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Occurrence and multilocus genotyping of Giardia duodenalis from post-weaned dairy calves in Sichuan province, China


Authors: Jiaming Dan aff001;  Xueping Zhang aff001;  Zhihua Ren aff001;  Liqin Wang aff002;  Suizhong Cao aff001;  Liuhong Shen aff001;  Junliang Deng aff001;  Zhicai Zuo aff001;  Shumin Yu aff001;  Ya Wang aff001;  Xiaoping Ma aff001;  Haifeng Liu aff001;  Ziyao Zhou aff001;  Yanchun Hu aff001;  Hualin Fu aff001;  Changliang He aff001;  Yi Geng aff001;  Xiaobin Gu aff001;  Guangneng Peng aff001;  Zhijun Zhong aff001
Authors place of work: College of Veterinary Medicine, Sichuan Agricultural University, Key Laboratory of Animal Disease and Human Health of Sichuan, Chengdu, China aff001;  The Chengdu Zoo, Institute of Wild Animals, Chengdu, China aff002
Published in the journal: PLoS ONE 14(11)
Category: Research Article
doi: https://doi.org/10.1371/journal.pone.0224627

Summary

Giardia duodenalis is a zoonotic parasitic protist and poses a threat to human and animal health. This study investigated the occurrence of G. duodenalis infection in post-weaned calves from Sichuan province, China. Faecal samples were collected from a total of 306 post-weaned calves (3–12 months old) from 10 farms, including 4 intensive feeding farms and 6 free-ranging farms. The overall infection rate of G. duodenalis was 41.2% (126/306) based on the PCR results at any of the three genetic loci: beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia duodenalis assemblages E (n = 115, 91.3%), A (n = 3, 2.4%), and A mixed with E (n = 8, 6.3%) were identified among the 126 positive specimens. Multilocus sequence typing of G. duodenalis revealed 34 assemblage E multilocus genotypes (MLGs), 1 assemblage A MLG and 7 mixed assemblage (A and E) MLGs. The eBURST data showed a high degree of genetic diversity within assemblage E MLGs. The phylogenetic tree revealed that MLG E3 was the primary MLG subtype in Sichuan province and also the most widely distributed in China.

Keywords:

Genetic loci – Phylogenetic analysis – Polymerase chain reaction – Zoonoses – China – DNA extraction – Farms – Giardia

Introduction

Giardia is one of the most common parasitic protists that infects both humans and animals, poses a considerable threat to human and animal health globally [1, 2]. Among the six species of Giardia, only Giardia duodenalis can infect humans and animals (domestic, farmed and wild animals) [3, 4]. The life cycle of Giardia is relatively simple; involving two developmental stages of rapid multiplying trophozoites and infectious cysts, transmitted via the faecal-oral route (i.e., faeces, contaminated water or food) [1, 5]. Humans and animals infected with Giardia usually show symptoms such as diarrhoea, abdominal cramps, weight loss, malabsorption or recessive infections without obvious clinical symptoms [5, 6]. Young animals are more susceptible to giardiasis than adults and likely linked to the immature immune status, which lead to substantial production losses to the livestock industry [6].

Giardia duodenalis is recognised as a complex comprised of at least eight different assemblages (A–H) [4, 7]. Assemblage A and B can infect various mammals including humans, and are considered as the zoonotic assemblages. The other assemblages are either host specific or have narrow host ranges [6]. Cattle are dominantly infected with G. duodenalis assemblage E. Although there are fewer reports of zoonotic assemblages A and B, cattle are recognized as the contributor of the zoonotic sources of infection [6]. Many recent studies have focused on the infection of G. duodenalis in dairy calves, and the occurrence has been found to be significantly different between pre- and post-weaned stages [813]. In China, studies have also revealed different infection rates in pre- and post-weaned dairy calves, e.g., in Liaoning [14], Xinjiang [15], Hubei [16] and Guangdong [17] provinces. In our previous study, we conducted a preliminary study on G. duodenalis infection in pre-weaned calves in Sichuan province, China [18]. However, information regarding the occurrence in post-weaned dairy calves in Sichuan province is limited.

In this study, we further investigated the occurrence and genetic diversity of G. duodenalis in post-weaned calves from Sichuan province by using multilocus genotype (MLG) data and by assessing the zoonotic potential.

Materials and methods

Sample collection

A total of 306 faecal samples were collected from post-weaned calves (3–12 months old) from 10 farms in 10 regions in Sichuan province, southwestern China, from May to November 2018 (Fig 1). At the time of faecal collections, there were no reported cases of diarrhoea in the herds but with a history of diarrhoea. The collection sites included seven of the areas from our previous study [18]: Anyue (105°33′E, 30°10′N), Chengdu (104°06′E, 30°57′N), Deyang (104°39′E, 31°13′N), Meishan (103°84′E, 30°08′N), Mianyang (104°67′E, 31°47′N), Qingbaijiang (104°25′E, 30°88′N), and Qionglai (103°46′E, 30°41′N); and three additional areas: Nanchong (106°72′E, 31°01′N), Xichang (102°51E, 28°64′N), and Ya’an (103°08′E, 30°18′N). Of the 10 farms, four (Chengdu, Mianyang, Nanchong and Qionglai) were intensive feeding farms, while the other six were free-ranging. Specific information on intensive and free-ranging farming and the specific sampling protocols used in the present study were consistent with those stipulated in our previous study [18]. A city-level map was provided by the National Geomatics Centre of China (National Geomatics Centre of China, Beijing, China, http://ngcc.sbsm.gov.cn/).

Distribution of sampling sites in Sichuan province in this study.
Fig. 1. Distribution of sampling sites in Sichuan province in this study.
Sampling sites from the present study are indicated by black triangles.

Fresh faecal samples (~25 g per calf) were collected directly from the rectum of the study calves using disposable gloves, transferred into disposable plastic bags, and then stored in 2.5% potassium dichromate at 4°C prior to DNA extraction.

This study was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Sichuan Agricultural University (DYY-S20174604). Permission was obtained from the farm owners before collecting the fecal samples.

DNA extraction

Before DNA extraction, stored faeces were washed with distilled water to remove the potassium dichromate. Genomic DNA was extracted from ~250 mg of the individual samples using the PowerSoil DNA isolation kit (MOBIO, USA), according to the manufacturer’s instructions. All DNA samples were stored at −20°C prior to analysis by Giardia PCR.

PCR amplification

Giardia duodenalis DNA was detected by nested PCR amplification of the beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. The primers and amplification conditions used in this study have been described previously [19]. Positive and negative controls were included in each test. The secondary PCR products were visualized under UV light after electrophoresis on 1% agarose gel mixed with Golden View.

Sequence analysis

All positive secondary PCR products were sent to BGI Tech Solutions (Liuhe Beijing) Co., Limited and were sequenced in both directions. Sequences were aligned with reference sequences from GenBank using BLAST (http://blast.ncbi.nlm.nih.gov) and Clustal X (http://www.clustal.org/).

Specimens that were successfully subtyped at all three loci were used to investigate the MLGs of G. duodenalis. Sequences were concatenated for each positive isolate to form a multilocus sequence in accordance with (bg + tpi + gdh). All the concatenated MLGs were used in a neighbour-joining analysis, with the Kimura-2 parameter model calculated using the Molecular Evolutionary Genetics Analysis (MEGA) version 7 (http://www.megasoftware.net/). The genetic pedigree of the assemblage E MLGs in Sichuan was assessed by using eBURST 3.0 (http://eBURST.mlst.net).

The novel G. duodenalis genotypes obtained at bg, tpi and gdh loci in this study were deposited in GenBank under the accession numbers: MK642904-MK642913.

Statistical analysis

The variation in G. intestinalis prevalence among the different regions was analysed by χ2 test using SPSS Statistics version 20.0. Differences were considered significant at P < 0.05.

Results and discussion

Infected animals were detected from all of the 10 examined farms, with prevalence ranging from 6.7% to 63.3% (Table 1). This study revealed G. duodenalis as a common and widespread pathogen in post-weaned dairy calves in Sichuan province, China. Based on the PCR results at any of the 3 genetic loci (bg, tpi and gdh), 126 (41.2%) of the 306 faecal specimens tested positive for G. duodenalis, which was similar to the occurrence in Hubei (37.8%, 28/74) [16], but much higher than the majority of provinces in China. These provinces included Jilin (4.4%, 5/114) [14], Liaoning (3.1%, 3/98) [14], Heilongjiang (12.5%, 3/24) [14], Shaanxi (17.54%, 30/171) [20], Xinjiang (16.6%, 46/277) [15] and Guangdong (1.1%, 5/533) [17]. Compared with studies in other countries, the overall infection rate in the present study was higher than in Maryland, USA (32.1%, 125/390) [11]; Malaysia (8.3%, 10/120) [8]; India (12.5%, 9/72) [10] and New Zealand (2%, 2/100) [9], but lower than that in another study in the USA (52%, 237/456) [12]. The reasons for these differences in infection rates is still unclear, however, they may be related to geo-ecological conditions, management factors and health status [1, 15, 21, 22]. Nested PCR amplification is a sensitive method and widely used in the detection of G. duodenalis [3, 6]. In this study, nested PCR amplification was used directly instead of using microscopic examination which led to low detection and also cannot genotype G. duodenalis. Numerous studies have reported that the prevalence of G. duodenalis is inversely associated with animal age [1, 8, 11]. However, we measured a significantly higher occurrence of G. duodenalis in post-weaned calves (41.2%) compared with previous measurements of pre-weaned calves (26/278, 9.4%) [18] (P < 0.01, X2 = 76.623, df = 1); which was similar to some reports from China [15, 16] and the USA [12, 13]. Moreover, we analysed the infection rates between intensive feeding and free-ranging farms and found consistent results with our previous study [18], i.e., no significant differences between the two breeding systems (P = 0.179, X2 = 1.809, df = 1). Similarly, a study conducted in Malaysia also showed no significant differences in the occurrence of G. duodenalis infection between intensive and semi-intensive farms [8].

Tab. 1. Occurrence and assemblages of Giardia duodenalis in post-weaned dairy calves in Sichuan province.
Occurrence and assemblages of <i>Giardia duodenalis</i> in post-weaned dairy calves in Sichuan province.

Of the 126 G. duodenalis-positive specimens, 121 were positive in bg gene, 101 in tpi gene, and 111 in gdh gene. Giardia duodenalis assemblage E (n = 115, 91.3%), assemblage A (n = 3, 2.4%), and mixed assemblage (A and E) (n = 8, 6.3%) were identified among the 126 genotyped specimens (Table 1), which is consistent with other studies conducted in China [14], the USA [11] and India [10]. All of the assemblage A isolates identified in the present study belonged to subtype A1, which has mostly been detected in animals [1, 5]. However, there have been a few reports of human infections of subtype A1, e.g., in China [23], Portugal [24], Mexico [25] and Brazil [26], which suggests that dairy calves may potentially play a role in the zoonotic transmission of G. duodenalis infection from cattle to humans. Sequence analyses revealed 12, 9 and 15 subtypes identified at the bg, tpi and gdh loci, respectively. Of the bg subtypes, eight had previously been identified. The remaining four sequences represented subtypes E17-E20 (MK642904-MK642907) that were previously unpublished. Of the tpi subtypes, eight subtypes identified before and the remaining one sequence subtype E25 (MK642913) was previously unpublished. Of the gdh subtypes, ten were known and five were previously unpublished: E21–E25 (MK642908-MK642912). Of these subtypes, the most common were E9 (n = 20, bg subtype), E3 (n = 64, tpi subtype) and E10 (n = 48, gdh subtype). The high genetic diversity of assemblage E in this study was consistent with previous studies [18, 27, 28], which may be related to intra-assemblage genetic recombination [19, 29].

Furthermore, MLG analysis of the bg, tpi and gdh genes was used to systematically characterize intra-assemblage genetic diversity and to determine the genotype of G. duodenalis. A total of 94 specimens were successfully sequenced at the bg, tpi and gdh loci, which formed 34 assemblage E MLGs (Table 2), one assemblage A MLG and seven mixed assemblage (A and E) MLGs. The most common MLGs were the MLG E48 (n = 11) and MLG E80 (n = 11), followed by MLG E94 (n = 9) and MLG E3 (n = 6). Among them, the most widely distributed MLG was MLG E3, which was detected in four regions (Chengdu, Deyang, Meishan and Ya’an). To reveal the genetic relationship between MLGs, we constructed clonal pedigree maps of the 34 assemblage E MLGs in the present study and the 19 assemblage E MLGs in our previous study [18] using eBURST software. Two clonal complexes and five singletons were observed (S1 Fig). MLG E3 is the primary founder of clonal complex 1, which is consistent with the results found in Shanghai [28]. Clonal complex 2 was formed by three assemblages E: MLG E94-E96. MLG E69, MLG E78, MLG E83, MLG E87 and MLG E98 were singletons. To better understand the diversity between assemblage E MLGs in Sichuan and in the other provinces of China (Henan [30], Gansu [31], Ningxia [31], Shaanxi [20], Xinjiang [15], Shanghai [28] and Guangdong [17, 27]), a phylogenetic evolutionary tree was constructed. The phylogenetic tree (S2 Fig) showed that MLG E3 has also been found in Gansu [31], Xinjiang [15], Shanghai [28] and Guangdong [27], which indicates the wide distribution of this MLG subtype in China. However, the predominant subtype needs further elucidation. As seen from the phylogenetic tree, the 218 assemblage E MLGs identified in China were highly diverse and formed multiple evolutionary branches. The 53 assemblage E MLGs from Sichuan province showed a scattered distribution in the phylogenetic tree, which indicated that geographical segregation was not strict in our study.

Tab. 2. Multilocus sequence genotypes of Giardia duodenalis assemblage E in post-weaned dairy calves in Sichuan province.
Multilocus sequence genotypes of <i>Giardia duodenalis</i> assemblage E in post-weaned dairy calves in Sichuan province.

Conclusion

The present study demonstrated a high occurrence of G. duodenalis in post-weaned calves in Sichuan province, China. Both assemblage E and zoonotic assemblage A were detected. MLG analysis revealed a high genetic diversity in assemblage E. MLG E3 was shown to be not only the primary MLG subtype in Sichuan province but also the most widely distributed MLG subtype throughout China.

Supporting information

S1 Fig [tif]
eBURST networks for . assemblage E isolated from Sichuan province.

S2 Fig [tif]
Phylogenetic relationships between assemblage E MLGs.


Zdroje

1. Li J, Wang H, Wang R, Zhang L. Giardia duodenalis Infections in Humans and Other Animals in China. Front Microbiol. 2017;8:2004. doi: 10.3389/fmicb.2017.02004 29081771

2. Thompson RC, Palmer CS, O'Handley R. The public health and clinical significance of Giardia and Cryptosporidium in domestic animals. Vet J. 2008;177(1):18–25. doi: 10.1016/j.tvjl.2007.09.022 18032076.

3. Ryan U, Caccio SM. Zoonotic potential of Giardia. Int J Parasitol. 2013;43(12–13):943–56. Epub 2013/07/17. doi: 10.1016/j.ijpara.2013.06.001 23856595.

4. Monis PT, Caccio SM, Thompson RC. Variation in Giardia: towards a taxonomic revision of the genus. Trends Parasitol. 2009;25(2):93–100. doi: 10.1016/j.pt.2008.11.006 19135417.

5. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24(1):110–40. doi: 10.1128/CMR.00033-10 21233509;

6. Abeywardena H, Jex AR, Gasser RB. A perspective on Cryptosporidium and Giardia, with an emphasis on bovines and recent epidemiological findings. Advances in parasitology. 2015;88:243–301. doi: 10.1016/bs.apar.2015.02.001 25911369.

7. Koehler AV, Jex AR, Haydon SR, Stevens MA, Gasser RB. Giardia/giardiasis—a perspective on diagnostic and analytical tools. Biotechnology advances. 2014;32(2):280–9. doi: 10.1016/j.biotechadv.2013.10.009 24189092.

8. Muhid A, Robertson I, Ng J, Yang R, Ryan U. Prevalence of Giardia spp. infection in pre-weaned and weaned calves in relation to management factors. Vet J. 2012;191(1):135–7. doi: 10.1016/j.tvjl.2011.01.007 21339075.

9. Abeywardena H, Jex AR, Nolan MJ, Haydon SR, Stevens MA, McAnulty RW, et al. Genetic characterisation of Cryptosporidium and Giardia from dairy calves: discovery of species/genotypes consistent with those found in humans. Infect Genet Evol. 2012;12(8):1984–93. doi: 10.1016/j.meegid.2012.08.004 22981927.

10. Khan SM, Debnath C, Pramanik AK, Xiao L, Nozaki T, Ganguly S. Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm workers in West Bengal, India. Vet Parasitol. 2011;178(3–4):342–5. doi: 10.1016/j.vetpar.2011.01.029 21324592.

11. Santin M, Trout JM, Fayer R. A longitudinal study of Giardia duodenalis genotypes in dairy cows from birth to 2 years of age. Vet Parasitol. 2009;162(1–2):40–5. doi: 10.1016/j.vetpar.2009.02.008 19264407.

12. Trout JM, Santin M, Greiner E, Fayer R. Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves. Vet Parasitol. 2005;130(3–4):177–83. doi: 10.1016/j.vetpar.2005.03.032 15925721.

13. Trout JM, Santin M, Greiner E, Fayer R. Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves. Vet Parasitol. 2004;124(3–4):179–86. doi: 10.1016/j.vetpar.2004.07.010 15381298.

14. Liu G, Su Y, Zhou M, Zhao J, Zhang T, Ahmad W, et al. Prevalence and molecular characterization of Giardia duodenalis isolates from dairy cattle in northeast China. Exp Parasitol. 2015;154:20–4. doi: 10.1016/j.exppara.2015.03.020 25845754.

15. Qi M, Wang H, Jing B, Wang R, Jian F, Ning C, et al. Prevalence and multilocus genotyping of Giardia duodenalis in dairy calves in Xinjiang, Northwestern China. Parasit Vectors. 2016;9(1):546. doi: 10.1186/s13071-016-1828-3 27737706.

16. Fan Y, Wang T, Koehler AV, Hu M, Gasser RB. Molecular investigation of Cryptosporidium and Giardia in pre- and post-weaned calves in Hubei Province, China. Parasit Vectors. 2017;10(1):519. doi: 10.1186/s13071-017-2463-3 29070070.

17. Cui Z, Wang L, Cao L, Sun M, Liang N, Wang H, et al. Genetic characteristics and geographic segregation of Giardia duodenalis in dairy cattle from Guangdong Province, southern China. Infect Genet Evol. 2018;66:95–100. doi: 10.1016/j.meegid.2018.09.019 30244091.

18. Zhong Z, Dan J, Yan G, Tu R, Tian Y, Cao S, et al. Occurrence and genotyping of Giardia duodenalis and Cryptosporidium in pre-weaned dairy calves in central Sichuan province, China. Parasite. 2018;25:45. doi: 10.1051/parasite/2018046 30178744.

19. Caccio SM, Beck R, Lalle M, Marinculic A, Pozio E. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int J Parasitol. 2008;38(13):1523–31. doi: 10.1016/j.ijpara.2008.04.008 18571176.

20. Wang XT, Wang RJ, Ren GJ, Yu ZQ, Zhang LX, Zhang SY, et al. Multilocus genotyping of Giardia duodenalis and Enterocytozoon bieneusi in dairy and native beef (Qinchuan) calves in Shaanxi province, northwestern China. Parasitol Res. 2016;115(3):1355–61. doi: 10.1007/s00436-016-4908-6 26782809.

21. Bartley PM, Roehe BK, Thomson S, Shaw HJ, Peto F, Innes EA, et al. Detection of potentially human infectious assemblages of Giardia duodenalis in fecal samples from beef and dairy cattle in Scotland. Parasitology. 2018:1–8. doi: 10.1017/s0031182018001117 29978772.

22. Hu S, Liu Z, Yan F, Zhang Z, Zhang G, Zhang L, et al. Zoonotic and host-adapted genotypes of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in dairy cattle in Hebei and Tianjin, China. Vet Parasitol. 2017;248:68–73. doi: 10.1016/j.vetpar.2017.10.024 29173544.

23. Wang R, Zhang X, Zhu H, Zhang L, Feng Y, Jian F, et al. Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China. Exp Parasitol. 2011;127(1):42–5. doi: 10.1016/j.exppara.2010.06.034 20599984.

24. Sousa MC, Morais JB, Machado JE, Poiares-da-Silva J. Genotyping of Giardia lamblia human isolates from Portugal by PCR-RFLP and sequencing. J Eukaryot Microbiol. 2006;53 Suppl 1:S174–6. doi: 10.1111/j.1550-7408.2006.00221.x 17169050.

25. Lalle M, Jimenez-Cardosa E, Caccio SM, Pozio E. Genotyping of Giardia duodenalis from humans and dogs from Mexico using a beta-giardin nested polymerase chain reaction assay. J Parasitol. 2005;91(1):203–5. doi: 10.1645/GE-293R 15856905.

26. Volotao AC, Costa-Macedo LM, Haddad FS, Brandao A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta tropica. 2007;102(1):10–9. doi: 10.1016/j.actatropica.2007.02.010 17428432.

27. Feng Y, Gong X, Zhu K, Li N, Yu Z, Guo Y, et al. Prevalence and genotypic identification of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in pre-weaned dairy calves in Guangdong, China. Parasit Vectors. 2019;12(1):41. doi: 10.1186/s13071-019-3310-5 30654832.

28. Wang X, Cai M, Jiang W, Wang Y, Jin Y, Li N, et al. High genetic diversity of Giardia duodenalis assemblage E in pre-weaned dairy calves in Shanghai, China, revealed by multilocus genotyping. Parasitol Res. 2017;116(8):2101–10. doi: 10.1007/s00436-017-5509-8 28550644.

29. Aguiar JM, Silva SO, Santos VA, Taniwaki SA, Oliveira TM, Ferreira HL, et al. Evidence of heterozygosity and recombinant alleles in single cysts of Giardia duodenalis. Rev Bras Parasitol Vet. 2016;25(2):187–95. doi: 10.1590/S1984-29612016031 27334819.

30. Wang H, Zhao G, Chen G, Jian F, Zhang S, Feng C, et al. Multilocus genotyping of Giardia duodenalis in dairy cattle in Henan, China. PLoS One. 2014;9(6):e100453. doi: 10.1371/journal.pone.0100453 24971639.

31. Zhang XX, Tan QD, Zhao GH, Ma JG, Zheng WB, Ni XT, et al. Prevalence, risk factors and multilocus genotyping of Giardia intestinalis in dairy cattle, northwest China. J Eukaryot Microbiol. 2016;63(4):498–504. doi: 10.1111/jeu.12293 26729604.


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