Experimental Parasitology 154 (2015) 20–24

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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

Research Brief

Prevalence and molecular characterization of Giardia duodenalis isolates from dairy cattle in northeast China Gang Liu a, Yan Su a, Mengjiao Zhou a, Jixue Zhao c, Tianyu Zhang a, Waqas Ahmad a, Huijun Lu a, Ning Jiang a, Qijun Chen a, Mei Xiang b,*, Jigang Yin a,** a

Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun 130062, China The Second Hospital of Jilin University, Changchun 130041, China c The First Hospital of Jilin University, Changchun 130021, China b

H I G H L I G H T S

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In this study, the prevalence of G. duodenalis in cattle is 7.9% (52/655). Livestock-specific assemblage E is the major genotype (50/52). E-XI, E-I and E-III are the major subtypes of assemblage E.

A R T I C L E

I N F O

Article history: Received 8 January 2015 Received in revised form 5 March 2015 Accepted 20 March 2015 Available online 3 April 2015 Keywords: Giardia duodenalis Cattle Assemblage Subtype

A B S T R A C T

Giardia duodenalis is an important zoonotic intestinal parasite responsible for diarrhea in humans and other animals worldwide. The present study was conducted to assess the prevalence of bovine giardiosis and to perform molecular characterization of Giardia duodenalis in the northeast of China. A total of 655 fecal specimens were collected from dairy cattle in 15 farms located in three different provinces. G. duodenalis assemblages and subtypes were determined by sequence analysis of the triosephosphate isomerase (TPI) gene. As a whole, the G. duodenalis infection rate in dairy cattle was 7.9% (52/655), as determined by Lugol’s iodine staining. Two assemblages were identified, namely, the potentially zoonotic assemblage A (n = 1), the livestock-specific assemblage E (n = 50), and a mixed infection case of assemblages A and E. Seven distinct subtypes of E assemblages were identified and E-XI, E-I and E-III are the major subtypes. Only subtype A-I was identified in assemblage A. Findings relevant to assemblage A are of public health importance. The results indicated the livestock-specific assemblage E is the major genotype and zoonotic assemblage A or B occurs very seldomly which is significantly different with previous report in the same area. So that determination of genotypes in individual epidemiological setting can make important contributions to public health. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Giardiosis is a major diarrheal disease in humans, ruminants, companion animals and wild mammals worldwide (Ballweber et al., 2010; Feng and Xiao, 2011; Thompson and Smith, 2011). As one of the most common intestinal protozoan parasites, Giardia can infect humans, domestic livestock, and wildlife. Thus, Giardiosis is

* Corresponding authors. Fax: +86 431 88796212 E-mail address: [email protected] (M. Xiang). ** Corresponding authors. Fax: +86 431 87835730. E-mail address: [email protected] (J. Yin). http://dx.doi.org/10.1016/j.exppara.2015.03.020 0014-4894/© 2015 Elsevier Inc. All rights reserved.

considered to be a zoonotic disease and has veterinary health importance. Among six known Giardia species, only Giardia duodenalis (also known as Giardia lamblia or Giardia intestinalis) has been found to infect humans and most mammals. Giardia duodenalis consists of at least eight (A–H) genetically different assemblages (Monis et al., 2009). They are genetically and morphologically distinguishable and can infect humans and mammals, with some being host-specific and others having low host specificity. Among them, assemblages A and B appear to have the widest host ranges, possibly infecting humans and many other species of animals, and these are considered to be potentially zoonotic, whereas the remaining assemblages represent host-specific lineages, with assemblages C and D being mostly found in dogs. Assemblage E infects domestic ruminants and

G. Liu et al./Experimental Parasitology 154 (2015) 20–24

pigs, assemblages F and H infect cats and marine mammals, respectively, while assemblage G only infects mice and rats (Ballweber et al., 2010; Feng and Xiao, 2011; Monis et al., 2003; Thompson and Smith, 2011; Xiao and Fayer, 2008). Diarrhea is the common clinical symptom found in human and animal giardiosis, while Giardia is responsible for abdominal cramps, bloating, weight loss, malabsorption and ill thrift in children and young calves (Carvalho-Costa et al., 2007; Nematian et al., 2008), which result in growth and developmental retardation, even in asymptomatic cases (Prado et al., 2005). Some studies have revealed that close contact with farm animals is associated with the high prevalence of human giardiosis (Ekramul Hoque et al., 2002; Hoque et al., 2003). Due to the great number of cattle, the large feces output, and the high prevalence of Giardia infection, bovine giardiosis is of great concern. Therefore, calves are thought to be of public health significance, both as a source of waterborne outbreaks of giardiosis in humans and as a risk to animal handlers (Khan et al., 2011; Thompson and Monis, 2012). To date, few studies have been conducted to determine the prevalence of bovine giardiosis in China, and current data on the assemblage distribution and prevalence of the different Giardia genotypes in Chinese calves remain unclear. It is essential to understand the potential transmission of cross-assemblages of G. duodenalis between humans and animals in China. Thus, this study was conducted with the aim to obtain recent data about the frequency of different Giardia species affecting dairy cattle of different ages in different provinces of northeast China.

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2.2. DNA extraction and genotyping Fecal samples were washed twice in distilled water, and total genomic DNA was isolated from 200 mg of washed fecal samples using a QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany) according to manufacturer-recommended procedures, and stored at −20 °C until further use. Giardia cysts presented in the specimens were genotyped by nested-PCR amplification of a 532-bp fragment of the TPI gene (Sulaiman et al., 2003). All secondary PCR products were sequenced to identify the genotype and subtype present. Each specimen was analyzed at least twice by PCR. 2.3. DNA sequence analysis All secondary PCR products were sequenced on an ABIPRISMTM 3730 DNA Analyzer (Applied Biosystems, USA) using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The sequence accuracy of the PCR products was confirmed by sequencing directly in both directions and by sequencing a new PCR product as necessary. The nucleotide sequences obtained in the present study were aligned with G. duodenalis reference nucleotide sequences from GenBank and were analyzed using Clustal omega. Representative nucleotide sequences were obtained and deposited in the GenBank database under accession numbers KM434066–KM434081 and KM486622. 2.4. Statistical analysis Statistical analysis was conducted using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). Infection rates with G. duodenalis were compared using Chi-square analysis at a significance of P < 0.05.

2. Materials and methods 2.1. Specimens A fresh fecal sample was collected from each animal using a sterile disposal latex glove immediately after its defecation onto the ground, and was then placed individually into a disposable plastic bag marked with the date, age, and geographic origin. A total of 655 fecal samples of calves were randomly obtained during June 2011 to May 2014 from Jilin, Changchun, Shenyang, Jiutai and Daqing, in Northeast China. Among those, 52 samples were collected in June 2011 from three farms in Daqing. A second collection was conducted in August 2013 including 115 samples from three farms in Jilin City and 134 samples from three farms in Changchun. A third collection of 226 samples was conducted from three farms in Shenyang during November 2013. The last collection of 128 samples was obtained from three farms in Jiutai in May 2014. Fats from all of the fecal specimens were removed by formalin-ethyl acetate sedimentation and smear three sides for each specimen prior to iodine wet mount staining (Rajurkar et al., 2012), and examined by microscopic examination at 400× magnification within 48 h of collection, and all G. duodenalispositive specimens were stored in 2.5% potassium dichromate solution at 4 °C prior to DNA extraction.

3. Results 3.1. Prevalence of G. duodenalis on cattle farms In the present study, 655 bovine fecal specimens from three provinces of the northeast of China were examined microscopically. Overall, 52 [7.9%; 95% confidence interval (CI): 6.1%–10.3%] were positive for G. duodenalis by PCR and were later on successfully sequenced. Among the 655 specimens, 271 (41.4%; 95% CI: 37.7%–45.2%) were less than 2-month-old calves, 236 (36.0%; 95% CI: 32.5%–39.8%) were 3- to 12-month-old calves, 148 (22.6%; 95% CI: 19.6%–26.0%) were 13- to 24-month-old heifers. The infection rates at different ages were 13.3% (36/271; 95% CI: 9.8%–17.8%), 4.7% (11/236; 95% CI: 2.6%–8.2%), 3.4% (5/148; 95% CI: 1.5%–7.7%), respectively (Table 1). The G. duodenalis infection rate in pre-weaned dairy calves (

Prevalence and molecular characterization of Giardia duodenalis isolates from dairy cattle in northeast China.

Giardia duodenalis is an important zoonotic intestinal parasite responsible for diarrhea in humans and other animals worldwide. The present study was ...
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