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Article

Molecular Detection of Kinetoplastid Species in Ticks and Fleas Associated with Dogs and Humans in Mexico

by
Héctor M. Zazueta-Islas
1,†,
Beatriz Salceda-Sánchez
2,†,
Herón Huerta-Jiménez
2,
Carlos I. Miranda-Caballero
1,
Marlene Solis-Cortés
1,
Yaretzi de la Cruz-Pacheco
3,
Ana Cristina Luquín-García
3,
Laura V. Mondragon-Peña
3,
Jair Reyes-Hernández
3,
José L. Bravo-Ramos
4,
María-Guadalupe Sánchez-Otero
4,
Javier C. Huerta-Peña
3,
Rosa I. Hernández-Herrera
5,
Pablo San Martin-del Angel
5,
André Luiz Rodrigues Roque
6,
Ángel Rodríguez-Moreno
7,
Víctor Sánchez-Cordero
7,
Héctor Abelardo Rodríguez Martínez
8,
Estefania Grostieta
1,*,
Ingeborg Becker
1,* and
Sokani Sánchez-Montes
1,3,*add Show full author list remove Hide full author list
1
Centro de Medicina Tropical, División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
2
Laboratorio de Entomología, Instituto de Diagnóstico y Referencia Epidemiológicos, Secretaría de Salud, Mexico City 01480, Mexico
3
Facultad de Ciencias Biológicas y Agropecuarias, Región Poza Rica-Tuxpan, Universidad Veracruzana, Tuxpan de Rodríguez Cano, Veracruz 92870, Mexico
4
Facultad de Bioanálisis, Región Veracruz, Universidad Veracruzana, Veracruz 91700, Mexico
5
Laboratorio de Biotecnología Ambiental, Facultad de Ciencias Biológicas y Agropecuarias, Región Poza Rica-Tuxpan, Universidad Veracruzana, Tuxpan de Rodríguez Cano, Veracruz 92870, Mexico
6
Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro 21040-900, Brazil
7
Laboratorio de Geografía de la Biodiversidad, Pabellón Nacional de la Biodiversidad Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
8
Laboratorio de Investigaciones Anatomopatológicas, Unidad de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
*
Authors to whom correspondence should be addressed.
†
These authors contribute equally to the development of the manuscript.
Pathogens 2024, 13(12), 1072; https://doi.org/10.3390/pathogens13121072
Submission received: 14 November 2024 / Revised: 30 November 2024 / Accepted: 4 December 2024 / Published: 6 December 2024
(This article belongs to the Special Issue Zoonotic Pathogens in the Tropics: From the Forest to the Cities)
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Abstract

:
The Trypanosomatidae family encompasses around 24 genera of unicellular protozoans, many of which are transmitted by various hematophagous arthropods, particularly members of the Orders Diptera and Hemiptera. Fleas and ticks—an understudied group of ectoparasites—have been shown to be hosts of a wide and crescent variety of trypanosomatid species. Further, fleas and ticks of companion animals have been particularly neglected in trypanosomatid surveillance despite the proximity to human populations and the anthropophagous habits of many of these arthropods, which can potentially act as vectors of zoonotic trypanosomatids. We aimed to identify the presence, characterize the species, and establish the prevalence of Kinetoplastids, including members of the Trypanosomatidae family, in ectoparasites collected from dogs and humans from Mexico. A total of 537 ectoparasite specimens belonging to six ectoparasite taxa (Amblyomma mixtum, A. tenellum, Ctenocephalides felis felis, Pulex simulans, Rhipicephalus linnaei, and Rh. sanguineus s.s.) were collected from 15 States of Mexico. An 800 bp fragment of the 18S-rDNA gene from kinetoplastids was amplified and sequenced. The presence of two agents (Trypanosoma caninum and Parabodo sp.) was detected in R. linnaei ticks and one (Blechomonas lauriereadi) in the cat flea Ct. felis felis. This is the first record of genetic material of kinetoplastid species in ectoparasites from dogs and humans in Mexico.

1. Introduction

The Trypanosomatidae family encompasses around 24 genera of unicellular protozoans that show an elongated morphology, the presence of a unique flagellum, and a parasitic life cycle, which can be monoxenous in invertebrate hosts, or alternate between invertebrate and vertebrate hosts or plants [1,2,3]. Several genera contain species of importance for public health and veterinary medicine, such as members of the genera Leishmania and Trypanosoma, which are the etiological agents of the zoonotic tropical diseases leishmaniasis and Chagas disease, respectively [4].
The heteroxenous representatives of these trypanosomatids are transmitted by various groups of arthropods, such as the Orders Diptera and Hemiptera [1,5]. Some efforts have recently been made to identify the diversity of trypanosomatids associated with other groups of ectoparasites, such as members of the orders Siphonaptera and Ixodida (fleas and ticks, respectively) [6,7,8].
In the last 20 years, researchers have identified a diverse and complex array of trypanosomatids associated with fleas, primarily belonging to the genera Trypanosoma (subgenus Herpetosoma), Leptomonas, and Blechomonas [6,8,9,10]. These trypanosomatids are generally considered to be specialized flea parasites, with Trypanosoma (Herpetosoma) species known to be transmitted to vertebrate hosts, such as rodents and lagomorphs. Some species have regained significance from a public health perspective, including Trypanosoma lewisi, an agent of an opportunistic rat- and flea-borne human emergent zoonosis in southeast Asia [11]. Further, a recent review showed a wide range of records of trypanosomatids associated with ticks, mainly with members of the family Ixodidae, commonly known as hard ticks [12]. The presence of six trypanosomatid species associated with 20 ticks in three zoogeographic regions were identified, highlighting the presence of parasites responsible for human cutaneous and visceral leishmaniasis, as well as African trypanosomiasis. In the American continent, studies on the identification of trypanosomatid species associated with ticks have been conducted only in Argentina, Brazil, Chile, Mexico and Peru, revealing seven trypanosomatid species linked to six hard ticks of the genera Amblyoma and Rhipicephalus [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28].
However, trypanosomatids in ectoparasites associated with dogs, cats and humans have remained virtually unexplored. Since ticks and fleas are the two primary groups of ectoparasites infesting dogs and cats—companion animals of humans—it is important to monitor the trypanosomatid species associated with these ectoparasites. Such monitoring is essential not only to prevent the transmission of potentially zoonotic trypanosomatid species, but also to establish a comprehensive biological inventory of the species circulating among ectoparasites of these companion animals. Further, DNA of two species of the Trypanosoma genus (Trypanosoma cruzi and Trypanosoma evansi) and two of the Leishmania genus (Leishmania brasiliensis and Leishmania infantum) were detected in four species of hard ticks (Amblyomma cajennense, Amblyomma tigrinum and Rhipicephalus sanguineus s.s.) parasitizing dogs in Brazil and Chile [16,17,18,19,20,21,27]. Other studies have recorded T. lewisi and T. cruzi DNA in rodent flea guts from Venezuela [29], and a negative report of the presence of T. cruzi in three dog flea species (Ctenocephalides canis, Ctenocephalides felis felis, and Pulex irritans) in Chile [27]. Here, we aimed to document the presence and determine the prevalence of kinetoplastids, including members of the Trypanosomatidae family, in ectoparasites that infest dogs and humans from a wide geographical range in Mexico.

2. Materials and Methods

As part of the routine detection of Rickettsia by the National Network of Public Health Laboratories of the Ministry of Health and private veterinary clinics, ectoparasites from 76 localities in 49 municipalities, including 15 States of Mexico, were collected (Table 1, Figure 1).
215 dogs and 1 cat were externally examined for ectoparasites; additionally, ticks were manually collected from 8 persons who were incidentally bitten (Table 1). Ectoparasites were then placed in 1.5 mL polypropylene conical tubes containing 70% ethanol. The samples were transported to the Laboratorio de Entomologia from Instituto de Diagnóstico y Referencia Epidemiológicos and examined for morphological identification using a Carl Zeiss Stemi 305 stereomicroscope and an Axiocam 208 camera. Identification was performed using specialized taxonomic keys from SENASICA [30] and Bermudez et al. [31] for ticks and Salceda-Sánchez [32] for fleas.
Genomic DNA was extracted from individual specimens, which were macerated using a new pestle for each sample to prevent cross-contamination and the commercial kit DNeasy Blood & Tissue Kit, (QIAGEN, Inc., Hilden, Germany), using the protocol for purification of total DNA from animal tissues, with an elution in a final volume of 100 µL. Once the DNA was recovered, it was frozen at −20 °C until use.
As an endogenous control of extraction and for the molecular identification of ticks of the Rhipicephalus sanguineus complex [33], a 400 bp segment of the 16S-rDNA ribosomal gene was amplified using the primers 16S − 1 (5′-CTGCTCAATGATTTTTTAAATTGCTGTGG-3′) and 16S + 2 (5′-CGCTGTTATCCCTAGAGTATT-3′) and thermal conditions described by Norris et al. [34]. The same procedure was repeated for the cytochrome oxidase subunit I (COI) of fleas using the primers LCO1-1490 (5′-GGTCAACAAATCATAAAGATATTG -3′) and HCO1-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) following the thermal conditions described by Folmer et al. [35].
For the detection of kinetoplastids, including those species of the Trypanosomatidae family, a fragment of 800 bp of the 18S small subunit of the ribosomal gene (SSU rDNA) was amplified using oligonucleotides 609F (5′-CACCCGCGGTAATTCCAGC-3′) and 706R (5′-CTGAGACTGTAACCTCAA-3′) [36], following the thermal conditions described by Borghesan et al. [37]. DNA from the ectoparasite samples was analyzed by conventional PCR by pooling three to five specimens of the same species from the same collection period and locality in a total of 80 pools. The reaction mixture consisted of 12.5 μL of GoTaq® Green Master Mix (2X) from Promega Corporation (Madison, WI, USA), 1 µL of each oligonucleotide (2 µM each), 6.5 µL of DNase-free water, and 4 µL of DNA (The total DNA was quantified using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently adjusted to a concentration of 200–300 ng) in a final volume of 25 μL. Positive (DNA from Rhiphicephalus microplus, Ctenocephalies felis felis, and T. cruzi) and negative controls (nuclease-free water) were included. Amplicons were visualized on 2% agarose gels stained with Syto 60 fluorochrome using TAE buffer at 85 V for 45 min.
The positive PCR products were sent to Macrogen® (Seoul, Republic of Korea), for sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Cat. 4337455, Thermo Fisher, Waltham, MA, USA). The samples were purified by the BigDye XTerminator (Cat. 4376486, Thermo Fisher, Waltham, MA, USA) prior to loading on the ABI 3730 × L DNA analyzer (Thermo Fisher, Waltham, MA, USA).
The resulting sequences were compared with references deposited in GenBank using the BLASTn tool. Subsequently, a global alignment was performed using the Clustal W algorithm, and phylogenetic reconstruction was carried out based on the Maximum Likelihood inference method in IQ-TREE [38] with 10,000 bootstrap replicates, eliminating any gaps during the analysis.

3. Results

A total of 537 ectoparasites were collected. These included four species of hard ticks: 422 (214♀, 198♂ and 10 nymphs) specimens of Rhipicephalus linnaei, 19 (5♀, 9♂, 5 nymphs) speciments of Rhipicephalus sanguineus s.s., 14 (5♀, 5♂, 4 nymphs) specimens of Amblyomma mixtum and 11 (10♀, 1♂) specimens of Amblyomma tenellum. Furthermore, 66 (42♀, 24♂) fleas of Ctenocephalides felis felis species and 5 (4♀, 1♂) fleas of Pulex simulans were collected from 215 hosts, including 206 dogs, 1 cat, and 8 persons from 76 localities of 49 municipalities of 15 States of Mexico (including Aguascalientes, Baja California, Campeche, Chihuahua, Guanajuato, Hidalgo, Jalisco, Mexico City, Morelos, Nayarit, Nuevo León, Puebla, San Luis Potosí, Sinaloa, Sonora) (Table 1). The recovered sequences confirmed the morphological identification of three tick species, exhibiting a similarity of 99.51% (404/406 bp), 99.05% (420/424 bp) and 97.80% (444/454 bp) with sequences of Rh. linnaei, Rh. sanguineus s.s. and A. mixtum from Cuba, Germany, and USA (GenBank accession numbers KP830114.1, OP326204.1 and KM519935.1, respectively). The phylogenetic reconstruction supported the identification of the members of the R. sanguineus complex, grouping our sequences with those of references of R. linnaei, and R. sanguineus s.s. deposited in GenBank in two monophyletic groups, with support values above 90 (Figure 2).
Of the 537 ectoparasites grouped into 80 pools, only 15% (12 pools: 3 of Ct. felis felis and 9 of Rh. linnaei) recovered from Chihuahua, Guanajuato, Nayarit, Nuevo León, Puebla, and Sonora tested positive for the presence of Kinetoplastid DNA (Table 1). The molecular identification of the recovered sequences confirmed the presence of three taxa: (1) Trypanosoma caninum in six pools of R. linnaei from Nuevo León, Puebla and Sonora with a similarity of 99% with sequences from Brazil (KF805453.1); (2) Parabodo sp. in three pools of R. linnaei from Jalisco and Morelos with a similarity of 96% from sequences of Parabodo caudatus from Germany (JF754435.1), and (3) Blechomonas lauriereadi in three pools from Ct. felis felis from Nuevo León and Puebla with a similarity of 99% from a sequence from Czech Republic (KF054127.1). The Maximum Likelihood analysis confirms that the trypanosomatid species corresponded to Trypanosoma caninum and Blechomonas lauriereadi, showing support values above 90 (Figure 3 and Figure 4).
Due to the low similarity of the recovered Parabodo sequences, as well as the branch length of the phylogenetic reconstruction (Figure 5), it is possible to assume that this may represent a still not described species within the genus.
Sequences were deposited in GenBank under the following accession numbers: Trypanosomatids (PQ496669- PQ496680) and Rhipicephalus sanguineus s.l. (PQ495570-PQ495590).

4. Discussion

The study of trypanosomatids associated with ticks has gained interest in the last decades [7,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28]. At least six species of trypanosomatids associated with 20 species of ticks have been identified worldwide, where most of the studies are concentrated in the Afrotropical and Palearctic regions [12]. The monitoring of trypanosomatids in ticks associated with dogs is still limited. In the Neotropical region, only T. cruzi and T. evansi genetic material was detected in Amblyomma cajennense, Amblyomma tigrinum and Rhipicephalus sanguineus s.s. parasitizing dogs in Brazil and Chile [17,18,19,20,21,27]. The first study to identify Trypanosoma species in ticks from Mexico was conducted in haemolymph samples from Rhipicephalus microplus ticks associated with cattle from the State of Yucatan, in which the presence of Trypanosoma theileri was detected by light microscopy [14].
To our knowledge, our present work represents the first systematic approach to studying trypanosomatids from ticks associated with dogs and people in Mexico.
Although species of the Rhipicephalus sanguineus s.l. complex represent the group of ticks most widely monitored for the detection of pathogens in Mexico [39,40,41,42,43], it is also relevant to highlight that surveillance has focused primarily on the identification of species of the genera Rickettsia, Ehrlichia and Anaplasma, neglecting other groups, especially protozoa [44]. For example, Rh. linnaei is a tick closely associated with domestic canids and is also recognized as a highly anthropophagous ectoparasite [45]. Since this tick has an intra-domiciliary life cycle and is in close contact with human populations, it poses a potential risk for the pathogen’s transmission. The confirmation of Rh. sanguineus s.s. in Guanajuato is a significant finding, as it indicates the range of expansion of this tick species in Mexico [46]. This tick was previously documented in the northern States of Baja California, Sonora, and Chihuahua, and has now been detected in central Mexico, which highlights its adaptability to a wide range of habitats [33,47]. This geographic expansion raises concerns about the potential for the increased transmission of tick-borne diseases, as Rh. sanguineus s.s. is a known vector for pathogens such as Rickettsia massiliae and Ehrlichia canis (causing Rickettsia massiliae-spotted fever and canine monocytic ehrlichiosis) [33]. The sympatry of Rh. sanguineus with Rh. linnaei in Guanajuato is particularly noteworthy, as the two species have distinct vectorial competencies. Their coexistence in the same environment needs further research to understand the implications for pathogen transmission dynamics, especially in areas where both ticks may be present on the same hosts. This study underscores the importance of ongoing surveillance and research into the ecological and epidemiological impacts of tick species distribution in Mexico [48].
Further, Trypanosoma caninum is one of the most recently recognized species of the genus, which was first described in Brazil in 2009 from dog skin samples [49,50,51,52,53,54]. Although it is apparently restricted to domestic canids in Brazil, a related species was found in Ixodes ricinus from Slovakia in recent years [55]. Our study provides the first record of this species of trypanosomatid DNA in Rh. linnaei ticks worldwide. However, it is still premature to establish a role for this tick in the life cycle of the parasite, since the individuals examined were collected from dogs, so it cannot be established whether the parasite DNA was naturally present in the tick or whether it came from the feeding of an infected dog. Therefore, it is a priority to conduct studies to evaluate the vector competence of this hard tick species. Although T. caninum is considered a non-pathogenic species, it is important to understand its life cycle and the possible immune response it exerts on the host. In an experimental study conducted in Brazil, it was shown that sera from dogs diagnosed with T. cruzi infection did not show cross-reactivity with T. caninum lysates [56]. However, this phenomenon should be evaluated in larger canine populations, as well as in humans, particularly in communities with a high abundance and biting rate of species of the Rh. sanguineus s.l. complex, to identify the degree of cross-reactivity and the possible confounding effect that may occur in sero-epidemiological surveys [51,57].
The telmophagous habits of this tick, which degrade the outer layer of the skin by using various enzymes, facilitate the exposure to T. caninum that lives in the dermis of the dog [49,58]. Previous studies with other species such as Trypanosoma rhipicephalis have demonstrated the trans-ovarian transmission of these protozoa in tick progeny [59]. Future studies should monitor the presence of genetic material and characteristic forms of trypanosomatids in Rh. linnaei populations.
Although T. cruzi DNA has been detected in brown dog ticks (Rh. sanguineus s.s.) associated with dogs from Chile [27], the role of ticks as potential vectors has not been explored to date.
Further, Parabodo caudatus was first isolated from a human urine sample in Czechoslovakia in 1950 [60]. However, its role as a symbiont or pathogen has not been widely discussed. It was suspected that it was an agent that contaminated urine samples from humans, although a recent report detected the presence of this protozoan in the urine of a laboratory dog with haematuria [60]. This possibility was supported in a recent trial in which its presence was detected in the blood of domestic canines living in Bangkok [61]. A similarity of 96% is too low to define a kinetoplastid species, so we decided to name the detected lineage as Parabodo sp. Additionally, the phylogenetic analysis showed that there are several branches that could indicate that they may be different species, not yet formally described. Since the knowledge of the Parabodo genus is very limited, several sequences that have been deposited as P. caudatus in GenBank may represent different (not yet described) species, mainly because this is a database where there is no curation of information. Therefore, it is possible to consider that, in a more solid taxonomic study, it would be concluded that these P. caudatus sequences are, in fact, sequences from different species close to this species. Additionally, since P. caudatus was detected from urine samples in a dog, it is possible to suspect that the microorganism detected in the present study, as well as in dog blood in Bangkok [61], corresponds to an undescribed species, which requires priority in further studies. Since our study now reports Parabodo DNA in ticks, it is tempting to propose that the parasite possibly remains in dogs’ blood circulation long enough for an ectoparasite, such as Rh. linnaei, to acquire it.
The species belonging to the subfamily Blechomonadinae have only been described from the Palearctic and Afrotropical regions [10]. Blechomonas lauriereadi has been isolated and described from the hindgut of the fleas Ct. canis and Ct. felis felis from Vulpes vulpes in the Czech Republic in 2013 [10]. Our study represents the first record of B. lauriereadi outside the Palearctic region and the first for the American continent, which notoriously increases the geographic distribution of this trypanosomatid species. Additionally, it expands the range of vertebrate host species that harbor infected fleas by documenting positive results for Ct. felis felis collected in dogs, given that this species of fleas has a cosmopolitan distribution [62]. Clearly, it is a priority to implement studies on these ectoparasites to determine the distribution and prevalence of this protozoan in Ct. felis felis populations worldwide. Additionally, it is a priority to evaluate whether this species can infect dogs, since there are no previous studies aimed at monitoring blood and/or skin samples from canids that are hosts of fleas testing positive for protozoan pathogens.
The findings of this study increase the inventory of trypanosomatids associated with ectoparasites of dogs in Mexico and the American continent, as well as the inventory of this family nationwide. Further, the presence of two genera, Parabodo and Blechomonas, are documented for the first time in Mexico.

5. Conclusions

The present study demonstrates the detection of DNA from three kinetoplastid species, which may be the result of the presence of these parasites in these ectoparasites and/or in the blood of the animals on which they fed. In this sense, two approaches should be taken in future studies: (1) separate the digestive tract of fleas and ticks and perform the diagnosis separately. Detection of DNA outside the digestive tract may be an indication of infection; (2) experimental studies with artificial infected blood feeding with the subsequent follow-up of their detection in the ectoparasites, both by dissection and direct observation, and molecular diagnosis. Due to the challenges of dissecting ectoparasites while preventing cross-contamination of their organs with pathogen DNA, alternative methods to PCR should be considered. Immunohistochemistry, for instance, can be employed to precisely identify the pathogen′s localization within specific anatomical sites of the ectoparasite. Similar experimental strategies have been described for Mycoplasma surveillance in ticks [63].
Given the limited knowledge of the detected kinetoplastid species, it is a priority to establish studies aimed at identifying the biology, ecology, biogeography, and epidemiology of these agents to understand their life history and contextualize them as part of the biodiversity of protozoa nationwide.

Author Contributions

Conceptualization, H.M.Z.-I., B.S.-S., H.H.-J., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P., J.R.-H., J.L.B.-R., M.-G.S.-O., J.C.H.-P., R.I.H.-H., P.S.M.-d.A., A.L.R.R., Á.R.-M., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; methodology, H.M.Z.-I., B.S.-S., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P. and J.R.-H.; validation, H.M.Z.-I., B.S.-S., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P. and J.R.-H.; formal analysis, H.M.Z.-I., B.S.-S., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P. and J.R.-H.; investigation, H.M.Z.-I., B.S.-S., H.H.-J., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P., J.R.-H., J.L.B.-R., M.-G.S.-O., J.C.H.-P., R.I.H.-H., P.S.M.-d.A., A.L.R.R., Á.R.-M., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; resources, B.S.-S., H.H.-J., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; data curation, H.M.Z.-I., B.S.-S., H.H.-J., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P., J.R.-H., J.L.B.-R., M.-G.S.-O., J.C.H.-P., R.I.H.-H., P.S.M.-d.A., A.L.R.R., Á.R.-M., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; writing—original draft preparation, H.M.Z.-I., B.S.-S., H.H.-J., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P., J.R.-H., J.L.B.-R., M.-G.S.-O., J.C.H.-P., R.I.H.-H., P.S.M.-d.A., A.L.R.R., Á.R.-M., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; writing—review and editing, H.M.Z.-I., B.S.-S., H.H.-J., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P., J.R.-H., J.L.B.-R., M.-G.S.-O., J.C.H.-P., R.I.H.-H., P.S.M.-d.A.A, A.L.R.R., Á.R.-M., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; visualization, H.M.Z.-I., B.S.-S., H.H.-J., C.I.M.-C., M.S.-C., Y.d.l.C.-P., A.C.L.-G., L.V.M.-P., J.R.-H., J.L.B.-R., M.-G.S.-O., J.C.H.-P., R.I.H.-H., P.S.M.-d.A., A.L.R.R., Á.R.-M., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; supervision, B.S.-S., H.H.-J., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; project administration, B.S.-S., H.H.-J., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M.; funding acquisition, B.S.-S., H.H.-J., V.S.-C., H.A.R.M., E.G., I.B. and S.S.-M. All authors have read and agreed to the published version of the manuscript.

Funding

The project was partially financed by UNAM-PAPIIT IG2000924 and CONACYT Ciencia de Frontera 6682 and CF-2023-G-174.

Institutional Review Board Statement

The study was approved by the Ethics Board of the Universidad Nacional Autónomande México (FM/DI/71/2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the results of this study are available in GenBank, under the following accession numbers Kinetoplastids (PQ496669-PQ496680) and Rhipicephalus sanguineus s.l. (PQ495570-PQ495590).

Acknowledgments

We thank the State Laboratories of Public Health and give special thanks to the Vectors group from the Health Department of the state for field assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Sampling sites of ectoparasites of dogs and humans selected for kinetoplastid molecular detection in Mexico.
Figure 1. Sampling sites of ectoparasites of dogs and humans selected for kinetoplastid molecular detection in Mexico.
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Figure 2. Maximum Likelihood inference phylogenetic compress analysis of the selected 16S rDNA partial gene for Rhipicephalus sanguineus s.s. and Rhipicephalus linnaei collected in the present study.
Figure 2. Maximum Likelihood inference phylogenetic compress analysis of the selected 16S rDNA partial gene for Rhipicephalus sanguineus s.s. and Rhipicephalus linnaei collected in the present study.
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Figure 3. Maximum Likelihood inference phylogenetic compress analysis of the selected 18S rDNA partial gene for several members of the genus Blechomonas. The sequences obtained in the present study are presented in bold and blue.
Figure 3. Maximum Likelihood inference phylogenetic compress analysis of the selected 18S rDNA partial gene for several members of the genus Blechomonas. The sequences obtained in the present study are presented in bold and blue.
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Figure 4. Maximum Likelihood inference phylogenetic compress analysis of the selected 18S rDNA partial gene for several members of the genus Trypanosoma. The sequences obtained in the present study are presented in bold and orange.
Figure 4. Maximum Likelihood inference phylogenetic compress analysis of the selected 18S rDNA partial gene for several members of the genus Trypanosoma. The sequences obtained in the present study are presented in bold and orange.
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Figure 5. Maximum Likelihood inference phylogenetic compress analysis of the selected 18S rDNA partial gene for several members of the genus Parabodo. The sequences obtained in the present study are presented in bold and red.
Figure 5. Maximum Likelihood inference phylogenetic compress analysis of the selected 18S rDNA partial gene for several members of the genus Parabodo. The sequences obtained in the present study are presented in bold and red.
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Table 1. Collection sites of ticks and fleas associated with domestic animals and people in Mexico. Abbreviations: BL: Blechomonas lauriereadi; ND: Not determined; TC: Trypanosoma caninum; PS: Parabodo sp.
Table 1. Collection sites of ticks and fleas associated with domestic animals and people in Mexico. Abbreviations: BL: Blechomonas lauriereadi; ND: Not determined; TC: Trypanosoma caninum; PS: Parabodo sp.
StateMunicipalityLocalityCoordinatesHostCollection DateEctoparasite SpeciesTotalFemaleMaleNymphTCPSBL
AguascalientesAguascalientesPeñuelas21°43′44′′ N, 102°16′19′′ W3 Dogs28 July 2021Ctenocephalides felis felis4220−−−
2 Dogs28–29 July 2021Pulex simulans5410−−−
CalvilloCalvillo21°50′49″ N, 102°42′52″ W2 Dogs15,20 May 2021Rhipicephalus linnaei8530−−−
Ventanillas21°57′53″ N, 102°40′59″ W4 Dogs20 August 2021Rh. linnaei4220−−−
Rincón de RomosRincón de Romos22°13′44″ N, 102°18′18″ W6 Dogs28 April 2021Rh. linnaei10172−−−
Baja CaliforniaMexicaliColonia Hidalgo32°36′54″ N, 115°27′03″ W1 Dog09 September 2019Rh. linnaei2020−−−
Ejido Cuernavaca32°33′52″ N, 115°18′11″ W9 Dogs09 September 2019Rh. linnaei166100−−−
Ejido Sinaloa32°32′51″ N, 115°16′11″ W4 Dogs19 September 2019Rh. linnaei6420−−−
CampecheChampotònChampotòn19°20′40″ N, 90°43′28″ W19 Dogs12 August 2021Rh. linnaei261970−−−
ChihuahuaChihuahuaColonia El Porvenir31°14′22″ N, 105°52′31″ W2 Human04 February 2020Rh. linnaei2020+−−
Colonia Tarahumara28°44′05″ N, 106°06′45″ W6 Dogs28 August 2019Rh. linnaei6600−−−
Colonia Zaarco28°36′43″ N, 106°04′55″ W1 Human25 September 2023Rh. linnaei1100−−−
Chihuahua28°38′13″ N, 106°04′37″ W1 Human27 January 2020Rh. linnaei1100−−−
Granjas del Valle28°45′10″ N, 106°7′3″ W3 Human19/09/2023Rh. linnaei6510−−−
Rinconada los Nogales28°37′27″ N, 105°59′40″ W1 Human11 February 2020Rh. linnaei1010−−−
JuárezND28°36′39″ N, 106°00′51″ W4 Dogs14–15 August 2019Rh. linnaei4400−−−
MeoquiLázaro Cárdenas28°23′23″ N, 105°37′25″ W1 Human11 August 2023Rh. linnaei2200−−−
GuanajuatoComonfortComonfort20°42′54″ N, 100°45′24″ W9 Dogs23–24 May 2019Rhipicephalus sanguineus16475−−−
PurísimaPurísima21°01′50″ N, 101°52′45″ W9 Dogs23 May 2023Rh. linnaei15780+−−
San Miguel de AllendeSan Miguel de Allende20°54′55″ N, 100°44′38″ W2 Dogs31 May 2019Rh. sanguineus3120−−−
HidalgoIxmiquilpanLa Reforma20°28′42″ N, 99°13′52″ W2 Dogs05 April 2019Rh. linnaei2200−−−
HuazalingoTzapotitla20°58′46″ N, 98°28′55″ W2 Dogs15 August 2019Amblyomma mixtum2020−−−
JaliscoPuerto VallarataMandarina20°31′51″ N, 105°17′32″ WNDNDA. mixtum11434−−−
Rh. linnaei2200−+−
Mexico CityIztacalcoCampamento 2 de Octubre19°23′05″ N, 99°06′59″ W1 Human06 January 2023A. mixtum1100−−−
MorelosEmiliano ZapataEl Calvario18°50′58″ N, 99°10′57″ W1 Dog05 February 2019Ct. felis felis3300−−−
Puente de IxtlaXoxocotla18°41′06″ N, 99°14′38″ W5 Dogs05 March 2019Rh. linnaei11245−+−
TemixcoLomas del Carril18°51′55″ N, 99°14′00″ W4 Dogs19 February 2019; 14 February 2024Ct. felis felis181260−−−
12 Dogs17 December 2018; 13 February 2019; 10 March 2019Rh. linnaei17890−+−
XochitepecLoma Bonita18°56′02″ N, 99°10′23″ W1 Dog30 January 2019Ct. felis felis3210−−−
NayaritCompostelaChacala21°10′00″ N, 105°13′28″ WNDNDRh. linnaei15663+−−
Nuevo LeónAllende Comunidad los Sabinos25°19′06.903″ N, 99°59′09.816″ W 1 Dog02 September 2021Ct. felis felis5320−−+
Cadereyta Valle del Roble25°36′08″ N, 100°04′32″ W3 Dogs10 September 2021Rh. linnaei6420−−−
Dr. ArroyoLa Chiripa25°37′56″ N, 100°10′14″ W2 Dogs21 September 2021Rh. linnaei4310−−−
Jesús María de Berrones23°55′02″ N, 100°01′49″ W3 Dogs02 September 2021Rh. linnaei4310−−−
La Unión y El Cardonal23°50′51″ N, 100°05′29″ W2 Dogs14 September 2021Rh. linnaei3120−−−
Escobedo Colinas del Topo25°47′50″ N, 100°22′27″ W2 Dogs13 August 2021Rh. linnaei5410−−−
Fuentes de Escobedo25°47′14″ N, 100°18′41″ WEnvironment31 August 2021Rh. linnaei8170−−−
Palmiras25°50′36″ N, 100°25′22″ W2 Dogs09 August 2021Rh. linnaei3120−−−
Pedregal de Topo Chico25°47′05″ N, 100°19′42″ W2 Dogs14 September 2021Rh. linnaei4220−−−
Santa Lucía25°48′10″ N, 100°22′01″ W2 Dogs13 August 2023Rh. linnaei4220−−−
Villas de Francisco25°47′41″ N, 100°20′12″ W2 Dogs20 September 2021Rh. linnaei4220−−−
García Paseo de las Torres25°47′20″ N, 100°35′50″ WEnvironment14 September 2021Rh. linnaei2200−−−
General ZuazuaValle de Santa Elena25°51′18″ N, 100°08′32″ W4 Dogs08 September 2021Rh. linnaei6240−−−
Guadalupe Jardínes de Santa Clara25°40′09″ N, 100°12′34″ W1 Dog12 August 2023Rh. linnaei1010−−−
Residencial Dos Ríos25°40′39″ N, 100°15′35″ W2 Dogs25 August 2021Rh. linnaei2200−−−
Tierra Propia25°38′23″ N, 100°11′01″ W5 Dogs26 August 2021Rh. linnaei5320−−−
Unidad Reforma25°40′08″ N, 100°08′00″ W2 Dogs27 August 2021Rh. linnaei2020−−−
JuárezReal de San Josè25°39′28″ N, 100°04′56″ W8 Dogs29 March 2021Rh. linnaei8620−−−
Terranova25°39′41″ N, 100°06′07″ W3 Dogs15 July 2021Rh. linnaei3210−−−
LinaresEjido San José25°47′41″ N, 100°27′49″ W1 Dog14 April 2021Amblyomma tenellum10910−−−
Villegas25°51′27″ N, 99°33′38″ W11 Dogs12 August 2021Rh. linnaei15870−−−
MinaEl Pedregal26°00′13″ N, 100°31′57″ W2 Dogs21 September 2021Rh. linnaei3120−−−
MontemorelosMaranatha25°11′50″ N, 99°51′16″ W1 Dog15 April 2021A. tenellum1100−−−
MonterreyCentro25°40′31″ N, 100°18′42″ W1 Dog07 September 2021Rh. linnaei4130−−−
Los Nogales25°44′02″ N, 100°19′19″ W11 Dogs08 July 2021; 14 August 2021; 24 August 2021Rh. linnaei18990−−−
Morelos25°43′14″ N, 100°20′53″ W1 Dog27 July 2021Rh. linnaei6330−−−
San Antonio25°39′26″ N, 100°17′30″ W4 Dogs22 July 2021Rh. linnaei6600−−−
Terminal25°41′16″ N, 100°17′50″ W2 Dogs11 August 2021Rh. linnaei4220−−−
Valle de Santa Lucía25°44′41″ N, 100°21′20″ W3 Dogs24 August 2021Rh. linnaei3300−−−
San Nicolás de los GarzaConstituyentes de Querétaro25°43′02″ N, 100°15′09″ W2 Dogs13 September 2021Rh. linnaei4220−−−
San Cristobal25°44′07″ N, 100°12′49″ W2 Dogs15 September 2021Rh. linnaei3120−−−
Santa CatarinaEnrique Rangel25°44′39″ N, 100°21′09″ W2 Dogs09 September 2021Rh. linnaei4220−−−
San Gilberto25°41′53″ N, 100°27′37″ W2 Dogs15 September 2002Rh. linnaei2110−−−
PueblaAtlixco Tolometla18°55′34″ N, 98°23′52″ W1 Dog10 May 2019Ct. felis felis4400−−+
San Pedro YeloixtlahuacaSan Juan Llano Grande18°03′50″ N, 98°05′04″ W5 Dogs20 November 2019Rh. linnaei10640−−−
−−−
Santiago MiahuatlánSan Isidro Labrador18°32′30″ N, 97°26′27″ W11 Dogs28 June 2019–13/September/2019Rh. linnaei181080−−−
TehuacánTehuacán18°27′29″ N, 97°24′26″ W14 Dogs, 1 Cat11, 14, 20, 22, 24 November 2019; 08 December 2023Rh. linnaei3113180−−−
5 Dogs24 November 2019Ct. felis felis14860−−+
TenampulcoTenampulco20°10′11″ N, 97°24′23″ W6 Dogs03 March 2021Rh. linnaei10550−−−
Tepexi de RodríguezEstrella Roja18°34′45″ N, 97°55′39″ W3 Dogs21 May 2019Rh. linnaei4220−−−
Tlacotepec de Benito JuárezTlacotepec de Benito Juárez18°40′35″ N, 97°39′03″ W3 Dogs04 December 2019Ct. felis felis12840−−−
Venustiano CarranzaEstrella Roja20°30′18″ N, 97°40′15″ W1 Dog21 May 2019Rh. linnaei1100−−−
ZongozotlaZotik19°58′52″ N, 97°43′44″ W2 Dogs11 June 2019Ct. felis felis3030−−−
San Luis PotosíLagunillas Mirador21°53′40″ N, 100°51′13″ W1 Dog07 September 2023Rh. linnaei2020−−−
SinaloaAhomeNuevo San Miguel25°57′52″ N, 109°03′22″ W7 Dogs17 March 2021Rh. linnaei207130−−−
SonoraAgua PrietaAgua Prieta31°17′44″ N, 109°32′29″ W2 Dogs27 June 2019Rh. linnaei8260+−−
HermosilloHermosillo29°05′00″ N, 110°59′17″ W5 Dogs07 September 2023Rh. Linnaei7430+−−
ImurisImuris30°47′21″ N, 110°50′37″ W4 Dogs03 July 2019Rh. Linnaei8260−−−
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MDPI and ACS Style

Zazueta-Islas, H.M.; Salceda-Sánchez, B.; Huerta-Jiménez, H.; Miranda-Caballero, C.I.; Solis-Cortés, M.; de la Cruz-Pacheco, Y.; Luquín-García, A.C.; Mondragon-Peña, L.V.; Reyes-Hernández, J.; Bravo-Ramos, J.L.; et al. Molecular Detection of Kinetoplastid Species in Ticks and Fleas Associated with Dogs and Humans in Mexico. Pathogens 2024, 13, 1072. https://doi.org/10.3390/pathogens13121072

AMA Style

Zazueta-Islas HM, Salceda-Sánchez B, Huerta-Jiménez H, Miranda-Caballero CI, Solis-Cortés M, de la Cruz-Pacheco Y, Luquín-García AC, Mondragon-Peña LV, Reyes-Hernández J, Bravo-Ramos JL, et al. Molecular Detection of Kinetoplastid Species in Ticks and Fleas Associated with Dogs and Humans in Mexico. Pathogens. 2024; 13(12):1072. https://doi.org/10.3390/pathogens13121072

Chicago/Turabian Style

Zazueta-Islas, Héctor M., Beatriz Salceda-Sánchez, Herón Huerta-Jiménez, Carlos I. Miranda-Caballero, Marlene Solis-Cortés, Yaretzi de la Cruz-Pacheco, Ana Cristina Luquín-García, Laura V. Mondragon-Peña, Jair Reyes-Hernández, José L. Bravo-Ramos, and et al. 2024. "Molecular Detection of Kinetoplastid Species in Ticks and Fleas Associated with Dogs and Humans in Mexico" Pathogens 13, no. 12: 1072. https://doi.org/10.3390/pathogens13121072

APA Style

Zazueta-Islas, H. M., Salceda-Sánchez, B., Huerta-Jiménez, H., Miranda-Caballero, C. I., Solis-Cortés, M., de la Cruz-Pacheco, Y., Luquín-García, A. C., Mondragon-Peña, L. V., Reyes-Hernández, J., Bravo-Ramos, J. L., Sánchez-Otero, M.-G., Huerta-Peña, J. C., Hernández-Herrera, R. I., San Martin-del Angel, P., Roque, A. L. R., Rodríguez-Moreno, Á., Sánchez-Cordero, V., Rodríguez Martínez, H. A., Grostieta, E., ... Sánchez-Montes, S. (2024). Molecular Detection of Kinetoplastid Species in Ticks and Fleas Associated with Dogs and Humans in Mexico. Pathogens, 13(12), 1072. https://doi.org/10.3390/pathogens13121072

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