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Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China
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Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China Jing Jiang a,1 , Jian-Gang Ma d,g,1 , Nian-Zhang Zhang c , Peng Xu e , Guangyu Hou b,∗ , Quan Zhao a,∗ , Xiao-Xuan Zhang f a
College of Animal Science and Technology, Chanchun Sci-Tech University, Shuangyang, Jilin Province 130600, PR China College of Basic Medicine, Mudanjiang Medical College, Mudanjiang, Heilongjiang Province 157011, PR China c State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China d College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province, 712100, PR China e College of Animal Husbandry and Veterinary Medicine, Jinzhou Medical University, Jinzhou, Liaoning Province 121001, PR China f College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China g College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin Province 130118, PR China b
a r t i c l e
i n f o
Article history: Received 6 October 2017 Received in revised form 21 January 2018 Accepted 21 February 2018 Keywords: Domestic rabbits Prevalence Genotyping Northern China
a b s t r a c t Background: Giardia duodenalis is one of the most important intestinal parasites that can infect virtually all animals, including rabbits and humans. However, there is little information regarding the prevalence and genotypes of G. duodenalis in domestic rabbits in China. Methods: A total of 426 rabbit fecal samples (136 from Shenyang City, 174 from Changchun City, and 116 from Jilin City) were examined by Lugol’s iodine staining with microscopy analysis, and the positive samples were genotyped at the triosephosphate isomerase (tpi) and the beta giardin (bg) gene loci using nested PCR. Results: Forty-two (9.86%) out of 426 rabbit fecal samples were G. duodenalis-positive under microscopy analysis, and the highest G. duodenalis infection rate was 23.08% on farm 6. The prevalence of G. duodenalis in rabbits from different cities ranged from 1.47% to 14.37%. Among different age groups, G. duodenalis prevalence in rabbits ranged from 5.41% to 12.58%. The prevalence of G. duodenalis in outdoor rabbits and indoor rabbits was 14.29% and 6.77%, respectively. In the present study, region and farming mode were highly correlated with G. duodenalis infection in rabbits. All 42 G. duodenalis isolates were successfully amplified and sequenced at the tpi and bg loci, and only G. duodenalis assemblage B were identified. Conclusion: This study not only further confirmed the dominance of G. duodenalis assemblage B in rabbits, but also further improved the foundation data concerning the distribution of G. duodenalis assemblages in China. © 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Giardiasis is caused by Giardia duodenalis (syn. Giardia intestinalis, G. lamblia), a widespread enteric parasite of mammalian species [1–4]. Giardiasis is transmitted through the fecal-oral route [5], and shows symptoms of chronic diarrhea [4]. However, G. duo-
∗ Corresponding authors. E-mail addresses: [email protected] (G. Hou), [email protected] (Q. Zhao). 1 These authors have contributed equally to this work.
denalis coinfection with immune-compromised patients can cause significant morbidity and mortality [6]. In general, giardiasis affects around 2.0 × 108 people worldwide each year [7]. A wide range of animals and humans were reported as hosts for G. duodenalis [8]. Phylogenetic analysis revealed that eight assemblages (A–H) of G. duodenalis could be identified worldwide at present [1,9]. Among them, G. duodenalis assemblages A and B are responsible for the majority of human infections [10]. More importantly, some assemblages of G. duodenalis (e.g., assemblages A and B) can infect both humans and animals, raising public health concerns.
https://doi.org/10.1016/j.jiph.2018.02.008 1876-0341/© 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Table 1 Summarized the prevalence and assemblages of Giardia duodenalis in rabbits in the world (available data). Regions
No. tested
No. positive
Prevalence
Assemblages
References
Central China Heilongjiang Europe Australia Ecuador Germany California
955 378 528 97 20 232 4
80 28 40 1 4 0 0
8.4% 7.41% 7.6% 1.03% 20% 0% 0%
B and E B B A NA – –
[4] [14] [18] [19] [20] [28] [29]
Recently, studies have suggested that rabbits are an important reservoir for many pathogens [11–13], but only a small number of genotypes/assemblages of G. duodenalis in rabbits were reported around the world (listed in Table 1), especially in China. In China, information regarding the prevalence and genotypes of G. duodenalis infection in rabbits was only reported in Heilongjiang Province [14,15] and Henan province [4]. However, information concerning the prevalence and genotypes of G. duodenalis in rabbits in Jilin and Liaoning Province remains unclear. The present study aimed to investigate the prevalence of G. duodenalis in rabbits and to identify their genotypes/assemblages. We also aimed to estimate the zoonotic potential of G. duodenalis from rabbits to humans. Methods Specimen collection and preparation The study population comprised of 426 rabbits collected from three cities in China, in which nearly 3,437,000 rabbits were raised in 2014. The samples collected from nine farms, raising more than 3000 rabbits. According to the reported prevalence of Giardia in rabbit populations of 7.41% in 2012 [14], the expected prevalence was 7.5% (P) with an accepted deviation of the true prevalence of 5% (d) and a confidence level of 95% (z = 1·96). The sample size was calculated as 107 [according to n = P (1 − P)z2 /d2 ]. This study was carried out in Jilin province (41◦ –46◦ N, 122◦ –131◦ E) and Liaoning province (38◦ –43◦ N, 118◦ –125◦ E), Northeast China, between May 2015 and June 2015. A total of 426 rabbit fecal samples were randomly collected from the ground after defection using sterile gloves, and were then put into box containing ice, immediately. We only used fresh and clean samples from the middle of the fecal sample for further analysis. Information regarding region, farming mode, and age were acquired by a questionnaire filled in by the farmers. Collection and preparation of feces samples Giardia cysts in the fecal materials were detected with Lugol’s iodine staining. All Giardia-positive fecal specimens were washed three times, and genomic DNA was extracted using an E.Z.N.A. Stool DNA kit (OMEGA Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer’s procedures. The extracted DNA samples were stored at −20 ◦ C until PCR analysis. PCR amplification G. duodenalis assemblages were determined by nested PCR of an approximately 530 bp fragment of the triosephosphate isomerase (tpi) locus [16] and a 511 bp fragment of the beta giardin (bg) gene locus [17]. The PCR reactions for all genes were performed in 25l PCR reaction composed of 1 × Ex Taq buffer (Mg2+ free), 2 mM MgCl2 , 200 M each dNTP, 0.4 M each primer, 0.625 U of Takara Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), and 2 l of DNA. The cycling conditions were: 94 ◦ C for 3 min; 35 cycles of 94 ◦ C for 45 s, 55 ◦ C for 45 s, and 72 ◦ C for 60 s; followed by 72 ◦ C for 10 min.
Both positive (Giardia-positive isolates collected from cattle and stored at the laboratory) and negative controls were included in each test. All the secondary PCR products were examined by electrophoresis in 1.5% agarose gels and observed under UV light after GoldViewTM (Solarbio, Beijing, China) staining. Sequencing and phylogenetic analyses All of the positive secondary PCR products were sent to the Genscript Company (Nanjing, China) for bidirectional sequencing. Meanwhile, genotypes that produced sequences with mutations, such as single nucleotide substitutions, deletions, or insertions, and that were confirmed by the DNA sequencing of at least two PCR products, were considered as novel genotypes. The genotypes/assemblages of G. duodenalis were determined by alignment with reference sequences available in GenBank using the computer program Clustal X 1.83 and BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/). Statistical analysis Statistical analysis of the prevalence of G. duodenalis infection in rabbits from different region (x1), farming mode (x2), and age (x3) was performed a Chi-square test in SAS (SAS Institute Inc., Cary, NC, USA, Version 9.1). In the multivariable regression analysis, each of these variables was included in the binary Logit model as an independent variable. The best model was judged by Fisher’s scoring algorithm. All tests were two-sided, and the results were considered statistically significant if P < 0.05. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were provided to explore the strength of the association between G. duodenalis-positivity and the conditions investigated. Accession numbers for nucleotide sequences All the representative nucleotide sequences obtained in this study were deposited in GenBank with following accession numbers: KT372238 for the tpi gene, and KT715813 for the bg gene. Results A total of 42 (9.86%) out of 426 rabbit samples were detected as G. duodenalis-positive (Table 2), and the highest G. duodenalis infection rate was 23.08% on farm 6 (Table 3). Rabbits collected from Changchun City had a highest G. duodenalis infection rates compared with those collected from Jilin City and Shenyang City (Table 2). Moreover, in different age groups, the G. duodenalis prevalence in rabbits ranged from 5.41% to 12.58% (Table 3), and G. duodenalis prevalence in outdoor rabbits and indoor rabbits was 14.29% and 6.77%, respectively. All 42 G. duodenalis isolates were successfully amplified and sequenced at the tpi and bg loci. Sequencing analyses of the tpi and bg genes revealed that only G. duodenalis assemblage B was identified in this study.
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Table 2 Factors associated with prevalence of Giardia duodenalis in rabbits in Jilin Province and Liaoning Province, northeastern China. Factor
No. tested
No. positive
% (95% CI)
OR (95% CI)
Region
136 174 116 251 175 37 172 159 58
2 25 15 17 25 2 16 20 4
1.47 (0.00–3.49) 14.37 (9.16–19.58) 12.93 (6.83-19.04) 6.77 (3.66–9.88) 14.29 (9.10–19.47) 5.41 (0.00–12.69) 9.30 (4.96–13.64) 12.58 (7.42–17.73) 6.90 (0.38–13.42)
Reference 11.24 (2.61–48.36) 9.95 (2.23–44.50) Reference 2.29 (1.20–4.39) Reference 1.80 (0.40–8.17) 2.52 (0.56–11.29) 1.30 (0.23–7.46)
426
42
9.86 (7.03–12.69)
Farming mode Age
Total
Table 3 Distribution of Giardia duodenalis assemblages in rabbits in different farms in Jilin Province and Liaoning Province, northeastern China. Region
Farm ID
Farm mode
Sample size
No. positive
Prevalence (%) (95% CI)
Changchun City
Farm 1 Farm 2 Farm 3 Farm 4 Farm 5 Farm 6 Farm 7 Farm 8 Farm 9
Indoor Indoor Outdoor Outdoor Indoor Outdoor Indoor Outdoor Indoor
62 33 47 32 77 39 36 57 43
6 4 10 5 6 9 – 1 1
9.68 (2.32–17.04) 12.12 (0.99–23.26) 21.28 (9.58–32.98) 15.63 (3.04–28.21) 7.79 (1.81–13.78) 23.08 (9.85–36.30) 0 (–) 1.75 (0.00–5.16) 2.33 (0.00–6.83)
426
42
9.86 (7.03–12.69)
Jilin City Shenyang City
Total
Discussion In the present study, the overall infection rates for G. duodenalis was 9.86% (42/426, 95% CI 7.03–12.69), which was higher than that in Europe (7.6%, 40/528) [18], Melbourne, Australia (1.03%, 1/97) [19], Henan province (8.4%, 80/955) [4], and Heilongjiang province (7.41%, 28/378) [14], but lower than that the 20.0% (4/20) in rabbits in Ecuador [20]. Fecal samples containing low cyst intensity might lead to the low detection rates of the parasite by microscopy [21]. Moreover, many factors, such as test methods, sample sizes, animal health status, and geo-ecological conditions could also influence the result of different studies. The Giardia cysts were also found in water [22]; therefore, outbreaks of water-born Giardia should be monitored. Table 2 shows the exposure regarding farming mode and collection region associated with G. duodenalis-positivity in domestic rabbits based on univariate analysis. The impacts of multiple variables on G. duodenalis were evaluated by the forward stepwise logistic regression analysis using Fisher’s scoring technique. In the final model, two variables had effects on the infectious disease, described by the following equation y = 0.7891 × 1 + 0.7956 × 2 − 0.3218. Region and farming mode had positive effects on the risk of G. duodenalis, for which the ORs were 2.20 (95% CI 1.40–3.46) and 2.22 (95% CI 1.15–4.29), respectively. G. duodenalis can be transmitted through the ingestion of Giardia cysts [5]; therefore, the higher breeding density in Jilin Province compared with that in Liaoning Province is one of the major reasons why rabbits collected from Changchun City (14.37%, 95% CI 9.16–19.58) and Jilin City (12.93%, 95% CI 6.83–19.04) has a significantly higher G. duodenalis prevalence than that collected from Shenyang City (1.47%, 95% CI 0.00–3.49) (Table 2). Moreover, many factors, such as ecological and geographical factors, sanitation, animal welfare, and management mode may also be related to the infection rates of G. duodenalis in different regions. Farming mode was another risk factor for G. duodenalis infection in rabbits in this study. Outdoor rabbits had a significantly higher G. duodenalis prevalence than that in indoor rabbits. Outdoor-reared rabbits have more opportunities to contact with environmental specimens con-
taminated by cysts compared to those reared indoors, especially when it rains. The small subunit ribosomal RNA (SSUrRNA), glutamate dehydrogenase (gdh), elongation factor 1 alpha (ef-1 a), tpi, and bg gene loci have been used in genotypic analysis of G. duodenalis [21]. In the present study, all G. duodenalis isolates were amplified and sequenced at the tpi and bg loci. Only assemblage B was identified, which further confirmed the dominance of G. duodenalis assemblage B in rabbits [4,14,15,18,23]. However, the present result was different from the result reported in rabbits in Henan, Central China [4], where assemblage E was also found. G. duodenalis assemblage B has been widely reported in cattle [24], sheep, goats [25], rabbits [4,14,15], diarrheal outpatients [26], dogs, and cats [27] in China. Furthermore, one new subtype and one known subtype (Bb-7) were observed at the tpi and bg loci within the assemblage B, respectively, in the present study. The obtained tpi sequences (accession no. KT372238) had 99% homology with the assemblage B reference sequence from a rabbit in Harbin City (accession no. HQ397719), and only one insertion of a single nucleotide (-/C) presented at 514 sites. Moreover, the obtained bg gene sequences were identical to the published assemblage B bg sequences isolated from a Lemur catta (Ring-tailed lemur) (accession no. KJ888980, subtype: Bb-7) in China, and a Homo sapiens in Canada (accession no. KM190805). These results indicate a low diversity of G. duodenalis in domestic rabbits in Jilin and Liaoning province, Northeastern China. The results also suggested that attention should be paid to interspecies transmission of G. duodenalis among rabbits, humans, and other animals. In conclusion, the present study indicated the existence of G. duodenalis (9.86%) infection in rabbits in Jilin and Liaoning province, Northeastern China. Risk factors for G. duodenalis infection wee highly associated with the farming mode and collection region of the rabbits. Furthermore, the present study further confirmed the dominance of G. duodenalis assemblage B in rabbits, which might play a major role as zoonotic reservoirs for G. duodenalis. Effective control and prevention measures should be undertaken to prevent G. duodenalis infection in domestic rabbits, other animals, and humans.
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Funding No funding sources. Competing interests None declared. Ethical approval All operations were handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. This study was approved by the Animal Ethics Committee of Jilin Agricultural University. References [1] Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;24(1):110–40. [2] Zheng G, Hu W, Liu Y, Luo Q, Tan L, Li G. Occurrence and molecular identification of Giardia duodenalis from stray cats in Guangzhou, southern China. Korean J Parasitol 2015;53(1):119–24. [3] Shin JC, Reyes AW, Kim SH, Kim S, Park HJ, Seo KW, et al. Molecular detection of Giardia intestinalis from stray dogs in animal shelters of Gyeongsangbuk-do (Province) and Daejeon, Korea. Korea J Parasitol 2015;53(4):477–81. [4] Qi M, Xi J, Li J, Wang H, Ning C, Zhang L. Prevalence of zoonotic Giardia duodenalis assemblage B and first identification of assemblage E in rabbit fecal samples isolates from Central China. J Eukaryot Microbiol 2015;62(6):810–4. [5] Adamska M. Molecular characterization of Cryptosporidium and Giardia occurring in natural water bodies in Poland. Parasitol Res 2015;114(2):687–92. [6] Kaplan JE, et al. Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm Rep 2009;58(RR-4):1–207. [7] Lane S, Lloyd D. Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol 2002;28(2):123–47. [8] Elfadaly HA, Hassanain NA, Hassanain MA, Barakat AM, Shaapan RM. Evaluation of primitive ground water supplies as a risk factor for the development of major waterborne zoonosis in Egyptian children living in rural areas. J Infect Public Health 2017, http://dx.doi.org/10.1016/j.jiph.2017.07.025 [Epub ahead of print]. [9] Santin M, Fayer R. Enterocytozoon bieneusi, Giardia, and Cryptosporidium infecting white-tailed deer. J Eukaryot Microbiol 2015;62(1):34–43. [10] Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;24(1):110–40. [11] Shin JC, Kim DG, Kim SH, Kim S, Song KH. Seroprevalence of Encephalitozoon cuniculi in pet rabbits in Korea. Korean J Parasitol 2014;52(3):321–3. [12] Xie X, Bil J, Shantz E, Hammermueller J, Nagy E, Turner PV. Prevalence of lapine rotavirus, astrovirus, and hepatitis E virus in Canadian domestic rabbit populations. Vet Microbiol 2017;208:146–9.
[13] Ni X, Qin S, Lou Z, Ning H, Sun X. Seroprevalence and risk factors of Chlamydia infection in domestic rabbits (Oryctolagus cuniculus) in China. Biomed Res Int 2015;2015:460473. [14] Zhang W, Shen Y, Wang R, Liu A, Ling H, Li Y, et al. Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission. PLoS One 2012;7(2):e31262. [15] Liu A, Yang F, Shen Y, Zhang W, Wang R, Zhao W, et al. Genetic analysis of the Gdh and Bg genes of animal-derived Giardia duodenalis isolates in Northeastern China and evaluation of zoonotic transmission potential. PLoS One 2014;9(4):e95291. [16] Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 2003;9(11):1444–52. [17] Cacciò SM, De Giacomo M, Pozio E. Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol 2002;32(8):1023–30. [18] Pantchev N, Broglia A, Paoletti B, Globokar Vrhovec M, Bertram A, Nöckler K, et al. Occurrence and molecular typing of Giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in Europe during 2006–2012. Vet Rec 2014;175(1):18. [19] Koehler AV, Haydon SR, Jex AR, Gasser RB. Cryptosporidium and Giardia taxa in faecal samples from animals in catchments supplying the city of Melbourne with drinking water (2011 to 2015). Parasit Vectors 2016;9(1):315. [20] Vasco K, Graham JP, Trueba G. Detection of zoonotic Enteropathogens in children and domestic animals in a semirural community in Ecuador. Appl Environ Microbiol 2016;82(14):4218–24. [21] Bouzid M, Halai K, Jeffreys D, Hunter PR. The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples. Vet Parasitol 2015;207(3–4):181–202. [22] Imre K, Morar A, Ilie MS, Plutzer J, Imre M, Emil T, et al. Survey of the occurrence and human infective potential of Giardia duodenalis and Cryptosporidium spp. in wastewater and different surface water sources of Western Romania. Vector Borne Zoonotic Dis 2017;17(10):685–91. [23] Lebbad M, Mattsson JG, Christensson B, Ljungström B, Backhans A, Andersson JO, et al. From mouse to moose: multilocus genotyping of Giardia isolates from various animal species. Vet Parasitol 2010;168(3–4):231–9. [24] 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. [25] Zhang W, Zhang X, Wang R, Liu A, Shen Y, Ling H, et al. Genetic characterizations of Giardia duodenalis in sheep and goats in Heilongjiang Province, China and possibility of zoonotic transmission. PLoS Negl Trop Dis 2012;6(9):e1826. [26] Liu H, Shen Y, Yin J, Yuan Z, Jiang Y, Xu Y, et al. Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China. BMC Infect Dis 2014;14(25). [27] Li W, Li Y, Song M, Lu Y, Yang J, Tao W, et al. Prevalence and genetic characteristics of Cryptosporidium, Enterocytozoon bieneusi and Giardia duodenalis in cats and dogs in Heilongjiang province, China. Vet Parasitol 2015;208(3–4):125–34. [28] Epe C, Coati N, Schnieder T. Results of parasitological examinations of faecal samples from horses, ruminants, pigs, dogs, cats, hedgehogs and rabbits between 1998 and 2002. Dtsch Tierarztl Wochenschr 2004;111(6):243–7 [Article in German]. [29] Roug A, Byrne BA, Conrad PA, Miller WA. Zoonotic fecal pathogens and antimicrobial resistance in county fair animals. Comp Immunol Microbiol Infect Dis 2013;36(3):303–8.
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Contents lists available at ScienceDirect
Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph
Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China Jing Jiang a,1 , Jian-Gang Ma d,g,1 , Nian-Zhang Zhang c , Peng Xu e , Guangyu Hou b,∗ , Quan Zhao a,∗ , Xiao-Xuan Zhang f a
College of Animal Science and Technology, Chanchun Sci-Tech University, Shuangyang, Jilin Province 130600, PR China College of Basic Medicine, Mudanjiang Medical College, Mudanjiang, Heilongjiang Province 157011, PR China c State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China d College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province, 712100, PR China e College of Animal Husbandry and Veterinary Medicine, Jinzhou Medical University, Jinzhou, Liaoning Province 121001, PR China f College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China g College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin Province 130118, PR China b
a r t i c l e
i n f o
Article history: Received 6 October 2017 Received in revised form 21 January 2018 Accepted 21 February 2018 Keywords: Domestic rabbits Prevalence Genotyping Northern China
a b s t r a c t Background: Giardia duodenalis is one of the most important intestinal parasites that can infect virtually all animals, including rabbits and humans. However, there is little information regarding the prevalence and genotypes of G. duodenalis in domestic rabbits in China. Methods: A total of 426 rabbit fecal samples (136 from Shenyang City, 174 from Changchun City, and 116 from Jilin City) were examined by Lugol’s iodine staining with microscopy analysis, and the positive samples were genotyped at the triosephosphate isomerase (tpi) and the beta giardin (bg) gene loci using nested PCR. Results: Forty-two (9.86%) out of 426 rabbit fecal samples were G. duodenalis-positive under microscopy analysis, and the highest G. duodenalis infection rate was 23.08% on farm 6. The prevalence of G. duodenalis in rabbits from different cities ranged from 1.47% to 14.37%. Among different age groups, G. duodenalis prevalence in rabbits ranged from 5.41% to 12.58%. The prevalence of G. duodenalis in outdoor rabbits and indoor rabbits was 14.29% and 6.77%, respectively. In the present study, region and farming mode were highly correlated with G. duodenalis infection in rabbits. All 42 G. duodenalis isolates were successfully amplified and sequenced at the tpi and bg loci, and only G. duodenalis assemblage B were identified. Conclusion: This study not only further confirmed the dominance of G. duodenalis assemblage B in rabbits, but also further improved the foundation data concerning the distribution of G. duodenalis assemblages in China. © 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Giardiasis is caused by Giardia duodenalis (syn. Giardia intestinalis, G. lamblia), a widespread enteric parasite of mammalian species [1–4]. Giardiasis is transmitted through the fecal-oral route [5], and shows symptoms of chronic diarrhea [4]. However, G. duo-
∗ Corresponding authors. E-mail addresses: [email protected] (G. Hou), [email protected] (Q. Zhao). 1 These authors have contributed equally to this work.
denalis coinfection with immune-compromised patients can cause significant morbidity and mortality [6]. In general, giardiasis affects around 2.0 × 108 people worldwide each year [7]. A wide range of animals and humans were reported as hosts for G. duodenalis [8]. Phylogenetic analysis revealed that eight assemblages (A–H) of G. duodenalis could be identified worldwide at present [1,9]. Among them, G. duodenalis assemblages A and B are responsible for the majority of human infections [10]. More importantly, some assemblages of G. duodenalis (e.g., assemblages A and B) can infect both humans and animals, raising public health concerns.
https://doi.org/10.1016/j.jiph.2018.02.008 1876-0341/© 2018 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Table 1 Summarized the prevalence and assemblages of Giardia duodenalis in rabbits in the world (available data). Regions
No. tested
No. positive
Prevalence
Assemblages
References
Central China Heilongjiang Europe Australia Ecuador Germany California
955 378 528 97 20 232 4
80 28 40 1 4 0 0
8.4% 7.41% 7.6% 1.03% 20% 0% 0%
B and E B B A NA – –
[4] [14] [18] [19] [20] [28] [29]
Recently, studies have suggested that rabbits are an important reservoir for many pathogens [11–13], but only a small number of genotypes/assemblages of G. duodenalis in rabbits were reported around the world (listed in Table 1), especially in China. In China, information regarding the prevalence and genotypes of G. duodenalis infection in rabbits was only reported in Heilongjiang Province [14,15] and Henan province [4]. However, information concerning the prevalence and genotypes of G. duodenalis in rabbits in Jilin and Liaoning Province remains unclear. The present study aimed to investigate the prevalence of G. duodenalis in rabbits and to identify their genotypes/assemblages. We also aimed to estimate the zoonotic potential of G. duodenalis from rabbits to humans. Methods Specimen collection and preparation The study population comprised of 426 rabbits collected from three cities in China, in which nearly 3,437,000 rabbits were raised in 2014. The samples collected from nine farms, raising more than 3000 rabbits. According to the reported prevalence of Giardia in rabbit populations of 7.41% in 2012 [14], the expected prevalence was 7.5% (P) with an accepted deviation of the true prevalence of 5% (d) and a confidence level of 95% (z = 1·96). The sample size was calculated as 107 [according to n = P (1 − P)z2 /d2 ]. This study was carried out in Jilin province (41◦ –46◦ N, 122◦ –131◦ E) and Liaoning province (38◦ –43◦ N, 118◦ –125◦ E), Northeast China, between May 2015 and June 2015. A total of 426 rabbit fecal samples were randomly collected from the ground after defection using sterile gloves, and were then put into box containing ice, immediately. We only used fresh and clean samples from the middle of the fecal sample for further analysis. Information regarding region, farming mode, and age were acquired by a questionnaire filled in by the farmers. Collection and preparation of feces samples Giardia cysts in the fecal materials were detected with Lugol’s iodine staining. All Giardia-positive fecal specimens were washed three times, and genomic DNA was extracted using an E.Z.N.A. Stool DNA kit (OMEGA Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer’s procedures. The extracted DNA samples were stored at −20 ◦ C until PCR analysis. PCR amplification G. duodenalis assemblages were determined by nested PCR of an approximately 530 bp fragment of the triosephosphate isomerase (tpi) locus [16] and a 511 bp fragment of the beta giardin (bg) gene locus [17]. The PCR reactions for all genes were performed in 25l PCR reaction composed of 1 × Ex Taq buffer (Mg2+ free), 2 mM MgCl2 , 200 M each dNTP, 0.4 M each primer, 0.625 U of Takara Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), and 2 l of DNA. The cycling conditions were: 94 ◦ C for 3 min; 35 cycles of 94 ◦ C for 45 s, 55 ◦ C for 45 s, and 72 ◦ C for 60 s; followed by 72 ◦ C for 10 min.
Both positive (Giardia-positive isolates collected from cattle and stored at the laboratory) and negative controls were included in each test. All the secondary PCR products were examined by electrophoresis in 1.5% agarose gels and observed under UV light after GoldViewTM (Solarbio, Beijing, China) staining. Sequencing and phylogenetic analyses All of the positive secondary PCR products were sent to the Genscript Company (Nanjing, China) for bidirectional sequencing. Meanwhile, genotypes that produced sequences with mutations, such as single nucleotide substitutions, deletions, or insertions, and that were confirmed by the DNA sequencing of at least two PCR products, were considered as novel genotypes. The genotypes/assemblages of G. duodenalis were determined by alignment with reference sequences available in GenBank using the computer program Clustal X 1.83 and BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/). Statistical analysis Statistical analysis of the prevalence of G. duodenalis infection in rabbits from different region (x1), farming mode (x2), and age (x3) was performed a Chi-square test in SAS (SAS Institute Inc., Cary, NC, USA, Version 9.1). In the multivariable regression analysis, each of these variables was included in the binary Logit model as an independent variable. The best model was judged by Fisher’s scoring algorithm. All tests were two-sided, and the results were considered statistically significant if P < 0.05. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were provided to explore the strength of the association between G. duodenalis-positivity and the conditions investigated. Accession numbers for nucleotide sequences All the representative nucleotide sequences obtained in this study were deposited in GenBank with following accession numbers: KT372238 for the tpi gene, and KT715813 for the bg gene. Results A total of 42 (9.86%) out of 426 rabbit samples were detected as G. duodenalis-positive (Table 2), and the highest G. duodenalis infection rate was 23.08% on farm 6 (Table 3). Rabbits collected from Changchun City had a highest G. duodenalis infection rates compared with those collected from Jilin City and Shenyang City (Table 2). Moreover, in different age groups, the G. duodenalis prevalence in rabbits ranged from 5.41% to 12.58% (Table 3), and G. duodenalis prevalence in outdoor rabbits and indoor rabbits was 14.29% and 6.77%, respectively. All 42 G. duodenalis isolates were successfully amplified and sequenced at the tpi and bg loci. Sequencing analyses of the tpi and bg genes revealed that only G. duodenalis assemblage B was identified in this study.
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Table 2 Factors associated with prevalence of Giardia duodenalis in rabbits in Jilin Province and Liaoning Province, northeastern China. Factor
No. tested
No. positive
% (95% CI)
OR (95% CI)
Region
136 174 116 251 175 37 172 159 58
2 25 15 17 25 2 16 20 4
1.47 (0.00–3.49) 14.37 (9.16–19.58) 12.93 (6.83-19.04) 6.77 (3.66–9.88) 14.29 (9.10–19.47) 5.41 (0.00–12.69) 9.30 (4.96–13.64) 12.58 (7.42–17.73) 6.90 (0.38–13.42)
Reference 11.24 (2.61–48.36) 9.95 (2.23–44.50) Reference 2.29 (1.20–4.39) Reference 1.80 (0.40–8.17) 2.52 (0.56–11.29) 1.30 (0.23–7.46)
426
42
9.86 (7.03–12.69)
Farming mode Age
Total
Table 3 Distribution of Giardia duodenalis assemblages in rabbits in different farms in Jilin Province and Liaoning Province, northeastern China. Region
Farm ID
Farm mode
Sample size
No. positive
Prevalence (%) (95% CI)
Changchun City
Farm 1 Farm 2 Farm 3 Farm 4 Farm 5 Farm 6 Farm 7 Farm 8 Farm 9
Indoor Indoor Outdoor Outdoor Indoor Outdoor Indoor Outdoor Indoor
62 33 47 32 77 39 36 57 43
6 4 10 5 6 9 – 1 1
9.68 (2.32–17.04) 12.12 (0.99–23.26) 21.28 (9.58–32.98) 15.63 (3.04–28.21) 7.79 (1.81–13.78) 23.08 (9.85–36.30) 0 (–) 1.75 (0.00–5.16) 2.33 (0.00–6.83)
426
42
9.86 (7.03–12.69)
Jilin City Shenyang City
Total
Discussion In the present study, the overall infection rates for G. duodenalis was 9.86% (42/426, 95% CI 7.03–12.69), which was higher than that in Europe (7.6%, 40/528) [18], Melbourne, Australia (1.03%, 1/97) [19], Henan province (8.4%, 80/955) [4], and Heilongjiang province (7.41%, 28/378) [14], but lower than that the 20.0% (4/20) in rabbits in Ecuador [20]. Fecal samples containing low cyst intensity might lead to the low detection rates of the parasite by microscopy [21]. Moreover, many factors, such as test methods, sample sizes, animal health status, and geo-ecological conditions could also influence the result of different studies. The Giardia cysts were also found in water [22]; therefore, outbreaks of water-born Giardia should be monitored. Table 2 shows the exposure regarding farming mode and collection region associated with G. duodenalis-positivity in domestic rabbits based on univariate analysis. The impacts of multiple variables on G. duodenalis were evaluated by the forward stepwise logistic regression analysis using Fisher’s scoring technique. In the final model, two variables had effects on the infectious disease, described by the following equation y = 0.7891 × 1 + 0.7956 × 2 − 0.3218. Region and farming mode had positive effects on the risk of G. duodenalis, for which the ORs were 2.20 (95% CI 1.40–3.46) and 2.22 (95% CI 1.15–4.29), respectively. G. duodenalis can be transmitted through the ingestion of Giardia cysts [5]; therefore, the higher breeding density in Jilin Province compared with that in Liaoning Province is one of the major reasons why rabbits collected from Changchun City (14.37%, 95% CI 9.16–19.58) and Jilin City (12.93%, 95% CI 6.83–19.04) has a significantly higher G. duodenalis prevalence than that collected from Shenyang City (1.47%, 95% CI 0.00–3.49) (Table 2). Moreover, many factors, such as ecological and geographical factors, sanitation, animal welfare, and management mode may also be related to the infection rates of G. duodenalis in different regions. Farming mode was another risk factor for G. duodenalis infection in rabbits in this study. Outdoor rabbits had a significantly higher G. duodenalis prevalence than that in indoor rabbits. Outdoor-reared rabbits have more opportunities to contact with environmental specimens con-
taminated by cysts compared to those reared indoors, especially when it rains. The small subunit ribosomal RNA (SSUrRNA), glutamate dehydrogenase (gdh), elongation factor 1 alpha (ef-1 a), tpi, and bg gene loci have been used in genotypic analysis of G. duodenalis [21]. In the present study, all G. duodenalis isolates were amplified and sequenced at the tpi and bg loci. Only assemblage B was identified, which further confirmed the dominance of G. duodenalis assemblage B in rabbits [4,14,15,18,23]. However, the present result was different from the result reported in rabbits in Henan, Central China [4], where assemblage E was also found. G. duodenalis assemblage B has been widely reported in cattle [24], sheep, goats [25], rabbits [4,14,15], diarrheal outpatients [26], dogs, and cats [27] in China. Furthermore, one new subtype and one known subtype (Bb-7) were observed at the tpi and bg loci within the assemblage B, respectively, in the present study. The obtained tpi sequences (accession no. KT372238) had 99% homology with the assemblage B reference sequence from a rabbit in Harbin City (accession no. HQ397719), and only one insertion of a single nucleotide (-/C) presented at 514 sites. Moreover, the obtained bg gene sequences were identical to the published assemblage B bg sequences isolated from a Lemur catta (Ring-tailed lemur) (accession no. KJ888980, subtype: Bb-7) in China, and a Homo sapiens in Canada (accession no. KM190805). These results indicate a low diversity of G. duodenalis in domestic rabbits in Jilin and Liaoning province, Northeastern China. The results also suggested that attention should be paid to interspecies transmission of G. duodenalis among rabbits, humans, and other animals. In conclusion, the present study indicated the existence of G. duodenalis (9.86%) infection in rabbits in Jilin and Liaoning province, Northeastern China. Risk factors for G. duodenalis infection wee highly associated with the farming mode and collection region of the rabbits. Furthermore, the present study further confirmed the dominance of G. duodenalis assemblage B in rabbits, which might play a major role as zoonotic reservoirs for G. duodenalis. Effective control and prevention measures should be undertaken to prevent G. duodenalis infection in domestic rabbits, other animals, and humans.
Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008
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Funding No funding sources. Competing interests None declared. Ethical approval All operations were handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. This study was approved by the Animal Ethics Committee of Jilin Agricultural University. References [1] Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;24(1):110–40. [2] Zheng G, Hu W, Liu Y, Luo Q, Tan L, Li G. Occurrence and molecular identification of Giardia duodenalis from stray cats in Guangzhou, southern China. Korean J Parasitol 2015;53(1):119–24. [3] Shin JC, Reyes AW, Kim SH, Kim S, Park HJ, Seo KW, et al. Molecular detection of Giardia intestinalis from stray dogs in animal shelters of Gyeongsangbuk-do (Province) and Daejeon, Korea. Korea J Parasitol 2015;53(4):477–81. [4] Qi M, Xi J, Li J, Wang H, Ning C, Zhang L. Prevalence of zoonotic Giardia duodenalis assemblage B and first identification of assemblage E in rabbit fecal samples isolates from Central China. J Eukaryot Microbiol 2015;62(6):810–4. [5] Adamska M. Molecular characterization of Cryptosporidium and Giardia occurring in natural water bodies in Poland. Parasitol Res 2015;114(2):687–92. [6] Kaplan JE, et al. Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm Rep 2009;58(RR-4):1–207. [7] Lane S, Lloyd D. Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol 2002;28(2):123–47. [8] Elfadaly HA, Hassanain NA, Hassanain MA, Barakat AM, Shaapan RM. Evaluation of primitive ground water supplies as a risk factor for the development of major waterborne zoonosis in Egyptian children living in rural areas. J Infect Public Health 2017, http://dx.doi.org/10.1016/j.jiph.2017.07.025 [Epub ahead of print]. [9] Santin M, Fayer R. Enterocytozoon bieneusi, Giardia, and Cryptosporidium infecting white-tailed deer. J Eukaryot Microbiol 2015;62(1):34–43. [10] Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;24(1):110–40. [11] Shin JC, Kim DG, Kim SH, Kim S, Song KH. Seroprevalence of Encephalitozoon cuniculi in pet rabbits in Korea. Korean J Parasitol 2014;52(3):321–3. [12] Xie X, Bil J, Shantz E, Hammermueller J, Nagy E, Turner PV. Prevalence of lapine rotavirus, astrovirus, and hepatitis E virus in Canadian domestic rabbit populations. Vet Microbiol 2017;208:146–9.
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Please cite this article in press as: Jiang J, et al. Prevalence and risk factors of Giardia duodenalis in domestic rabbbits (Oryctolagus cuniculus) in Jilin and Liaoning province, northeastern China. J Infect Public Health (2018), https://doi.org/10.1016/j.jiph.2018.02.008