Seroprevalence of Tick-Borne Pathogens and Tick Infestation in Dogs from Tapirapé and Karajá Indigenous Communities,Brazil

Abstract

With the aim of studying some tick-borne diseases, a total of 327 dogs (114 from Tapirapé and 213 from Karajá indigenous ethnicity, Mato Grosso, MT, Brazil) were sampled. Serum samples were submitted to the indirect fluorescence antibody test (IFAT) to detect antibodies against Babesia vogeli, Ehrlichia canis, and Rickettsia spp. Possible associations of risk factors and the occurrence of seroreativity to tick-borne agents and tick infestations were analyzed through chi-squared tests. Among 327 dogs, 46 (13.15%) were seropositive for B. vogeli and 47 (14.37%) for E. canis. The B. vogeli seroprevalence was higher for Karajá and for adult dogs (p>0.05). No association was found for E. canis seroprevalence. From 103 serum samples tested with rickettsial antigens, 90 (87%) dogs were seropositive to Rickettsia spp., with highest reactivity to Rickettsia amblyommii. Canine seropositivity to Rickettsia spp. was associated (p<0.05) with ethnicity (higher seroprevalence in Tapirapé dogs), age (higher in adults), and hunting (higher among hunting dogs). From the 327 dogs, 39 (11.9%) were infested by ticks (Amblyomma cajennense sensu stricto, Amblyomma ovale, Amblyomma oblongoguttatum, Amblyomma tigrinum, and Rhipicephalus sanguineus). Infestations by Amblyomma spp. ticks were higher in dogs from Tapirapé community and in hunting dogs (p<0.05). Regarding R. sanguineus, infestations were higher (p<0.05) among young dogs.

Introduction

In recent years, there has been a substantial improvement in our knowledge of tick-borne diseases among domestic dogs in Brazil. On the other hand, because Brazil occupies nearly half of the South American continent, there are still many areas where tick-borne diseases have been poorly investigated, such as the Indian communities of the Amazon region. Canine babesiosis is a tick-borne disease of domestic and wild canids characterized by fever, depression, and anemia (Kuttler 1988). Parasitological and serological studies show that babesiosis, due to Babesia vogeli, is widely distributed in many Brazilian states, with seropositive ranging from 1.9% to 66.9% (Ribeiro et al. 1990, Dell'Porto et al. 1993, O'Dwyer et al. 2001, Bastos et al. 2004, Soares et al. 2006, Trapp et al. 2006, Spolidorio et al. 2011). From the Amazon region, Spolidorio et al. (2013) reported a 42.6% overall prevalence among urban and rural dogs from Pará State.

Canine monocytic ehrlichiosis, caused by Ehrlichia canis, is the most important tick-borne disease of dogs in Brazil. E. canis is currently the only ehrlichial species that has been isolated from dogs in South America (Vieira et al. 2011). A preliminary investigation of Ehrlichia species was developed in the northern and southeastern regions of Brazil, including various tick species, humans, dogs, and capybaras, but no Ehrlichia DNA was found in human samples (Labruna et al. 2007). In contrast, ehrlichial DNA compatible with Ehrlichia chaffeensis and Ehrlichia ewingii were reported in animal blood samples in Brazil (Machado et al. 2007, Oliveira et al. 2009). A previous report from Pará State revealed an overall canine seroprevalence of 16.2% for E. canis (Spolidorio et al. 2013)

Brazilian spotted fever (BSF), a rickettsial disease caused by Rickettsia rickettsii, is the most important tick-borne zoonosis in Brazil, where the disease has been reported since 1929 (Piza et al. 1932). In contrast, only recently was natural infection of dogs by R. rickettsii reported in South America (Labruna et al. 2009). Regarding the Brazilian Amazon region, a few published studies reported the occurrence of antibodies reactive to R. parkeri, R. amblyommii, and R. rhipicephali in dogs from Western Amazon, State of Rondônia (Labruna et al. 2007), and recently R. bellii, R. rhipicephali, R. rickettsii, and R. amblyommii in dogs from Pará State, Eastern Amazon (Spolidorio et al. 2013).

The indigenous population of Brazil is estimated to be 734,137 people from 225 ethnicities, generally sharing many cultural traits regardless of geographic distances. This population inhabited 107 millions of hectares distributed among 653 registered indigenous communities, of which 98% are located in the Brazilian Amazon. Many communities live by subsistence, using nature as their food supply through hunting and fishing (Malheiros et al. 2011). The Indians also have pets, which include dogs, cats, and some wild animals, especially birds. Dogs are important because they are used for hunting activities (Malheiros et al. 2011, Minervino et al. 2012).

Very few studies have investigated tick-borne diseases in humans or dogs from indigenous communities. In a study in Mato Grosso State, immunoglobulin G (IgG) antibodies anti–spotted fever group (SFG) rickettsiae and anti–Ehlichia spp. were detected in sera from 12 (8%) and four (6.8%) Indians, respectively (Barros Lopes et al. 2014). In another study, domestic dogs from two indigenous communities in Amazonas State were tested serologically to Rickettsia spp. and by molecular methods for tick-borne agents of the genera Rickettsia, Ehrlichia, Babesia, and Hepatozoon. Only a single dog in each of the communities was serologically reactive to Rickettsia spp., whereas none of the 26 dogs was found through molecular methods to be infected by tick-borne agents (Soares et al. 2014).

To our knowledge, there has been no additional study on tick-borne diseases in indigenous communities in Brazil. Therefore, the purpose of the present study was to determine the risk-factors associated with the seroprevalence of B. vogeli, E.canis, and Rickettsia spp. and the tick infestation on dogs from two indigenous ethnicities in the Brazilian Amazon.

Materials and Methods

Ethical approval

This work was authorized by the Indian National Foundation, Brazil (FUNAI authorization no. 45/AAEP/10, process no. 2433/07) and was approved by the Ethical Committee of Animal Use of the Faculty of Veterinary Medicine of the University of São Paulo (protocol no. 1747/2009).

Study site and sample collection

The study was conducted in two different indigenous ethnicities from Brazil— Tapirapé, restricted to seven small villages located in the North of Mato Grosso State, and Karajá, which occupies a larger area with villages spread in the Pará, Tocantins, and Mato Grosso States. For the Tapirapé ethnicity, seven villages were visited (10°40′16.1″S/51°21′13.8″W; 10°48′12.2″S/51°17′12.8″W; 10°42′27.5″S/51°18′49″W; 10°39′33.6″S/51°17′41.2″W; 10°52′09.6″S/51°15′37.8″W; 10°51′20.6″S/51°18′19.8″W; 10°36′23.9″S/51°10′47.9″W). For the Karajá, the four villages selected occupied the same region of Tapirapé (10°38′26″S/50°37′20″W; 10°28′18″S/50°28′59″W; 10°39′15.36″S/50°36′10.39″W; 10°37′30.97″S/50°37′30.73″W).

All dogs present in the villages during the visits had blood samples collected, and were included in the study. The dogs received a microchip (Destron Fearing™, São Caetano do Sul, SP, Brazil) for identification. Independent variables considered to be potential risk factors for tick-borne diseases (age, hunting activity, and gender) were assessed with the assistance of a native inhabitant for translation. In the Tapirapé and Karajá villages, a total of 114 and 213 dogs were sampled, respectively, resulting in a total of 327 dogs. Blood samples were collected from the jugular or cephalic vein of the dogs and sera were separated by centrifugation and stored at −20°C until tested. All dogs were examined for infestation by ticks, which were collected and preserved in absolute ethanol until taxonomic identification following Barros-Battesti et al. (2006), Martins et al. (2010), and Nava et al. (2014).

Serological analysis

Serum samples were submitted to the indirect fluorescent antibody test (IFAT) with antigens of B. vogeli (blood smear from experimentally infected dog) according to Bicalho (2004) using a cutoff of 1:64. To detect antibodies against E. canis, the serum samples were tested by IFAT against antigens of E. canis strain São Paulo (Aguiar et al. 2008). Reactions were performed with fluorescein isothiocyanate (FITC)-labeled anti-dog IgG (Sigma-Aldrich, São Paulo, Brazil) that had previously been titrated to the best working dilution (1:700), as described previously (Ristic et al. 1972, Aguiar et al. 2007). Serum was considered to contain antibodies reactive to E. canis if it presented a reaction at the 1:80 dilution (Krawczak et al. 2012).

For anti–Rickettsia spp. antibodies, a proportional stratified sampling of 30% of total dogs was taken considering the 11 villages. Thus, 30% of the dogs from each one of the villages were randomly selected, resulting in a total of 103 blood samples tested by IFAT against six Rickettsia species (R. rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, R. bellii, and R. felis). Rickettsial antigen was produced in Vero cells or C6/36 cells as described (Labruna et al. 2007). Sera were tested in the 1:64 screening dilution (Horta et al. 2004, Ogrzewalska et al. 2012). A serum showing a titer for a Rickettsia species at least four-fold higher than that observed for any other Rickettsia species was considered homologous for the first Rickettsia species or a very closely related genotype (Horta et al. 2004, Labruna et al. 2007).

Statistical analysis

Possible associations between ethnicity (Tapirapé or Karajá), gender, age (young dogs, ≤1 year old; adult dogs, >1 year old), or hunting activity with the prevalence of antibodies anti–B. vogeli, anti–E. canis, or anti–Rickettsia spp. in dogs. Tick infestations were analyzed with the chi-squared or Fisher exact tests. Seroreactivity to Rickettsia spp. was analyzed qualitatively at two dichotomous levels (0, nonreactive at the 1:64 dilution to any Rickettsia sp. or nonreactive at the 1:1024 dilution to R. amblyommii; 1, end point titer ≥64 to any Rickettsia sp. or end point titer ≥1024 to R. amblyommii, respectively). A 5% significance level was adopted.

Results

From the 327 sampled dogs, 46 (13.15%) were seropositive for B. vogeli and 47 (14.37%) were seropositive for E. canis. In the risk factor analysis, the B. vogeli seroprevalence was significantly higher (p<0.05) for Karajá dogs than for Tapirapé dogs and higher for adults than for young dogs (Table 1). No significant risk factor was found for E. canis seroprevalence.

Table 1.

Risk Factor Analysis for Babesia vogeli and Ehrlichia canis in Dogs from Tapirapé and Karajá Indigenous Communities, Mato Grosso State, Brazil

	Babesia vogeli			Ehrlichia canis
Risk factor	Positive/tested	Prevalence (%)	p^a	Positive/tested	Prevalence (%)	p^a
Ethnicity			0.037			0.391
Karajá	36/213	16.90		28/213	13.15
Tapirapé	10/114	8.77		19/114	16.67
Gender			0.460			0.774
Male	26/201	13.94		28/201	13.93
Female	20/126	15.87		19/126	15.08
Age^b			0.010			0.091
Young	3/62	4.84		5/62	8.06
Adult	43/263	16.35		42/263	15.97
Hunting^b			0.950			0.083
Yes	21/174	12.07		31/174	17.82
No	16/130	12.31		14/130	10.77
Total	46/327	13.15		47/327	14.37

Statistical analysis throughout Pearson chi-square of Fisher exact test.

Age or hunting information could not be verified for one or six dogs, respectively

Regarding Rickettsia antibodies, 90 (87%) out of the 103 tested dogs were positive to at least one Rickettsia tested, with end point titers varying from 64 to 8196. Among the seropositive dogs, 83 (80%) were reactive to R. amblyommii, 80 (77.6%) to R. rhipicephali, 58 (56.3%) to R. rickettsii, 61 (59.2%) to R. parkeri, 53 (51.4%) to R. bellii, and 27 (26.2%) to R. felis. A total of 27 (26.2%) canine sera showed titers to R. amblyommii at least four-fold higher than those to any of the other five antigens. The antibody titers in these 27 dogs were considered to have been stimulated by R. amblyommii or a very closely related species. On the basis of this same criterion, anti–Rickettsia spp. antibodies in 10 sera samples were considered to be stimulated by R. bellii, one serum was considered to be stimulated by R. parkeri, and one serum sample was considered to be stimulated by R. felis. For the remaining 51 seroreactive dogs, it was not possible to discriminate the infection agent because they displayed similar titers (less than four-fold difference) for two or more Rickettsia species or had a single titer of 64 for a single Rickettsia species.

Risk factor analyses revealed that canine seropositivity to Rickettsia spp. (any of the six Rickettsia antigens evaluated) was significantly associated (p<0.05) with ethnicity (higher seroprevalence in Tapirapé dogs), age (higher seroprevalence in adults), and hunting activity (higher seroprevalence in hunting dogs). The frequency of dogs with end point titers ≥1024 to R. amblyommii was significantly associated (p<0.05) with age (higher seroprevalence in adults) and hunting activity (higher seroprevalence in hunting dogs) (Table 2).

Table 2.

Factors Associated with the Prevalence of Rickettsia spp. or R. amblyommii in Dogs from Indigenous Communities from Amazon, Mato Grosso State, Brazil

	Rickettsia spp.			R. amblyommii
	(end point titer ≥64)			(end point titer ≥1024)
Risk factor	Positive/tested	Prevalence (%)	p^a	Positive/tested	Prevalence (%)	p^a
Ethnicity			0.007			0.084
Karajá	51/63	80.95		30/63	47.62
Tapirapé	39/40	97.50		26/40	65.00
Gender			0.739			0.549
Male	51/59	86.44		34/59	57.63
Female	39/44	88.64		22/44	50.00
Age^b			0.001			0.001
Young	12/21	57.14		3/21	14.29
Adult	77/81	95.06		52/81	64.20
Hunting^b			0.008			0.015
Yes	52/54	96.30		36/54	66.67
No	34/43	73.07		18/43	41.86
Total	90/103	87.40		56/103	54.37

Statistical analysis throughout Pearson chi-square of Fisher exact test.

Age or hunting information could not be verified for one or six dogs, respectively.

Among the 327 dogs, 39 (11.9%) were found infested by ticks (Table 3). Five different tick species were found infesting dogs as follows: Amblyomma cajennense (115 nymphs and 23 adults infesting 27 dogs); Amblyomma ovale (eight adults on six dogs); Amblyomma oblongoguttatum (two adults on two dogs); Amblyomma tigrinum (one adult); and Rhipicephalus sanguineus (30 adults and three nymphs on 10 dogs). All adult females of A. cajennense were identified as A. cajennense sensu stricto (s.s), following the morphological criteria of Nava et al. (2014) for ticks of the A. cajennense complex.

Table 3.

Tick Species Infesting Dogs Raised in the Two Indigenous Ethnicities, Mato Grosso State, Brazil

	No. infested dogs (%) ^a
Tick species	Tapirapé ethnicity No. of dogs, 114	Karajá ethnicity No. of dogs, 213
Amblyomma cajennense	22 (19.3)	5 (2.3)
Amblyomma ovale	5 (4.4)	1 (0.5)
Rhipicephalus sanguineus	3 (2.6)	7 (3.2)
Amblyomma oblongoguttatum	2 (1.7)	0 (0)
Amblyomma tigrinum	1 (0.9)	0 (0)
Overall tick infestation^b	39 (11.9%)

When the same dog presented two species of ticks, it was counted twice,

Number of dogs infested by at least one tick species.

The dogs raised in the Tapirapé community had more Amblyomma spp. ticks than dogs from Karajá (p<0.05). Infestations by R. sanguineus ticks were similar among dogs of both communities. Risk factor analyses revealed that infestations by Amblyomma spp. ticks were higher (p<0.05) in dogs from the Tapirapé community and in hunting dogs (Table 4). Regarding R. sanguineus, infestations were higher (p<0.05) on young than on adult dogs.

Table 4.

Risk Factors for Tick Species Infesting Dogs from Two Indigenous Communities from Amazon, Brazil

	Amblyomma spp. ^a			Rhipicephalus sanguineus^a
Risk factor	Infested/examined	Prevalence (%)	p^a	Infested/examined	Prevalence (%)	p^a
Ethnicity			<0.001			0.740
Karajá	6/213	2.82		7/213	3.29
Tapirapé	25/114	21.93		3/114	2.36
Gender			0.446			0.445
Male	21/201	10.45		5/201	2.49
Female	10/126	7.94		5/126	3.97
Age^b			0.229			0.025
Young	3/62	4.84		5/62	8.06
Adult	28/263	10.65		5/263	1.90
Hunting^b			0.001			0.104
Yes	25/174	14.37		7/130	5.38
No	5/130	3.85		3/174	1.72
Total	31/327	9.48		10/327	3.06

Statistical analysis made throughout chi-square of Fischer exact test.

Some of the information was missing since the owner did not know and it not could be verified.

Discussion

In the present study, serological analyses of domestic dogs from two indigenous communities indicate that these animals have been exposed to tick-borne agents of the genera Babesia, Ehrlichia, and Rickettsia. In Brazil, B. vogeli and E. canis have been reported in all regions, where they are primarily transmitted by the tick R. sanguineus (Dantas-Torres 2008, Vieira et al. 2011). R. sanguineus is an exotic species that was introduced in Brazil, possibly during European colonization, after the 15^th century (Aragão 1936). Since then, this tick species has spread out to all Brazilian regions, where it is found especially in urban areas and in human constructions in rural areas. In contrast to native species of the genus Amblyomma, R. sanguineus is not found inside forests, such as the Amazon rainforest (Aragão 1936, Labruna and Pereira 2001). Although infestations by R. sanguineus were found in both Indian communities at similar prevalence levels (Table 4), the higher seroprevalence of B. vogeli in Karajá dogs could be related to the fact that the main village of Karajá, where most of the dogs were sampled, was geographically much closer to urban areas than the Tapirapé villages, which were much more isolated. In addition, whereas Tapirapé Indians would seldom visit urban areas, the Karajá Indians used to go frequently to urban areas, where they used to sell fish (data not shown). This closer contact with urban areas may have enhanced the introduction B. vogeli–infected dogs or/and R. sanguineus in Karajá village.

Interestingly, the seroprevalence of E. canis was statistically similar among the two Indian communities. Although the presence of E. canis infection is likely to occur in both Indian communities (due to the presence of the vector R. sanguineus), it is possible that other ehrlichial agents, associated with native tick species, also occur in the region and therefore could be causing serological cross-reactions with E. canis antigens. In fact, recent studies have reported a variety of ehrlichial agents different from E. canis infecting ticks and animals in different regions of Brazil, indicating that the diversity of Ehrlichia spp. in Brazil is much greater than previously thought (Machado et al. 2007, Widmer et al. 2011, André et al. 2012, Cruz et al. 2012, Machado et al. 2012, Almeida et al. 2013, Aguiar et al. 2014).

During the present study, a parallel study evaluated rickettsial infection in ticks collected from wild animals that were hunted by the Tapirapé Indians. R. amblyommii was found infecting most of the A. cajennense s.s. ticks collected from different wild animals (Soares et al. 2015). Therefore, the significantly higher canine seroprevalence for Rickettsia spp. (notably to R. amblyommii at end point titers ≥1024) among dogs from Tapirapé than from Karajá communities is related to the predominance and much higher prevalence of A. cajennense s.s. infestations on dogs from the former community. This fact also corroborates our findings that at least 27 dogs were considered to be infected by R. amblyommii because they presented end point titers four-fold or more higher for R. amblyommii than for the other Rickettsia antigens. Similarly, our findings of 10 dogs that were considered to have been infected by R. rhipicephali in the present study are corroborated by the presence of this rickettsia infecting the tick Haemaphysalis juxtakochi in the Tapirapé Village, as reported by Soares et al. (2015). We did not collect H. juxtakochi from the dogs of the present study, but it is known that this tick can infest domestic dogs under natural conditions (Labruna et al. 2005).

The significantly higher seroprevalence for B. vogeli and Rickettsia spp. in older than in young dogs could be related merely to the greater exposure time of adult dogs, which would have experienced much higher previous exposure to ticks than young dogs. In addition, young dogs are usually not used for hunting until they reach adulthood. In the present study, hunting activity was significantly associated with higher seroprevalence for Rickettsia spp. and for R. amblyommii at end point titers ≥1024, which could be related to greater exposure to ticks within the forest, the habitat of R. amblyommii–infected ticks. This result is corroborated by a study with domestic dogs in the Pantanal biome of Mato Grosso State, where canine seroprevalence for Rickettsia spp. (notably by R. amblyommii) was significantly associated with hunting activity (Melo et al. 2011).

In contrast to a recent study with dogs from two indigenous communities in the state of Amazonas, where no dog was found to be infected by Ehrlichia or Babesia organisms (Soares et al. 2014), the present study provides serological evidence of the circulation of tick-borne agents of the genera Ehrlichia and Babesia organisms among dogs from two Indian communities in the Amazon region. Whereas R. sanguineus was not present in the Indian villages of the study of Soares et al. (2014), this tick species was present within the two Indian communities of the present study, providing epidemiological evidence that at least part of the seropositive dogs have been infected by E. canis and B. vogeli. Finally, the presence of many dogs with higher antibody titers to R. amblyommii, and infestations by A. cajennense s.s. ticks, in conjunction with the report of this Rickettsia species infecting most of the A. cajennense s.s. ticks collected from wildlife in the study area (Soares et al. 2015), provides epidemiological evidence for canine infection by R. amblyommii, which is a spotted fever agent suspected to cause infection or even illness in humans (Apperson et al. 2008, Blanton et al. 2014). Because serological reactivity to SFG rickettsiae have been found in another indigenous community of the Amazon region where R. amblyommi–infected ticks were also found (Barros Lopes et al. 2014), further investigations on the surveillance of SFG rickettsial infection among indigenous populations of the Brazilian Amazon are warranted.

Footnotes

Acknowledgments

We would like to thank Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Brazil, for a postdoctoral fellowship to A.H.H. Minervino and A.F. Malheiros, and Research productivity to S.M. Gennari and M.B. Labruna. This research was financially supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).

Author Disclosure Statement

No competing financial interests exist.

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