Rhipicephalus (Boophilus) microplus: Rickettsiae infection in Brazil

ABSTRACT The tick Rhipicephalus (Boophilus) microplus has been considered a major pest of cattle in tropical regions of the world, inducing significant economic impact on livestock activity. In Brazil, the scenarios of Spotted Fever (SF) epidemic cycles involve Amblyomma sculptum and Amblyomma aureolatum, transmitting Rickettsia rickettsii, as well as Amblyomma ovale, transmitting R. parkeri strain Atlantic Rainforest. However, other potentially pathogenic species of Rickettsia, as well as new species of ticks with vector potential, have been reported for Brazil. Thus, during SF focus investigation and environmental surveillance performed by Brazilian National Network of Environment Surveillance for Tick-borne Diseases between 2011 to 2017, ectoparasites were collected from vertebrate hosts and the environment in rural and urban areas of different Brazilian biomes. Here we analysed Rickettsiae infection in Rh. microplus and possible roles of this tick species in the maintenance and circulation of Rickettsia sp. in SF endemic areas. Cattle tick samples were naturally infected with R. rickettsii, R. parkeri, R. felis, R. tamurae, R. rhipicephali and R. bellii. The data in this study demonstrate that Rh. microplus acquires Rickettsia infection, including pathogenic species, and indicates this tick as suggested marker of Rickettsiae potential presence or circulation.


Introduction
Rhipicephalus (Boophilus) microplus, also known as the cattle tick, has a broad geographic distribution, being found mainly in tropical and subtropical regions (Estrada-Peña et al. 2006). Although Rh. microplus demonstrates a high degree of host-specificity and requires only one host to complete its lifecycle (on which it performs all the blood meals), Rh. microplus can parasitize alternative hosts, including humans (Soares et al. 2007).
The main importance of Rh. microplus is related to its significant economic impact on livestock. In Brazil, the cattle tick is distributed throughout the country and it is estimated that 80% of the national cattle herd is infested, causing an economic loss of three billion dollars a year. Under such circumstances, direct spoilage and the participation in the epizootic cycle of different pathogens (e.g. Babesia bovis, B. bigemina, Anaplasma marginale) are the main ways in which these ticks interact with disease development and deleterious influence on domestic bovines (Peter et al. 2005;de la Fuente et al. 2008;Grisi et al. 2014).
Due to its wide distribution in the country, Rh. microplus is found in Spotted Fever (SF) endemic regions. However, even though it has been reported as being infected by different species of Spotted Fever Group Rickettsiae (SFGR) (Bermúdez et al. 2009;Moura-Martiniano et al. 2014;Pesquera et al. 2015), the involvement of this tick in transmitting these bacteria remains incipient (Cordeiro et al. 2018).
In Brazil, known SF epidemic cycle scenarios involve Amblyomma sculptum and A. aureolatum transmitting R. rickettsia, and A. ovale transmitting R. parkeri strain Atlantic Rainforest (Szabó et al. 2013;Oliveira et al. 2016a;Nieri-Bastos et al. 2018). However, SF human cases recorded in new outbreaks in Brazil indicate clinical profiles that differ from those already established. Without already recognized vectors or detection of Rickettsia in these vectors, it is difficult to characterize the bioagent circulation mechanism involved in such outbreaks (e.g. Oliveira et al. 2016aOliveira et al. , 2016bOliveira et al. , 2017Oliveira et al. , 2018. In addition, in recent decades, other potentially pathogenic species of Rickettsia, as well as new species of ticks with vector potential, have been reported from Brazil (e.g. Labruna et al. 2011;Nieri-Bastos et al. 2014;Nunes et al. 2015;Moerbeck et al. 2016;Weck et al. 2016;Silva et al. 2017aSilva et al. , 2018, indicating SF cycle complexity and the existence of various distinct ecoepidemiological scenarios within the national territory. Furthermore, doubts still remain concerning the role of Rh. microplus in Rickettsiae circulation.
Here, we aim to investigate and molecularly characterize the presence of Rickettsia sp. in Rh. microplus specimens from areas of different ecological features in Brazil.

Materials and methods
During SF focus investigation and environmental surveillance performed between 2011 to 2017, State and Municipal Health Secretaries collected ectoparasites from vertebrate hosts and the environment (Figure 1) in rural and urban areas (pasture, farms and river bank) across a variety of Brazilian regions (Supplementary Table 1).
To confirm tick identification, we analysed the sequences of 12S rDNA (MN081899) and COI genes (MN088852) amplified from randomly chosen adult (sample code LIC 3764A) and pooled larval samples (sample code LIC 7156), and BLAST analysis revealed 100% identity sequences (380/380 for 12S and 645/645 for COI) with sequences of Rh. microplus (KP143546 and KC503261) deposited in GenBank.
In addition, phylogenetic inferences were performed with concatenated sequences of gltA, ompA, ompB, htrA and sca4 genes, using sequences corresponding to a sample of each Rickettsiae identity, and the resulting tree grouped: sequences from Ceará State with R. rickettsia, R. tamurae and R. bellii clusters; sequences from São Paulo State with R. rhipicephali cluster; sequences from Paraná State with R. parkeri strain Atlantic rainforest (identified in Barbieri et al. 2014 as strain Aa46) cluster; and sequences from Rio Grande do Sul State with R. felis cluster ( Figure 2).
Due to the obtained sequence sizes, bootstrap values were low in some tree clusters, indicating similar portions in analysed species fragments. However, the observed BLAST values mostly showed 100% overlap with the respective Rickettsiae sequence, and of the total sequences, one ompB sequence showed 98% similarity with R. felis and two gltA sequences showed 99% similarity with R. rhipicephali and with R. bellii (Table 1).  the mechanism by which cattle ticks are infected by this bacterium is still unknown.

Discussion
R. rhipicephali is commonly reported in Rhipicephalus sanguineus, with sporadic encounters in species of Dermacentor in the United States (Wikswo et al. 2008). In Brazil, it was detected in the tick Amblyomma sp. from Minas Gerais state (Zeringóta et al. 2016), and also in Haemaphysalis juxtakochi from Rondônia, São Paulo and Mato Grosso states (Labruna et al. 2005(Labruna et al. , 2007Soares et al. 2015). Although H. juxtakochi displays an affinity for cervid hosts, it has been reported parasitizing a variety of other vertebrate species (Guimarães et al. 2001;Guglielmone et al. 2014), including domestic dogs and humans (Labruna et al. 2005). Demonstrating the proximity of H. juxtakochi to anthropic areas, allowing a possible Rh. microplus acquisition of Rickettsia by co-feeding on the vertebrate hosts. Therefore, Rh. microplus infection by R. rhipicephali may not be as random depending on the area.
Another possibility for introduction of R. rhipicephali into a local tick population is by migrating birds, which serve as hosts for a variety of tick species (Ogrzewalska et al. 2010;Luz et al. 2017) and potential vectors for pathogens, including Rickettsia (Ogrzewalska and Pinter 2016). It is known that birds play an important role as carriers of parasites over long distances, including across geographic barriers. An infected tick inserted by birds in a population of otherwise uninfected ticks can transmit pathogens by co-feeding on a new host. However, further studies are needed to understand this form of Rickettsia circulation within Brazil and throughout its host range.
As discussed for R. rhipicephali, bird ticks represent a valuable resource for understanding the bioecology of these tick-borne pathogens. In Brazil, the potential role of birds in dispersing of A. ovale infected with R. parkeri was suggested (Luz et al. 2017), and results obtained here show a novel occurrence area for this bacterium. The samples LIC7001B and LIC7001E (Table 1) are Rh. microplus ticks collected feeding on cattle from a Cerrado biome area of Tocantins state, Northern region of the country, evidencing different eco-epidemiological characteristics of the pattern usually observed for R. parkeri in the country.
In several regions of the Americas, R. parkeri is reported as an inducer of a mild SF clinical form (Parola et al. 2013). In Brazil, R. parkeri occurs in multiple regions, being involved in SF human cases in southern, southeastern and northeastern states (Szabó et   in an enzootic cycle associated with A. ovale and dogs (Szabó et al. 2013;Moerbeck et al. 2016;Vizzoni et al. 2016;Acosta et al. 2018). The capacity of Rh. microplus to transmit R. parkeri is experimentally inconclusive (Cordeiro et al. 2018) and, considering the ecological scenario of this bioagent´s cycle, may have no epidemiological importance. Cattle tick acquisition of pathogenic Rickettsia may also occur by a co-feeding process, already established for Rickettsiae (Socolovschi et al. 2009). Rickettsia parkeri has been reported from Amblyomma triste, an Ixodid tick with the capacity to parasitize several vertebrates, including cervids and bovines (Labruna et al. 2003;Mertins et al. 2010), host species that share the same environments, allowing infection of Rh. microplus by R. parkeri.
Additionally, here we also report for the first time R. felis in cattle ticks, a bacterium with wide geographical distribution, including all Brazilian regions (Horta et al. 2014). However, besides being detected in various species of blood-feeding arthropods, the majority of reports are associated with the flea genus Ctenocephalides (Reif and Macaluso 2009;Parola 2011;Mediannikov et al. 2012), common worldwide as ectoparasites of various carnivores, especially dogs. Thus, this Rickettsiae detection in other species of dog ectoparasites seems not to be uncommon (Gehrke et al. 2009;Parola et al. 2013). However, reports, from various states of Brazil of its presence in Rh. microplus (Table 1) are important, especially considering that cattle ticks do not commonly parasitize carnivores. The specimens found in this study infected with R. felis were collected from cattle, horses and the environment (pasture), without evidence of the infection mechanisms.
Other sequences from Ceará state, northeastern Brazilian, were identified as R. rickettsii and R. bellii. The samples infected by R. rickettsii were collected in Maciço do Baturité, an SF endemic area (Moerbeck et al. 2016), with high-altitude forest, with conditions of high humidity and mild temperature conditions within the otherwise semiarid climate of the Caatinga biome. Although SF cases reported in the region were associated with R. parkeri (= Rickettsia sp. strain Atlantic rainforest) transmitted by A. ovale (Moerbeck et al. 2016), and despite having no reports of SF cases clinically associated with R. rickettsii, this species was also isolated from Rh. sanguineus collected on a dog from Maciço do Baturité (Silva et al. 2017b). Together with the results here obtained, this detection indicates the circulation of R. rickettsii in the region, and in a variety of tick species. Thus, due to the public health importance of R. rickettsii, it is necessary to understand the enzootic cycle throughout the region, in order to understand the potential epidemiological scenario and take preventive measures to prevent new occurrences of R. rickettsii SF in Brazil. In this context, Rh. microplus becomes even more important as an object of study, since its potential for R. rickettsii transmission has been experimentally demonstrated (Monteiro and Fonseca 1932).
Besides pathogenic Rickettsiae, the generally considered nonpathogenic R. bellii is a widely distributed species, having been detected from a great variety of hosts, indicating a broad circulation capacity (Labruna et al. 2004a;Tomassone et al. 2010;Parola et al. 2013) and possibly playing an important role in the ecology and epidemiology of other Rickettsiae species (Macaluso et al. 2002). The first record of this species in Maciço do Baturité was in an A. nodosum tick collected from a wild animal (Moerbeck et al. 2016). Here we presented the first report for Brazil of R. bellii detected in Rh. microplus, indicating the circulation of this bacterium throughout the studied region.
It is known that a primary infection with one species of Rickettsia would prevent transovarian transmission of a second Rickettsia species (Burgdorfer 1988;Macaluso et al. 2002). This was recently corroborated by an experimental study conducted with A. dubitatum, where primary R. bellii infection appeared to decrease the effectiveness of subsequent R. rickettsii transovarian transmission (Sakai et al. 2014).

Conclusion
In spite of the wide distribution of R. microplus throughout Brazil, because this species has rarely been reported on humans, the participation of the cattle tick in Rickettsiae enzootic and epidemic cycles remains unknown. The data from the current study demonstrate that Rh. microplus can naturally acquire Rickettsia infection, including infection with pathogenic species, and suggests that this tick could serve as an indicator of Rickettsiae presence or circulation.