Rickettsia felis in Ctenocephalides felis from Guatemala and Costa Rica

Abstract

Rickettsia felis is an emerging human pathogen associated primarily with the cat flea Ctenocephalides felis. In this study, we investigated the presence of Rickettsia felis in C. felis from Guatemala and Costa Rica. Ctenocephalides felis were collected directly from dogs and cats, and analyzed by polymerase chain reaction for Rickettsia-specific fragments of 17-kDa protein, OmpA, and citrate synthase genes. Rickettsia DNA was detected in 64% (55 of 86) and 58% (47 of 81) of flea pools in Guatemala and Costa Rica, respectively. Sequencing of gltA fragments identified R. felis genotype URRWXCal₂ in samples from both countries, and genotype Rf2125 in Costa Rica. This is the first report of R. felis in Guatemala and of genotype Rf2125 in Costa Rica. The extensive presence of this pathogen in countries of Central America stresses the need for increased awareness and diagnosis.

Rickettsia felis is an emerging pathogen, which was first detected in the cat flea, Ctenocephalides felis. It was later associated with human disease manifesting with fever, headaches, myalgia, and occasionally rash.¹ Even though R. felis has also been detected in domestic and wild animals, their susceptibility to this bacterium and their role as reservoirs has not been well established.²

Rickettsia felis has a cosmopolitan distribution associated with fleas. In the Americas, human disease caused by R. felis has been described from the United States, Mexico, and Brazil.^2,3 Furthermore, DNA of R. felis has also been detected in fleas from Peru, Uruguay, Chile, Argentina, and more recently in the West Indies, Panamá, and Costa Rica.^2–7 There is no evidence yet of human disease caused by R. felis in Central America, although human exposure to pathogenic spotted fever group rickettsiae different from R. rickettsii, may occur in this region.^8,9 As part of an ongoing project to characterize rickettsial diseases in Guatemala and Costa Rica, we assessed the presence of R. felis in sites where cases of rickettsioses have been previously reported.

Entomological surveys were carried out at several locations in Guatemala and Costa Rica throughout 2009 and 2010, including wet and dry seasons. Collection sites in Guatemala were located in the Southeastern region, departments of Santa Rosa (14°16′N, 90°18′W) and Jutiapa (14°16′N, 89°53′W), an area suspected of a spotted fever outbreak in 2007 (Eremeeva ME, unpublished data). In Costa Rica, collections were performed at sites from the Caribbean slope of the country, where cases of Rocky Mountain spotted fever and uncharacterized spotted fevers have been documented,¹⁰ specifically in the districts of Turrialba (9°54′N, 83°41′W), La Virgen (10°23′N, 84°08′W), Limón (9°59′N, 83°02′W), Cahuita (9°44′N, 82°50′W), Guápiles (10°13′N, 83°47′W), Guácimo (10°12′N, 83°41′W), and Jiménez (10°12′N, 83°44′W). Additional samples of C. felis from two locations in San José (9°55′N, 84°04′W), obtained by the laboratory of Medical Arthropodology (University of Costa Rica) through the general public as part of inquiries and identification services, were also analyzed.

At collection locations (households, farms, etc.) fleas were collected from household cats and dogs, and from opossums captured using live animal traps. Ctenocephalides felis were grouped into lots according to host species, collection site, and location.

For the preliminary detection of DNA of Rickettsia spp., pools of 1–10 C. felis from each lot were analyzed using nested and semi-nested polymerase chain reaction (PCR) assays targeting specific fragments of the 17-kDa protein and OmpA genes. Primers R17-122 and R17-500 were used for the primary PCR of Rickettsia-specific 17-kDa protein gene, and nested PCRs were performed using primers TZ15 and TZ16 or RP2 and RPID, which detect fragments specific for Rickettsia of the spotted fever group and typhus group, respectively.^11,12 For ompA, primers Rr190-70 and Rr190-701 were used in the first PCR, and Rr190-70 and Rr190-602 for the semi-nested PCR.¹² Detection of positive samples was further confirmed in samples from Guatemala using a TaqMan assay for the citrate synthase (gltA) gene that is species specific for detection of R. typhi and R. felis,¹³ or by the Rickettsia spp. wide-range gltA assay using primers CS-78 and CS-323 in samples from Costa Rica.¹⁴

Three-hundred thirty-three C. felis were collected from two sites in Guatemala and grouped into 86 pools, including 73 flea pools from dogs and 13 from cats (Table 1). The DNA of Rickettsia was detected in 55 pools (64%), 54 from Jutiapa (78% collected from dogs and 22% from cats) and one pool from Santa Rosa collected from a dog.

Table 1.

Rickettsia felis frequency and gltA genotypes in Ctenocephalides felis pools from different areas of Guatemala and Costa Rica

Country	Site	C. felis collected	Flea pools			Positive pools (%)	Samples sequenced	R. felis genotype
			Dogs	Cats	Total
Guatemala	Jutiapa	269	57	13	70	54 (77)	22	URRWXCal2
	Santa Rosa	64	16	0	16	1 (6)	1*	URRWXCal2
	All sites	333	73	13	86	55 (65)	23	–
Costa Rica	Cahuita	96	11	1	12	6 (50)	6	Rf2125
	Guápiles/Jiménez/Guácimo	107	20	1	21	12 (57)	7	Rf2125
	La Virgen	71	11	3	14	3 (21)	2	Rf2125
	Limón	59	13	2	15	10 (67)	9	Rf2125
	Turrialba	73	14	0	14	13 (93)	11	Rf2125
	San José	33	5	0	5	3 (60)	3	URRWXCal2
	All sites	416	74	7	81	47 (58)	38	–

^*ompA amplicon.

In Costa Rica, a total 439 C. felis was collected from the different sites, all samples collected from dogs and cats (Table 1). Forty-seven pools (58%) contained Rickettsia DNA by positive PCR for at least two of the three genes analyzed, and positivity varied between sites. Forty-four of 74 pools from dogs (59%) and 3 of 7 pools from cats (43%) were positive. No C. felis was found on two Didelphis marsupialis and three Philander opossum captured in Cahuita, Guácimo, Limón, and Turrialba.

The DNA of R. typhi was not detected during this study. The presence of R. felis DNA in C. felis from Guatemala was confirmed by multiplex TaqMan gltA assay. Only one genetic type of gltA was found by sequencing of the TaqMan product, and it was the same as the URRWXCal₂ reference strain of R. felis from California (CP000053). In contrast, two genotypes were identified in fleas from Costa Rica after sequencing gltA amplicons of 38 of 47 positive samples (81%), Rf2125 (AF516333) and URRWXCal₂ of R. felis (Table 1). The gltA fragments were identical between the three sequences of R. felis URRWXCal₂ analyzed from Costa Rica, and similarity was 99.25% (399 of 402) with the sequence reported in GenBank (CP000053). The only Costa Rican fleas containing R. felis URRWXCal₂ were from dogs from the capital city, San José. Two different sequences were detected in gltA fragments of R. felis Rf2125 from positive samples of all other sites in Costa Rica, and they were both 99.25% (399 of 402) similar to the corresponding fragment of the sequence reported in GenBank (AF516333). GenBank accession nos. for fragments of R. felis gltA obtained in this study are JF523341 (Guatemala) and JN982948-JN982950 (Costa Rica).

To our knowledge, we describe the first detection of R. felis in C. felis from Guatemala. This common and widespread occurrence of R. felis in fleas in Costa Rica and Guatemala is similar to findings previously reported from other countries in Latin America.^15–17 For example, in a study that analyzed pools of C. felis from Iquitos, Peru, 71 of 74 pools contained R. felis.¹⁷

In both countries, R. felis was detected in C. felis from both dogs and cats, although more dogs were sampled. Furthermore, R. felis was detected frequently on fleas from dogs, suggesting this may be a relevant host in maintaining C. felis and possibly R. felis in the areas studied. Considering the close relationship with pet owners, dogs may indirectly pose a risk for human infection, because they may promote exposure by transporting the infected fleas to the resident environment. Although Didelphis virginiana opossums have been associated with a life cycle of R. felis in wild-caught C. felis in Texas and California,¹⁸ C. felis were not found on the few opossums captured during this study. Therefore, additional trapping of opossums is needed to elucidate their role in circulation of R. felis in Costa Rica and Guatemala.

Human infection with R. felis has not been documented in Central America, although R. felis was reported recently in Panamá and Costa Rica.^5–7 There are previous reports of R. felis Rf2125 in Latin America and other countries, but as no human disease has yet been associated with this genotype, its pathogenic potential warrants further evaluation.^19,20 Moreover, this study shows the presence of at least two different genotypes of R. felis, including the pathogenic URRWXCal₂ strain, in regions of Central America. Whether either of these genetic types has an adaptive advantage in infecting fleas has not yet been evaluated.

Because C. felis was frequently found on cats and dogs in this study, and substantial numbers of fleas tested were infected with R. felis, humans may have a high probability of exposure to this pathogenic Rickettsia. Clinical signs and symptoms of R. felis infection are very similar to those of other rickettsioses and resemble other more commonly diagnosed tropical diseases, such as dengue and malaria.^3,21,22 Therefore, it is likely that infections caused by this pathogen are underestimated and misdiagnosed by the medical community throughout Central America. Because R. felis may cause severe illness in some individuals,^3,23 proper physician education, disease awareness, and adequate diagnosis are essential.

Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services of the United States, or the institutions with which the authors are affiliated.