequences of Blastocystis small subunit ribosomal RNA (SSU rRNA) gene using the neighbor-joining method (panel A) and the maximum parsimony method (panel B). The trees were rooted with GenBank sequence U37107. Bootstrap values greater than 50% from 1000 pseudoreplicates were shown. The sequences indicated by filled triangles are new SSU rRNA nucleotide sequences found in this study.
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Table 3 Host and geographic ranges of Blastocystis subtypes identified in this study. Subtype
Hosta (locationb)
Reference
ST4
Human (China, Australia, Colombia, Denmark, France, Germany, Greece, Iran, Ireland, Italy, Japan, KSA, Laos, Lebanon, Liberia, Malaysia, Netherlands, Nigeria, North Cyprus, Pakistan, Philippines, Romania, Senegal, Singapore, Spain, Sweden, Thailand, Tunisia, Turkey, UK and USA), NHP (Denmark, France, Japan, Spain and UK), Cattle (China), Buffalo (Nepal), Sheep (Nepal), Deer (Australia), Pig (Nepal), Dog (India and Philippines), Leopard (Australia), Kangaroo (Australia), Rat (Denmark, France, Japan, Singapore and USA), Chicken (Japan), Ostrich (Australia and France), Bird (Japan) Human (Bolivia, Germany, Iran, Malaysia, Pakistan, Philippines, Turkey and USA), NHP (China, Denmark, France, Tanzania and UK), Cattle (China, Denmark, Iran, Libya and UK), Rhinoceros (UK), Camel (Libya), Deer (UK), Pig (China, Australia, Cambodian, Denmark, Japan, Spain, UK, USA and Vietnam), Dog (India and Philippines), Capybara (France), Rat (UK), Tapir (France), Ostrich (China and France) NHP (China, Denmark), Cattle (China, Denmark, Libya, UK and USA), Anoa (UK), Yak (China), Bison (France), Takin (China), Bushbuck (China, France), Eland (China), Oryx (China and France), Sheep (Denmark, France, Libya and UK), Deer (China, Denmark, France and Mauritius), Camel (China, France, Libya and UK), Horse (China), Pig (China), Dog (France), Kangaroo (China), Ostrich (China) Cattle (China, Libya and USA), Yak (China), Bison (France), Takin (China), Bushbuck (China and France), Eland (China and France), Wildebeest (France), Camel (China and Libya), Sheep (Czech Republic and France), Deer (France), Lechwe (China)
(Alfellani et al., 2013a; Bart et al., 2013; Belleza et al., 2016; Ben Abda et al., 2017; Cian et al., 2017; El Safadi et al., 2014; El Safadi et al., 2013; Forsell et al., 2016; Lee et al., 2012; Matiut and Hritcu, 2014; Mohamed et al., 2017; Noel et al., 2005; Noradilah et al., 2016; Ramirez et al., 2017; Roberts et al., 2013; Sanchez et al., 2017; Santin et al., 2011; Seyer et al., 2017; Stensvold et al., 2012; Stensvold et al., 2009; Wang et al., 2013; Zhan et al., 2014), this study
ST5
ST10
ST14
a b
(Alfellani et al., 2013a; Alfellani et al., 2013b; Belleza et al., 2016; Cian et al., 2017; Khademvatan et al., 2017; Koltas and Eroglu, 2016; Petrasova et al., 2011; Ramirez et al., 2016; Roberts et al., 2013; Santin et al., 2011; Song et al., 2017; Stensvold et al., 2009; Wang et al., 2013; Zhao et al., 2017), this study
(Alfellani et al., 2013b; Cian et al., 2017; Noradilah et al., 2016; Osman et al., 2015; Song et al., 2017; Stensvold et al., 2009; Zhao et al., 2017), this study
(Alfellani et al., 2013b; Cian et al., 2017; Zhao et al., 2017), this study
NHP: nonhuman primates. KSA: Kingdom of Saudi Arabia.
ST4 was extended. Susceptible populations should be alert to the possibility of transmission of Blastocystis subtypes ST4 and ST5 from cattle sources.
ST4, and ST5) of which were the contributors to cattle infections in China. Of those, ST10 and ST14 represent the most widely distributed subtypes in cattle. A previous study indicated the unique presence of Blastocystis subtypes ST10 and ST14 in cattle from the USA (Fayer et al., 2012). As seen in Table 2, ST10 was the most dominant subtype identifed in cattle from Libya, Denmark, and the UK (Alfellani et al., 2013b; Stensvold et al., 2009). Table 3 displayed the host and geographic distributions of the four Blastocystis subtypes identified in this study. As shown in Table 3, over 80 percent of cases of ST10 infection and 100 percent of cases of ST14 infection appeared in ruminants (cattle, sheep, camel, deer, etc.), in which bovine animals possess the highest infection frequency. The data revealed some host-adapted potential in the two subtypes. It is of interest to note two novel SSU rRNA nucleotide sequences were obtained in this study, which differed by less than 5% from the existing ST10 sequences in GenBank. In this study, 5% nucleotide sequence divergence was considered as a benchmark for naming a new subtype as previously instructed (Alfellani et al., 2013b). Therefore, the two isolates should go to subtype ST10 groups, and this is supported by observing characteristics of the subtype clustering in phylogenetic analysis (Fig. 1). In addition to ST10 and ST14, we also determined two potentially zoonotic subtypes ST4 and ST5 with broad host and geographic ranges as reflected in Table 3. Subtype ST5 frequently appeared in human Blastocystis infections around the world, which can also colonize nonhuman primates, mammals, rodents, and birds (Table 3). Its repeated presence in cattle infections in China, Denmark, Iran, Libya, and the UK as summarized in Table 2 implied zoonotic transmission from cattle origin was a possibility. This is the first study reporting ST4 in cattle. Compared to ST5, ST4 appears to have much wider host spectrum and global distribution as observed in Table 3. This subtype has been documented in human infections in south China (Zhan et al., 2014). Taken together, the two subtypes are of public health importance. Moreover, the capability of bovine animals in carrying human-pathogenic Blastocystis subtypes ST1, ST3, and ST6 as shown in Table 2 may also constitute a risk for zoonotic transmission. Nevertheless, further efforts are needed to fully understand the exact role of cattle in zoonotic transmission of Blastocystis. In conclusion, here is the first study that explored the prevalence and genetic traits of Blastocystis in cattle from China. The host range of
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Hyg. 88, 1203–1206. El Safadi, D., Gaayeb, L., Meloni, D., Cian, A., Poirier, P., Wawrzyniak, I., Delbac, F., Dabboussi, F., Delhaes, L., Seck, M., Hamze, M., Riveau, G., Viscogliosi, E., 2014. Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect. Dis. 14, 164. Elwakil, H.S., Hewedi, I.H., 2010. Pathogenic potential of Blastocystis hominis in laboratory mice. Parasitol. Res. 107, 685–689. Fayer, R., Santin, M., Macarisin, D., 2012. Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitol. Res. 111, 1349–1355. Forsell, J., Granlund, M., Samuelsson, L., Koskiniemi, S., Edebro, H., Evengard, B., 2016. High occurrence of Blastocystis sp. subtypes 1–3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania. Parasite Vector 9, 370. Khademvatan, S., Masjedizadeh, R., Yousefi-Razin, E., Mahbodfar, H., Rahim, F., Yousefi, E., Foroutan, M., 2017. PCR-based molecular characterization of Blastocystis hominis subtypes in southwest of Iran. J. Infect. Public Health. http://dx.doi.org/10.1016/j. jiph.2017.03.009. Koltas, I.S., Eroglu, F., 2016. Subtype analysis of Blastocystis isolates using SSU rRNA-DNA sequencing in rural and urban population in southern Turkey. Exp. Parasitol. 170, 247–251. Lee, L.I., Chye, T.T., Karmacharya, B.M., Govind, S.K., 2012. Blastocystis sp.: waterborne zoonotic organism, a possibility? Parasite Vector 5, 130. Leelayoova, S., Rangsin, R., Taamasri, P., Naaglor, T., Thathaisong, U., Mungthin, M., 2004. Evidence of waterborne transmission of Blastocystis hominis. Am. J. Trop. Med. Hyg. 70 (76), 658–662. Leelayoova, S., Siripattanapipong, S., Thathaisong, U., Taamasri, P., Naaglor, T., Piyaraj, P., Mungthin, M., 2008. Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand. Am. J. Trop. Med. Hyg. 79 (73), 401–406. Matiut, D.S., Hritcu, L., 2014. The pathogenic role of Blastocystis isolated from patients with irritable bowel syndrome and colitis from Iasi, Romania. Acta Parasitol. 60, 116–123. Mohamed, A.M., Ahmed, M.A., Ahmed, S.A., Al-Semany, S.A., Alghamdi, S.S., Zaglool, D.A., 2017. Predominance and association risk of Blastocystis hominis subtype I in colorectal cancer: a case control study. Infect. Agent. Cancer 12, 21. Navarro, C., Dominguez-Marquez, M.V., Garijo-Toledo, M.M., Vega-Garcia, S., FernandezBarredo, S., Perez-Gracia, M.T., Garcia, A., Borras, R., Gomez-Munoz, M.T., 2008. High prevalence of Blastocystis sp. in pigs reared under intensive growing systems: frequency of ribotypes and associated risk factors. Vet. Parasitol. 153, 347–358. Noel, C., Dufernez, F., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Ho, L.C., Singh, M., Wintjens, R., Sogin, M.L., Capron, M., Pierce, R., Zenner, L., Viscogliosi, E., 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43, 348–355. Noradilah, S.A., Lee, I.L., Anuar, T.S., Salleh, F.M., Abdul Manap, S.N., Mohd Mohtar, N.S., Azrul, S.M., Abdullah, W.O., Moktar, N., 2016. Occurrence of Blastocystis sp: in water catchments at Malay villages and Aboriginal settlement during wet and dry seasons in Peninsular Malaysia. Peer J. 4, e2541. Osman, M., Bories, J., El Safadi, D., Poirel, M.T., Gantois, N., Benamrouz-Vanneste, S., Delhaes, L., Hugonnard, M., Certad, G., Zenner, L., Viscogliosi, E., 2015. Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk. Vet. Parasitol. 214, 167–170. Parkar, U., Traub, R.J., Vitali, S., Elliot, A., Levecke, B., Robertson, I., Geurden, T., Steele, J., Drake, B., Thompson, R.C., 2010. Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Vet. Parasitol. 169, 8–17. Petrasova, J., Uzlikova, M., Kostka, M., Petrzelkova, K.J., Huffman, M.A., Modry, D., 2011. Diversity and host specificity of Blastocystis in syntopic primates on Rubondo Island, Tanzania. Int. J. Parasitol. 41, 1113–1120. Ramirez, J.D., Sanchez, L.V., Bautista, D.C., Corredor, A.F., Florez, A.C., Stensvold, C.R.,
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Short communication
First report of Blastocystis infections in cattle in China Weining Zhu ⁎ Wei Lia, a b
a,1
a,1
, Wei Tao
b
a
a
MARK a
a
, Binbin Gong , Hang Yang , Yijing Li , Mingxin Song , Yixin Lu ,
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, China School of Chemical Engineering & Biotechnology, Xingtai University, Xingtai, Hebei 054001, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Blastocystis Prevalence Dairy cattle Subtyping Zoonotic potential
Blastocystis is one of the most common intestinal protists of humans and can also infect a variety of other mammals and birds. Blastocystis infections and subtype distribution in cattle have been documented, while nothing is known about those in China. Herein, a total of 526 dairy cattle from northeast China were sampled and investigated for the prevalence and genetic characteristics of Blastocystis and the potential role of bovine animals in zoonotic transmission of Blastocystis. The parasite was identified in 54 (10.3%) fecal specimens by nested PCR and DNA sequencing of the small subunit ribosomal RNA gene. Sequence analysis enabled identification of four Blastocystis subtypes (STs). Among those, subtype ST10 (75.9%, 41/54) has the highest frequency, followed by ST14 (18.5%, 10/54), ST4 (3.7%, 2/54), and ST5 (1.9%, 1/54). High prevalence and widespread distribution of ST10 and ST14 in cattle observed herein, together with analysis of their host distribution patterns in earlier studies, indicated some host-adapted potential in the two subtypes. The identification of human-pathogenic subtypes ST4 and ST5 might imply a potential zoonotic risk of cattle origin. This is the first study exploring the prevalence and genetic characteristics of Blastocystis in cattle in China. The host range of subtype ST4 was extended. The findings of this study should be helpful for a better understanding of the epidemiology and public health potential of Blastocystis.
1. Introduction Blastocystis, one of the most ubiquitous parasites of mammalian intestinal tracts, was reported in a variety of vertebrate hosts including humans (Fayer et al., 2012; Parkar et al., 2010; Roberts et al., 2013). The pathogenicity of Blastocystis is related to such factors including subtype variation and host immune status, while the exact mechanism remains controversial (Cirioni et al., 1999; Elwakil and Hewedi, 2010). Blastocystis is commonly transmitted through sewage-contaminated water and food that contains cysts and the fecal-oral route is a major mode of transmission (Leelayoova et al., 2004; Leelayoova et al., 2008; Yoshikawa et al., 2004b). Close contacts with infected animals may constitute risks of zoonotic transmission of Blastocystis (Osman et al., 2015; Wang et al., 2014). Genetic polymorphisms of the small subunit ribosomal RNA (SSU rRNA) gene enabled identification of 17 distinct Blastocystis subtypes (STs) in different human and animal species (Alfellani et al., 2013b; Stensvold et al., 2009; Wang et al., 2014). Nine subtypes (ST1–ST9) have been reported in human Blastocystis infections, with ST3 and ST1 most frequently examined. The variation in human Blastocystis subtype
⁎
1
by geographical region has been analyzed, with predominance of ST1 (followed by ST2 and ST3) in America, ST3 (ST4 and ST1) in Europe, ST3 (ST1 and ST6) in Africa, ST3 (ST1 and ST4) in Australia, and ST3 (ST1 and ST2) in Asia (Alfellani et al., 2013a). The host specificity and pathogenic potential among the various Blastocystis subtypes differ considerably as well, which might represent contributors to the symptom variability in patients with Blastocystis (Dominguez-Marquez et al., 2009; Khademvatan et al., 2017; Mohamed et al., 2017; Souppart et al., 2010). The rest eight subtypes such as ST10 and ST14 usually circulate in specific animal hosts and have never appeared in human infections. Most of the animal species surveyed can harbor potentially zoonotic Blastocystis subtypes and thus can be potential reservoirs of human infections (Khademvatan et al., 2017; Stensvold and Clark, 2016; Wang et al., 2014). In recent decades, Blastocystis infections have been repeatedly reported in a wide range of domestic animals and some factors associated with zoonotic transmission have been evaluated as well (Cian et al., 2017; Osman et al., 2015). Several reports have indicated the prevalence and distribution of Blastocystis subtypes ST1, ST3, ST5, ST6, ST10, and ST14 in cattle from Iran, USA, UK, Libya, Denmark, Japan,
Corresponding author. E-mail address: [email protected] (W. Li). These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.vetpar.2017.09.001 Received 26 July 2017; Received in revised form 31 August 2017; Accepted 1 September 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.
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screen positive PCRs.
and Colombia (Alfellani et al., 2013b; Badparva et al., 2015; Ramirez et al., 2014; Santin et al., 2011; Stensvold et al., 2009; Yoshikawa et al., 2004a). This study was conducted to investigate 526 dairy cattle of various ages in northeast China for the prevalence and genetic characteristics of Blastocystis as well as to assess the potential role of bovine animals in zoonotic transmission of Blastocystis.
2.4. Sequence analysis and phylogeny PCR amplicons of expected size were sequenced in both directions at the Beijing Genomics Institute. Raw sequences were proofread and edited manually with Chromas Pro 1.33 (Technelysium, Queensland, Australia) and BioEdit 7.0 (http://www.mbio.ncsu.edu/BioEdit/ bioedit.html). To determine Blastocystis subtypes, each of the corrected sequences was compared with GenBank sequences by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/). All the sequences obtained in this study were aligned with the reference sequences of known Blastocystis subtypes using MAFFT 7 (http://mafft.cbrc.jp/ alignment/software/). Their phylogenetic relationships were analyzed by the neighbor-joining (NJ) method under the Kimura 2-parameter model and the maximum parsimony (MP) method with bootstrap values out of 1000 replicates, using the MEGA 5.0 (http://www. megasoftware.net/). Unique SSU rRNA sequences gained herein were deposited in GenBank under accession numbers of MF573940 to MF573945.
2. Materials and methods 2.1. Ethics statement The protocol of the current study was reviewed and approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (no. SRM-08). Before the experiment, we contacted the farm owners for their permissions. No specific permits were required for the described field studies. And the locations where we sampled are not privately-owned or protected in any way. 2.2. Specimen collection A total of 526 fecal specimens were collected from cattle in cities Harbin (n = 196) in October 2013, Qiqihar (n = 190) in April 2014, and Daqing (n = 140) in July 2014. The three cities are closely linked and located in northeast China. Three age categories of animals were seen in Harbin, with preweaned animals aged < 3 months (n = 69), weaned animals aged 3–12 months (n = 61), and yearlings and adults aged > 12 months (n = 66) (Table 1). Specimens from Harbin were transferred individually into 50-ml bottles filled with 2.5% (m/v) potassium dichromate and stored at 4 °C. Only preweaned calves were kept on the farm in Qiqihar, and weaned cattle in Daqing likewise (Table 1). Specimens from the two locations were stored frozen in disposable plastic bags at −20 °C. All the specimens were collected as quickly as possible after defecation. One specimen per animal was used in this study.
2.5. Statistical analysis We used chi-square test in SPSS 17.0 (SPSS Inc., Chicago, IL, USA) to analyze the prevalence difference between animal groups. P values of < 0.05 were considered statistically significant. 3. Results 3.1. Prevalence of Blastocystis in cattle Nested PCRs and sequence analysis of the SSU rRNA gene identified Blastocystis in 54 of 526 (10.3%) cattle fecal specimens, with a prevalence of 23.0% (45/196) in Harbin and 6.4% (9/140) in Daqing (Table 1). The prevalence difference between the two cities was significant (p < 0.01, χ2 = 16.5) (Table 1). In addition, weaned cattle (36.1%, 22/61) from Harbin had a significantly higher prevalence than those (6.4%, 9/140) within the same age range from Daqing (p < 0.01, χ2 = 28.6) (Table 1). There were no Blastocystis-positive isolates found in preweaned cattle from Qiqihar (Table 1). In Harbin, the prevalence of Blastocystis in weaned cattle (36.1%, 22/61) was significantly higher than that in preweaned calves (18.8%, 13/69; p < 0.05, χ2 = 4.9) and that in yearlings and adults (15.2%, 10/66; p < 0.01, χ2 = 7.4) (Table 1). When the other two study areas were considered, the differences in the overall prevalence between weaned and preweaned cattle (15.4%, 31/201 versus 5.0%, 13/259) and between adult and preweaned cattle (15.2%, 10/66 versus 5.0%, 13/259) were significant (p < 0.01, χ2 = 14.2 and 6.7, respectively) (Table 1).
2.3. DNA extraction and PCR Prior to DNA extraction, fecal specimens stored in potassium dichromate were washed twice in double-distilled H2O by centrifugation at 12,000 × g for 5 min at room temperature. Genomic DNA was extracted from 0.2 g of feces using Stool DNA rapid extraction kits (TIANGEN, China) as instructed by the manufacturer. Nested PCR based on the SSU rRNA gene was performed using the primers RD3 (5′GGGATCCTGATCCTTCCGCAGGTTCACCTAC-3′) and RD5 (5′GGAAGCTTATCTGGTTGATCCTGCCAGTA-3′) that amplified a fragment of about 1780 bp in length in the first round PCR (Parkar et al., 2010) and the primers Blasto 2F (5′-TCTGGTTGATCCTGCCAGT-3′) and Blasto 2R (5′-AGCTTTTTAACTGCAACAACG-3′) that amplified a fragment of about 600 bp in length in the second round PCR (Souppart et al., 2009). PCR reaction systems used herein were exactly the same as those described (Souppart et al., 2009). Two parallel PCRs were conducted for each specimen using 2 μl DNA extract per reaction. Suitable positive and negative controls were placed with each plate. All PCRs were conducted in a GeneAmp PCR System 9700 (Applied Biosystems) thermal cycler. Agarose gel electrophoresis (1.5%) was performed to
3.2. Subtype distribution Sequencing is available for all 54 Blastocystis-positive isolates. Four Blastocystis subtypes were identified by sequence polymorphisms of the SSU rRNA gene (Table 1). Among those, subtype ST10 (75.9%, 41/54) has the highest frequency, followed by ST14 (18.5%, 10/54), ST4
Table 1 Prevalence and subtype distribution of Blastocystis in dairy cattle from northeast China. City
Harbin Qiqihar Daqing Total
No. of cattle
196 190 140 526
No. of subtype
Prevalence in age groups (% [positive no./total no.])
ST4
ST5
ST10
ST14
< 3 months
3–12 months
> 12 months
2 0 0 2
1 0 0 1
38 0 3 41
4 0 6 10
18.8 (13/69) 0.0 (0/190)
36.1 (22/61)
15.2 (10/66)
6.4 (9/140) 15.4 (31/201)
15.2 (10/66)
5.0 (13/259)
39
Total prevalence (% [positive no./total no.])
23.0 (45/196) 0.0 (0/190) 6.4 (9/140) 10.3 (54/526)
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Table 2 Blastocystis subtypes and their frequency in cattle around the world. Country
China Colombia Denmark Iran Japan Libya UK USA Total
No. of subtype (frequency [%]) ST1
ST3
12 (60.0)
8 (40.0)
2 (12.5)
2 (10.5) 3 (12.5)
ST4
ST5
2 (3.7)
1 (1.9) 3 (12.0) 9 (47.4)
13 (8.0)
2 (1.2)
16 (9.8)
ST10
ST14
41 (75.9)
10 (18.5)
Reference
10.3 (54/526) 80.0 (20/25)
This study (Ramirez et al., 2014) (Stensvold et al., 2009) (Badparva et al., 2015) (Yoshikawa et al., 2004a) (Alfellani et al., 2013b) (Alfellani et al., 2013b) (Fayer et al., 2012; Santin et al., 2011)
Mixed
22 (88.0) 2 (10.5) 6 (75.0)
2 (13.3) 1 (14.3)
1 (14.3)
15 (9.2)
ST6
Total prevalence (% [positive no./total no.])
8 (4.9)
2 (10.5)
9.7 (19/196) 41.7 (15/36) 22.6 (7/31) 19.0 (16/84)
6 (40.0) 3 (42.9) 13 (81.3)
2 (13.3) 1 (6.3)
5 (33.3) 2 (28.6) 2 (12.5)
85 (52.1)
13 (8.0)
11 (6.7)
worldwide and population characteristics of the parasite varied by host types and geographical locations (Navarro et al., 2008; Ramirez et al., 2014; Wang et al., 2014). However, the relevant studies were rarely seen in China. This study surveyed Blastocystis infections in cattle and presented an overall prevalence of 10.3%. The prevalence is similar with that (9.7%) reported in cattle from Iran (Badparva et al., 2015), but lower than those from USA (19%), UK (22.6%), Libya (41.7%), and Colombia (80%) (Alfellani et al., 2013b; Fayer et al., 2012; Ramirez et al., 2014; Santin et al., 2011). Likewise, we found significant difference in Blastocystis prevalence among animal groups from different cities in this study. The prevalence difference may result from variations of animal ages, seasonality, and ecological environments. A significantly lower prevalence observed in preweaned calves than that in weaned, yearling, and adult cattle may be attributed to immune protection accomplished by maternal antibodies (Stenzel and Boreham, 1996; Zierdt, 1991). This would also be responsible for the absence of detection of Blastocystis in preweaned cattle from Qiqihar. Thus far, subtyping on the basis of the SSU rRNA gene has facilitated definition of 17 valid Blastocystis subtypes (ST1–ST17) (Alfellani et al., 2013b; Stensvold et al., 2009; Wang et al., 2014), four (ST10, ST14,
(3.7%, 2/54), and ST5 (1.9%, 1/54) (Table 2). Forty-five cattle in Harbin were affected with subtypes ST4 (n = 2), ST5 (n = 1), ST10 (n = 38), and ST14 (n = 4), and nine cattle in Daqing with subtypes ST10 (n = 3) and ST14 (n = 6) (Table 1). Subtypes ST4 (n = 2) and ST5 (n = 1) with zoonotic potential parasitized three preweaned calves in Harbin (Table 1). There were no mixed infections observed. 3.3. Phylogenetic analysis BLAST comparison to known sequence databases in GenBank recognized two novel partial SSU rRNA sequences from isolates HC104 and HC158 (Fig. 1). Sequences of isolates HC158 and HC104 differed from a GenBank sequence under accession number of KC148207 by one single nucleotide polymorphism (SNP) and four SNPs, respectively. They were both grouped with the known isolates that belong to subtype ST10 in phylogenetic analysis using NJ and MP methods (Fig. 1). 4. Discussion Blastocystis infections occurred in lots of human and animal species
Fig. 1. Phylogenetic relationships among nucleotide sequences of Blastocystis small subunit ribosomal RNA (SSU rRNA) gene using the neighbor-joining method (panel A) and the maximum parsimony method (panel B). The trees were rooted with GenBank sequence U37107. Bootstrap values greater than 50% from 1000 pseudoreplicates were shown. The sequences indicated by filled triangles are new SSU rRNA nucleotide sequences found in this study.
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Table 3 Host and geographic ranges of Blastocystis subtypes identified in this study. Subtype
Hosta (locationb)
Reference
ST4
Human (China, Australia, Colombia, Denmark, France, Germany, Greece, Iran, Ireland, Italy, Japan, KSA, Laos, Lebanon, Liberia, Malaysia, Netherlands, Nigeria, North Cyprus, Pakistan, Philippines, Romania, Senegal, Singapore, Spain, Sweden, Thailand, Tunisia, Turkey, UK and USA), NHP (Denmark, France, Japan, Spain and UK), Cattle (China), Buffalo (Nepal), Sheep (Nepal), Deer (Australia), Pig (Nepal), Dog (India and Philippines), Leopard (Australia), Kangaroo (Australia), Rat (Denmark, France, Japan, Singapore and USA), Chicken (Japan), Ostrich (Australia and France), Bird (Japan) Human (Bolivia, Germany, Iran, Malaysia, Pakistan, Philippines, Turkey and USA), NHP (China, Denmark, France, Tanzania and UK), Cattle (China, Denmark, Iran, Libya and UK), Rhinoceros (UK), Camel (Libya), Deer (UK), Pig (China, Australia, Cambodian, Denmark, Japan, Spain, UK, USA and Vietnam), Dog (India and Philippines), Capybara (France), Rat (UK), Tapir (France), Ostrich (China and France) NHP (China, Denmark), Cattle (China, Denmark, Libya, UK and USA), Anoa (UK), Yak (China), Bison (France), Takin (China), Bushbuck (China, France), Eland (China), Oryx (China and France), Sheep (Denmark, France, Libya and UK), Deer (China, Denmark, France and Mauritius), Camel (China, France, Libya and UK), Horse (China), Pig (China), Dog (France), Kangaroo (China), Ostrich (China) Cattle (China, Libya and USA), Yak (China), Bison (France), Takin (China), Bushbuck (China and France), Eland (China and France), Wildebeest (France), Camel (China and Libya), Sheep (Czech Republic and France), Deer (France), Lechwe (China)
(Alfellani et al., 2013a; Bart et al., 2013; Belleza et al., 2016; Ben Abda et al., 2017; Cian et al., 2017; El Safadi et al., 2014; El Safadi et al., 2013; Forsell et al., 2016; Lee et al., 2012; Matiut and Hritcu, 2014; Mohamed et al., 2017; Noel et al., 2005; Noradilah et al., 2016; Ramirez et al., 2017; Roberts et al., 2013; Sanchez et al., 2017; Santin et al., 2011; Seyer et al., 2017; Stensvold et al., 2012; Stensvold et al., 2009; Wang et al., 2013; Zhan et al., 2014), this study
ST5
ST10
ST14
a b
(Alfellani et al., 2013a; Alfellani et al., 2013b; Belleza et al., 2016; Cian et al., 2017; Khademvatan et al., 2017; Koltas and Eroglu, 2016; Petrasova et al., 2011; Ramirez et al., 2016; Roberts et al., 2013; Santin et al., 2011; Song et al., 2017; Stensvold et al., 2009; Wang et al., 2013; Zhao et al., 2017), this study
(Alfellani et al., 2013b; Cian et al., 2017; Noradilah et al., 2016; Osman et al., 2015; Song et al., 2017; Stensvold et al., 2009; Zhao et al., 2017), this study
(Alfellani et al., 2013b; Cian et al., 2017; Zhao et al., 2017), this study
NHP: nonhuman primates. KSA: Kingdom of Saudi Arabia.
ST4 was extended. Susceptible populations should be alert to the possibility of transmission of Blastocystis subtypes ST4 and ST5 from cattle sources.
ST4, and ST5) of which were the contributors to cattle infections in China. Of those, ST10 and ST14 represent the most widely distributed subtypes in cattle. A previous study indicated the unique presence of Blastocystis subtypes ST10 and ST14 in cattle from the USA (Fayer et al., 2012). As seen in Table 2, ST10 was the most dominant subtype identifed in cattle from Libya, Denmark, and the UK (Alfellani et al., 2013b; Stensvold et al., 2009). Table 3 displayed the host and geographic distributions of the four Blastocystis subtypes identified in this study. As shown in Table 3, over 80 percent of cases of ST10 infection and 100 percent of cases of ST14 infection appeared in ruminants (cattle, sheep, camel, deer, etc.), in which bovine animals possess the highest infection frequency. The data revealed some host-adapted potential in the two subtypes. It is of interest to note two novel SSU rRNA nucleotide sequences were obtained in this study, which differed by less than 5% from the existing ST10 sequences in GenBank. In this study, 5% nucleotide sequence divergence was considered as a benchmark for naming a new subtype as previously instructed (Alfellani et al., 2013b). Therefore, the two isolates should go to subtype ST10 groups, and this is supported by observing characteristics of the subtype clustering in phylogenetic analysis (Fig. 1). In addition to ST10 and ST14, we also determined two potentially zoonotic subtypes ST4 and ST5 with broad host and geographic ranges as reflected in Table 3. Subtype ST5 frequently appeared in human Blastocystis infections around the world, which can also colonize nonhuman primates, mammals, rodents, and birds (Table 3). Its repeated presence in cattle infections in China, Denmark, Iran, Libya, and the UK as summarized in Table 2 implied zoonotic transmission from cattle origin was a possibility. This is the first study reporting ST4 in cattle. Compared to ST5, ST4 appears to have much wider host spectrum and global distribution as observed in Table 3. This subtype has been documented in human infections in south China (Zhan et al., 2014). Taken together, the two subtypes are of public health importance. Moreover, the capability of bovine animals in carrying human-pathogenic Blastocystis subtypes ST1, ST3, and ST6 as shown in Table 2 may also constitute a risk for zoonotic transmission. Nevertheless, further efforts are needed to fully understand the exact role of cattle in zoonotic transmission of Blastocystis. In conclusion, here is the first study that explored the prevalence and genetic traits of Blastocystis in cattle from China. The host range of
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