de

Pe

bin

d nmental Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, GA 30333, USA

fected warm dognotic su

e prevalence of G. duodenalis in dogs was 13.2% (27/205). The potentially

2011; Thompson and Smith, 2011). Giardia duodenalis (also known

and B infect both humans and many species of animals, thus are

represent host-specic lineages, with assemblages C and D beingmostly found in dogs, assemblage E in domestic ruminants andpigs, assemblage F in cats, assemblage G in mice and rats, andassemblage H in marine mammals (Ballweber et al., 2010; Feng

Karanis et al., 2007; Lobo et al., 2009).monly used ge-tes at theiman et al

Xiao and Fayer, 2008). For example, in a recent study in the United

on tpi sequence analysis (Scorza et al., 2012). Characterization ofG. duodenalis tpi and other genetic loci has revealed a highincidence of dog-specic assemblages C and D in kennel andhousehold canines in Croatia (Beck et al., 2012). Another studycompared three genetic loci (SSU-rDNA, elongation factor 1-alpha,and tpi) for genotyping and subtyping G. duodenalis isolates andconcluded that tpi was the most appropriate marker for assessingthe zoonotic transmission of G. duodenalis between dogs andhumans (Traub et al., 2004).

Corresponding authors. Fax: +1 404 718 4197 (L. Xiao), fax: +86 431 8798 1351(X. Zhang).

Experimental Parasitology 135 (2013) 223226

Contents lists availab

Experimental

.eE-mail addresses: lxiao@cdc.gov (L. Xiao), zhangxic@public.cc.jl.cn (X. Zhang).considered to be potentially zoonotic, whereas the remaining ones States, three main assemblages A, C, and D were seen in dogs basedas Giardia lamblia or Giardia intestinalis) is the species infectinghumans and most mammals and consists of at least eight geneti-cally different assemblages A to H. Among them, assemblages A

Triosephosphate isomerase (tpi) gene is a comnetic marker for differentiating G. duodenalis isolablage and subtype levels (Sprong et al., 2009; Sula0014-4894/$ - see front matter Published by Elsevier Inc.http://dx.doi.org/10.1016/j.exppara.2013.07.009assem-., 2003;Available online 25 July 2013

Keywords:Giardia duodenalisDogTriosephosphate isomeraseGenotypingSubtyping

zoonotic assemblage A and the dog-specic assemblage C was identied in 25 (12.2%) and two (1.0%)dogs, respectively. All assemblage A isolates belonged to sub-assemblage AI, genotype AI-1. Likewise,one subtype was found in assemblage C. The high occurrence of potentially zoonotic G. duodenalis sub-type AI-1 in dogs that are in close contact with humans is of public health concern.

Published by Elsevier Inc.

1. Introduction

Giardiasis is a major diarrheal disease in humans and domesticand wild animals worldwide (Ballweber et al., 2010; Feng and Xiao,

and Xiao, 2011; Monis et al., 2003; Thompson and Smith, 2011;Xiao and Fayer, 2008). The contamination of source water byG. duodenalis from animal reservoir hosts is of increasing publichealth concern (Baldursson and Karanis, 2011; Feng et al., 2011;Received in revised form 1 July 2013Accepted 11 July 2013

imens from dogs were screphate isomerase gene. ThDivision of Foodborne, Waterborne and Enviro

h i g h l i g h t s

The police and farm dogs have been in Signicantly higher infection rates of f The high occurrence of potentially zoo

a r t i c l e i n f o

Article history:Received 15 February 2013ith Giardia duodenalis in Shenyang.s than police dogs were seen.btype AI-1 in dogs is of public health concern.

a b s t r a c t

To assess the potential zoonotic transmission of giardiasis from dogs in China, a total of 205 fecal spec-ened for Giardia duodenalis using PCR and sequence analysis of the triosephos-bCollege of Animal Science and Veterinary Medicine, Jilin University, 5333 Xian Road, Changchun 130062, Chinac Shenyang Police Dog Technical College, 4 Baishan Road, Shenyang 110034, ChinaResearch Brief

Molecular characterization of Giardia duoand farm dogs in China

Wei Li a,b, Chengwu Liu c, Yuqiang Yu b, Jianhua Li b,Xichen Zhang b,aCollege of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Har

journal homepage: wwwnalis isolates from police

ngtao Gong b, Mingxin Song a, Lihua Xiao d,,

150030, China

le at ScienceDirect

Parasitology

lsevier .com/locate /yexpr

Dogs, as the most common companion animals, are in close contact

Prior to the collection of canine fecal specimens, permission

were kept in cages. Only one specimen per dog was used in the

PCR. Non-acetylated bovine serum albumin (TaKaRa, Japan) at a

directions. The nucleotide sequences obtained were edited using

or 16.5%; 95% CI: 0.0890.241) was slightly higher than males (9/

female adult police dogs (Table 1). In contrast, assemblage C was

rasiChromas Pro 1.33 (Technelysium Pty Ltd, Helensvale, Queensland,Australia) and aligned with reference sequences using the ClustalX1.81 package (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) to iden-tify Giardia species, genotypes, and subtypes.

2.5. Statistical analysisconcentration of 400 ng/ll was used in primary PCR to neutralizeresidual PCR inhibitors in the extracted DNA. PCR products werevisualized by electrophoresis in 1.5% agarose containing ethidiumbromide.

2.4. Genotyping and subtyping of G. duodenalis

The secondary PCR products of the anticipated size were sent tothe Sangon Company (Shanghai, China) for DNA sequencing at bothstudy. All the dogs sampled had frequent contact with their keep-ers or trainers, and the farm dogs had access to source water. Thedogs were assigned into two age groups: adult group with animalsolder than one year and juvenile group with animals aged betweentwo months to one year.

2.3. Detection of Giardia infection by PCR

Fecal specimens were washed twice in distilled water, andgenomic DNA was extracted from 0.3 g of washed specimens usinga QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) andmanufacturer-recommended procedures. G. duodenalis in speci-mens was detected by nested PCR amplication of a 532-bp frag-ment of the tpi gene as described (Sulaiman et al., 2003). Eachspecimen was analyzed twice using 2 ll of the DNA extract perwas obtained from the farm owners and the police canine manag-ers. Dogs were caged, fed alone in each cage and on the followingday, fecal specimens were collected in plastic bags. The animalswere not harmed in any way during the procedure.

2.2. Specimens

A total of 205 fecal specimens were obtained during October toDecember 2011 from police and farm dogs in Shenyang, China.Among them, 52 specimens were collected in October and Novem-ber 2011 from ve farms (canine breeding facilities) in suburb andrural areas of Shenyang, where the dogs were free ranging. A sec-ond collection of 153 specimens was done in December 2011 ata police canine training station in urban Shenyang, where dogswith humans in China. In this study, we have examined the occur-rence of G. duodenalis in 205 police and farm dogs and character-ized the parasite at both the genotype and subtype levels usingPCR and sequence analysis of the tpi gene.

2. Material and methods

2.1. Ethical considerationsThus far, few studies have been conducted to assess the occur-rence of G. duodenalis genotypes and subtypes in animals in China.

224 W. Li et al. / Experimental PaStatistical analysis was conducted using SPSS version 17.0 (SPSSInc., Chicago, IL, USA). Infection rates with G. duodenalis were com-pared using Chi-square analysis at a signicance of p < 0.05.only seen in two (1.0%; 95% CI: 0.0040.023) specimens fromfarm dogs (Table 1). The two assemblage C specimens had tpisequences that were identical to a sequence previously identiedin a dog in Atlanta, USA (GenBank accession No. AY228643)(Sulaiman et al., 2003) (Table 2). The subtype identied was namedas CI-1 in this study (Tables 1 and 2).

4. Discussion

Infection rates of G. duodenalis in dogs were reportedly high insome countries: 36.8% in Brazil, 26.6% in Italy, 23.4% in Japan,22.7% in Belgium, and 21.0% in United Kingdom (Claereboutet al., 2009; Itoh et al., 2011; Paoletti et al., 2008; Upjohn et al.,2010; Volotao et al., 2007). Lower infection rates were reportedin some other countries such as the Netherlands (15.2%), Italy(15.0%), Peru (14.5%), China (11.0%), Australia (9.4%), Nicaragua(8.0%), Thailand (7.9%), Finland (5.3%), and Poland (2.0%) (Berrilliet al., 2004; Cooper et al., 2010; Inpankaew et al., 2007; Lebbadet al., 2008; Li et al., 2012; Overgaauw et al., 2009; Palmer et al.,2008; Rimhanen-Finne et al., 2007; Solarczyk and Majewska,2010). The data obtained in this study clearly showed higher infec-tion rates of G. duodenalis in free range dogs on farms than cageddogs in a police canine training station. This may result from themore frequent contact among farm dogs. Most earlier studies haveshown lower infection rates in adult animals than in juveniles96 or 9.4%; 95% CI: 0.0330.155) (p > 0.05). On farms, male dogs(9/35 or 25.7%; 95% CI: 0.0890.425) had an infection rate higherthan females (3/17 or 17.6%; 95% CI: 0.0230.376) (p > 0.05). Inthe police canine station, 15 of 92 (16.3%; 95% CI: 0.0810.246) fe-male dogs were positive, whereas none of the 61 male dogs werepositive.

3.2. G. duodenalis genotypes and subtypes

Sequence analysis of tpi PCR products identied G. duodenalisassemblages A and C. The former was detected in 25 (12.2%; 95%CI: 0.0740.170) specimens, of which 10 were from farm dogsand 15 from police canines. Within assemblage A, one subtypewas identied: sub-assemblage AI, genotype AI-1, previouslyfound in humans, cattle, water buffaloes, cats, pigs, dogs, and sheep(Feng and Xiao, 2011). Subtype AI-1 was found in seven male adultdogs, one male juvenile, and two female adult dog on farms and 153. Results

3.1. G. duodenalis occurrence

Overall, 27 of 205 specimens [13.2%; 95% condence interval(CI): 0.0820.181] produced the expected tpi PCR product, includ-ing 15 (9.8%; 95% CI: 0.0480.148) from 153 police canines and 12(23.1%; 95% CI: 0.1000.361) from 52 farm dogs. The difference ininfection rates between farm dogs (23.1%) and police canines(9.8%) was signicant (p < 0.05). A signicantly higher infectionrate was seen in adult dogs (26 of 103 or 25.2%; 95% CI: 0.1550.349) than in juvenile dogs (1 of 102 or 1.0%; 95% CI: 0.0090.029) (p < 0.01). In the police canine training station, 15 of 70(21.4%; 95% CI: 0.1060.323) adult dogs examined were positive,whereas none of the 83 juvenile dogs were positive. On farms, 11of 33 (33.3%; 95% CI: 0.1360.530) adult dogs and one of 19(5.3%; 95% CI: 0.0510.156) juvenile dogs examined were posi-tive (p < 0.05). The overall infection rate in female dogs (18/109

tology 135 (2013) 223226(Ballweber et al., 2010; Feng and Xiao, 2011; Xiao and Fayer,2008). Thus, one recent study from China reported that the infec-tion rate of G. duodenaliswas signicantly higher in young pet dogs

olic

itive

J, an

J, an

lis a

sitio

8

GGGGGGGGGGG

rs

rasiless than six months (25.58%) than in dogs of six months to threeyears (7.37%) and adults over three years (7.04%) (Li et al., 2012).The reason for the difference between the current and earlier stud-ies is not clear.

In most studies on G. duodenalis in dogs, assemblages A, C, and Dwere the dominant assemblages (see the review by Feng and Xiao,2011 on earlier data). The dog-specic assemblages C and D werefar more prevalent than the zoonotic assemblage A in most areas(Beck et al., 2012; Berrilli et al., 2012; Feng and Xiao, 2011; Itohet al., 2011; Liang et al., 2012; McDowall et al., 2011). In contrast,

Locations No. of specimens Assemblage A

# Positive Subtype (# pos

Farms 52 10 AI-1 (7 MA, 1 MPolice canine training station 153 15 AI-1 (15 FA)Total 205 25 AI-1 (7 MA, 1 M

a MA: male adult, MJ: male juvenile, and FA: female adult.

Table 2Nucleotide sequence differences in the partial tpi gene among subtypes of G. duodena

Sequences Locations References Nucleotide at po

36 72

AY228641 USA Sulaiman et al. (2003) T GAY228642 USA Sulaiman et al. (2003) T GAY228643 USA Sulaiman et al. (2003) T GAY228644 USA Sulaiman et al. (2003) T THQ696782 USA Wang et al. (2012) T GAF069563 Australia Monis et al. (1999) T GEU781004 Sweden Lebbad et al. (2010) T GEU781005 Sweden Lebbad et al. (2010) T TEU781006 Sweden Lebbad et al. (2010) C GEU781007 Sweden Lebbad et al. (2010) C GCI-1 China This study T G

a Nucleotide position numbers according to AY228642, with the beginning of theTable 1Giardia duodenalis genotypes and subtypes based on the tpi gene characterization in p

W. Li et al. / Experimental Pain this study, assemblage A was more commonly found in dogsthan assemblage C. Assemblage D was not found in any of the dogs,but this could be due to the non-amplication of the tpi gene ofthis groups of parasites by the PCR primers used (Lebbad et al.,2010). In a few recent studies, assemblage A was also the dominantgenotype of G. duodenalis in dogs (Schurer et al., 2012; Volotaoet al., 2011). A recent study on G. duodenalis in pet dogs in Chinahad also identied both assemblages A (in 5 animals) and D (in18 animals) (Li et al., 2012).

Subtyping of assemblage A at various loci has identied at leastthree broad sub-assemblages: AI, AII, and AIII. Within each of thesesub-assemblages, various genotypes have been identied, withsub-assemblage AI (genotype A1) detected in a range of hostsincluding humans, cattle, water buffaloes, sheep, dogs, cats, pigs,and other animals. Various genotypes of AII circulate mainly in hu-mans and AIII mostly wild ruminants (Caccio et al., 2008; Feng andXiao, 2011). In China, both AI-1 and AII have been identied in hu-mans (Wang et al., 2011). In this study, most of the assemblage Ainfections in dogs was caused by subtype AI-1. Although this sub-type is more commonly seen in animals than in humans (Feng andXiao, 2011), due to the close contact between dogs and humans inChina, the frequent nding of subtype AI-1 is of potential publichealth signicance. Nevertheless, the observations at the tpi locusneed to be substantiated using other genetic loci.

Acknowledgments

This work was supported by Major Program of Preventive andControl for National Severe Infective Diseases (No. 2008ZX10004-References

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226 W. Li et al. / Experimental Parasitology 135 (2013) 223226

Molecular characterization of Giardia duodenalis isolates from police and farm dogs in China1 Introduction2 Material and methods2.1 Ethical considerations2.2 Specimens2.3 Detection of Giardia infection by PCR2.4 Genotyping and subtyping of G. duodenalis2.5 Statistical analysis

3 Results3.1 G. duodenalis occurrence3.2 G. duodenalis genotypes and subtypes

4 DiscussionAcknowledgmentsReferences