Experimental infection of the bat tick Carios fonsecai ( Acari : Ixodidae ) with the rabies virus

Introduction: This study assessed the viability of the rabies virus in the argasid tick Carios fonsecai following experimental infection. Methods: The mouse inoculation test (MIT), fl uorescent antibody test (FAT) and polymerase chain reaction (PCR) were used. The rabies virus was administered to ticks via the intra-coelomic route, and the ticks were sacrifi ced at different time points. Results: The inoculated ticks were negative for rabies according to the MIT. Ticks macerated with rabies virus were positive according to the MIT and FAT. All of the tick lots tested by PCR were positive. Conclusions: The rabies virus became unviable shortly after its inoculation into tick bodies. Ticks are not likely to play an important role in the epidemiology of rabies.

Ticks belong to the order Acari, which is divided into the Ixodidae (≈700 species), Argasidae (≈200 species) and Nuttalliellidae (one species) families.Although a few species of Ixodidae have occasionally been reported to infest bats, approximately one-third of the Argasidae species have been observed to feed primarily on different bat species around the world 1,2 .All tick species are hematophagous during one or more phases of their life cycles.For this reason, these arthropods have been examined for their role as the vectors of different agents (viruses, bacteria, protozoa and helminths) that infect vertebrate hosts.In fact, it has been reported that a greater variety of agents are transmitted by ticks than by any other vector group, including mosquitoes¹.In Brazil, certain argasid ticks (Carios spp.) that are associated with bats have been reported to infest humans, primarily under indoor conditions, as these ticks are maintained by bats that live in the roofs of human dwellings 3,4 .
Bats belong to the order Chiroptera and are present throughout almost the entire world.Bats comprise the mammalian group with the second-largest number of species; in particular, there are 1,120 species of bats.There are nine families, 64 genera and 167 species of bats that live in Brazil 5 .
According to antigenic and genetic characterization studies of rabies, bats are among the most important reservoirs and vectors of the rabies virus around the world 6,7 .In recent years, however, certain human rabies cases associated with bat variants have been detected in the absence of skin lesions that were attributable to bat bites and in cases that lacked any documented history of bat bites.
Based on this information, the present study sought to examine the viability of the rabies virus in an argasid tick species, despite ticks never having been observed to be infected naturally by rabies viruses.
The ticks used in this study were derived from a laboratory colony of Carios fonsecai (Acari: Argasidae) that was originally collected in April 2005 in the São Miguel cave in the State of Mato Grosso do Sul, which is in the central-western region of Brazil.Within this cave, the hematophagous bat Desmodus rotundus was observed to be a host of C. fonsecai under natural conditions 4 .The tick colony in question has been maintained in the laboratory.In that setting, naïve rabbits and/or mice have been used to feed the ticks.For the present study, nymphs and adult ticks of the F 2 to F 4 lab generations were utilized.
In this study, a fl uorescent antibody test (FAT) 8 and a mouse inoculation test (MIT) 9 were used to diagnose the presence of rabies.The genetic characterization was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), followed by deoxyribonucleic acid (DNA) sequencing 10 .
The rabies virus variant that was used in this study (Brldr2918) was isolated in 1997 from the brain of a D. rotundus bat.The fi rst mouse passage of this bat isolate was preserved  at -70°C until it was used for the present study.The viral doses were previously tested in rabies studies by the vaccination and experimental infection of D. rotundus bats 11 , and the doses were in the present study for the inoculation of ticks.
The rabies virus was administered (0.03mL) to adult ticks or nymphs by an intra-coelomic route through the articulation of the coxa IV and trochanter IV of each tick (Figure 1), using a 28-gauge microfi ne insulin needle, in accordance with previously described procedures 12 .
The inoculated ticks were maintained alive at room temperature until different time points (from 0.5h to 48h postinoculation), until they were assessed for the presence of rabies virus by RT-PCR and MIT.The FAT was conducted to confi rm rabies in symptomatic mice.
The RT-PCR tests were performed using the rabies virus oligonucleotides 'SeqN' and '304', which generate a 765-bp

Favoretto
SR et al -Experimental infection of ticks with the rabies virus

FIGURE 1 -
FIGURE 1 -Scanning electron microscopy of Carios fonsecai, ventral view: in the ellipse is the detail of the intra-coelomic route through the articulation of the coxa VI and trochanter IV, which is the region where the rabies virus was inoculated (Labruna & Venzal, 2009 authorized).

TABLE 1 -
The number of ticks (Carios fonsecai) inoculated with the rabies virus, according to the post-inoculation time at which they were killed for rabies diagnoses.