Molecular and Serological Survey of the Cat-Scratch Disease Agent (Bartonella henselae) in Free-Ranging Leopardus geoffroyi and Leopardus wiedii (Carnivora: Felidae) From Pampa Biome, Brazil.

The genus Bartonella comprises emerging bacteria that affect humans and other mammals worldwide. Felids represent an important reservoir for several Bartonella species. Domestic cats are the main reservoir of Bartonella henselae, the agent of cat scratch disease (CSD). It can be transmitted directly by scratches and bites from infected cats and via cat fleas. This study aims to investigate the circulation of Bartonella spp. in free-ranging Neotropical wild felids from Southern Brazil using serological and molecular methods. In this study, 53 live-trapped free-ranging wild felids were sampled, 39 Leopardus geoffroyi and 14 Leopardus wiedii, from five municipalities in the Rio Grande, do Sul state, southern Brazil. All captured animals were clinically healthy. Two blood samples of L. geoffroyi were positive, by PCR, for the presence of B. henselae DNA. Conversely, none of L. wiedii blood samples were positive when tested using PCR. Indirect immunofluorescence assay (IFA) showed that 28% of serum samples of wild felids were reactive (seropositive) for B. henselae by immunofluorescence, with titers ranging from 64 to 256. The results presented here provide the first evidence of a Bartonella-enzootic cycle involving L. geoffroyi and L. wiedii, which may account for the spillover of the emerging zoonotic pathogen B. henselae for the indigenous fauna in Southern Brazil.

Detection of Rangelia vitalii (Piroplasmida: Babesiidae) in asymptomatic free-ranging wild canids from the Pampa biome, Brazil.

Canine rangeliosis is a tick-borne disease caused by the protozoan Rangelia vitalii, which has only been reported in South America. With this knowledge, we hypothesized that neotropical foxes could act as asymptomatic natural carriers of R. vitalii. To test this, we captured 44 free-ranging foxes and investigated the presence of R. vitalii DNA, and whether the infected animals presented any clinical findings or hematological changes. Eight foxes (18%), seven Cerdocyon thous (7/27-25%), and one Lycalopex gymnocercus (1/17-5%) were positive for R. vitalii. All foxes were clinically healthy and showed no hematological abnormalities. Thus, we propose that neotropical canids, particularly C. thous, could be the natural carriers of R. vitalii.

Clinical Findings Associated with Ornithodoros brasiliensis Tick Parasitism in Travelers, Southern Brazil.

We report a series of clinical cases associated with parasitism by the Ornithodoros brasiliensis tick in a group of travelers in the Caxias do Sul municipality, Southern Brazil. These cases draw attention to underdiagnosed noninfectious syndromes caused by ticks with restricted local distributions.

Molecular survey of Rickettsia spp. in the Neotropical deer tick Haemaphysalis juxtakochi from Brazilian Pampa.

Spotted fever (SF) is a tick-borne disease associated with Rickettsia spp.. In the Pampa biome, Southern Brazil, cases of SF seem to be strongly linked with the practice of hunting wild animals. An investigation of rickettsiae in tick species found on wild animals could provide more information regarding the rickettsiosis enzootic cycle. The aim of this study is to describe the results of a molecular survey of Rickettsia spp. in the Neotropical deer tick, Haemaphysalis juxtakochi Cooley, 1946 (Acari: Ixodidae), from the Brazilian Pampa. Ticks were obtained from 14 road-killed gray brocket deer, Mazama gouazoubira (Artiodactyla: Cervidae), found in nine different municipalities of Rio Grande do Sul state, Southern Brazil. Ticks were processed individually to obtain genomic DNA, and then Rickettsia spp. was investigated using a set of PCR reactions that amplified the rickettsial fragments of the gltA, ompA, and htrA genes. Of the 24 tick samples tested, DNA of Rickettsia parkeri sensu stricto (s.s.) was found in 11 H. juxtakochi specimens collected in two different areas of the Brazilian Pampa. This is the first report of R. parkeri s.s. (the main agent associated with SF in the Uruguayan, Argentinian, and Brazilian Pampa) in H. juxtakochi ticks. These findings indicate that R. parkeri s.s. may be much more common and widely distributed in the Pampa biome than previously assumed. Moreover, H. juxtakochi ticks and gray brocket deer could participate in the potential spillover of R. parkeri s.s. from endemic to non-endemic areas in the South American Pampa.

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of Metarhizium anisopliae on Rhipicephalus microplus tick larvae.

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 106, 107 and 108 conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT50 values obtained were 134.6, 27.2 and 24.8 days for concentrations of 106, 107 and 108 conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT50 values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 106, 107 and 108 conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.

DETECTION OF BARTONELLA SP. IN DEER LOUSE FLIES (LIPOPTENA MAZAMAE) ON GRAY BROCKET DEER (MAZAMA GOUAZOUBIRA) IN THE NEOTROPICS.

Louse flies or deer keds, Lipoptena spp., are widespread in Neotropical cervids, but the vector-borne pathogens of louse flies had only been previously reported in the Northern hemisphere. This is the first report of Bartonella spp. in deer louse flies (Lipoptena mazamae) in the neotropics collected from gray brocket deer ( Mazama gouazoubira ) in Southern Brazil. DNA from Bartonella sp. was detected in all 429 L. mazamae collected from 11 road-killed gray brocket deer. The same sequences of DNA of Bartonella spp. were identified in samples. Gray brocket deer are abundant in Brazil, so Bartonella-infected Lipoptena spp. may be widely distributed in the neotropics.

Comparison of Seroprevalence and Identification of Risk Factors for Theileria equi in Horses From Vector-Free and Infested Areas in Southern Brazil.

The apicomplexan hemoprotozoan Theileria equi is a tick-borne pathogen that causes disease in equids, and together with Babesia caballi, causes equine piroplasmosis (EP). Many ticks are associated with EP around the world, and in South America three species may be related: Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus microplus, as they are commonly found in horses. Among the species cited above, only R. microplus is found in Rio Grande do Sul state, Southern Brazil. In addition, this state has the only area legally free of R. microplus in Brazil. This study aimed to compare the seroprevalence for T. equi on farms located in a tick-free area (23 farms, 215 horses) and a tick-infested (25 farms, 141 horses) adjacent areas, as well as to identify potential risk factors for exposure to T. equi. Blood was collected from 356 horses from both areas, and later an indirect enzyme-linked immunosorbent assay was performed to detect anti-T. equi antibodies. Besides the blood collection, questionnaires and interviews were conducted in an attempt to identify potential risk factors. The R. microplus-free zone had 6% seropositive horses against 70% in the tick-infested zone. Previous observation of ticks on horses of the herd increased four times the odds of a horse to be seropositive for T. equi, and by three times if ticks were observed on cattle which share paddocks with horses on the farm. The results showed a large difference in T. equi seroprevalence between tick-infested and free areas, and also emphasized the relevance of R. microplus in the epidemiology of T. equi. The study also reveals the potential of the establishment of a T. equi-free zone for horse breeding in Southern Brazil.

Rotational and selective protocols using acaricides to control a multi-resistant strain of Rhipicephalus microplus under field conditions in Southern Brazil.

The cattle tick, Rhipicephalus microplus, causes severe economic losses to beef and dairy production in several countries. The control of the cattle tick is based mostly on chemical acaricides. Currently, there are reports of tick populations resistant to all six classes of acaricides available on the market. The aim of this work was to evaluate two protocols for the control of the cattle tick, R. microplus, under field conditions. For that, an acaricide rotational-based approach and selective (partial)-based protocols were tested against a multi-resistant tick strain. Eighteen Brangus cattle were divided into three experimental groups: Group 1, a rotational-based approach aiming at six treatments per year (spring-spring period) using four different classes of acaricide (IPV6 protocol); Group 2, a selective (partial) and rotational-based protocol, in which only half of the animals with the highest tick counts were treated (IPV7S protocol); and Group 3 (non-treated). The protocols were able to maintain low tick infestation during the 57 weeks of study. The two experimental groups showed lower tick counts than the non-treated group, but not between them. Weekly efficacy of the experimental protocols reached 95% in Group 1 and 88% in Group 2. Herein, we proposed two protocols of cattle tick control (IPV6 and IPV7S) for use in multi-resistant tick populations in Southern Brazil.

The Influence of Landscape Structure on the Occurrence of Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in Free-Living Neotropical Primates.

PURPOSE: Habitat fragmentation is the main threat to primate survival in the world. Additionally, changes in the environments in which they live can also contribute to exposure to pathogens. To investigate some pathogens that free-living primates may be exposed to in Rio Grande do Sul State (RS; southern Brazil) and characterize the forest remnants in which they live, we investigated anti-Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. antibodies in the serum of the animals. METHODS: We analyzed 105 serum samples from 63 black howler monkeys (Alouatta caraya), 39 southern brown howler monkeys (Alouatta guariba clamitans), and 03 capuchin monkeys (Sapajus nigritus cucullatus), which were captured in forest fragments of RS. Indirect fluorescence antibody test (IFAT) and indirect hemagglutination assay (IHA) were used to detect antibodies to the agents. We then characterized the landscapes in a multiscale approach in radii from 200 to 1400 m to investigate the relationship of the presence of the agents with landscape elements. RESULTS: In the IFAT-IgG, 13.3% (14/105) of the samples were seropositive for N. caninum, 4.8% (5/105) for T. gondii, and 5.7% (6/105) for Sarcocystis spp. In the IHA-IgM/IgG, 24.8% (26/105) were seropositive for T. gondii. The metrics that best explained exposure to agents were edge and patch density, forest cover, urban cover, and average Euclidean distance to the nearest patch. CONCLUSIONS: This study indicated that the primates were exposed to the agents studied, demonstrating that some landscape features are associated with exposures to the investigated pathogens.

Mechanisms of amitraz resistance in a Rhipicephalus microplus strain from southern Brazil.

Amitraz is one of the most used acaricides for the control of ticks of domestic animals, however, extensive use of this active ingredient has favored the development of resistant populations of Rhipicephalus microplus worldwide. The possible mechanisms of metabolic and/or target-site alterations mechanisms of amitraz resistance were investigated in a Brazilian field population of R. microplus (São Gabriel strain). Bioassays with the synergists piperonylbutoxide, triphenylphosphate and diethyl-maleate were used to evaluate the metabolic mechanisms involved. Target-site insensitivity was investigated by amplification and sequencing of a fragment of the octopamine/tyramine (OCT/TYR) receptor gene. Piperonylbutoxide synergism (synergism ratio = 2.8) indicated the participation of the P450 pathway in the detoxification of amitraz. Previously reported single nucleotide polymorphisms that confer amino acid changes in the OCT/TYR receptor, threonine to proline (T8P) and leucine to serine (L22S), were found in the amitraz-resistant strain but not in the susceptible reference strain. The results suggest that amitraz resistance in the studied strain is multi-factorial and may result from cytochrome P450 detoxification and mutations in octopamine receptors.

The deletion of chiMaD1, a horizontally acquired chitinase of Metarhizium anisopliae, led to higher virulence towards the cattle tick (Rhipicephalus microplus).

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.

Transcriptomic Analysis of Salivary Glands of Ornithodoros brasiliensis Aragão, 1923, the Agent of a Neotropical Tick-Toxicosis Syndrome in Humans.

Tick salivary glands produce and secrete a variety of compounds that modulate host responses and ensure a successful blood meal. Despite great progress made in the identification of ticks salivary compounds in recent years, there is still a paucity of information concerning salivary molecules of Neotropical argasid ticks. Among this group of ticks, considering the number of human cases of parasitism, including severe syndromes and hospitalization, Ornithodoros brasiliensis can be considered one of the major Neotropical argasid species with impact in public health. Here, we describe the transcriptome analysis of O. brasiliensis salivary glands (ObSG). The transcriptome yielded ~14,957 putative contigs. A total of 368 contigs were attributed to secreted proteins (SP), which represent approximately 2.5% of transcripts but ~53% expression coverage transcripts per million. Lipocalins are the major protein family among the most expressed SP, accounting for ~16% of the secretory transcripts and 51% of secretory protein abundance. The most expressed transcript is an ortholog of TSGP4 (tick salivary gland protein 4), a lipocalin first identified in Ornithodoros kalahariensis that functions as a leukotriene C4 scavenger. A total of 55 lipocalin transcripts were identified in ObSG. Other transcripts potentially involved in tick-host interaction included as: basic/acid tail secretory proteins (second most abundant expressed group), serine protease inhibitors (including Kunitz inhibitors), 5' nucleotidases (tick apyrases), phospholipase A2, 7 disulfide bond domain, cystatins, and tick antimicrobial peptides. Another abundant group of proteins in ObSG is metalloproteases. Analysis of these major protein groups suggests that several duplication events after speciation were responsible for the abundance of redundant compounds in tick salivary glands. A full mitochondrial genome could be assembled from the transcriptome data and confirmed the close genetic identity of the tick strain sampled in the current study, to a tick strain previously implicated in tick toxicoses. This study provides novel information on the molecular composition of ObSG, a Brazilian endemic tick associated with several human cases of parasitism. These results could be helpful in the understanding of clinical findings observed in bitten patients, and also, could provide more information on the evolution of Neotropical argasids.

Molecular characterization of bacterial communities of two neotropical tick species (Amblyomma aureolatum and Ornithodoros brasiliensis) using rDNA 16S sequencing.

Ticks are one of the main vectors of pathogens for humans and animals worldwide. However, they harbor non-pathogenic microorganisms that are important for their survival, facilitating both their nutrition and immunity. We investigated the bacterial communities associated with two neotropical tick species of human and veterinary potential health importance from Brazil: Amblyomma aureolatum and Ornithodoros brasiliensis. In A. aureolatum (adult ticks collected from wild canids from Southern Brazil), the predominant bacterial phyla were Proteobacteria (98.68%), Tenericutes (0.70%), Bacteroidetes (0.14%), Actinobacteria (0.13%), and Acidobacteria (0.05%). The predominant genera were Francisella (97.01%), Spiroplasma (0.70%), Wolbachia (0.51%), Candidatus Midichloria (0.25%), and Alkanindiges (0.13%). The predominant phyla in O. brasiliensis (adults, fed and unfed nymphs collected at the environment from Southern Brazil) were Proteobacteria (90.27%), Actinobacteria (7.38%), Firmicutes (0.77%), Bacteroidetes (0.44%), and Planctomycetes (0.22%). The predominant bacterial genera were Coxiella (87.71%), Nocardioides (1.73%), Saccharopolyspora (0.54%), Marmoricola (0.42%), and Staphylococcus (0.40%). Considering the genera with potential importance for human and animal health which can be transmitted by ticks, Coxiella sp. was found in all stages of O. brasiliensis, Francisella sp. in all stages of A. aureolatum and in unfed nymphs of O. brasiliensis, and Rickettsia sp. in females of A. aureolatum from Banhado dos Pachecos (BP) in Viamão municipality, Brazil, and in females and unfed nymphs of O. brasiliensis. These results deepen our understanding of the tick-microbiota relationship in Ixodidae and Argasidae, driving new studies with the focus on the manipulation of tick microbiota to prevent outbreaks of tick-borne diseases in South America.

Genomic analysis on Brazilian strains of Anaplasma marginale.

Anaplasma marginale is a vector-borne pathogen that causes a disease known as anaplasmosis. No sequenced genomes of Brazilian strains are yet available. The aim of this work was to compare whole genomes of Brazilian strains of A. marginale (Palmeira and Jaboticabal) with genomes of strains from other regions (USA and Australia strains). Genome sequencing of Brazilian strains was performed by means of next-generation sequencing. Reads were mapped using the genome of the Florida strain of A. marginale as a reference sequence. Single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) were identified. The data showed that two Brazilian strains grouped together in one particular clade, which grouped in a larger American group together with North American strains. Moreover, some important differences in surface proteins between the two Brazilian isolates can be discerned. These results shed light on the evolutionary history of A. marginale and provide the first genome information on South American isolates. Assessing the genome sequences of strains from different regions is essential for increasing knowledge of the pan-genome of this bacteria.

Serosurvey of antibodies against zoonotic pathogens in free-ranging wild canids (Cerdocyon thous and Lycalopex gymnocercus) from Southern Brazil.

The expansion of urbanization on natural areas is increasing contact between human populations with wild animals. Wild carnivores can act as sentinel hosts or environmental health indicators. Thus, the aim of this work was to investigate the exposure of two major species of wild canids from Southern Brazil to selected pathogens. For that, we live-trapped free-ranging Cerdocyon thous and Lycalopex gymnocercus in five localities and determined the frequency of animals with antibodies against Toxoplasma gondii, Trypanosoma cruzi, Leishmania infantum, Neospora caninum, and Leptospira spp. Among the canids sampled, 23% (12/52) (95%CI: 13-36%) had antibodies against T. gondii, with titers ranging from 64 to 512. For T. cruzi, 28% (15/52) (95%CI: 18-42%) of sampled canids were seropositive, with titers ranging from 8 to 64. Concerning the protozoan pathogen N. caninum, a total of 5% (3/52) (95%CI: 2-15%) of wild canids had antibodies against it. None of the sampled canids showed the presence of antibodies against L. infantum. On the other hand, 44% (23/52) (95%CI: 31-57%) of the wild canids showed antibodies against Leptospira spp. The set of results presented here, show that free-ranging neotropical wild canids are exposed and have antibodies against to T. gondii, T. cruzi, Leptospira spp., and to a lesser degree to N. caninum. We found no evidence of L. infantum circulation among the studied populations. These results highlight some of the major pathogens which may represent risks for populations of these wild canids. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Serosurvey of West Nile virus (WNV) in free-ranging raptors from Brazil.

West Nile virus (WNV) is a mosquito-borne Flavivirus that can affect birds, horses, and humans, and is the only zoonotic Flavivirus that has been identified in six continents. In Brazil, until 2010, there was no evidence of WNV circulation. Recently, the virus was isolated from a horse with encephalitis, and the first human cases were registered in Brazil. Despite that, there is still no information on the enzootic cycle of this virus in birds or wildlife. This study aimed to investigate whether there is evidence of WNV circulation among wild birds from Southern Brazil. For this, we used free-living wild raptors (live-trapped or rescued) as potential sentinels to investigate the presence of WNV antibodies using ELISA and plaque reduction neutralization test (PRNT) assay. In addition, the presence of nucleic acids from Flavivirus family members was investigated. None of the birds sampled presented clinical findings compatible with WNV. Of the 200 serum samples from birds of prey belonging to 21 species, ten (5%) were positive for the presence of WNV antibodies on ELISA testing. The PRNT test did not confirm the ELISA results, but indicated that three birds had possibly been exposed to Saint Louis encephalitis virus (SLEV). All samples were negative for Flavivirus RNA. The results presented here evince the need for permanent surveillance for emerging flaviviruses in Brazil, as well as for a contingency policy in the case of human/animal outbreaks, particularly in high-risk areas.

Molecular detection and phylogenetic relationship of Haemosporida parasites in free-ranging wild raptors from Brazil.

The order Haemosporida is widely distributed parasitizing members of the Aves class. In birds of prey, infection with Plasmodium spp. parasites varies from an apathogenic form to a clinical syndrome. However, studies on Haemosporida in raptors from the neotropical region are scarce. The aim of this study was to investigate natural infection by Plasmodium spp., Haemoproteus spp. and Leucocytozoon spp. in free-ranging wild raptors from southern Brazil. For this, we sampled 206 individuals of 21 species: 94 live-trapped Southern Caracaras (Caracara plancus) and 112 raptors from other species that were brought to rehabilitation centers. The presence of infection was investigated using a nested-PCR for Haemosporida performed on blood samples. Overall, 56 out of 206 birds were positive for Plasmodium spp./Haemoproteus spp. Twenty-two percent (21/94) of the C. plancus samples were positive. Of the 112 wild raptors rescued, 31% (35/112) of those belonging to 15 other species tested positive. No sample was positive for Leucocytozoon spp. Herein, we demonstrated nine lineages of Haemosporidian parasites (eight Plasmodium sp. and one Haemoproteus sp.) in free-living species of Brazilian birds of prey, being six of them potential novel lineages. It suggests that information currently available on South-American haemosporidian from these birds greatly underestimate the potential lineage diversity in this region.

Ticks, mites, fleas, and vector-borne pathogens in free-ranging neotropical wild felids from southern Brazil.

The study of vectors and vector-borne diseases associated with ectoparasites from free-living Neotropical small wild felids is scarce, and the few existing studies on this theme focused on the genus Panthera, Paleartic species or in captive animals. For this reason, the aim of this study was to identify the diversity of ectoparasites and potentially associated Rickettsia and Bartonella species in free-ranging neotropical wild cats collected in the State of Rio Grande do Sul, southern Brazil. A total of 82 ticks, 10 fleas and 22 mites were collected from 18 Geoffroy's cats (Leopardus geoffroyi), 11 margays (Leopardus wiedii), two southern tiger cats (Leopardus guttulus), two jaguarundis (Herpailurus yagouaroundi), three ocelots (Leopardus pardalis) and two pumas (Puma concolor). We identified four tick species: Rhipicephalus microplus and three species from genus Amblyomma, the most frequent being Amblyomma aureolatum; three flea species corresponding to Ctenocephalides felis, Xenopsylla cheopis, and Pulex irritans; and one mite of genus Eutrombicula. In ectoparasites we found DNA of Rickettsia parkeri and Rickettsia asembonensis in ticks and DNA of Bartonella clarridgeiae, Bartonella sp. and R. asembonensis in fleas. Our results highlight the evidences of vectors and vector-borne agents in wild animals, and their potential wide distribution in the Pampa biome and the southernmost portion of the Atlantic Rainforest in Brazil.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to ivermectin in Argentina.

Resistance to ivermectin in populations of the cattle tick Rhipicephalus microplus in Argentina was diagnosed in this work. The in vitro larval immersion test (LIT) was used to determine quantitatively the levels of resistance to ivermectin in different populations of R. microplus. Additionally, field trials to control natural infestations of R. microplus on cattle with a commercial formulation of ivermectin 3.15% were carried and jointly analyzed with the in vitro assays. The phenotypic response of the populations analyzed was not uniform. Five of them were classified as susceptible, four populations as resistant, and one in the category "incipient resistance". Regarding the field trials, the therapeutic efficacy in a population classified with LIT as susceptible achieved values higher than 94% two weeks after treatment, and no reproductively viable females were observed after the second day post-treatment. Conversely, the values of efficacy percentage in a population (named as "San Martín") classified with LIT in the category "incipient resistance" never exceeded the 70.8%, and engorged females were collected in practically all counts. The population "San Martín" was classified in the category "incipient resistant" with LIT analysis, but the field trial unambiguously shows that this tick population is resistant. The comparison of the results obtained with LIT in vitro assays and through field trials shows that biased estimations of resistance levels may occur when resistance ratios (RR) values are ≤2, and additional field efficacy trials could be needed to know with precision the status of the tick populations evaluated.