Serological Survey of Rickettsia japonica Infection in Dogs and Cats in Japan

Abstract

Antibodies against Rickettsia japonica in 20 of 1,207 dogs and 5 of 584 cats in Japan were detected using immunofluorescence. Some antibody-positive animals were detected in Niigata and Kagawa Prefectures, areas in which Japanese spotted fever in human patients has never been identified. Some animals were positive for antibodies against other new Rickettsia species.

Rickettsiae belong to the order Rickettsiales and are obligate intracellular, gram-negative bacteria. Several species cause disease in humans and other animals and have a worldwide distribution. The genus Rickettsia comprises the spotted-fever group rickettsiae and the typhus group (TG) rickettsiae (14). In Japan, Rickettsia japonica, classified within the spotted-fever group, is the causative agent of Japanese spotted fever (JSF) (12). In 1984, the first JSF patient was reported in Tokushima Prefecture, and since then most JSF patients have been identified in the western part of Japan (12). Recent epidemiological studies clarified both the vectors and the reservoirs for JSF in Japan. Dermacentor taiwanensis and Haemaphysalis flava are confirmed vectors of R. japonica (10), and Haemaphysalis longicornis may also be a potential vector of the pathogen (17). The isolation of R. japonica from wild mice indicated that mice are a mammalian reservoir of the pathogen (19). Furthermore, dogs are also thought to be a mammalian reservoir of R. japonica, as antibodies against R. japonica have been detected in canine serum (7, 9). Dogs and cats are often exposed to a large number of tick species, depending on the distribution of these arthropod vectors in the environment (15, 16), and likely have an increased risk of tick bites compared to humans due at least in part to their activity in woodland and bush areas. Thus, dogs and cats can serve as valid indicators of the prevalence of JSF in a given area. In this study, the prevalence of antibodies against R. japonica in dogs and cats were surveyed in 35 prefectures in Japan.

Sera were collected from 1,207 dogs and 584 cats, all of which were domestic, examined in animal hospitals located within 35 prefectures (Hokkaido, Aomori, Akita, Miyagi, Fukushima, Tochigi, Ibaraki, Saitama, Tokyo, Chiba, Kanagawa, Niigata, Fukui, Nagano, Yamanashi, Shizuoka, Aichi, Gifu, Mie, Osaka, Kytoto, Nara, Wakayama, Hyogo, Tottori, Okayama, Yamaguchi, Kagawa, Tokushima, Kochi, Fukuoka, Nagasaki, Kumamoto, Miyazaki, and Okinawa) between July 2005 and July 2006. All the animals examined had experienced outdoor activity. The clinical status and epidemiological information, including the sex, age, and breed of each animal, the level of tick infestation, any history of travel, the clinical history, and the main location of activity, were recorded by the veterinarians treating these animals.

The sera were evaluated by indirect immunofluorescence analysis (IFA) using the R. japonica (strain Aoki) antigen, which was provided by H. Fujita. The bacteria were cultivated at 32°C in a tissue culture flask containing L929 cell monolayers. Heavily infected L929 cells were harvested and centrifuged at 10,000 × g for 10 min, and the pellet was suspended in phosphate-buffered saline with 3% fetal bovine serum. The suspension was applied to each well of the 18-well microscope slides. After the slides had been air dried, the cells were fixed in acetone for 15 min at room temperature. The detection of antibodies was carried out as described previously (18). The serum samples were screened at a 1:20 dilution in phosphate-buffered saline (pH 7.2) with 0.5% Tween 20. Fluorescein isothiocyanate-labeled rabbit anti-canine immunoglobulin G (Fc) conjugates (Rockland Inc., Gilbertsville, PA) or fluorescein isothiocyanate-labeled rabbit anti-feline immunoglobulin G (Fc) conjugates (Rockland Inc., Gilbertsville, PA) were used as the secondary antibodies for the IFA. Reactive antibodies were then detected using a fluorescence light microscope. End point titers were determined for those samples that reacted with the R. japonica antigen at the screening dilution. In this study, antibody titers of 1:40 or above were considered positive, as in a previous survey (7). The samples that were positive for antibodies against R. japonica were also examined for titers of antibodies against other domestic Rickettsia species, including R. helvetica (strain IP-1; H. Fujita), R. tamurae (strain AT-1; H. Fujita), and R. asiatica (strain IO-1; H. Fujita). The method was exactly the same as that described above.

Among the 1,207 dogs and 584 cats examined, 20 dogs (1.7%) and 5 cats (0.9%) had antibodies against R. japonica. Profiles of the positive animals are shown in Table 1. The end point titers of antibodies against R. japonica in the samples from the positive animals ranged from 1:40 to 1:320. Among the 25 animals that were positive for antibodies against R. japonica, 14 dogs and 5 cats also had antibodies against one or more of the other Rickettsia antigens (Table 1). Eight dogs (D1 to D8) showed the highest titers of antibodies against R. japonica, suggesting that these animals may indeed be infected with R. japonica. Furthermore, dogs D1 to D8 lived in seven prefectures, including Niigata, Mie, Osaka, Wakayama, Kagawa, Tokushima, and Fukuoka, and JSF patients have already been identified in six of these seven prefectures. In the present study, two dogs (D1 and D4) in Niigata Prefecture and another dog (D7) in Kagawa Prefecture, where JSF has not previously been reported, had significant titers of antibodies to R. japonica. These three dogs did not have histories of travel to other prefectures, indicating that R. japonica may exist in both Niigata and Kagawa Prefectures.

TABLE 1.

Profiles of dogs and cats with sera that reacted to R. japonica antigen with titers of 1:40 or more^a

Animal	IFA titer of antibodies to antigen from:				Prefecture	Age (yr)	Breed	Sex	Present or past illness(es) or condition(s)
	R. japonica	R. helvetica	R. asiatica	R. tamurae
D1	80	<40	<40	<40	Niigata	13	Beagle	F	Mammary gland tumor
D2	80	<40	<40	<40	Mie	2	Shiba	F	None
D3	80	40	<40	<40	Osaka	12	Mix	M	Diabetes mellitus
D4	40	<40	<40	<40	Niigata	3	Pomeranian	F	Otitis externa
D5	40	<40	<40	<40	Wakayama	3	Mix	F	Anemia
D6	40	<40	<40	<40	Tokushima	7	Mix	F	Heart failure
D7	40	<40	<40	<40	Kagawa	9	Mix	M	Testis tumor
D8	40	<40	<40	<40	Fukuoka	8	Mix	F	None
D9	320	320	160	320	Mie	14	Mix	F	Insect bite
D10	320	80	160	320	Kochi	5	Mix	M	Filariasis
D11	320	160	80	80	Nagasaki	9	Mix	F	IVDP
D12	80	80	80	<40	Mie	5	Toy poodle	M	Otitis externa, diarrhea
D13	80	40	80	40	Hyogo	9	German shepherd	M	None
D14	40	40	<40	<40	Chiba	13	Mix	F	None
D15	40	<40	40	40	Kanagawa	2	Miniature dachshund	M	None
D16	40	40	<40	<40	Kagawa	8	Beagle	M	Filariasis
D17	40	<40	<40	80	Mie	11	Great Pyrenees	M	Otitis externa
C1	80	40	80	40	Hyogo	7	Mix	M	FIV infection
C2	80	40	80	40	Miyazaki	17	Mix	F	Renal failure
C3	40	40	<40	<40	Chiba	11	Mix	M	Head tilt
C4	40	<40	<40	80	Mie	10	Mix	M	None
C5	40	<40	<40	80	Mie	8	Mix	M	Flea dermatitis
D18	40	<40	<40	160	Shizuoka	2	Cocker spaniel	M	None
D19	40	40	40	320	Wakayama	11	Maltese	M	Wound
D20	80	640	320	640	Yamaguchi	6	Akita	M	Filariasis

^aAnimal designations D1 to D20 represent dogs and C1 to C5 represent cats. Titers of ≥40 are listed in bold. F, female; M, male; IVDP; intervertebral disk protrusion; FIV, feline immunodeficiency virus.

Another 14 animals (D9 to D17 and C1 to C5) were positive for antibodies against R. japonica, with end point titers ranging from 1:40 to 1:320; however, their sera also cross-reacted with other antigens at no more than one dilution. The Rickettsia species that stimulated the antibodies in these samples could not be determined because there may be cross-reactivity between closely related species (6). Because of the limited amounts of sera, cross-absorption analysis to determine the stimulant (6, 11) was not performed.

The remaining three positive animals (D18 to D20) showed titers of antibodies against R. helvetica, R. tamurae, or R. asiatica at least two dilutions higher than those of antibodies against R. japonica, indicating that the positive results for antibodies against R. japonica in these three animals may be attributable to the cross-reactivity of other Rickettsia antigens. For example, D18 and D19, from Shizuoka and Wakayama Prefectures, respectively, showed the highest titers of antibodies against R. tamurae, with titers ranging from 1:80 to 1:320. The antibodies titers in these two animals were considered to have been stimulated by R. tamurae. Another dog (D20) in Yamaguchi Prefecture also showed a positive titer of antibodies against R. japonica; however, the highest titers recorded were those of antibodies against R. helvetica and R. tamurae (1:640), suggesting that the dog may be infected with R. helvetica or R. tamurae, although there have been no reports of the existence of these Rickettsia species in Yamaguchi Prefecture.

Recently, new Rickettsia species have been isolated or detected in Japan. R. helvetica was previously known to exist only in European countries (2), but R. helvetica is now widespread in Japan, from Hokkaido, the northern island, to Kyusyu, a southern island (1, 5, 10). Indeed, the first human case of R. helvetica infection was reported in the Fukui Prefecture (13). In the present study, some dogs and cats were positive for R. helvetica antibodies, although the species that stimulated the antibodies in these samples could not be determined. Furthermore, other Rickettsia species, including R. tamurae (3) and the closely related species “Candidatus Rickettsia tarasevichiae,” were also detected in ticks isolated in Japan (5, 8). R. asiatica was also isolated from Ixodes ovatus (4). Despite the wide variety of Rickettsia species in Japan, little information is available on their pathogenesis and epidemiology, including vectors and reservoir animals for these Rickettsia species. Additionally, cross-reactivity with TG rickettsiae should be considered, although the numbers of patients with TG rickettsia infection are presently very low in Japan.

Some of the antibody-positive animals analyzed in this study had present or past illnesses (Table 1). Because there is little information available on the pathogenesis of the Rickettsia species that were detected in Japan, the relationships between positive antibody titers and illness are difficult to determine. Although none of these animals had fever and erythema, typical symptoms related to spotted fever, some of them had severe conditions such as heart (D6) and renal (C2) failure. Other animals suffered from diabetes mellitus (D3) or feline immunodeficiency virus infection (C1). Such diseases can cause immunodeficiencies, which may result in the appearance of the clinical symptoms of Rickettsia infection. Further studies are required to clarify the pathogenesis of these Rickettsia species in domestic animals.

Finally, the present findings demonstrate Rickettsia antibody detection in cats in Japan, suggesting that cats may be possible carriers of the agents. Most cats have free-roaming habitats, and they suffer from tick infestation (19). As cats also live in close contact with humans, they may be important reservoir animals for tick-borne Rickettsia.

In conclusion, this study showed that blood or sera isolated from dogs and cats may supply much-needed information on the epidemiology of Rickettsia infection in Japan.