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Communication

First Molecular Evidence of Theileria and Anaplasma Genospecies in Subulo gouazoubira Exhibiting Clinical Symptoms from Entre Ríos Province, Argentina

by
Patrick S. Sebastian
1,*,
Paula J. Vaschalde
2,
María C. Pelosi
3,
Fernando S. Flores
4,5,
Julio Yedro
6 and
Evelina L. Tarragona
1,*
1
Instituto de Investigación de la Cadena Láctea (IdICaL), Consejo Nacional de Investigaciones Científicas y Técnicas-Instituto Nacional de Tecnología Agropecuaria (CONICET-INTA), Rafaela 2300, Argentina
2
Laboratorio de Ecología de Enfermedades, Instituto de Ciencias Veterinarias del Litoral (ICIVET Litoral) Universidad Nacional del Litoral (UNL)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Esperanza 3080, Argentina
3
Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral (UNL), Esperanza 3080, Argentina
4
Centro de Investigaciones Entomológicas de Córdoba (CIEC), Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba 5000, Argentina
5
Instituto de Investigaciones Biológicas y Tecnológicas (IIByT)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Córdoba 5000, Argentina
6
Dirección de Recursos Naturales, Gobierno de la Provincia de Entre Ríos, Paraná 3100, Argentina
*
Authors to whom correspondence should be addressed.
Wild 2025, 2(3), 35; https://doi.org/10.3390/wild2030035
Submission received: 12 May 2025 / Revised: 29 July 2025 / Accepted: 27 August 2025 / Published: 4 September 2025
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Abstract

In November 2022, a male specimen of Subulo gouazoubira was found with signs of obnubilation and recumbency without the ability to maintain sternal or ambulatory posture. Based on the symptoms, serological, biochemical and molecular analyses were performed. The results of the biochemical blood analysis and the clinical signs were compatible with theileriosis. DNA of Theileria were detected in the blood sample by PCR analysis. The phylogenetic analysis of the obtained partial sequences of the 18S rDNA gen resulted in the identification of the detected strain as a Theileria genospecies closely related to Theileria spp. detected in other deer species. Further, a genospecies of Anaplasma was detected in the blood sample. This genospecies is located phylogenetically near to Anaplasma phagocytophilum. The results of this study demonstrate co-infection with two novel genospecies of Anaplasma and Theileria in a clinical case of a free-living S. gouazoubira for the first time in Argentina. However, the pathogenicity of these strains and specific role of S. gouazoubira in their enzootic transmission cycles remains unclear. To improve epidemiological understanding, assess risks and develop targeted control strategies, future studies should prioritize the isolation, characterization and cultivation of these genospecies and the assessment of vector competence.

1. Introduction

The gray brocket deer (Subulo gouazoubira) is a small ruminant found across a wide environmental range, spanning the forest, savanna, shrubland and wetlands of Argentina, Bolivia, Brazil, Paraguay and Uruguay [1,2]. This deer faces a concerning decline in population due to increased human activities, high hunting pressure, agricultural expansion, habitat fragmentation and the introduction of exotic species [3]. Subulo gouazoubira infected with microorganisms such as Anaplasma, Babesia and Theileria species have been reported in recent years [4,5,6].
The genus Theileria belongs to the order Piroplasmida within the phylum Apicomplexa, which are obligate intracellular hemoparasite that affects a wide range of mammals, both domestic and wild, but ruminants act as the main hosts [7]. It is known that some Theileria spp. are transmitted by ixodid ticks (Acari: Ixodidae) of the genera Amblyomma, Haemaphysalis, Hyalomma and Rhipicephalus [7]. Theileria species are characterized by a heteroxenous cycle, involving asexual reproduction in the vertebrate host (indirect host) and sexual reproduction in the tick that acts as a vector (direct host). Recent studies suggest that Lipoptena spp. (Insecta: Hippoboscidae) could also transmit Theileria spp. [8,9]. In Argentina, Theileria spp. were detected in horses, marsh deer (Blastocerus dichotomus) and Amblyomma neumanni [10,11,12,13,14].
The genus Anaplasma (Rickettsiales: Anaplasmataceae) contains Gram-negative obligate intracellular alphaproteobacteria that infect different cell types in vertebrate hosts, including erythrocytes, granulocytes, monocytes and platelets. Anaplasma spp. are transmitted by ticks of five genera: Amblyomma, Dermacentor, Ixodes, Hyalomma and Rhipicephalus (Ixodidae) [15]. In Argentina, several strains of Anaplasma have been reported recently [16,17,18].
The aim of the present study was to report the molecular detection of two genospecies of Theileria and Anaplasma related to a clinical case in a free-living S. gouazoubira specimen from Entre Rios Province, Argentina.

2. Materials and Methods

2.1. Case Report

On 22nd November 2022, a free-living adult male specimen of S. gouazoubira was found with signs of obnubilation and recumbency without the ability to maintain sternal or ambulatory posture in the protected area “Reserva Natural Provincial de Uso Multiple Parque General San Martín” (−31.72586, −60.32618), Entre Ríos, Argentina. The specimen was confined to a farmyard for clinical examination. At the semiology, the following were observed: decline in sensorium, pale oral and eyelid mucosa, exophthalmia, dehydration and hindquarter paralysis (Figure 1). Antibiotic and palliative treatment were applied.
On 24 November 2022, no effect of the applied treatment could be observed. The animal died on 28 November 2022.

2.2. Sample Collection

Blood samples were obtained by puncture of the jugular vein on the 24th of November (Sample ID: HS11). The animal was further examined for ectoparasites, which were collected and stored in ethanol 96º for morphological identification following Nava et al. [19] and Pinheiro et al. [20].

2.3. Molecular, Biochemical, Serological and Phylogenetic Analysis

DNA was extracted from whole blood samples (200 µL) and complete ectoparasites using a High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany), according to the manufacturer’s instructions. DNA quality and quantity were checked post-extraction by Nanodrop® (Thermo Fisher Scientific, Waltham, MA, USA) measuring. As theileriosis was suspected based on the symptoms, the samples were subjected to a series of PCRs as described by Thompson et al. [21] to generate nearly complete 18S rDNA gene sequences. Hematology and blood biochemistry were also performed (Biovet Lab. S.A., Rafaela, Santa Fe, Argentina). Additionally, the nearly complete sequence of the 16S rRNA gene from bacteria of the Anaplasmataceae family by PCR was amplified using the primers fD1/EHR16SR and EHR16SD/Rp2 [22]. DNA of Anaplasma marginale and Babesia bigemina (provided by Instituto Nacional de Tecnología Agropecuaria (INTA) Rafaela, Santa Fe, Argentina) were used as positive controls in the correspondent PCR assay, while ultra-pure water acted as the negative control. PCR amplicons were purified and sent for sequencing (Sanger method, (INTA) Castelar, Unidad Genómica, Hurlingham, Buenos Aires, Argentina). The DNA sequences obtained were edited using BioEdit Sequence Alignment Editor [23] and aligned with the program Clustal W [24]. A phylogenetic tree was constructed using the Maximum-likelihood method and the best model was determined with the Akaike Information Criterion with 1.000 replicates in MEGA X [25].

2.4. Ethical Statement

This study was approved by the “Secretaría de ambiente de la Provincia de Entre Ríos” (Gobierno de la Provincia de Entre Ríos; resolution: 2983-2021) and the Ethics and Biosafety Advisory Committee (C.A.E.S., Universidad Nacional del Litoral, Esperanza, Santa Fe; protocol: 676/21).

3. Results and Discussion

According to the observed symptoms, the first treatment applied on 22nd of November was Doramectin 1% (0.2 mg/kg, SC), Calcium Gluconate 23% (500 mL, IV), Enrofloxacin 5% (5 mg/kg, SC) and Dexamethasone 0.2% (0.2 mg/kg).
The day after the blood was drawn, the PCR results for the 18S rDNA gene were obtained, giving positive results. Treatment with Imidocarb IM injections of 4 mg/kg was immediately started.
The results of the hemogram and biochemical blood analysis performed on the same day, compared with the reference values for this species reported by Camargo et al. [26], showed decreased values of hematocrit, erythrocyte count, hemoglobin, creatinine and total protein. This occurred in association with an increase in MCHC (mean corpuscular hemoglobin concentration), liver enzymes (AST, ALT and GGT) and CPK (creatine kinase) (Table 1).
A total of ten ticks were collected in the neck and head area. All ticks were identified as male specimens of Haemaphysalis juxtakochi and were subsequently deposited in the Tick Collection of INTA E.E.A. Rafaela (accession number INTA-2533). Although S. gouazoubira was parasitized by a high number of flies, only 16 were collected. All flies were determined as Lipoptena mazamae adults based on the presence of one fronto-orbital setae on the left antimere of the head and tibia II with two apical setae on the ventral surface. No Piroplasmida or Anaplasmataceae DNA was amplified in any of the collected ectoparasites.
The obtained 18S rDNA gene fragment (1794 bp; GenBank Acc. No: PX251302) shows up to 99.01% sequence identity to a sequence of Theileria sp. detected in a white-tailed deer (Odocoileus virginianus) from the USA (GenBank Acc. No.: AY735132). Also, other sequences of Theileria sp.—some named as Theileria cervi—detected in deer or deer-related ticks showed sequence identities from over 98.80% to the obtained sequence of this study. The phylogenetic analysis of the sequence with reference sequences of other Theileria spp. showed that the detected Theileria sp. is located in a clade with other Theileria sp. detected in O. virginianus, Rangifer tarandus [27] and Dermacentor nitens [28] from the USA (see Figure 2). This clade separates from the next clade, which includes sequences of Theileria sp. cf. T. velifera strains from Myanmar, with a bootstrap value of 76. Also, in phylogenetical proximity but clearly separated, a clade of Theileria sp. strains detected in Cervus nippon from China and Japan is located. Although these Asian reports were deposited as T. cervi in the GenBank by the authors, previous studies [13,29] and the results of this study show that these strains must belong to a different species. The T. cervi clade, which includes sequences of strains mainly detected in the USA, is located next to the clades of Theileria annulata/Theileria parva and Theileria ovis and clearly separated from the Asian strains and the strain detected in this study. Therefore, it must be assumed that the strain detected in S. gouazoubira from Argentina belongs to an underdetermined Theileria genospecies, which is related to deer species. In Brazil, various studies were carried out according to the presence of Theileria sp. in S. gouazoubira [4,30]. Here, different genospecies of Theileria sp. are described; some of them are classified as T. cervi while others are of unclear species affiliation. A phylogenetic analysis including the Brazilian strains was not applicable due to the lack of long 18S rDNA gene sequences of these strains. However, the results of the present study and the Brazilian reports allow the assumption that at least two different genospecies of Theileria sp. are circulating in S. gouazoubira populations in South America. Therefore, it is essential to execute more phylogenetic analysis, including other genes, to determine the exact species affiliation of these strains, which can lead to a better understanding of the possible pathogenicity of these hemoparasites.
Besides the 18S rDNA partial gene sequence of Theileria sp., a 16S rDNA gene sequence was also obtained (1136 bp; GenBank Acc. No.: PX251301). This sequence was 98.86% identical to different uncultured Anaplasma sp. and strains of A. phagocytophilum that were detected in various ruminant species, including deer, goats and sheep from Asia (e.g., GenBank Acc. Nos.: MG869519, MN319542, AB196721, AB454076). The maximum-likelihood tree constructed with the obtained sequence and other Anaplasma spp. illustrate that the sample from S. gouazoubira of Argentina is located in the main clade of A. phagocytophilum and forms part of a sub-clade, named the A. phagocytophilum-like 1 group by Rar et al. [15] (see Figure 3). Within this group, other Anaplasma sp. strains related to ruminants from Asia are situated. Anaplasma phagocytophilum-like 1 strains, with an unknown pathogenicity, were firstly described in sika deer (C. nippon), cattle, and Haemaphysalis douglasii from Japan [31,32]. Further reports exist from other Asian countries (e.g., Seo et al. [33]) in cattle and cattle-associated ticks, but also in sheep and goats from the Mediterranean area [34].
Particularly in South America, Anaplasma sp. have been reported in Subulo spp. from Brazil [4,6] and Uruguay [5]. However, these strains seem to belong to a novel Anaplasma sp., closely related to Anaplasma bovis (this study, Figure 4; [5]) or Anaplasma platys (this study, Figure 4; [4]). Da Silveira et al. [4] describe the molecular detection of A. phagocytophilum in S. gouazoubira from Brazil. Unfortunately, only short partial sequences (GenBank Acc. Nos.: KF790919; 400 bp, and KF790920; 382 bp) of these findings are available, so these sequences could not be included in the phylogenetic analyses of the large sequences. Nevertheless, the maximum-likelihood tree constructed with short sequences (alignment length 340 bp) shows that the Brazilian strains are within the clade of A. phagocytophilum and closely related to the Argentinian strain of this study (see Figure 4). The sequence identities of the Argentinian strain are 98.64% (362/367 bp) and 98.39% (366/372 bp) with the GenBank Acc. Nos. KF790919 and KF790920, respectively. Based on the shortness of the used gene fragment, a separation of the A. phagocytophilum clades could not be achieved. However, in concordance with the Brazilian report, the results of the present study confirm the circulation of these A. phagocytophilum-related strains in S. gouazoubira populations in South America.

4. Conclusions

The co-infection of genospecies of Anaplasma and Theileria was observed in S. gouazoubira, although their pathogenicity remains unclear and even less is known about the potential arthropod vector. In the present study, a high load of L. mazamae and a lower load of H. juxtakochi parasitizing S. gouazoubira are reported. It is possible that co-infection combined with high parasitic loads may have triggered the death of the individual. The effects of parasitic infestation are determined by the intensity of infestation, the host animal’s resistance and the parasite’s virulence [35].
The specific role of S. gouazoubira in the enzootic transmission cycles of Anaplasma and Theileria remains unclear. To improve epidemiological understanding, assess risks and develop targeted control strategies, future studies should prioritize the isolation, characterization and cultivation of these genospecies and the assessment of vector competence. These investigations are essential to determine the veterinarian and ecological impact of these microorganisms on wild S. gouazoubira populations.

Author Contributions

Conceptualization, P.S.S. and E.L.T.; methodology, P.J.V., M.C.P., F.S.F., J.Y. and P.S.S.; software, P.S.S.; validation, P.S.S. and E.L.T.; formal analysis, P.S.S. and E.L.T.; investigation, P.J.V., M.C.P. and P.S.S.; resources, P.J.V., M.C.P., J.Y. and E.L.T.; data curation, P.S.S. and E.L.T.; writing—original draft preparation, P.S.S. and P.J.V.; writing—review and editing, P.S.S., P.J.V., F.S.F. and E.L.T.; visualization, P.S.S. and P.J.V.; supervision, P.S.S. and E.L.T.; project administration, E.L.T.; funding acquisition, E.L.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Agencia Nacional de Promoción Científica y Tecnológica, (BID PICT 2020-1633), Consejo Nacional de Investigaciones Científicas y Técnicas (PIBAA 1199), Instituto Nacional de Tecnología Agropecuaria (INTA, PE I044), Estación Experimental Agropecuaria Rafaela and Asociación Cooperadora INTA Rafaela.

Data Availability Statement

All collected data are provided within this manuscript.

Acknowledgments

The authors would like to thank to the park director Griselda Urich and Jorgelina Torrents and Johann Barolin for their help during field work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Wild 02 00035 g001
Figure 1. Male specimen of Subulo gouazoubira with clinical symptoms found in “Reserva Natural Provincial de Uso Multiple Parque General San Martín”, Entre Ríos, Argentina. Signs of dehydration and pale eyelid are visible.
Figure 1. Male specimen of Subulo gouazoubira with clinical symptoms found in “Reserva Natural Provincial de Uso Multiple Parque General San Martín”, Entre Ríos, Argentina. Signs of dehydration and pale eyelid are visible.
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Figure 2. Maximum-likelihood tree constructed from 18S rDNA sequences for different Theileria species (substitution model: generalized time-reversible model with Gamma distribution and invariant sites (GTR G+I); alignment length 1440 bp). The partial sequence generated in this study is written in bold letters. Numbers represent bootstrap support generated from 1000 replications and GenBank Accession numbers are given in brackets.
Figure 2. Maximum-likelihood tree constructed from 18S rDNA sequences for different Theileria species (substitution model: generalized time-reversible model with Gamma distribution and invariant sites (GTR G+I); alignment length 1440 bp). The partial sequence generated in this study is written in bold letters. Numbers represent bootstrap support generated from 1000 replications and GenBank Accession numbers are given in brackets.
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Figure 3. Maximum-likelihood tree constructed from 16S rDNA sequences for different Anaplasma species (substitution model: generalized time-reversible model with Gamma distribution and invariant sites (GTR G+I); alignment length 1101 bp). The partial sequence generated in this study is written in bold letters. Numbers represent bootstrap support generated from 1000 replications and GenBank Accession numbers are given in brackets.
Figure 3. Maximum-likelihood tree constructed from 16S rDNA sequences for different Anaplasma species (substitution model: generalized time-reversible model with Gamma distribution and invariant sites (GTR G+I); alignment length 1101 bp). The partial sequence generated in this study is written in bold letters. Numbers represent bootstrap support generated from 1000 replications and GenBank Accession numbers are given in brackets.
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Figure 4. Maximum-likelihood tree constructed from 16S rDNA sequences of Anaplasma sp. related to Subulo gouazoubira and other Anaplasma species (substitution model: Hasegawa–Kishino–Yano model with Gamma distribution (HKY+G); alignment length 340 bp). The partial sequence generated in this study is written in bold letters. Numbers represent bootstrap support generated from 1000 replications and GenBank Accession numbers are given in brackets.
Figure 4. Maximum-likelihood tree constructed from 16S rDNA sequences of Anaplasma sp. related to Subulo gouazoubira and other Anaplasma species (substitution model: Hasegawa–Kishino–Yano model with Gamma distribution (HKY+G); alignment length 340 bp). The partial sequence generated in this study is written in bold letters. Numbers represent bootstrap support generated from 1000 replications and GenBank Accession numbers are given in brackets.
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Table 1. Results of hematology and blood biochemistry of the male specimen of Subulo gouazoubira with theileriosis clinical symptoms found in “Reserva Natural Provincial de Uso Multiple Parque General San Martín”, Entre Ríos, Argentina, with reference values.
Table 1. Results of hematology and blood biochemistry of the male specimen of Subulo gouazoubira with theileriosis clinical symptoms found in “Reserva Natural Provincial de Uso Multiple Parque General San Martín”, Entre Ríos, Argentina, with reference values.
Parameter * Reference Values 1
Hematogram
Hematocrit (%)9.746.33 ± 4.39
Erythrocytes/mm3 (×106)2.5914.63 ± 1.53
Leukocytes/mm351204.48 ± 1.14
Hemoglobin (g/dL)11.816.38 ± 1.32
MCV (µm3)3832.48 ± 6.47
MCH (%)45.611.49 ± 2.17 2
MCHC (g/dL)121.335.35 ± 1.93
Biochemical blood profile
Urea (mg/dL)5063.25 ± 22.21
Creatinine (mg/dL)0.671.75 ± 0.27
Glucose (g/L)0.51N/A
Total protein (g/dL)5.178.22 ± 0.80
AST/ASA/GOT (IU)97797.71 ± 79.70
ALT/ALA/GPT (IU)5216.5 ± 5.13
γGT/GGT (IU)8062.65 ± 17.89
Alkaline phosphatase (IU)73N/A
Bilirubin (total, mg/dL)0.05N/A
Bilirubin (direct, mg/dL)0.03N/A
Bilirubin (indirect, mg/dL)0.02N/A
CPK (IU)6332171 ± 150
* IU = international units; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; AST/ASA/GOT = aspartate aminotransferase; ALT/ALA/GPT = alanine aminotransferase; γGT/GGT = Gamma-glutamyl transferase; CPK = Creatine kinase; 1 Reference values for male specimens of Subulo gouazoubira according to Camargo et al. [26]; 2 pg; N/A: not available.
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MDPI and ACS Style

Sebastian, P.S.; Vaschalde, P.J.; Pelosi, M.C.; Flores, F.S.; Yedro, J.; Tarragona, E.L. First Molecular Evidence of Theileria and Anaplasma Genospecies in Subulo gouazoubira Exhibiting Clinical Symptoms from Entre Ríos Province, Argentina. Wild 2025, 2, 35. https://doi.org/10.3390/wild2030035

AMA Style

Sebastian PS, Vaschalde PJ, Pelosi MC, Flores FS, Yedro J, Tarragona EL. First Molecular Evidence of Theileria and Anaplasma Genospecies in Subulo gouazoubira Exhibiting Clinical Symptoms from Entre Ríos Province, Argentina. Wild. 2025; 2(3):35. https://doi.org/10.3390/wild2030035

Chicago/Turabian Style

Sebastian, Patrick S., Paula J. Vaschalde, María C. Pelosi, Fernando S. Flores, Julio Yedro, and Evelina L. Tarragona. 2025. "First Molecular Evidence of Theileria and Anaplasma Genospecies in Subulo gouazoubira Exhibiting Clinical Symptoms from Entre Ríos Province, Argentina" Wild 2, no. 3: 35. https://doi.org/10.3390/wild2030035

APA Style

Sebastian, P. S., Vaschalde, P. J., Pelosi, M. C., Flores, F. S., Yedro, J., & Tarragona, E. L. (2025). First Molecular Evidence of Theileria and Anaplasma Genospecies in Subulo gouazoubira Exhibiting Clinical Symptoms from Entre Ríos Province, Argentina. Wild, 2(3), 35. https://doi.org/10.3390/wild2030035

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