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Research in Veterinary Science 85 (2008) 125–127

Nested PCR-based detection of Toxoplasma gondii in Germanshepherd dogs and stray cats in South Korea

J.Y. Lee a, S.E. Lee a, E.G. Lee b, K.H. Song c,*

a Research Institute of Veterinary Medicine, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Koreab National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro,

Hokkaido 080-8555, Japanc Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Korea

Accepted 6 September 2007

Abstract

The prevalence of Toxoplasma gondii was surveyed by using a nested polymerase chain reaction (PCR) that was targeted to T. gondii

B1 gene in German shepherd dogs and stray cats. Sixty-four (46.3%) out of 138 German shepherd dogs and 50 (47.2%) out of 106 straycats were tested positive by the nested PCR assay, respectively. There was no significant difference in gender or age in German shepherddogs and stray cats. In the five positive dogs and five positive cats, the nucleotide partial sequence of the T. gondii B1 gene was identifiedby direct sequence analysis. All the sequences were identical to each other and the corresponding sequence, T. gondii B1 gene (AccessionNo. AF179871). The results suggest that the prevalence of T. gondii is high, and the nested PCR assay is useful for early detection of T.

gondii for asymptomatic dogs and cats.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Toxoplasma gondii; Nested PCR; German shepherd dog; Stray cat; Prevalence

Toxoplasma gondii is an obligate intracellular protozoanparasite that has a global distribution in animals andhuman. In South Korea, T. gondii infection has beenreported in dogs (Lee et al., 1992), cats (Suh and Joo,1999), pigs (Lee et al., 1993), horses (Gupta et al., 2002)and humans (Song et al., 2005). This agent is transmittedmainly through the ingestion of oocysts excreted in thefeces of infected cats or by meat from an intermediate hostcontaining cysts (Ettinger and Feldman, 2005). Free-livinganimals such as stray cats can be used as sentinels for theenvironmental spread of T. gondii in densely built urbanareas because they are exposed to all the infective formsof the parasite without any protection (Meireles et al.,2004). Although dogs do not produce T. gondii oocysts likecats, they can mechanically transmit oocysts after ingestingfeline feces. Ground-feeding animals such as outdoor living

0034-5288/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.rvsc.2007.09.006

* Corresponding author. Tel.: +82 42 8216789; fax: +82 42 8216703.E-mail address: songkh@cnu.ac.kr (K.H. Song).

dogs would likely become infected by oocysts from soilcontaminated with T. gondii. Canine and feline toxoplas-mosis might be an important epidemiological indicator ofthe risk of toxoplasmosis to humans.

This study examined the prevalence of T. gondii in Ger-man shepherd dogs and stray cats by nested PCR in theprovince of Kyunggi, which is a mountainous area locatedin the western part of South Korea, and Daejeon city,which is located in the mid-western of South Korea. The138 dogs (65 females and 73 males) were examined fromMarch 2006 to September 2006, which used for guardingor hunting in rural areas of Kyunggi province, which is amountainous area located in the western part of SouthKorea. The 106 stray cats (61 females and 45 males) inves-tigated were collected from March 2005 to October 2006 inDaejeon city, which is located in the mid-western of SouthKorea. The dog samples were chosen randomly from thoseentering the veterinary clinics for a Dirofilaria immitis

examination and/or annual vaccination. All the dogs and

Fig. 1. Nested PCR (B) results of clinical samples. All samples arenegative in first PCR but 96 bp DNA band is amplified in four samples innested PCR.

126 J.Y. Lee et al. / Research in Veterinary Science 85 (2008) 125–127

cats were asymptomatic, and had not received any prophy-laxis and/or treatment for T. gondii. The ages of the dogsranged from less than 1 year to more than 7 years (mean7.1 years old), and the ages of the stray cats are unknown.The blood samples were collected in a tube containingEDTA and then were stored at minus 20 �C until DNA iso-lation. 400 ll of blood was lysed in 0.1 M Tris–HCl (pH8.0) containing 1% SDS, 0.1 M NaCl and 10 mM EDTAand treated with proteinase K (100 lg/ml) for 2 h at55 �C. The DNA was then extracted with phenol/chloro-form, precipitated with ethanol, dissolved in 50 ll of aTE buffer [10 mM Tris–HCl (pH 8.0) 1 mM EDTA], andstored at 4 �C until analyzed by PCR.

In this study, nested PCR was performed using the pre-viously described primers (Jones et al., 2000), which weredesigned to detect the B1 gene of T. gondii. The targetedB1 gene is highly conserved in all T. gondii strains and ismultiple copy gene within the T. gondii genome. In the pre-vious studies, the nested PCR assay of B1 gene detected50 fg of T. gondii DNA in water and B1 gene showedhigher sensitivity and specificity than other genes in T. gon-

dii such as P30 and 18S rDNA (Jones et al., 2000). For thefirst-round amplification, the PCR reaction was performedin a 20 ll reaction mixtures, containing 1 ll templateDNA, 20 pmol of outer primers, 10 mM of dNTP mixturesand 1 unit of Prime Taq DNA polymerase (Genet Bio Ltd.,South Korea). The cycling conditions were 95 �C for 5 min,40 cycles of 95 �C for 30 sec, 57 �C for 30 sec, 72 �C for30 sec and a final extension of 72 �C for 1 min. NestedPCR was performed in 1 ll first-round PCR product astemplate DNA and 20 pmol of the inner primers. Thecycling conditions were 95 �C for 5 min, 40 cycles of95 �C for 30 sec, 62 �C for 30 sec, 72 �C for 30 sec and afinal extension of 72 �C for 1 min. For the positive control,the genomic DNA was purified from the T. gondii RHstrain.

The first and nested PCR products were analyzed byelectrophoresis using 2% TBE agarose gel and were visu-alized using ethidium bromide staining and UV radiation(Fig. 1). For the confirmation of the T. gondii B1 gene,the DNA sequences of five positive dogs and cats wereanalyzed, respectively. The amplified DNA was purifiedusing the Geneclean II kit (BIO 101 Inc., CA, USA).

Table 1Nested PCR-based prevalence of Toxoplasma gondii in German shepherd dog

German shepherd dogs

NE NP PR (%)

Sex

Females 65 31 47.7Males 73 33 45.2

Age (yrs)

<3 27 13 48.1>3 111 51 45.9

Total 138 64 46.3

NE: numbers of examined; NP: numbers of positives; PR: positive rate.

The PCR products were excised from TAE gels, dissolvedin sodium iodide and recovered into solution accordingto the manufacturer’s instructions. The purified DNAwas directly cycle sequenced in both directions usingthe inner primers on an ABI prism DNA sequencer(Applied Biosystems, CA, USA). The determinedsequence data were analyzed using the program, Genetyxversion 7.0 (Genetyx Co., Tokyo, Japan). The statisticalanalysis of the prevalence between the age, genders anddogs and stray cats, was carried out using a v2-test (SPSSver. 10.0, IL, USA). A p-value < 0.05 was consideredsignificant.

Sixty-four (46.3%) of 138 German shepherd dogsshowed positive results by using nested PCR (Table 1).Female dogs showed a higher prevalence than male dogs,but there was no statistically significant difference. In ages,although <3 group (48.1%) had a higher prevalence than>3 group (45.9%), there was no significant difference ingenders (females, 47.7% vs. males, 45.2%), They have ahigh probability of becoming exposed to a T. gondii infec-tion because investigated German shepherd dogs are usedfor hunting or guarding purposes in rural areas. It is alsobelieved that exposure to the environment or infected ani-mals has a larger effect on the molecular prevalence oftoxoplasmosis than age and gender.

In PCR assay, Suh and Joo (1999) reported that theprevalence of toxoplasmosis was 5.3% in healthy cats inSouth Korea. In the present study, 50 (47.2%) of 106 straytested positive by the nested PCR assay. There was no sig-nificant difference between genders (females, 50.1% vs.males, 42.2%), as reported by others (Ali et al., 2003). Ina previous report, the prevalence for feline toxoplasmosiswas considered to be strongly age-dependent, with the

s and stray cats

Stray cats

NE NP PR (%)

61 31 50.145 19 42.2

unknown unknown unknownunknown unknown unknown

106 50 47.2

J.Y. Lee et al. / Research in Veterinary Science 85 (2008) 125–127 127

highest rates being associated with older cats (Childs andSeegar, 1986). However, in this study, no age-dependentdifference was detected in the stray cats. In South Korea,stray cats have many chances to come in contact otherinfected wild animals as they are at the top of the foodchain. The nucleotide partial sequence of the B1 gene ofT. gondii from each of the five samples of 64 positive dogsand five samples from 50 positive cats was successfullydetermined. All the sequences were identical both to eachother and to the corresponding sequence, the B1 gene ofT. gondii (Accession No. AF179871).

In conclusion, the nested PCR assay demonstrated ahigh prevalence of T. gondii, and the nested PCR assay isuseful for early detection of T. gondii in asymptomatic dogsand cats.

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