Parasitism on domestic cats by Amblyomma auricularium and serological evidence of exposure to Rickettsia amblyommatis

Abstract The domestic cat is not considered a primary host for any specific tick species; however, it can be affected by some Ixodidae species, such as Rhipicephalus sanguineus sensu lato and Amblyomma spp. The study reports parasitism by Amblyomma auricularium and the detection of anti-Rickettsia spp. antibodies in domestic cats from a rural property in the Afrânio municipality, Pernambuco, Brazil. Amblyomma auricularium (24 nymphs, six females, and four males) and Amblyomma sp. (42 larvae) parasitized three cats, and 73 free-living ticks were captured in armadillo burrows: A. auricularium (36 nymphs, six females, five males) and Amblyomma sp. (26 larvae). Blood samples from cats were collected and the obtained plasma were subjected to indirect immunofluorescence assay (IFA) to detect antibodies against Rickettsia antigens. Thus, anti-Rickettsia spp. antibodies were determined (titers ranging from 128 to 512) and showed a predominant antibody response to Rickettsia amblyommatis or a very closely related genotype. This study reports the first infestation of nymphs and adults of A. auricularium on cats in a new area of occurrence in the semi-arid region of Northeastern Brazil and reports for the first time the presence of anti-Ricketsia antibodies in cats in the region, with R. amblyommatis as the probable infectious agent.

Braz J Vet Parasitol 2024; 33(1): e018123 2/5 Amblyomma auricularium on domestic cats Domestic cats (Felis catus domesticus) are parasitized by a variety of ectoparasites including mites, lice, fleas, and ticks (Dantas-Torres & Otranto, 2014), which are of great importance in feline medicine (Ferreira et al., 2010).Felines are reservoirs for various pathogens with zoonotic potential for humans, including Bartonella henselae, which causes cat scratch disease and Rickettsia typhi, which causes murine typhus (Case et al., 2006), both of which are transmitted by fleas.
The genus Amblyomma has the greatest species diversity in Brazil, and its distribution has been reported worldwide (Barros-Battesti et al., 2006).Rickettsiae are a group of obligate intracellular Gram-negative bacteria that cause a variety of diseases called rickettsioses, transmitted by hematophagous arthropods (Hiraoka et al., 2005).
Thus, this study reports the parasitism by Amblyomma auricularium in three domestic cats from a farm in the municipality of Afrânio, state of Pernambuco, Brazil, in addition to the detection of anti-Rickettsia spp.antibodies in plasma samples.
From September to December 2021, 76 specimens of ticks were collected from the three domestic cats that used to live on a farm in the municipality of Afrânio.In addition, 73 tick specimens were collected from inside six burrows previously inhabited by six-banded armadillos (Euphractus sexcinctus) on the same farm.To collect ticks from the burrows, we used a flannel with a rod inserted into the burrows, and when the flannel was removed, the ticks were visualized on it.All the ticks were placed in tubes containing 70% ethanol and transported to the laboratory for taxonomic identification.Ticks were separated by stages and identified according to Barros-Battesti et al. (2006) and Martins et al. (2010).
Blood samples were collected through venipuncture of the cephalic vein, previously disinfected with ethanol 70%, using 0.55 × 20 mm needles and sterile syringes.Samples were stored in sterile tubes containing anticoagulants at room temperature.Blood samples were centrifuged at 5,000 rpm for 15 min to obtain plasma, which was stored at -20ºC until laboratory analysis.Plasma samples from cats were individually tested by IFA to verify their reactivity to Rickettsia spp., according to Horta et al. (2007), using crude antigens of Rickettsia rickettsii strain Taiaçu, Rickettsia parkeri strain At24, Rickettsia amblyommatis strain Ac37, Rickettsia rhipicephali strain HJ5, and Rickettsia bellii strain Mogi.Each Rickettsia strain was cultivated in Vero cells and harvested when nearly 100% of the cells were infected.The infected cells were centrifuged at 4,000 g for 10 min, and pellet was washed in 0.1 M phosphate-buffered saline (PBS), pH 7.4, centrifuged again, and resuspended in PBS containing 1% bovine calf serum and 0.1% sodium azide.Ten microliters of rickettsiae-infected cells were applied onto each of 12 wells on microscopic slides, air-dried, fixed in acetone for 10 min, and stored at -80ºC until used.Feline plasma was diluted in two-fold increments with PBS starting from a 1:64 dilution.Ten microliters of diluted plasma were added to each well of the antigen slides.The slides were incubated at 37ºC for 30 min in a humid chamber.The slides were rinsed once, and then washed twice for 15 min per wash in PBS.The slides were incubated with fluorescein isothiocyanate-labeled goat anti-cat IgG (Sigma, St Louis, USA) from a 1:1,000 dilution, and washed as described earlier.The slides were mounted with buffered glycerin under coverslips.The slides were read using an ultraviolet microscope (Olympus, Tokyo, Japan) at 400x magnification.Plasma was considered to contain antibodies against the rickettsiae if it displayed a reaction at the 1:64 dilution.End-point titers against each Rickettsia strain were determined by testing serial plasma dilutions.In each slide, a serum previously shown to be non-reactive (negative control) and a known reactive cat serum (positive control) were tested (Horta et al., 2007).
The ticks collected from cats were identified as A. auricularium (24 nymphs, six females, four males) and Amblyomma sp.(42 larvae).The ticks collected from the armadillo burrows were identified as A. auricularium (36 nymphs, six females, and five males) and Amblyomma sp.(26 larvae).The presence of anti-Rickettsia spp.
This study assessed one male and two female healthy adult felines.The three cats had free access to a wooded area where wild animals such as rodents, marsupials, and armadillos were present, and it was possible to visualize and count six empty armadillo burrows near the property.The coexistence of domestic animals with wildlife in forest areas can favor tick infestation, thus facilitating the transmission of pathogens from wild animals to domestic animals and humans (Little et al., 2018).
Studies reporting tick parasitism and serological evidence of exposure to Rickettsia spp. in cats are very rare in Brazil because cats are not the primary hosts of any tick species and thus far have no importance in the epidemiological cycle of Rickettsiae.A study carried out by da Silva et al. (2007) in the state of Rio Grande do Sul reported parasitism of a cat by Amblyomma triste, while Mendes et al. (2019) reported parasitism by Amblyomma sculptum in the state of Rio de Janeiro; however, no diagnosis was made to prove exposure to agents from the Rickettsiae group.
In a previous study conducted in the same region as the present study, Saraiva et al. (2013) demonstrated the infection and vectorial competence of A. auricularium to R. amblyommatis.In this study, we confirmed, for the first time, the exposure of cats to the spotted fever group Rickettsiae, likely R. amblyommatis, using IFA.Generally, studies that detect anti-Rickettsia antibodies included included dogs, horses, ruminants, or wild animals, and those dealing with domestic cats in Brazil are scarce.Studies from other countries have reported Rickettsiae in cats and ticks, highlighting their epidemiological importance (Hiraoka et al., 2005;Case et al., 2006;Segura et al., 2014;Little et al., 2018).Lopes et al. (2018) and Oliveira et al. (2020) reported the presence of anti-R.amblyommatis antibodies in Didelphis albiventris and Rattus rattus.Here, we found anti-R.amblyommatis antibodies in domestic cats, thus suggesting the likely circulation of R. amblyommatis in both wild and domestic animals.R. amblyommatis, initially considered a non-pathogenic bacterium for humans, has already been considered a possible human pathogen based on serological evidence of human infection in the United States (Apperson et al., 2008).
In Brazil, there are at least two distinct reports of A. auricularium parasitism on humans (Lopes et al., 2018;Szabó et al., 2020).As a result, the true zoonotic potential of these findings remains unclear.However, it underscores the importance of implementing epidemiological surveillance to monitor the prevalence of this spotted fever group Rickettsiae and its vector in the region, using felines with free access to forest areas and hunting habits as sentinels for this pathogen.
The study reports for the first time the infestation of nymphs and adults of A. auricularium on domestic cats in a new area of occurrence in the semi-arid region of Northeast.Additionally, the study also focused on the detection of anti-Rickettsia spp.antibodies in parasitized cats, with R. amblyommatis identified as the probable infectious agent.