Seroprevalence of Toxoplasma gondii and Bartonella ...

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Hyung-Jin Park, Sang-Eun Lee, Sung-Hee Hong, Won-Ja Lee, Kyoung-Won Seo, Kun-Ho Song. ELISA for T. gondii infection. An ELISA test was performed ...
大韓獸醫學會誌 (2014) 第 54 卷 第 2 號 Korean J Vet Res(2014) 54(2) : 87~89 http://dx.doi.org/10.14405/kjvr.2014.54.2.87



Seroprevalence of Toxoplasma gondii and Bartonella henselase infection in stray cats of the Daejeon City, Korea Hyung-Jin Park1,†, Sang-Eun Lee2,†, Sung-Hee Hong2, Won-Ja Lee2, Kyoung-Won Seo1, Kun-Ho Song1,* 1

Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea 2 Division of Malaria & Parasitic Diseases, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, 187 Osongsaengmyung 2-ro, Osong-eup 363-951, Korea

(Received: October 25, 2013; Revised: February 15, 2014; Accepted: February 24, 2014) Abstract : In this study, the seroprevalence of Toxoplasma (T.) gondii and Bartonella (B.) henselae infection among stray cats in Daejeon City, Korea was surveyed. A total of seven samples were positive (7/118, 5.93%) for T. gondii including three samples from female cats (3/58, 5.2%) and four samples from male cats (4/60, 6.7%). There was no significant difference between the genders. A total 22 samples (22/118, 18.6%) were positive for B. henselae; nine were from females and 13 were from males. There was no significant difference between genders. Nineteen samples had a titer of 1 : 50, two samples had a titer of 1 : 100, and one sample had a titer of 1 : 200. The present study is the first to use serological tests to analyze B. henselae prevalence among stray cats in Korea. Keywords : Bartonella henselae, cat, Toxoplasma gondii

troversial issue, due to their uncontrolled overpopulation, public health, animal welfare [11, 19]. Also, they disrupt people’s sleep, traffic accidents, and this has been a serious issue since 2000 [12]. The purpose of this study is re-evaluate the seroprevalence of T. gondii and B. henselae which are zoonotic pathogen that can be spread by cats in Daejeon by Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT).

Introduction Toxoplasma (T.) gondii is a worldwide endemic intracellular parasite that infects most warm-blooded vertebrates including cat, and human [12, 17, 18]. It is generally known that cats are a major spreader of this zoonotic agent by eating or direct contacting with feces, soil, food or water contaminated oocysts or tissue cysts of T. gondii over a period of 1~2 weeks [5]. Cat is the only animal that excretes resistant oocysts into the environment and a single cat may excrete 10 million oocysts [15]. Recently, several reports on the prevalence of T. gondii infection in feral cats in local areas, including Gyeonggi province [9], Seoul [13] and Daejeon [11] have been published. A syndrome of fever, malasise, and regional lymphadenopathy in people that was frequently associated with contact with kittens or cats was called cat scratch disease (CDS), which is a zoonosis caused by Bartonella(B.) henselase [2, 4, 6]. The natural infection with B. henselase is usually asymptomatic, or mild clinical signs such as lymphoadenopathy, stomatitis, renal, and neurological disease in cats [7, 8]. Infected cats, even if asymptomatic, may be highly bacteremic for several months, and can be a risk factor for human infection [3, 6]. In Korea, large numbers of stray cats become a con-

Materials and Methods Animals and sampling In total 118 feral cats were captured through the local ward government’s Trap, Neuter, and Return (TNR) program; the feral cats were captured safely, subjected to ovariohysterectomy in females and castration in males, and then returned to the territory from which they were captured. Blood was collected from each cat via cephalic or jugular puncture and separated equally into 2 parts; blood in a plain test tube was centrifuged for 5 min at 1,800 × g after clotting at room temperature for 30 min.

*Corresponding author Tel: +82-42-821-6789, Fax: +82-42-821-6703 E-mail: [email protected] † The first two authors contributed equally to this work. 87

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Hyung-Jin Park, Sang-Eun Lee, Sung-Hee Hong, Won-Ja Lee, Kyoung-Won Seo, Kun-Ho Song

Table 1. Seroprevalence of Toxoplasma gondii and Bartonella henselae infection in stray cats Toxoplasma gondii

Sex Female Male Total

Bartonella henselae

NE

NP

PR (%)

NE

NP

PR (%)

58 60 118

3 4 7

5.2 6.7 5.9

58 60 118

9 13 22

15.5 21.7 18.6

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

ELISA for T. gondii infection An ELISA test was performed according to Roqueplo et al. [16] method on serum from cats, using a commercial test kit (Multi-species ID Screen Toxoplasmosis Indirect; ID Vet, France) for the detection of antibodies against the T. gondii P30 protein according to the manufacturer’s instruction. All samples were examined in twice basically and the doubtful samples were examined in triple. IFAT for B. henselae infection The indirect immunofluorescence antibody test (IFAT) was performed on the B. henselae IFAT slides (MegaCor Diagnostik, Austria) in MegaScreen BARTONELLA kit. All sera were diluted 1 : 50 (cut off) in phosphate-buffered saline and incubated on wells of the slides in a humid chamber at 37oC for 30 min. The slides were rinsed two times in PBST (PBS + 0.4% Tween 80; Sigma Aldrich, USA), two times in distilled water and air-dried. Each well of the slides was surveyed with FITC-conjugated goat anti-Cat IgG (Santa Cruz Biotechnology, USA) diluted 1 : 50 in Evans Blue (Sigma Aldrich) solution and incubated at 37oC in a humid chamber for 30 min. The slides were washed and dried as described above and observed with a fluorescence microscope. Positive samples were two fold serially diluted to determine the endpoint titer. Basically, all samples were tested in twice and the doubtful samples were repeated in once. Statistical analysis Statistical analysis was performed by a commercially available computer-based software program (IBM SPSS Statistics 18.0.0; SPSS, USA). Differences in the prevalence of infected cats by sex was analyzed by Chi-square. Statistical significance was defined at p < 0.05.

Results ELISA for detection of T. gondii Three samples were positive (3/58, 5.2%) in female cats and 4 samples were positive (4/60, 6.7%) in male cats. Total 7 samples were positive (7/118, 5.93%), and there was no significant difference in gender (Table 1). IFAT for B. henselae Nine samples were positive (9/57, 15.8%) in female cats

Table 2. Immunofluorescence antibody titers for Bartonella henselae infection in stray cats Titer

Numbers of positive

Positive rate (%)

1 : 50 1 : 100 1 : 200

19 2 1

16.10 1.69 0.84

and 13 samples were positive (13/22, 21.3%) in male cats. Total 22 samples were positive (22/118, 18.6%) in IFAT. There was no significant difference between genders. Among this, 19 samples were positive in 1 : 50 titer, 2 samples were positive in 1 : 100 titer, and only 1 sample was positive in 1 : 200 titer (Table 2).

Discussion Internationally, stray cats are a controversial issue threating public health as a host for diseases such as toxoplasmosis and bartonellosis with their overpopulation [19]. Out of 118 serologically examined cats, 7 (5.9%) were positive on presence of specific antibodies for T. gondii and there is no significant difference between gender. Recently, several reports on the prevalence of T. gondii infection in feral cats in local regions, such as in Gyeonggi Province 13% [9] , and Seoul 15.8% [13] have been published. Seropositive rate in the present study is lower than previous studies. Although it could not compare directly because of different detecting method, this result was much lower than that of described by Lee et al. [11]. It may be caused an age, sex, detecting method and different environmental condition with regions. There are few reports on the presence and distribution of infection of cats with B. henselae in Korea. According to Kim et al. [10] B. henselae was detected in 41.8% of blood samples from feral cats. Different factors that lead to infection and distribution of the disease are responsible for the presence of this illness in humans and cats. Out of 118 serologically examined cats, 22 (18.6%) were positive on presence of specific antibodies for B. henselae. Males represent 13 (21.7%) of analyzed cats, females 9 (15.5%) in this study. Antibodies at 1 : 50 titer were present in 19 sera, 1 : 100 in 2 sera, 1 : 200 in 1 serum. We observed that the group of seropositive cats is dominated by male. A previous study reported that 41.8% of cats were B.henselae positive by

Seroprevalence of Toxoplasma gondii and Bartonella henselase infection

nested polymerase chain reaction (PCR) method [10]. Because of different method, we could not compare the result directly, but, low positive rate may be influnced from different method, survayed region and researched time. Our positive rate was lower than No significant difference was observed between males and females. Al-Majali [1] did not observe a connection between sex and seropositivity on B. henselae. Al-Majali [1] determined outdoor life and predator behavior as risk factors for B. henselae. In the present study, because all sampling cats are stray cats who living in outdoors and high predator in natural environment, Korea and they are assumed to one of a risk factor for B. henselae. In addition, it is considered that outdoor cats are more chance to be exposed to contact with fleas and other sources of infection [1, 14]. Further study for correlation between the presence of fleas and/or ticks and B. henselae seropositivity should be needed. We observed a prevalence of IgG antibodies against B. henselae of 18.6% on the territory of the Daejeon city. It is reported that seroprevalence for antibodies against B. henselae varies between 5 and 80% [4]. Although all cats in the current study did not show clinical signs of Bartonella infection, clinically healthy cats can be a reservoir for CSD for human [6]. Seropositive cats with IFAT and ELISA might be from a current bacteraemic phase, or a past infection [1, 16]. For this reasons, IFAT and ELISA can be an useful tool for epidemiologic survay but not for the current disease status or diagnosis [6]. In particular, because a first patient coinfected with T. gondii and B. henselae was reported in Korea [20], the screening investigation of this zoonotic pathogens should be continuously and regularly performed in a view of monitoring a public health threaten. In conclusion, this is a first report of serological investigation for B. henselae infection among stray cats in small population.

Acknowledgments This is supported by a fund (4847-302-210-13, 2013) from the Korea Centers for Disease Control and Prevention.

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