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Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan
G Model TTBDIS-163; No. of Pages 4
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan Hiromi Sasaki a , Yasuaki Ichikawa b , Yoshimi Sakata b , Yasuyuki Endo c , Kazuo Nishigaki d , Kotaro Matsumoto a , Hisashi Inokuma a,∗ a
Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro 080-8555, Hokkaido, Japan Merial Japan Ltd., Chiyoda-Ku, 100-0014 Tokyo, Japan c Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, 890-0065 Kagoshima, Japan d Laboratory of Molecular Immunology and Infectious Disease, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, 753-8515 Yamaguchi, Japan b
a r t i c l e Keywords: Rickettsia Ehrlichia Anaplasma Anaplasma bovis Cats Japan
i n f o
a b s t r a c t The prevalence of Rickettsia, Ehrlichia, and Anaplasma in 1764 DNA samples extracted from feline peripheral blood from all 47 prefectures in Japan was evaluated by screening real-time PCR, genus-specific PCR, and DNA nucleotide sequencing. The survey revealed that all cats were negative for Rickettsia infection. Two cats were positive for Ehrlichia or Anaplasma based on the screening PCR assay. Nucleotide sequence analysis of the partial 16S rRNA including the divergent region near the 3 -end revealed that the 2 positives were most similar to Anaplasma bovis with percent identities of 99.8% and 99.2%. This was the first detection of A. bovis DNA fragments in cats. Although these 2 cats showed stomatitis, both were also infected with feline immunodeficiency virus. The relationship between A. bovis carriage and clinical disease is not yet understood. © 2012 Elsevier GmbH. All rights reserved.
Introduction The organisms in the families Rickettsiaceae and Anaplasmataceae of the order Rickettsiales are obligate intracellular, Gram-negative bacteria that were reclassified in 2001 after phylogenetic analysis of the 16S rRNA and groESL gene sequences (Dumler et al., 2001). Several species of Rickettsia cause disease in humans and other animals and have a worldwide distribution. This genus comprises the spotted fever group (SFG) and the typhus group (TG) of rickettsiae (Raoult and Roux, 1997). R. japonica, classified within the SFG, is a well-known tick-borne pathogen in Japan and the causative agent of human Japanese spotted fever (JSF) (Mahara et al., 1985). Several animals have been reported as possible reservoirs for Rickettsia. For example, R. japonica has been isolated from wild mice (Yamamoto et al., 1992). The family Anaplasmataceae comprises several genera, including Ehrlichia, Anaplasma, Neorickettsia, Wolbachia, and ‘Candidatus Neoehrlichia’. Anaplasma and Ehrlichia are especially important emerging tickborne pathogens in both humans and animals (Dumler et al., 2005). DNA fragments of species such as Ehrlichia canis, E. muris, Anaplasma platys, A. phagocytophilum, A. centrale, A. marginale, and A. bovis have been detected in several animals in Japan, including
∗ Corresponding author. Tel.: +81 155 49 5370; fax: +81 155 49 5370. E-mail address: [email protected] (H. Inokuma).
mice, dogs, cattle, and deer (Kawahara et al., 1993, 2006; Inokuma et al., 2002; Unver et al., 2003; Ooshiro et al., 2008; Jilintai et al., 2009). Companion animals, such as dogs and cats, are potential reservoirs of these tick-borne pathogens because they are often exposed to several tick species, depending on the distribution of the arthropod vectors in the environment (Shimada et al., 2003a,b) and are likely at increased risk for tick bites compared to humans, due at least in part to their activity in woodland and bush areas. In fact, dogs are known potential reservoirs for R. rickettsii, the pathogen of Rocky Mountain spotted fever (Lissman and Benach, 1980). A. phagocytophilum, a zoonotic pathogen, is also detected in dogs (Madewell and Gribble, 1982). Although cats seropositive for R. japonica have been detected (Tabuchi et al., 2007), it is not clear whether clinical illness occurs in cats or whether they can be reservoirs. Several cases of A. phagocytophilum infection of cats have been reported in European countries and Ehrlichia-like bodies have been detected in peripheral lymphocytes or monocytes of naturally exposed cats (Bjöersdorff et al., 1999; Beaufils et al., 2002; Lappin et al., 2004; Tarello, 2005). In addition, cats become clinically ill when inoculated experimentally with Neorickettsia risticii (Dawson et al., 1988). No data are available for Ehrlichia or Anaplasma infection of cats in Japan. Thus, in this study, we surveyed the prevalence of Rickettsia, Ehrlichia, and Anaplasma in cats in all 47 prefectures using PCR assays.
1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.028
Please cite this article in press as: Sasaki, H., et al., Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan. Ticks Tick-borne Dis. (2012), http://dx.doi.org/10.1016/j.ttbdis.2012.10.028
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Materials and methods DNA samples from cats throughout Japan A total of 1764 DNA samples extracted from feline peripheral blood samples was analyzed with understanding and permission of the cat owners for this study. These blood samples were taken from cats admitted to 47 private veterinary hospitals located in each of the 47 prefectures in Japan from March to October 2008 (Nakamura et al., 2010). We included cats that were outdoors at least once a week and excluded cats kept strictly indoors. Age, gender, chief complaints, and other clinical information for each cat were recorded at each hospital. We determined the status of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infection by detection of anti-FIV antibody and FeLV p27 antigen in serum using a commercially available test kit (SNAP FeLV/FIV combo kit; IDEXX Laboratories Inc., Westbrook, ME, USA) (Nakamura et al., 2010).
Screening real-time PCR We used a real-time TaqMan PCR assay reported by Stenos et al. (2005) to screen for Rickettsia with small modification in some reagents. Briefly, the assay used oligonucleotide primers CS-F (5 TCG CAA ATG TTC ACG GTA CTT T-3 ) and CS-R (5 -TCG TGC ATT TCT TTC CAT TGT G-3 ), and the probe CS-P (5 -FAM-TGC AAT AGC AAG AAC CGT AGG CTG GAT G-MGB-3 ) based on the Rickettsia citrate synthase gene (gltA). Each reaction contained 20 mM of each primer and probe, 2× Fast Universal PCR buffer (Applied Biosystems), distilled water, and 2 l of extracted DNA in a total reaction volume of 20 l. Real-time TaqMan PCR was performed using the StepOneTM Real Time PCR system (Applied Biosystems) with a R. helvetica-positive control and distilled water as a negative control, an initial holding temperature of 95 ◦ C for 600 s, followed by 50 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 60 s. Emission was monitored at the end of every 60 ◦ C annealing step on a predetermined FAM channel. The screening for Ehrlichia and Anaplasma used primers EHR-417F (5 -ACA GAA GAA GTC CCG GCA AA-3 ) and EHR-473R (5 -TTG CCC CCT CCG TAT TAC C-3 ), and the probe EHR-439P (5 FAM-CCG TGC CAG CAG C-MGB-3 ) designed for this study based on sequence alignment of 16S rRNA genes from several Ehrlichia and Anaplasma species. The reaction conditions for the Ehrlichia and Anaplasma screening were the same as for Rickettsia screening, but E. canis DNA was used as a positive control. For both screening assays, samples that showed an increasing reaction curve with final delta Rn value of more than 0.2 were selected as possible positive reactions for the next step.
Fig. 1. Phylogenetic relationships of various Anaplasma spp. based on nucleotide sequences of the 16S rRNA gene. Scale bar indicates genetic distance (0.01 substitutions/site). The Anaplasma spp. detected in this study are in bold print.
Data analysis Homology searches based on sequences of the PCR products were performed using BLAST (National Center for Biotechnology Information). We performed distance matrix calculations and constructed phylogenetic trees using ClustalW version 1.8 in the DNA Data Bank of Japan (DDBJ; Mishima, Japan [http://www.ddbj.nig.ac.jp/htmls]). We used the Kimura twoparameter method for calculating distance matrices for the aligned sequences with all gaps ignored and the neighbor-joining method to construct a phylogenetic tree. The stability of the phylogenetic tree was estimated by bootstrap analysis of 100 replications using the same program. The tree figure was generated using TreeView version 1.6.6. The GenBank accession numbers of the 16S rRNA gene sequences used to construct phylogenetic trees and to analyze percent identities were as follows: A. bovis strain South Africa, U03775; A. bovis detected from a dog in Hiroshima, Japan, HN131217; A. bovis detected from a raccoon in Hokkaido, Japan, GU937023; A. bovis detected from cattle in Okinawa, Japan, EU368732; A. phagocytophilum strain Webster, U02521; A. ovis, AF414870; A. platys, AY077619; A. marginale, AF309867; A. centrale, AF283007; and E. canis, M73221.
Results and discussion Genus-specific nested PCR and sequencing We used genus-specific nested PCR and direct sequencing to determine the species detected in screening positive samples. The primer pairs RpCS.877p/RpCS.1273r and RpCS.896f/RpCS.1258n were used for the first and second amplifications of Rickettsia, respectively (Roux et al., 1997). A semi-nested PCR with primer pairs fD1/EHR16SR and fD1/GA1UR were used for the first and second amplifications, respectively, of Ehrlichia and Anaplasma (Little et al., 1997; Parola et al., 2000). These primers cover the divergent region of the 16S rRNA gene near the 3 end. The PCR conditions were described in our previous reports (Inokuma et al., 2001; Sashika et al., 2010). When positive reactions were obtained in the second PCR, we performed direct sequencing of the PCR products and analyzed the sequences obtained as described previously (Inokuma et al., 2003).
Forty-four (2.5%) of 1764 samples were positive in the Rickettsia screening real-time PCR. However, all these samples were negative in the genus-specific nested PCR. The real-time PCR used in this study can detect most Rickettsia species in the spotted fever and typhus groups, and the specificity was examined using many bacterial species commonly found in the environment (Stenos et al., 2005). The assay is also very sensitive, capable of detecting one target copy gene per PCR reaction (Stenos et al., 2005). The criteria for determining a positive result in the screening real-time PCR (delta Rn value of more than 0.2) might be a reason for the nonspecific positive reactions in this study. R. felis which is related to cat fleas has recently been detected from wild animals in Japan (Sashika et al., 2010). Although the assay used in this study could also detect R. felis, no positive results were found in the present study.
Please cite this article in press as: Sasaki, H., et al., Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan. Ticks Tick-borne Dis. (2012), http://dx.doi.org/10.1016/j.ttbdis.2012.10.028
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In contrast, 196 (11.1%) of 1764 samples were positive in the screening real-time PCR for Ehrlichia and Anaplasma. Among these 196 samples, 12 showed an appropriately sized band (approximately 450 bp) in the genus-specific semi-nested PCR. The higher detection rate of nonspecific results in the screening realtime PCR might be due to the lower specificity of the assay because the specificity of this system was not examined. It might also be due to the selection criteria of the assay as in the Rickettsia screening assay. The nucleotide sequences of these 12 PCR products, excluding the primer region, were determined by direct sequencing. Sequence analysis revealed that 2 products, Ehime-14 and -20, were similar to A. bovis (U03775) with percent identities of 99.8% and 99.2%, respectively (Fig. 1). The second closest species was A. platys (AY077619) with percent identities of 96.9 and 96.5%, respectively. These sequences detected from feline blood have been deposited in GenBank with accession numbers of AB648912 and AB648913, respectively. Sequences from the other 10 samples were similar to nonsignificant bacteria, including Acinetobacter spp. and Archangium spp. that are known to be environmental bacteria. These results suggest that the specificities of both PCR assays for Ehrlichia and Anaplasma used in this study were not high. The two A. bovis-positive cats were originally from Ehime Prefecture in western Japan. Although these cats were kept independently by 2 different owners, both cats lived in Ozu city. Ehime-14 was a mixed-breed female of unknown age and was FIV antibody-positive and FeLV antigen-negative. She was brought to an animal hospital for ovariohysterectomy without any obvious symptoms, but the physical examination revealed stomatitis. Ehime-20 was a 10-yearold intact male, positive for both FIV antibody and FeLV antigen. He was brought to the animal hospital with anorexia, diarrhea, and fever. The physical examination also revealed that the cat had stomatitis and periodontal disease. Although both A. bovis-positive cats had stomatitis and one showed apparent clinical symptoms, the relationship between A. bovis infection and clinical illness could not be determined based on the present data because stomatitis is a common clinical sign of cats with FIV infection (Ishida and Tomoda, 1990). Because the cats had access to outdoors at least once a week, they might have A. bovis infection from tick infestation. A. bovis was first found in cattle and causes an economically devastating disease in its bovine host. The principal symptoms include fever, anorexia, diarrhea, and infrequently, involvement of the central nervous system (Matson, 1967). Recently, DNA fragments of A. bovis have been detected in several animals in Japan, including cattle, deer, dogs, and raccoons (Kawahara et al., 2006; Ooshiro et al., 2008; Jilintai et al., 2009; Sakamoto et al., 2010; Sashika et al., 2011). The present data represent the first report of A. bovis DNA from peripheral blood of cats, suggesting that A. bovis has a broad host range. Although Haemaphysalis longicornis and H. megaspinosa are possible vectors of A. bovis in Japan (Kawahara et al., 2006; Yoshimoto et al., 2010), information regarding tick infestation on the positive cats was not available. More epidemiological studies are required to confirm the epidemiological role of cats in A. bovis infection. Furthermore, although the pathogenicity of A. bovis for cats is unknown, veterinarians should be alert to the possible health risks this agent poses to cats.
Acknowledgements The authors would like to thank local veterinarians for collecting and transporting feline blood samples. This study was supported in part by Grant H21-Shinkou-Ippan-014 for Research on Emerging and Re-Emerging Infectious Diseases and a grant from Merial Japan Ltd.
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ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan Hiromi Sasaki a , Yasuaki Ichikawa b , Yoshimi Sakata b , Yasuyuki Endo c , Kazuo Nishigaki d , Kotaro Matsumoto a , Hisashi Inokuma a,∗ a
Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro 080-8555, Hokkaido, Japan Merial Japan Ltd., Chiyoda-Ku, 100-0014 Tokyo, Japan c Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, 890-0065 Kagoshima, Japan d Laboratory of Molecular Immunology and Infectious Disease, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, 753-8515 Yamaguchi, Japan b
a r t i c l e Keywords: Rickettsia Ehrlichia Anaplasma Anaplasma bovis Cats Japan
i n f o
a b s t r a c t The prevalence of Rickettsia, Ehrlichia, and Anaplasma in 1764 DNA samples extracted from feline peripheral blood from all 47 prefectures in Japan was evaluated by screening real-time PCR, genus-specific PCR, and DNA nucleotide sequencing. The survey revealed that all cats were negative for Rickettsia infection. Two cats were positive for Ehrlichia or Anaplasma based on the screening PCR assay. Nucleotide sequence analysis of the partial 16S rRNA including the divergent region near the 3 -end revealed that the 2 positives were most similar to Anaplasma bovis with percent identities of 99.8% and 99.2%. This was the first detection of A. bovis DNA fragments in cats. Although these 2 cats showed stomatitis, both were also infected with feline immunodeficiency virus. The relationship between A. bovis carriage and clinical disease is not yet understood. © 2012 Elsevier GmbH. All rights reserved.
Introduction The organisms in the families Rickettsiaceae and Anaplasmataceae of the order Rickettsiales are obligate intracellular, Gram-negative bacteria that were reclassified in 2001 after phylogenetic analysis of the 16S rRNA and groESL gene sequences (Dumler et al., 2001). Several species of Rickettsia cause disease in humans and other animals and have a worldwide distribution. This genus comprises the spotted fever group (SFG) and the typhus group (TG) of rickettsiae (Raoult and Roux, 1997). R. japonica, classified within the SFG, is a well-known tick-borne pathogen in Japan and the causative agent of human Japanese spotted fever (JSF) (Mahara et al., 1985). Several animals have been reported as possible reservoirs for Rickettsia. For example, R. japonica has been isolated from wild mice (Yamamoto et al., 1992). The family Anaplasmataceae comprises several genera, including Ehrlichia, Anaplasma, Neorickettsia, Wolbachia, and ‘Candidatus Neoehrlichia’. Anaplasma and Ehrlichia are especially important emerging tickborne pathogens in both humans and animals (Dumler et al., 2005). DNA fragments of species such as Ehrlichia canis, E. muris, Anaplasma platys, A. phagocytophilum, A. centrale, A. marginale, and A. bovis have been detected in several animals in Japan, including
∗ Corresponding author. Tel.: +81 155 49 5370; fax: +81 155 49 5370. E-mail address: [email protected] (H. Inokuma).
mice, dogs, cattle, and deer (Kawahara et al., 1993, 2006; Inokuma et al., 2002; Unver et al., 2003; Ooshiro et al., 2008; Jilintai et al., 2009). Companion animals, such as dogs and cats, are potential reservoirs of these tick-borne pathogens because they are often exposed to several tick species, depending on the distribution of the arthropod vectors in the environment (Shimada et al., 2003a,b) and are likely at increased risk for tick bites compared to humans, due at least in part to their activity in woodland and bush areas. In fact, dogs are known potential reservoirs for R. rickettsii, the pathogen of Rocky Mountain spotted fever (Lissman and Benach, 1980). A. phagocytophilum, a zoonotic pathogen, is also detected in dogs (Madewell and Gribble, 1982). Although cats seropositive for R. japonica have been detected (Tabuchi et al., 2007), it is not clear whether clinical illness occurs in cats or whether they can be reservoirs. Several cases of A. phagocytophilum infection of cats have been reported in European countries and Ehrlichia-like bodies have been detected in peripheral lymphocytes or monocytes of naturally exposed cats (Bjöersdorff et al., 1999; Beaufils et al., 2002; Lappin et al., 2004; Tarello, 2005). In addition, cats become clinically ill when inoculated experimentally with Neorickettsia risticii (Dawson et al., 1988). No data are available for Ehrlichia or Anaplasma infection of cats in Japan. Thus, in this study, we surveyed the prevalence of Rickettsia, Ehrlichia, and Anaplasma in cats in all 47 prefectures using PCR assays.
1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.028
Please cite this article in press as: Sasaki, H., et al., Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan. Ticks Tick-borne Dis. (2012), http://dx.doi.org/10.1016/j.ttbdis.2012.10.028
G Model TTBDIS-163; No. of Pages 4
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Materials and methods DNA samples from cats throughout Japan A total of 1764 DNA samples extracted from feline peripheral blood samples was analyzed with understanding and permission of the cat owners for this study. These blood samples were taken from cats admitted to 47 private veterinary hospitals located in each of the 47 prefectures in Japan from March to October 2008 (Nakamura et al., 2010). We included cats that were outdoors at least once a week and excluded cats kept strictly indoors. Age, gender, chief complaints, and other clinical information for each cat were recorded at each hospital. We determined the status of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infection by detection of anti-FIV antibody and FeLV p27 antigen in serum using a commercially available test kit (SNAP FeLV/FIV combo kit; IDEXX Laboratories Inc., Westbrook, ME, USA) (Nakamura et al., 2010).
Screening real-time PCR We used a real-time TaqMan PCR assay reported by Stenos et al. (2005) to screen for Rickettsia with small modification in some reagents. Briefly, the assay used oligonucleotide primers CS-F (5 TCG CAA ATG TTC ACG GTA CTT T-3 ) and CS-R (5 -TCG TGC ATT TCT TTC CAT TGT G-3 ), and the probe CS-P (5 -FAM-TGC AAT AGC AAG AAC CGT AGG CTG GAT G-MGB-3 ) based on the Rickettsia citrate synthase gene (gltA). Each reaction contained 20 mM of each primer and probe, 2× Fast Universal PCR buffer (Applied Biosystems), distilled water, and 2 l of extracted DNA in a total reaction volume of 20 l. Real-time TaqMan PCR was performed using the StepOneTM Real Time PCR system (Applied Biosystems) with a R. helvetica-positive control and distilled water as a negative control, an initial holding temperature of 95 ◦ C for 600 s, followed by 50 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 60 s. Emission was monitored at the end of every 60 ◦ C annealing step on a predetermined FAM channel. The screening for Ehrlichia and Anaplasma used primers EHR-417F (5 -ACA GAA GAA GTC CCG GCA AA-3 ) and EHR-473R (5 -TTG CCC CCT CCG TAT TAC C-3 ), and the probe EHR-439P (5 FAM-CCG TGC CAG CAG C-MGB-3 ) designed for this study based on sequence alignment of 16S rRNA genes from several Ehrlichia and Anaplasma species. The reaction conditions for the Ehrlichia and Anaplasma screening were the same as for Rickettsia screening, but E. canis DNA was used as a positive control. For both screening assays, samples that showed an increasing reaction curve with final delta Rn value of more than 0.2 were selected as possible positive reactions for the next step.
Fig. 1. Phylogenetic relationships of various Anaplasma spp. based on nucleotide sequences of the 16S rRNA gene. Scale bar indicates genetic distance (0.01 substitutions/site). The Anaplasma spp. detected in this study are in bold print.
Data analysis Homology searches based on sequences of the PCR products were performed using BLAST (National Center for Biotechnology Information). We performed distance matrix calculations and constructed phylogenetic trees using ClustalW version 1.8 in the DNA Data Bank of Japan (DDBJ; Mishima, Japan [http://www.ddbj.nig.ac.jp/htmls]). We used the Kimura twoparameter method for calculating distance matrices for the aligned sequences with all gaps ignored and the neighbor-joining method to construct a phylogenetic tree. The stability of the phylogenetic tree was estimated by bootstrap analysis of 100 replications using the same program. The tree figure was generated using TreeView version 1.6.6. The GenBank accession numbers of the 16S rRNA gene sequences used to construct phylogenetic trees and to analyze percent identities were as follows: A. bovis strain South Africa, U03775; A. bovis detected from a dog in Hiroshima, Japan, HN131217; A. bovis detected from a raccoon in Hokkaido, Japan, GU937023; A. bovis detected from cattle in Okinawa, Japan, EU368732; A. phagocytophilum strain Webster, U02521; A. ovis, AF414870; A. platys, AY077619; A. marginale, AF309867; A. centrale, AF283007; and E. canis, M73221.
Results and discussion Genus-specific nested PCR and sequencing We used genus-specific nested PCR and direct sequencing to determine the species detected in screening positive samples. The primer pairs RpCS.877p/RpCS.1273r and RpCS.896f/RpCS.1258n were used for the first and second amplifications of Rickettsia, respectively (Roux et al., 1997). A semi-nested PCR with primer pairs fD1/EHR16SR and fD1/GA1UR were used for the first and second amplifications, respectively, of Ehrlichia and Anaplasma (Little et al., 1997; Parola et al., 2000). These primers cover the divergent region of the 16S rRNA gene near the 3 end. The PCR conditions were described in our previous reports (Inokuma et al., 2001; Sashika et al., 2010). When positive reactions were obtained in the second PCR, we performed direct sequencing of the PCR products and analyzed the sequences obtained as described previously (Inokuma et al., 2003).
Forty-four (2.5%) of 1764 samples were positive in the Rickettsia screening real-time PCR. However, all these samples were negative in the genus-specific nested PCR. The real-time PCR used in this study can detect most Rickettsia species in the spotted fever and typhus groups, and the specificity was examined using many bacterial species commonly found in the environment (Stenos et al., 2005). The assay is also very sensitive, capable of detecting one target copy gene per PCR reaction (Stenos et al., 2005). The criteria for determining a positive result in the screening real-time PCR (delta Rn value of more than 0.2) might be a reason for the nonspecific positive reactions in this study. R. felis which is related to cat fleas has recently been detected from wild animals in Japan (Sashika et al., 2010). Although the assay used in this study could also detect R. felis, no positive results were found in the present study.
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In contrast, 196 (11.1%) of 1764 samples were positive in the screening real-time PCR for Ehrlichia and Anaplasma. Among these 196 samples, 12 showed an appropriately sized band (approximately 450 bp) in the genus-specific semi-nested PCR. The higher detection rate of nonspecific results in the screening realtime PCR might be due to the lower specificity of the assay because the specificity of this system was not examined. It might also be due to the selection criteria of the assay as in the Rickettsia screening assay. The nucleotide sequences of these 12 PCR products, excluding the primer region, were determined by direct sequencing. Sequence analysis revealed that 2 products, Ehime-14 and -20, were similar to A. bovis (U03775) with percent identities of 99.8% and 99.2%, respectively (Fig. 1). The second closest species was A. platys (AY077619) with percent identities of 96.9 and 96.5%, respectively. These sequences detected from feline blood have been deposited in GenBank with accession numbers of AB648912 and AB648913, respectively. Sequences from the other 10 samples were similar to nonsignificant bacteria, including Acinetobacter spp. and Archangium spp. that are known to be environmental bacteria. These results suggest that the specificities of both PCR assays for Ehrlichia and Anaplasma used in this study were not high. The two A. bovis-positive cats were originally from Ehime Prefecture in western Japan. Although these cats were kept independently by 2 different owners, both cats lived in Ozu city. Ehime-14 was a mixed-breed female of unknown age and was FIV antibody-positive and FeLV antigen-negative. She was brought to an animal hospital for ovariohysterectomy without any obvious symptoms, but the physical examination revealed stomatitis. Ehime-20 was a 10-yearold intact male, positive for both FIV antibody and FeLV antigen. He was brought to the animal hospital with anorexia, diarrhea, and fever. The physical examination also revealed that the cat had stomatitis and periodontal disease. Although both A. bovis-positive cats had stomatitis and one showed apparent clinical symptoms, the relationship between A. bovis infection and clinical illness could not be determined based on the present data because stomatitis is a common clinical sign of cats with FIV infection (Ishida and Tomoda, 1990). Because the cats had access to outdoors at least once a week, they might have A. bovis infection from tick infestation. A. bovis was first found in cattle and causes an economically devastating disease in its bovine host. The principal symptoms include fever, anorexia, diarrhea, and infrequently, involvement of the central nervous system (Matson, 1967). Recently, DNA fragments of A. bovis have been detected in several animals in Japan, including cattle, deer, dogs, and raccoons (Kawahara et al., 2006; Ooshiro et al., 2008; Jilintai et al., 2009; Sakamoto et al., 2010; Sashika et al., 2011). The present data represent the first report of A. bovis DNA from peripheral blood of cats, suggesting that A. bovis has a broad host range. Although Haemaphysalis longicornis and H. megaspinosa are possible vectors of A. bovis in Japan (Kawahara et al., 2006; Yoshimoto et al., 2010), information regarding tick infestation on the positive cats was not available. More epidemiological studies are required to confirm the epidemiological role of cats in A. bovis infection. Furthermore, although the pathogenicity of A. bovis for cats is unknown, veterinarians should be alert to the possible health risks this agent poses to cats.
Acknowledgements The authors would like to thank local veterinarians for collecting and transporting feline blood samples. This study was supported in part by Grant H21-Shinkou-Ippan-014 for Research on Emerging and Re-Emerging Infectious Diseases and a grant from Merial Japan Ltd.
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Please cite this article in press as: Sasaki, H., et al., Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan. Ticks Tick-borne Dis. (2012), http://dx.doi.org/10.1016/j.ttbdis.2012.10.028