Ticks on birds in a savanna (Cerrado) reserve on the outskirts of Uberlândia, Minas Gerais, Brazil

We report tick infestations on birds, in the environment and on domestic animals in a non-forested phytophysiognomy, the savanna-like Cerrado sensu stricto , in a natural reserve on the outskirts of the urban area of Uberlândia, Minas Gerais, Brazil. Overall, 238 birds within 50 species, 15 families and six orders were caught. Passeriformes were the most numerous, with 216 birds (90.75%), among which 22 had ticks (n = 31). Within this order, the prevalence of tick infestation was 10.2%, and the abundance and mean intensity were 0.14 and 1.41, respectively. Only immature ticks of the species Amblyomma nodosum were found on the birds. The tick species found both on animals ( Rhipicephalus sanguineus , Rhipicephalus ( Boophilus ) microplus , Amblyomma cajennense and Dermacentor nitens ) and in the environment ( Amblyomma dubitatum , Rhipicephalus ( B. ) microplus and Amblyomma cajennense ) were as expected. This difference in tick species between the environment and birds possibly occurred because the sampling of the environment was limited to the ground. This study also highlights the importance of the diverse microenvironments used by ticks and hosts in the same area and the complex ecology of bird-tick relationships. Ecological and epidemiological aspects of the findings are discussed.


Introduction
Ticks are the most important vectors transmitting pathogens to animals and are only second to mosquitoes in the case of human beings (JONGEJAN; UILENBERG, 2004).Out of the approximately 61 species that occur in Brazil (DANTAS-TORRES et al., 2009), comprehensive information exists in relation to less than a dozen species.Generally these species are those affecting domestic animals, and there is a lack of basic information such as hosts, life cycle and pathogen transmission in relation to most others.
Emergence and re-emergence of tick-borne diseases is currently being witnessed worldwide (OSTFELD; KEESING, 2000;PADDOCK, 2009).Although the exact causes for this are complex, both environmental and human behavioral changes might have a role in it.Territorial mixing of wildlife, domestic animals and humans, in association with habitat fragmentation, increases microorganism circulation from and to natural areas (OSTFELD; KEESING, 2000;QUEIROGAS et al., 2012).Some of these microorganisms are pathogenic to humans and thus, knowledge of vector ecology and life cycles is essential for understanding the epidemiology of emerging diseases.
Recent reports have begun to reveal bird-tick relationships in various biomes and phytophysiognomies in Brazil in a more systematic fashion.Thus, there have been recent records of tick species on birds from the Araucaria forest (ARZUA et al., 2003;ARZUA;BARROS-BATTESTI, 1999); Atlantic forest (LABRUNA et al., 2007;OGRZEWALSKA et al., 2008OGRZEWALSKA et al., , 2009a)), Amazon (OGRZEWALSKA et al., 2010), Cerrado (ROJAS et al., 1999;TOLESANO-PASCOLI et al., 2010) andnortheastern Brazil (OGRZEWALSKA et al., 2011).These studies have shown a role for birds to feed immature ticks, and that these ticks may harbor microorganisms that are potentially pathogenic to humans, such as Rickettsia (OGRZEWALSKA et al., 2009b).These data also suggest that birds might spread ticks and tick-borne diseases between small fragments of natural environment.
The Brazilian "Cerrado" (or savanna) is considered to be a "hotspot" of global biodiversity.However, it is severely endangered by human activities and has suffered severe fragmentation (MYERS et al., 2000), mainly due to agricultural activities (SILVA et al., 2006).This biome is vast (only smaller than the Amazon) and comprises several phytophysiognomies ranging from open fields to forest vegetation.There are only a few reports of bird ticks in this area (MARINI; COUTO, 1997;ROJAS et al., 1999;KANEGAE, 2003;TOLESANO-PASCOLI, 2010;LUZ et al., 2012).Nonetheless, this biome is known to be very rich in birds, totaling as many as 837 species, of which 36 are considered endemic (SILVA, 1995).
We recently began a systematic study of bird-tick relationships in the Cerrado by evaluating hosts from a forest fragment (TOLESANO-PASCOLI et al., 2010).Here, we report on tick infestations on birds, in the environment and on home-dwelling domestic animals in a non-forested phytophysiognomy, the cerrado sensu stricto, in a natural reserve on the outskirts of the urban area of the city of Uberlândia, Minas Gerais, Brazil.

Study site
Sampling was performed in a savanna (Cerrado) reserve named Reserva Vegetal do Clube de Caça e Pesca Itororó, which is on the outskirts of the city of Uberlândia, state of Minas Gerais, Brazil.The reserve, which is adjacent to the urban area, comprises 127 ha and is approximately eight km from downtown.Several Cerrado phytophysiognomies are found in the reserve including "cerrado sensu stricto" (vegetation dominated by trees and shrubs that are often 3-8 m tall, giving more than 30% crown cover but still with a fair amount of herbaceous vegetation between them) and palm swamp ("vereda"), i.e. valley-side marshes where the water table reaches or almost reaches the surface during the rainy season.

Bird sampling
Birds were caught with the aid of 10 mist nets, each 12 meters long and 3 meters high, during four consecutive seasons: May 2008 (autumn); June 2008 (winter); September and December 2008 (spring); and February 2009 (summer).In each season, bird-catching was undertaken for four hours around dawn, on two consecutive days.Mist nets were set up in ¨Cerrado sensu stricto" areas.The birds that were caught were identified, weighed and measured, and received metallic bands provided by the National Research Center for Wild Bird Conservation and the Chico Mendes Institute for Biodiversity Conservation (CEMAVE/ICMBIO: Centro Nacional de Pesquisa para Conservação de Aves Silvestres/Instituto Chico Mendes de Conservação da Biodiversidade).Bird-catching was authorized by CEMAVE (license No. 2943).The birds were identified in accordance with Ridgely andTudor (1989, 1994) and Sick (2004), and the nomenclature followed the prescriptions of the Brazilian Committee for Ornithological Records (Comitê Brasileiro de Registros Ornitológicos) (CBRO, 2011).

Tick collection from birds
The search for ticks was done by blowing the feathers from birds to expose the skin.Ticks were collected with tweezers and placed in plastic containers with leaves to prevent desiccation.After this, the birds were released at the capture site.
Rev. Bras.Parasitol.Vet.20 m.Twenty CO 2 traps, five per site, and 160 minutes of dragging, 40 minutes per site, were used in each campaign.

Ticks from domestic animals
To detect domestic animal infestation with ticks from the reserve, dogs, cattle and horses on four neighboring farm properties were inspected every season from autumn 2008 to summer 2009.

Tick identification
Birds in Brazil are overwhelmingly parasitized by immature specimens of Amblyomma.Unfortunately, there is no reliable key for identifying Neotropical Amblyomma larvae, and until 2010, there was also no key for Amblyomma nymphs.Thus, identification was done initially by rearing ticks until the adult stage in the laboratory or by molecular methods as described previously (OGRZEWALSKA et al., 2009a, b).Adult ticks and, later on, nymphs were identified under a stereomicroscope, in accordance with morphological criteria and dichotomous keys (ONOFRIO et al., 2006;MARTINS et al., 2010).Voucher tick specimens collected during this study have been deposited in the FAMEV/UFU Tick Collection, Federal University of Uberlândia (accession numbers: 264-267, 325).
The laboratory procedures for tick identification (feeding of ticks on laboratory animals until the adult stage) were approved by the Ethics Committee for Animal Research of the Federal University of Uberlândia (protocol number 007/2008).

Hemolymph test
Rickettsia is an intracellular bacterium with several species that are pathogenic to humans and transmitted by ticks.Searches for Rickettsia in ticks were performed in the hemolymph of adult ticks.For this purpose, each tick specimen was individually tested using the hemolymph test, as described by Burgdorfer (1970).Briefly, a drop of hemolymph collected from the broken leg of each tick was spread onto a slide and then subjected to Gimenez staining.The slide was then scrutinized under an optical microscope for Rickettsia-like organisms, especially inside hemolymph cells.

Data analysis
The prevalence, abundance and mean intensity of bird tick infestations were determined (BUSH et al., 1997).Minimum and maximum numbers of ticks per host (variation) were also noted.

Ticks on vegetation
Fifty-eight ticks and also three larval clusters were collected over the two years of sampling (Table 4).The main tick species

Ticks from domestic animals
Overall, on each occasion, 2 to 9 horses, 2 to 9 cattle and 7 to 21 dogs were examined.thus resulting in 26, 28 and 67 evaluations of horses, cattle and dogs, respectively.The prevalence of tick infestation on horses, cattle and dogs was 23.1%, 75% and 25.4%, respectively.Four species of ticks were found on the animals (Table 5).Rhipicephalus (B.) microplus (n = 166) was the only species found on cattle, although this tick species was also found in lesser numbers on dogs and horses.Dermacentor nitens (Neumann, 1897) (n = 37) was found only on horses, whereas Rhipicephalus sanguineus (Latreille, 1806) (n = 11) was solely on dogs.Amblyomma cajennense (n = 12) was recovered from both dogs and horses.

Hemolymph test
The hemolymph test was performed on twelve adult ticks and none displayed Rickettsia-like structures.

Discussion
The sampling at the Reserva Vegetal do Clube de Caça e Pesca Itororó showed that the tick species on birds and in the environment were dissimilar.Whereas only A. nodosum was found on birds, A. cajennense, A. dubitatum and R. (B.) microplus was found on the vegetation.Similar results were obtained by Ogrzewalska et al. (2009a), who did not collect any specimens of A. nodosum questing tick (in contrast to hundreds of questing A. cajennense specimens) in one Atlantic forest area where nearly 10% of the passerine birds were infested by A. nodosum ticks.Such dissimilarity may be explained both by the sampling locations (microenvironment) and by the techniques, which might have been inadequate for ticks questing for birds.Whereas cloth dragging and CO 2 traps are more suitable for ambush and hunter ticks, it is possible that bird ticks in the Cerrado are nidicolous and restricted to particular spots.Thus, the precise questing locations of bird ticks should be determined in future observations.Immature specimens of Amblyomma nodosum, mostly nymphs, have been described previously on several passerine bird species (LABRUNA et al., 2007;OGRZEWALSKA et al., 2009a;TOLESANO-PASCOLI et al., 2010;LUZ et al., 2012), and these hosts seem to be important for its life cycle.Adult A. nodosum ticks feed almost exclusively on anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla) (ARAGÃO, 1936;JONES et al., 1972;BECHARA et al., 2002) and, as stated previously (LABRUNA et al., 2007), this tick's distribution seems to be associated with that of its adult hosts.Since anteaters are widely distributed in the Cerrado (REIS et al., 2006), A. nodosum is expected to be present throughout this biome.However, in bird tick surveys in the Araucaria forest (ARZUA et al., 2003), Amazon rainforest (OGRZEWALSKA et al., 2010) and Atlantic rainforest (OGRZEWALSKA et al., 2008) this tick species was lacking.This might be explained either by a lack of hosts for adults or by an inadequate environment for the off-host life phase of the ticks, a matter that should be investigated.
Our results and those described previously suggest that the life cycle of A. nodosum involves anteaters for the adult stage of the ticks to feed on, and various passerine bird species for larvae and nymphs to feed on.Labruna et al. (2007) suggested that groundfeeding passerine birds seem to be the most important species for A. nodosum, but in our work several arboreal passerine birds were common hosts as well.In fact, not only ground-feeding birds such as Turdus leucomelas and T. amaurochalinus, but also arboreal species such as Elaenia cristata and Casiornus rufus (SICK, 2004) were infested with A. nodosum.Since the observations of Labruna et al. (2007) were predominantly from the Atlantic forest and were restricted to two A. nodosum nymphs out of 568 tick specimens, environmental factors and/or low A. nodosum sampling might explain this discrepancy.Moreover, since the hosts for adults are terrestrial (Myrmecophaga tridactyla) but also have mixed terrestrial and arboreal habits (Tamandua tetradactyla) (REIS et al., 2006), host questing of A. nodosum may be successful in either location for all stages.Nonetheless, A. nodosum questing habits, particularly with regard to location, should be addressed by further research.
Finally, one important issue regarding A. nodosum is that it may be involved in the epidemiology of rickettsiosis.A spotted-fever group Rickettsia closely related to Rickettsia parkeri was isolated from A. nodosum ticks of Passeriformes birds caught in the Atlantic forest (OGRZEWALSKA et al., 2009b).
Another interesting feature of the present work was the lack of A. longirostre, the tick species that was most prevalent on birds in other reports (LABRUNA et al., 2007;OGRZEWALSKA et al., 2008OGRZEWALSKA et al., , 2010;;TOLESANO-PASCOLI et al., 2010;LUZ et al., 2012).The adult stage of this tick species feeds on porcupines (Coendou spp.) and the immature stages on passerine birds (ARAGÃO, 1936;JONES et al., 1972).Since porcupines are arboreal animals (REIS et al., 2006), forested phytophysiognomies provide a better habitat for both host and tick.Coincidently, in most surveys, A. longirostre prevailed over other tick species in forested habitats such as the Atlantic forest (LABRUNA et al., 2007;OGRZEWALSKA et al., 2008), Amazon forest (OGRZEWALSKA et al., 2010) and forested fragments within the Cerrado biome (TOLESANO-PASCOLI et al., 2010;LUZ et al., 2012).Thus, it is possible to speculate that the area sampled in our work ("cerrado sensu stricto"), which has a fair amount of herbaceous vegetation, is less appropriate for A. longirostre and its rodent host (porcupine).
The other tick species found on animals or in the environment did not present unexpected features.Rhipicephalus sanguineus is a common tick that infests dogs, R.(B.) microplus is the main cattle tick, and A. cajennense and D. nitens are ticks that infest horses in the Neotropical region (BARROS-BATTESTI et al., 2006).Even though unusual, infestation of dogs with A. cajennense and R. (B.) microplus and horses with R. (B.) microplus can occur if these hosts go through areas infested with these tick species.
Capybaras are the principal host for all parasitic stages of A. dubitatum (NAVA et al., 2010).Albeit in low numbers, this tick species was found in the vereda phytophysiognomy (valley-side marshes), a wetland habitat that is suitable for capybaras (REIS et al., 2006).This finding indicates that capybaras were present at this site.The finding of R. (B.) microplus larvae showed that cattle had access to the reserve, an observation also reinforced by the finding of cattle feces during the survey.A. cajennense and Amblyomma spp.nymphs were the main tick species found on the vegetation.Amblyomma is the main tick genus in the Neotropical region (BARROS-BATTESTI et al., 2006) and A. cajennense seems to be the most prevalent species in the cerrado biome (VERONEZ et al., 2010).However, on the whole, the environmental tick infestation of the reserve was very low, thus suggesting that the host density was low.
In conclusion, A. nodosum was the main tick species infesting passerine birds in the area.Dissimilar tick species were found on the ground, thus indicating varying host questing activities among the tick species within the cerrado.Nymphs of A. nodosum were found on both ground feeding and arboreal birds, and the questing sites of this species are still open to speculation.

Table 3 .
Tick species on birds in the reserve of the Clube de Caça e PescaItororó, period 2008-2009, in Uberlândia, MG, Brazil.

Table 4 .
Ticks from vegetation in the reserve of the Clube de Caça e PescaItororó, period 2007-2009, in Uberlândia, MG, Brazil.

Table 5 .
2008 species and numbers found on domestic animals on farm properties surrounding the cerrado reserve, period2008-2009,  in Uberlândia, MG,Brazil.