veterinary

sciences

Communication
Costa Rican Genotype of Ehrlichia canis: A Current Concern
Karla Irigaray Nogueira Borges 1 , Nathalia de Assis Pereira 2 , Daniel Moura de Aguiar 2 ,
Isis Indaiara Gonçalves Granjeiro Taques 3 , Bruna Samara Alves-Ribeiro 4 , Dirceu Guilherme de Souza Ramos 4
and Ísis Assis Braga 1,4, *

1 Veterinary Medicine College, Basic Unit of Bioscience, Mineiros University Center, 22nd Street s/n,
Mineiros 75833-130, GO, Brazil; karla@unifimes.edu.br
2 Virology and Rickettsioses Laboratory, Department of Clinical Veterinary Medicine,
Veterinary Medicine College, Federal University of Mato Grosso, Fernando Correa da Costa Avenue 2367,
Cuiabá 78060-900, MT, Brazil; nathaliaassis89@gmail.com (N.d.A.P.); danmoura@ufmt.br (D.M.d.A.)
3 EDUVALE College—Campus Jaciara, Caiçara Street, 2.114, Jaciara 78820-000, MT, Brazil;
isisgtaques@gmail.com
4 Laboratory of Veterinary Pathology and Parasitology, Academic Unit of Agricultural Sciences,
Federal University of Jataí, Jataí 75801-615, GO, Brazil; brunasamara@discente.ufj.edu.br (B.S.A.-R.);
dguilherme@ufj.edu.br (D.G.d.S.R.)
* Correspondence: isisbraga@ufj.edu.br

Simple Summary: Clinical and hematological characteristics of the blood of 125 dogs with different
Ehrlichia canis genotypes were analyzed. We observed that the Brazilian genotype is more prevalent
and adapted in dogs in the central-western region of Brazil, whereas the American genotype appears
to be more pathogenic, causing inflammatory signs. However, we highlight the high frequency of dogs
reactive to the Costa Rican genotype, which is suggested to be less adapted to the immune response
of the animals. In addition, this genotype has an imminent risk because of its zoonotic potential.

Abstract: Canine monocytic ehrlichiosis (CME) is endemic to Brazil, and studies have verified that
dogs have been exposed to different genotypes of Ehrlichia canis. This genetic divergence can influence
the clinical response of the animals. We aimed to describe clinical and hematological changes in
Citation: Borges, K.I.N.; 125 dogs that reacted to BrTRP36, USTRP36, and CRTRP36 genotypes through enzyme immunoassays
Pereira, N.d.A.; Aguiar, D.M.d.; and to highlight the current concern regarding infection by the Costa Rican genotype. The results
Taques, I.I.G.G.; Alves-Ribeiro, B.S.;
showed that 52.0% reacted to the Brazilian genotype, 22.4% reacted to the Costa Rican genotype,
Ramos, D.G.d.S.; Braga, Í.A. Costa
and 16.0% reacted to the American genotype, and some co-reactions were observed. Dogs reactive
Rican Genotype of Ehrlichia canis: A
to BrTRP36 were 1.24% more likely to present with medullary regeneration in cases of anemia and
Current Concern. Vet. Sci. 2023, 10,
316. https://doi.org/10.3390/
3% less likely to manifest hyperproteinemia, while dogs reactive to CRTRP36 were 0.7% less likely to
vetsci10050316 present with medullary regeneration. Febrile illness and neurological alterations were also statistically
associated, with an 85.7% and 231.2% increased likelihood, respectively, to occur in dogs that reacted
Academic Editors: Rodolfo Cordeiro
to USTRP36. The dogs with the American genotype developed clinical manifestations related to
Giunchetti and Lorena Lopes Ferreira
systemic inflammation, while those with the Brazilian genotype of E. canis were more dispersed in
Received: 7 March 2023 the region studied, showing greater adaptation to the hosts. We highlight the significant serocurrence
Revised: 7 April 2023 of the Costa Rican genotype, which has already been described to have zoonotic potential and which
Accepted: 18 April 2023 showed less adaptation.
Published: 27 April 2023

Keywords: Ehrlichia canis; BrTRP36; CRTRP36; USTRP36; immune adaptation; zoonotic potential

Copyright: © 2023 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and Canine monocytic ehrlichiosis (CME) is an endemic disease in Brazil, transmitted by
conditions of the Creative Commons Rhipicephalus sanguineus sensu lato ticks [1] associated with a complex symptomatology
Attribution (CC BY) license (https:// that varies according to the intensity and clinical stage of the disease [2]. New research has
creativecommons.org/licenses/by/ verified that infection by distinct Ehrlichia canis genotypes may lead to variations in the
4.0/). animal’s clinical response [3].

Vet. Sci. 2023, 10, 316. https://doi.org/10.3390/vetsci10050316 https://www.mdpi.com/journal/vetsci

Vet. Sci. 2023, 10, 316 2 of 6

Currently, four genotypes of E. canis have been identified based on the divergence
of the tandem repeat protein (TRP) sequences: USTRP36, BrTRP36, CRTRP36, and more
recently, CUBTRP36—the American, Brazilian, Costa Rican [4–6] and Cuban genotypes,
respectively [7]. TRPs are known to play important roles in the entry of E. canis into the
host cell, modulation of replication mechanisms, and facilitation of the exit of the bacterium
from the infected cell. Thus, they are essential for host adaptation and immune response
evasion [8].
In Brazil, the Brazilian and American genotypes are widely distributed, whereas
the Costa Rican genotype was observed in 0.6% of the exposed dogs [9]. In a previous
analysis, Taques et al. [3] found some correlations between animals exposed to USTRP36
and BrTRP36 genotypes with common changes in CME; however, gaps remain. Thus,
further evaluation of the clinical behavior of dogs with different genotypes is necessary,
specially due to the lack of data about Costa Rican genotype. We aimed to described clinical
and hematological changes in different genotypes of E. canis compared to CRTRP36 and to
highlight the current concern regarding infection by the Costa Rican genotype.

2. Materials and Methods

From March 2021 to June 2022, dogs at veterinary clinics in the city of Mineiros, in
the midwestern region of Brazil, were selected for inclusion in the study based on the
following criteria: a presence of hematological changes associated with CME (anemia and
thrombocytopenia), presence of Ehrlichia spp. morulae in blood smears and/or reactivity
to SNAP4Dx® Plus Test (IDEXX Laboratories ©, Fremont, CA, USA). Thus, 125 dogs were
selected with owner authorizing the use of clinical and hematological data of animals in
the research by filling out the informed consent form (ICF). The study was approved by
the Ethics Committee on Animal Research (CEUA) of the University Center of Mineiros
(protocol number 0088/2020) for biological sample manipulation.
Clinical data, such as age, sex, breed, presence of ticks, body temperature, mucosal
evaluation, palpation of lymph nodes and spleen, and data on parameters such as the
presence of gastroenteric, hemodynamic, neurological, and ophthalmic alterations, were
collected. Additionally, hematological data, such as hematometry, leukometry, and platelet
count, were verified, considering the reference values of Meyer and Harvey [10] and
Jain [11]. To evaluate medullary regeneration, the presence and intensity of anisocytosis
(RDW), polychromasia, and metarubrites was considered.
Subsequently, the canine serum samples were subjected to the enzyme immunoassay tech-
nique described by Aguiar et al. [12] using synthetic peptides (Bio-Synthesis Inc® , Lewisville,
TX, USA) of E. canis, with their respective diagnostic targets—TRP19 (protein): HFTGPTS-
FEVNLSEEEKMELQEVS (synthetic peptide; E. canis (genotype) [13]; TRP36US: TEDSVSAP-
ATEDSVSAPA, the American E. canis [4]; TRP36Br: ASVVPEAEASVVPEAEASVVPEAE, the
Brazilian E. canis [12]; and TRP36CR: EASVVPAAEAPQPAQQTEDEFFSDGIEA, the Costa
Rican E. canis [14]. The optical density cut-off point was 0.250 [12].
Clinical and hematological data were classified as qualitative variables, compared
to the presence or absence of each genotype and were subjected to the chi-square test of
association using a 95% confidence interval (CI). Statistical significance was set at p < 0.05.
Statistical analyses were performed using the Epi info vs. 7.2.5.0 software. This analysis
was conducted to demonstrate the main alterations in E. canis genotypes.

3. Results
Of the 125 dogs, 77.6% reacted to the TRP19 genotype, a highly conserved protein in
the E. canis species, 52.0% reacted to the Brazilian genotype, 22.4% reacted to the Costa
Rican genotype, and 16.0% reacted to the American genotype. Co-reactions were also
observed (Table 1). The optical densities ranged from 0.258 to 1.768 (mean, 0.834) for the
TRP19 peptide, 0.251 to 1.302 (mean, 0.691) for TRP36Br, 0.273 to 1.762 (mean, 1.061) for
TRP36CR, and 0.254 to 1.193 (mean, 0.691) for TRP36US.
Vet. Sci. 2023, 10, 316 3 of 6

Table 1. Number of dogs that reacted concomitantly to TRP19, BrTRP36, CRTRP36, and USTRP36
peptides from Ehrlichia canis from Mineiros city, midwestern region of Brazil.

Animals (n = 125)
Peptides
Quantity %
TRP19, TRP36Br, TRP36 CR e TRP36US 1 0.80
TRP19 e TRP36Br 49 39.20
TRP19 e TRP36CR 18 14.40
TRP19 e TRP36US 13 10.40
TRP36Br e TRP36CR 16 12.80
TRP36Br e TRP36US 6 4.80
TRP36US e TRP36CR 2 1.60
Total 105 84

The racial characteristics, sex, and age range of the seroreactive dogs are shown in
Table 2.
Table 2. Profile of dogs reacting to TRP19, BrTRP36, CRTRP36, and USTRP36 peptides of Ehrlichia
canis from Mineiros city, midwestern region of Brazil.

Animals (n = 125)
Profile
Positive % TRP19 TRP36 BR TRP36 CR TRP36 US
Female 62 49.6 49 32 12 8
Male 63 50.4 48 33 16 12
Adults 98 78.4 78 * 55 † 23 12
Puppy 27 21.6 19 10 5 8
Defined Race 79 63.2 66 + 40 15 16
SRD 46 36.8 31 25 13 4
* p = 0.010 (odds ratio = 4.105). † p = 0.006 (odds ratio = 5.5). + p = 0.024 (odds ratio = 1.955).

The hematological evaluations of the dogs are shown in Table 3. Clinical data are
presented in Table 4.

Table 3. Hematological analyses of the blood of dogs reacting to the peptides TRP19, BrTRP36,
CRTRP36, and USTRP36 of Ehrlichia canis, from Mineiros city, midwestern region of Brazil.

Animals (n = 125)
Alteration
Positive % TRP19 TRP36 BR TRP36 CR TRP36 US
Thrombocytopenia 114 91.2 89 61 27 19
Anemia 96 76.8 74 54 20 14
Medullar
73 58.4 57 44 * 10 † 11
regeneration
Hyperproteinaemia 59 47.2 49 + 32 ◦ 15 7
Activated
38 30.4 30 20 7 6
monocytes
Leukopenia 32 25.6 25 17 8 5
Leucocytosis 22 17.6 14 13 2 3
Presence of
11 8.8 7 5 2 1
morulae on smear
* p = 0.028 (odds ratio = 2.239). † p = 0.005 (odds ratio = 0.299). + p = 0.00003 (odds ratio = 1.021). ◦ p = 0.024
(odds ratio = 0.97).
Vet. Sci. 2023, 10, 316 4 of 6

Table 4. Clinical manifestation of dogs reacting to the peptides TRP19, BrTRP36, CRTRP36, and
USTRP36 of Ehrlichia canis from Mineiros city, midwestern region of Brazil.

Animals (n = 125)
Clinical Sign
Positive % TRP19 TRP36 BR TRP36 CR TRP36 US
Anorexia 112 89.6 86 60 26 16
Apathy 73 58.4 54 45 17 11
Fever 55 44 39 27 13 13 *
Dehydration 55 44 45 28 8 9
Mucosal pallor 54 43.2 41 32 6 11
Presence of ticks 37 29.6 28 16 † 6 8
Vomit 15 12 14 9 5 0
Diarrhea 15 12 11 8 2 3
Lymphadenomegalia 11 8.8 9 5 4 1
Epistaxis 10 8 10 11 3 3
Neurological changes 9 7.2 9 5 3 4+
Ophthalmic changes 8 6.4 8 3 2 1
* p = 0.026 (odds ratio = 1.857). † p = 0.038 (odds ratio = 0.496). + p = 0.047 (odds ratio = 3.3125).

4. Discussion
Corroborating what was described by Taques [9], it was found that the genotypes of
E. canis are widely distributed in the population of dogs in Brazil; however, we highlight
the relative frequency of exposure of dogs to the Costa Rican genotype in the studied area,
as previously, this genotype had been reported only in the southeast and northwest regions
of the country at a low frequency.
It is believed that the bacterium is undergoing a process of genetic recombination
through these proteins, which have already been characterized as targets for host-induced
immune pressures [5,15,16], seeking means of evasion of the immune response, and even
reaching new hosts. Navarrete [7] reported that the genotypic diversity of E. canis was
associated with the emergence of strains with greater virulence. In this study, co-reaction
with E. canis genotypes was observed in 84% of the samples evaluated (Table 1), which is
suggestive of co-infection by the different genotypes. This mechanism is commonly present
in microorganisms and is considered one of the main drivers of genetic diversity in obligate
intracellular microorganisms [17].
Bouza-Moura et al. [6] found that the TRP of the Costa Rican genotype is very similar to
that of the Brazilian genotype, with the addition of some amino acids, suggesting a possible
mutation. Another noteworthy factor is the association of the CRTPR36 genotype of E. canis
with infections in humans, which has been considered to have zoonotic potential [6]. It
was observed that 28.0% of the dogs reacted only to TRP19, suggesting that unidentified
genotypes of E. canis may be present in the region, such as the most recently described
Cuban genotype (CUBTRP36) or even other unreported recombinations.
When analyzing the characteristics of the animals and their respective clinical con-
ditions, it was observed that adult dogs were predominantly associated with TRP19,
indicating that this age group was 310.5% more likely to be exposed to E. canis than puppies
were, especially of the Brazilian genotype (Table 2). Although Harrus and Waner [18]
stated that there is no age predisposition to CME, we know that adult animals have longer
exposure to factors associated with E. canis transmission and can often act as carriers of the
bacterium. As for the racial characterization, it was verified that dogs of defined breeds
have a 95.5% higher chance of being exposed to E. canis, which is in agreement with the
literature [19,20], but there were no associations with the other genotypes, and TRP19 was
not correlated with sex.
Regarding the clinical and laboratory manifestations of the different genotypes, it was
possible to infer that the Brazilian genotype is more adapted to the animals and to the vector
R. sanguineus s.l. (currently known as Rhipicephalus linnaei) [21], as dogs exposed to the
Brazilian genotype presented 50.37% fewer chances of being parasitized by ticks at the time
of consultation, and these dogs were parasitized throughout their lives by infected ticks.
Vet. Sci. 2023, 10, 316 5 of 6

Furthermore, determining the vector competence of species and strains of R. saguineus s.l.
for each E. canis genotype requires further studies.
Moreover, dogs reactive to BrTRP36 were 1.24% more likely to present with medullary
regeneration in cases of anemia, suggesting a better response to infection, and 3% less likely
to manifest hyperproteinemia, which demonstrates less antigenic stimulation caused by
the presence of the bacteria and consequently a lower production of gamma globulins [18].
Taques et al. [3] described a lower correlation between BrTRP36 and hyperproteinemia in
another location in central-western Brazil.
Dogs exposed to the American genotype presented an 85.7% greater likelihood of
developing febrile illness, on top of neurological alterations such as convulsions and motor
incoordination, which were also statistically associated, with a 231.2% greater likelihood of
occurrence in dogs that reacted to USTRP36, demonstrating that it is a more pathogenic
and immunogenic genotype than the Brazilian genotype, which supports the findings of
Taques et al. [9].
For the first time, an analysis of dogs reacting to the Costa Rican genotype, which was
proven to be less adapted to the animal, could be performed, during which it was observed
that dogs presented a 0.7% decreased likelihood of medullary regeneration and that the
average optical density was higher than that of the other genotypes studied.

5. Conclusions
The results of this study allowed us to conclude that there are some phenotypic
differences associated with the different E. canis genotypes. Medullary regeneration was
more closely related to the Brazilian genotype and less closely related to the Costa Rican
genotype. The TRP19 genotype was associated with hyperproteinemia. Moreover, it was
observed that dogs exposed to the American genotype developed clinical manifestations
related to systemic inflammation, such as fever and neurological alterations, which in cases
of canine monocytic ehrlichiosis occur in an immune-mediated manner. The Brazilian
genotype of E. canis was more dispersed in the region studied, showing greater adaptation
to the hosts. Finally, we highlight the significant serocurrence of the Costa Rican genotype,
which showed less adaptation to dogs, and highlight concerns about the possible zoonotic
potential attributed to this genotype.

Author Contributions: Conceptualization, K.I.N.B., D.G.d.S.R. and Í.A.B.; methodology, K.I.N.B.,

N.d.A.P., D.M.d.A. and Í.A.B.; software, I.I.G.G.T., B.S.A.-R., D.G.d.S.R. and Í.A.B.; validation, K.I.N.B.,
D.M.d.A., D.G.d.S.R. and Í.A.B.; formal analysis, K.I.N.B., D.M.d.A., D.G.d.S.R. and Í.A.B.; investiga-
tion, K.I.N.B., N.d.A.P., D.M.d.A., I.I.G.G.T., B.S.A.-R., D.G.d.S.R. and Í.A.B.; resources, D.M.d.A. and
Í.A.B.; data curation, K.I.N.B., D.M.d.A., D.G.d.S.R. and Í.A.B.; writing—original draft preparation,
K.I.N.B., N.d.A.P., D.M.d.A., I.I.G.G.T., B.S.A.-R., D.G.d.S.R. and Í.A.B.; writing—review and editing,
K.I.N.B., D.M.d.A., D.G.d.S.R. and Í.A.B.; visualization, K.I.N.B., D.M.d.A., D.G.d.S.R. and Í.A.B.;
supervision, D.M.d.A., D.G.d.S.R. and Í.A.B.; project administration, D.M.d.A., D.G.d.S.R. and Í.A.B.;
funding acquisition, D.M.d.A. and Í.A.B. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: This study was approved by the Animal Research Ethics
Committee (CEUA) of the University Center of Mineiros under protocol no. 088/2020 for biological
sample manipulation.
Informed Consent Statement: The Informed Consent Statement was obtained from all animal
owners that are included in this study.
Data Availability Statement: Data sharing not applicable.
Acknowledgments: We thank the National Council for Scientific and Technological Development
(CNPq) for granting a scholarship to Daniel Moura de Aguiar.
Conflicts of Interest: The authors declare no conflict of interest.
Vet. Sci. 2023, 10, 316 6 of 6

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