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Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

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Abstract

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmaposis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.

Introduction

Anaplasma phagocytophilum (A. phagocytophilum) is a tick-borne obligate intracellular bacterium that infects the granulocytes of various mammals, including humans, sheep, goats, horses, dogs, cattle, llamas, and rodents, and first was identified as a human pathogen in 1994 (Chen et al., 1994, Dumler et al., 2005). Clinical symptoms include pyrexia, headache, respiratory symptoms and gastrointestinal symptoms in humans, and abortion, pyrexia and edema in cattle (Chen et al., 1994, Petrovec et al., 1997, Woldehiwet, 2006). Ixodes ticks are the main vector hosts of A. phagocytophilum, with Ixodes scapularis (I. scapularis) and I. pacificus harbouring the pathogen in USA, I. ricinus in Europe and I. persulcatus in Russia and Japan (Piesman and Eisen, 2008). In Japan, previous studies have reported the presence of A. phagocytophilum from reservoirs and vectors (Inokuma et al., 2007, Kawahara et al., 2006, Ohashi et al., 2005, Wuritu et al., 2009). In the present study, we have assessed the prevalence of A. phagocytophilum infections in ticks and cattle in the central area of Hokkaido by molecular epidemiological methods.

Section snippets

Detection of A. phagocytophilum in ticks

Host-seeking Ixodes adult ticks were collected by flagging with cotton flannel at the central area of Hokkaido, Japan, as described previously (Konnai et al., 2008). The ticks were transported alive to the laboratory and mechanically disrupted using pipette tips to extract DNA. As preliminary survey, a pooled salivary gland sample of I. persulcatus ticks (15 female ticks) was used for the detection of A. phagocytophilum by PCR. PCR was performed by using primers MSP2-3F (5′-CCA GCG TTT AGC AAG

Detection of A. phagocytophilum in field collected ticks

As preliminary survey, the detection of A. phagocytophilum infection was performed by a PCR method using pooled samples. As shown in Fig. 1, A. phagocytophilum was specifically detected in salivary glands of adult ticks, but not in midguts and tick carcasses. Thus, to further investigate the prevalence of A. phagocytophilum in ticks, a total of 325 unfed, host-seeking I. persulcatus and I. ovatus ticks were collected from the same area, and individually analyzed for the presence of A.

Discussion

The objectives of the present study were to assess the prevalence of A. phagocytophilum in cattle and ticks. In previous studies, molecular survey was conducted for I. persulcatus and I. ovatus in Honshu, Japan. In Hokkaido, however, molecular survey was mainly conducted for Haemophysalis ticks. In this study, A. phagocytophilum was found to be prevalent in I. persulcatus ticks (7.2%) in the surveyed area. In this surveillance, A. phagocytophilum was not detected in I. ovatus ticks, although

Conclusions

Although our study determined low prevalence rates among ticks collected from the sampling sites, the detection of infected cattle from the same areas indicates that these ticks are highly efficient in transmission of A. phagocytophilum, a significant finding given that this pathogen is zoonotic. Studies utilizing larger sample size and from different geographical regions would provide further insight on the transmission dynamics of A. phagocytophilum in these areas.

Acknowledgements

The vital information given by Prof. Hisashi Inokuma of Obihiro University of Agriculture and Veterinary Medicine, Japan, is gratefully appreciated. We are indebted to Prof. Yasuko Rikihisa, Ohio State University, for donating the control DNA, and Dr. William Harold Witola for the help in preparing the manuscript. This work was supported by grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), a grant for the Global COE (Centers of Excellence) Program

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The first two authors contributed equally to the report.

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