Serosurvey on rickettsiae of the spotted fever group and Rickettsia bellii among dogs in the state of Goiás, Brazil

Inquérito sorológico sobre riquétsias do grupo da febre maculosa e Rickettsia bellii entre cães no estado de Goiás, Brasil

Abstract

The purpose of this study was to do a serological survey on three rickettsial species: Rickettsia rickettsii and Rickettsia parkeri, two species of the spotted fever group (SFG) that are considered to be great importance for public health; and Rickettsia bellii, a species of unknown pathogenicity that infects a variety of human-biting ticks. Serum samples from 273 dogs were tested using the indirect immunofluorescence assay (IFA). A total of 52 samples (19.04%) were seropositive for at least one of the three Rickettsia spp. antigens. Thirty-eight (73.07%), twelve (23.07%) and one (1.92%) of these dogs showed homologous reactions to R. bellii, R. rickettsii and R. parkeri, respectively. Our results showed that the seroprevalence of Rickettsia spp. was relatively low. However, the positive serological tests indicated that these dogs had become infected by these agents at some point in their lives. Lastly, our study adds to the previous knowledge on the epidemiology of rickettsiosis in the state of Goiás by doing the first record of detection of anti-R. rickettsii, R. parkeri and R. bellii antibodies by IFA among dogs, thus indicating that these agents may be circulating in the dog population analyzed.

Keywords:
Rickettsia rickettsii; Rickettsia parkeri; IFA; Goiânia; Brazilian cerrado; tick-borne disease

Resumo

O objetivo deste estudo foi realizar um levantamento sorológico para três espécies de rickettsias: Rickettsia rickettsii e Rickettsia parkeri, duas espécies do grupo da febre maculosa (GFM) consideradas de grande importância para a saúde pública; e Rickettsia bellii, uma espécie de patogenicidade desconhecida que infecta uma variedade de carrapatos que parasitam seres humanos. Amostras de soro de 273 cães foram testadas, usando-se a técnica de reação de imunofluorescência indireta (RIFI). O total de 52 amostras (19,04%) foram soropositivas para pelo menos um dos três antígenos de Rickettsia spp. Trinta e oito (73,07%), doze (23,07%) e um (1,92%) desses cães apresentaram reações homólogas à R. bellii, R. rickettsii e R. parkeri, respectivamente. Esses resultados demonstraram uma baixa soroprevalência para Rickettsia spp. No entanto, as amostras positivas indicam que esses cães foram infectados por esses agentes em algum momento de suas vidas. Por fim, este estudo contribui para o conhecimento sobre a epidemiologia das rickettsioses no estado de Goiás, realizando a primeira detecção de anticorpos anti-Rickettsia rickettsii, R. parkeri e R. bellii pela RIFI em cães, indicando que esses agentes podem estar circulando na população canina analisada.

Palavras-chave:
Rickettsia rickettsii; Rickettsia parkeri; RIFI; Goiânia; cerrado brasileiro; doença transmitida por carrapatos

Rickettsia rickettsii is the etiological agent of Brazilian spotted fever (BSF), a reemerging zoonosis of extreme importance for public health because of the high fatality rate from this disease among humans (≥ 50%) (Brasil, 2017). In addition, Rickettsia parkeri strain Atlantic rainforest has been identified as the causative agent of an emerging spotted fever (SF) in different parts of Brazil (Spolidorio et al., 2010; Silva et al., 2011; Krawczak et al., 2016a). In particular, since 2010 in the state of Goiás - central-western region of Brazil, Cerrado biome - a total of 11 cases of SF without death records have been confirmed in the Disease Notification Information System (SINAN) in the following municipalities: Gameleira de Goiás (2), Goiânia (5), Guapó (1), Itumbiara (1), Jataí (1) and Mineiros (1) (Brasil, 2019), where knowledge about the disease is incipient.

Use of domestic dogs in seroepidemiological studies on rickettsiosis of the spotted fever group (SFG) constitutes a suitable approach for assessing possible infection caused by R. rickettsii and R. parkeri strain Atlantic rainforest in areas where the vectors of these agents, i.e. the ticks Amblyomma aureolatum and Amblyomma ovale, are present (Ogrzewalska et al., 2012; Szabó et al., 2013; Barbieri et al., 2014; Krawczak et al., 2016b). Domestic dogs may remain close to humans but may also enter natural areas where the vectors of SFG rickettsiosis are prevalent. If bitten by infected ticks, high titers of antibodies against Rickettsia spp. can be detected through the indirect immunofluorescence assay (IFA). Thus, dogs are excellent sentinels for assessing the epidemiological situation of SF in a single area (Horta et al., 2004; Ogrzewalska et al., 2012; Szabó et al., 2013; Barbieri et al., 2014; Krawczak et al., 2016c). Although cases of spotted fever have been confirmed in the state of Goiás (GO), current knowledge on the vectors, their associated hosts and the epidemiology of the disease in this state remains sparse (Brasil, 2019). In particular, no serological investigations among dogs from Goiânia, GO, Brazil, on SFG rickettsiae have yet been conducted.

Rickettsia bellii, a rickettsia that has been detected infecting more than 25 species of ticks, is considered non-pathogenic for animals and humans (Krawczak et al., 2018). However, it has been postulated that R. bellii could play a role in the ecology and epidemiology of several SFG rickettsiae through inhibiting their vertical transmission (transovarian transmission) to further generations of ticks (Parola et al., 2013; Sakai et al., 2014). In this context, the present study aimed to analyze the frequency of anti-R. rickettsii, -R. parkeri and -R. bellii antibodies in dogs from the cities in the South and Central mesoregions of Goiás that were attended at the Veterinary Hospital of the Federal University of Goiás (HV/EVZ/UFG), in Goiânia, GO.

Between November 2017 to February 2018, serum samples were collected from 273 dogs from the cities in the South and Central mesoregions of Goiás – Goiânia (243/273), Aparecida de Goiânia (11/273), Trindade (5/273), Santo Antônio de Goiás (3/273), Senador Canedo (3/273), Inhumas (1/273), Goianira (1/273), Itaguari (1/273), Itaguaru (1/273), Mossâmedes (1/273), Caturaí (1/273), Rio Quente (1/273) and Silvânia (1/273) (Table 1) – that were attended for routine clinical procedures, not necessarily related to vector-borne diseases. Serum samples from these animals were also assigned to other research projects that had been authorized by the Institutional Animal Care and Use Committee (IACUC) of the Federal University of Goiás (protocol 102/17). To detect anti-Rickettsia antibodies, serum samples were tested individually by means of the indirect immunofluorescence assay (IFA), using crude antigens derived from three Rickettsia isolates from Brazil (R. rickettsii strain Taiaçu, R. parkeri strain Atlantic rainforest and R. bellii strain Mogi), as previously described (Labruna et al., 2007). Briefly, the serum samples were diluted in twofold increments using phosphate-buffered saline (PBS), starting from the 1:64 dilution. Slides were incubated with fluorescein isothiocyanate-labelled rabbit anti-dog IgG (Sigma, St Louis, MO, USA).

Table 1
Results regarding dogs seroreactivity to three Rickettsia species in the state of Goiás, central-western region of Brazil (November 2017 to February 2018).

Nº of positive dogs/Nº tested dogs (% positivity)	Municipality (Nº tested dogs)	Number of positive dogs to each species of Rickettsia in each city/Nº tested dogs in the study			Total number of positive dogs to each species of Rickettsia/ Nº tested dogs (% positivity); Range of endpoint titres	Total number of dogs with PAIHR*/Nº positive dogs (%)
		Rickettsia rickettsii	Rickettsia parkeri	Rickettsia bellii
52/273 (19.04)	Goiânia (243)	16/273	2/273	33/273	39/273 (14.28);128-32768 R. bellii 18/273 (6.59); 64-2048 R. rickettsia 2/273 (0.73); 64-512 R. parkeri	38/52 (73.07) R. bellii 12/52 (23.07) R. rickettsii 1/52 (1.92) R. parkeri
	Trindade (5)	1/273	0	2/273
	Rio Quente (1)	1/273	0	1/273
	Silvânia (1)	0	0	1/273
	Senador Canedo (3)	0	0	2/273
	Aparecida de Goiânia (11)	0	0	0
	Santo Antônio de Goiás (3)	0	0	0
	Inhumas (1)	0	0	0
	Goianira (1)	0	0	0
	Caturaí (1)	0	0	0
	Mossâmedes (1)	0	0	0
	Itaguaru (1)	0	0	0
	Itaguari (1)	0	0	0

• ^*
  PAIHR: probable antigen involved in homologous reaction.

For each sample, the endpoint IgG titer reacting with each of the three Rickettsia antigens was determined. Endpoints titers at least fourfold higher for one Rickettsia species than for the other Rickettsia species were considered to probably be homologous to the first species (Labruna et al., 2007). On each slide, a serum that had previously been shown to be non-reactive (negative control) and a known reactive serum (positive control), both from the studies of Piranda et al. (2008), were tested at the 1:64 dilution. These samples were obtained from the serum bank of the Laboratory of Parasitic Diseases, Department of Preventive Veterinary Medicine and Animal Health (VPS), School of Veterinary Medicine and Animal Science (FMVZ), University of São Paulo (USP), São Paulo, SP, Brazil.

Overall, 19.04% (52) of the dogs reacted (titer ≥ 64) to at least one Rickettsia species. Totals of thirty-eight dogs (73.07%), twelve (23.07%) and one (1.92%) were considered to have elicited homologous reactions to R. bellii, R. rickettsii and R. parkeri, respectively (Table1).

The 19.04% overall rate of seroreactivity to Rickettsia spp. found in the present study was clearly lower than the overall canine seroprevalence rates (around 66%) that have been reported for SFG-endemic areas in the southeastern and southern regions of Brazil (Sangioni et al., 2005; Barbieri et al., 2014). However, it was considerably higher than the seroprevalences of 5.63% and 3% that were observed by Rotondano et al. (2017) and Lopes et al. (2019), respectively, in non-endemic areas in the northeastern region of Brazil. It was also higher than the 4.16% seropositivity for Rickettsia spp. that was found in a small population of 24 dogs that was analyzed in the city of Quirinópolis, GO, a region with suspected cases of SF (Martins et al., 2016a).

The finding of a low prevalence rate for circulation of SF bioagents does not downplay the importance of evaluating anti-Rickettsia spp. antibodies in sentinels such as dogs, which are common hosts for the ticks involved in SF epidemiology (Sangioni et al., 2005; Labruna et al., 2007; Brasil, 2019). Our results showed that 23.07% (12/52) and 1.92% (1/52) of the samples were seroreactive to R. rickettsii and R. parkeri, respectively, which suggested that these dogs had become infected at some point in their lives. These Rickettsia species can be transmitted to animals by the ticks Rhipicephalus sanguineus sensu lato, A. aureolatum, A. ovale and Amblyomma sculptum (Ogrzewalska et al., 2012; Szabó et al., 2013). Despite these two rickettsiae are etiological agents for SFG rickettsiosis in humans in Brazil, only R. rickettsii has generated fatal cases to date (Szabó et al., 2013).

The higher frequency of seroreactivity for R. rickettsii among dogs (23.07%) than for R. parkeri (1.92%) highlights the possible exposure to A. sculptum, the vector for R. rickettsii in the Cerrado biome (Szabó et al., 2013). Under natural conditions, immature stages of A. sculptum have low host specificity, being able to parasitize humans and domestic dogs in this area (Szabó et al., 2013). However, in the present study the rate of tick infestation in dogs was not measured.

The occurrence of A. sculptum in the state of Goiás (Martins et al., 2016b), in addition to our finding regarding the frequency of anti-R. rickettsii antibodies, demonstrates that there is a possibility of R. rickettsii circulation and risk of infection in this region. On the other hand, the relevance of R. parkeri in the area studied here should not be underestimated because of its low seroprevalence, given that it is related to human SF cases and is transmitted by the tick A. ovale. This tick is found in several regions of the Brazilian Cerrado, including the state of Goiás (Spolidorio et al., 2010; Szabó et al., 2013; Krawczak et al., 2016a; Bitencourth et al., 2019). Rickettsia parkeri strain Atlantic rainforest was recently detected in A. ovale from Goiás, and further studies are needed in order to ascertain the distribution of this rickettsial species and its vector tick within this state (Bitencourth et al., 2019). The high seropositivity of dogs to R. bellii (73.07%; 38/52) found in the present study can be attributed to the fact that this rickettsial species frequently infects different tick species in Brazil, including those that parasitize dogs such as A. ovale, as reported by Labruna et al. (2011) and Costa et al. (2015), but its pathogenicity to humans and dogs is unknown (Krawczak et al., 2018).

Despite the higher seropositivity to R. rickettsii than to R. parkeri, the titers to R. rickettsii were lower (≤ 2048) than those founded by Piranda et al. (2008) in experimentally infected dogs with R. rickettsii. Also, when comparing the seropositivity and titers to R. rickettsii (64-2048) and R. bellii (128-32768), there was a greater number of homologous dogs with higher titers for this second species, indicating more evidence of circulation and an exposure or reexposure recently of these animals to ticks infected with R. bellii (Piranda et al., 2008). These data may justify the absence of human SF fatal cases in the state of Goiás, since infections by R. parkeri are related to non-fatal cases and R. bellii has unknown pathogenicity for humans (Spolidorio et al., 2010; Silva et al., 2011; Krawczak et al., 2016a, 2018).

The findings from the present study underline the need for further investigations on the circulation of tick-borne rickettsiae in the region of Goiânia, either through using serological methods or by using molecular tools. Such studies would generate valuable data for determining occurrences of endemic areas for pathogenic Rickettsia species in the state of Goiás, which is important for public health given the severity of this disease. To our knowledge, this is the first study on detection of anti-R. rickettsii, R. parkeri and R. bellii antibodies by means of IFA among dogs in the state of Goiás. Our results indicate that these bioagents may be circulating in the dog population analyzed.

• How to cite: Neves LC, Barreto ALG, Souza MX, Martins DB, Barbieri ARM, Serpa MCA, et al. Serosurvey on rickettsiae of the spotted fever group and Rickettsia bellii among dogs in the state of Goiás, Brazil. Braz J Vet Parasitol 2020; 29(2): e021419. https://doi.org/10.1590/S1984-29612020018

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Publication Dates

• Publication in this collection
  26 June 2020
• Date of issue
  2020

History

• Received
  14 Nov 2019
• Accepted
  04 Mar 2020