Ehrlichia canis Seropositivity in a Hospital-Based Dog Population in Kars (Türkiye): IFAT–ELISA Comparison and Associated Demographic,Clinical,and Hematological Findings

Abstract

Background:

Ehrlichia canis is a common tick-borne pathogen in dogs and may occur without clear clinical signs, complicating diagnosis. Serological methods such as indirect immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) are therefore widely used. This study aimed to determine the seropositivity of E. canis in dogs from the Kars region of Türkiye and to evaluate associated demographic, clinical, and hematological findings.

Materials and Methods:

Blood samples were collected from 188 dogs of different ages, breeds, and clinical backgrounds presented to the Animal Health Education, Application, and Research Hospital of Kafkas University (Türkiye) for routine care or clinical complaints.

Results:

E. canis seropositivity was detected at 22.34% (42/188) by IFAT and 10.64% (20/188) by ELISA. Taking IFAT as the reference, ELISA showed 50% sensitivity, 100% specificity, and 87.14% diagnostic accuracy, with moderate agreement between tests (κ = 0.598). No statistically significant differences were found in hematological parameters between seropositive and non-dual-seropositive dogs (p values ranged from 0.065 to 0.343). Among demographic and clinical variables, only age showed a significant association (p = 0.008), with higher seropositivity in older dogs; however, this should be interpreted cautiously due to small subgroup sizes.

Conclusion:

The findings indicate notable exposure to E. canis in a hospital-based sample of dogs from the Kars region, even in the absence of clinical or hematological abnormalities. Serological testing alone reflects exposure rather than active infection. Using IFAT and ELISA together may provide a more cautious approach in identifying seroreactive animals, though not necessarily improving definitive diagnostic accuracy.

Keywords

seroprevalence IFAT ELISA canine ehrlichiosis hematological and clinical findings

Introduction

Ehrlichia canis is a member of the genus Ehrlichia within the family Rickettsiaceae and consists of pleomorphic, obligate intracellular bacteria. E. canis is of great importance in veterinary medicine due to its role as the causative agent of canine monocytic ehrlichiosis. The disease may present with acute, subacute, or chronic courses and can lead to both clinical and subclinical infections, thereby creating diagnostic challenges (Spickler, 2013). Canine monocytic ehrlichiosis is primarily transmitted by the brown dog tick (Rhipicephalus sanguineus), and the disease can spread among dogs through bites of infected ticks as well as via blood transfusions (Ferrolho et al., 2025). Although its zoonotic potential is limited, transmission to humans following contact with infected dogs or tick bites has been reported, and therefore, the disease is considered a minor zoonosis (Perez et al., 2006; Ferrolho et al., 2025).

The disease is particularly prevalent in tropical and subtropical regions; however, its prevalence varies across different geographical areas. Studies conducted worldwide have reported E. canis seropositivity rates ranging from 0.33% to 92.1% (Gomes dos Santos et al., 2020; Di Bella et al., 2024). In Türkiye, serological studies conducted in different regions have revealed that the prevalence of E. canis infections varies widely, ranging from 0.75% to 69.4% (Cihan et al., 2010; Başer et al., 2023). There are no available data regarding the prevalence of E. canis in dogs in the Kars region; however, seropositivity rates of 1% in dogs from the geographically closest region, Iğdır, and 0% in the Erzurum region have been reported (Sarı et al., 2013; Demir and Aktaş, 2020). Nevertheless, most of these studies were conducted in geographically limited areas, and up-to-date and comprehensive epidemiological data are lacking for certain regions of the country. In addition, the number of studies in which indirect immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA)—two serological methods widely used in the diagnosis of E. canis infections—have been comparatively evaluated within the same dog population is limited. This situation leads to uncertainties regarding which method is more reliable and applicable in clinical practice.

Canine monocytic ehrlichiosis in dogs is characterized in the acute phase by nonspecific clinical findings such as fever, depression, and thrombocytopenia. In the chronic phase, pancytopenia due to bone marrow hypoplasia and secondary infections may develop (Shipov et al., 2008). The variability of clinical manifestations and the presence of subclinical infections make it difficult to establish a definitive diagnosis based solely on clinical and hematological findings. Due to the obligate intracellular nature of E. canis, in vitro culture is not routinely performed and requires specialized laboratory systems; therefore, serological and molecular diagnostic methods are commonly used. In serological diagnosis, the presence of E. canis-specific IgG antibodies, which can be detected in the weeks following infection, represents an important indicator of exposure. The IFAT was used as the reference method for the serological assessment of E. canis exposure (Waner et al., 2000; Bélanger et al., 2002), whereas more recently developed ELISA methods have gained prominence due to their high diagnostic performance and practical applicability (Ferrero et al., 2025; Shepherdson et al., 2026). Nevertheless, comparative evaluation of the diagnostic performance of these methods is essential for establishing accurate diagnostic strategies.

This study aimed to determine E. canis seropositivity in dogs using IFAT and ELISA methods, to comparatively evaluate the results of these two serological techniques, and to identify demographic, clinical, and hematological findings associated with seropositivity.

Materials and Methods

Study material

The study was conducted between May 2023 and October 2024 on a hospital-based convenience sample of dogs presented to the Animal Health Education, Application, and Research Hospital of Kafkas University for various reasons, including routine check-ups, vaccination, and clinical complaints. The study material consisted of a total of 188 blood samples obtained from both clinically healthy-appearing dogs and dogs exhibiting various clinical and paraclinical findings such as depression, lethargy, weight loss, anorexia, pyrexia, lymphadenomegaly, splenomegaly, and bleeding tendency. Demographic and clinical data of the sampled dogs were also recorded (Table 1).

Table 1.

Distribution of Ehrlichia canis Seropositivity According to Demographic, Geographical, and Clinical Variables in Dogs

Demographic findings
Breed	n (%)^a	Seropositive (%)	p Value
Akbash	2 (1.06)	0	0.355
Anatolian Shepherd Dog	14 (7.45)	2 (14.29)
Belgian Malinois	2 (1.06)	0
Border Collie	1 (0.53)	0
Bull Terrier	1 (0.53)	1 (100)
Cairn Terrier	2 (1.06)	0
Cane Corso	2 (1.06)	0
Caucasian Shepherd Dog	16 (8.51)	2 (12.5)
Chihuahua	1 (0.53)	0
Dobermann	1 (0.53)	0
English Cocker Spaniel	1 (0.53)	0
English Setter	2 (1.06)	0
French Bulldog	1 (0.53)	1 (100)
German Shepherd Dog	4 (2.13)	1 (25)
Golden Retriever	3 (1.6)	1 (33.33)
Kangal Shepherd Dog	51 (27.13)	4 (7.84)
Labrador Retriever	1 (0.53)	0
Large Mixed-Breed Dog	23 (12.23)	3 (13.04)
Pekingese	1 (0.53)	0
Pomeranian	1 (0.53)	0
Poodle	1 (0.53)	0
Presa Canario	1 (0.53)	0
Pug	1 (0.53)	0
Rottweiler	1 (0.53)	0
Siberian Husky	2 (1.06)	0
Small Mixed-Breed Dog	24 (12.77)	0
Not recorded	28 (14.89)	3 (10.71)
Sex	n (%)	Seropositive (%)
Female	61 (32.45)	6 (9.84)	0.338
Male	109 (57.98)	12 (11.01)
Not recorded	18 (9.57)	0
Age (month)	n (%)	Seropositive (%)
0–24	103 (54.79)	14 (13.59)	0.008
25–96	62 (32.98)	1 (1.61)
97–120	6 (3.19)	2 (33.33)
Not recorded	17 (9.04)	1 (5.88)

Location findings
District	n (%)^a	Seropositive (%)
Akyaka	5 (2.66)	0	0.235
Arpaçay	2 (1.06)	0
Digor	4 (2.13)	0
Center	74 (39.36)	6 (8.11)
Sarıkamış	7 (3.72)	2 (28.57)
Selim	10 (5.32)	1 (10)
Susuz	25 (13.3)	0
Not recorded	61 (32.45)	9 (14.75)

Clinical findings
Pulse (beats/min)	n (%)	Seropositive (%)
70–89	15 (7.98)	3 (20)	0.580
90–100	26 (13.83)	2 (7.69)
101–120	43 (22.87)	5 (11.63)
≥121	63 (33.51)	4 (6.35)
Not recorded	41 (21.81)	4 (9.76)
Respiration (breaths/min)	n (%)	Seropositive (%)
12–40	90 (47.87)	12 (13.33)	0.314
41–60	37 (19.68)	2 (5.41)
>60	10 (5.32)	0
Not recorded	51 (27.13)	4 (7.84)
Body temperature (°C)	n (%)	Seropositive (%)
≤37.4	11 (5.85)	2 (18.18)	0.470
37.5–39.3	99 (52.66)	11 (11.11)
≥39.4	33 (17.55)	3 (9.09)
Not recorded	45 (23.94)	2 (4.44)
Coat structure	n (%) ^a	Seropositive (%)
Normal	44 (23.4)	2 (4.55)	0.170
Matted coat	78 (41.49)	9 (11.54)
Partially matted	36 (19.15)	2 (5.56)
Alopecia	1 (0.53)	1 (100)
Not recorded	29 (15.43)	4 (13.79)
Appetite	n (%)	Seropositive (%)
Normal	48 (25.53)	7 (14.58)	0.280
Reduced	31 (16.49)	1 (3.23)
Absent	88 (46.81)	7 (7.95)
Not recorded	21 (11.17)	3 (14.29)
Lethargy	n (%)	Seropositive (%)
Present	10 (5.32)	0	0.361
Absent	161 (85.64)	18 (11.18)
Not recorded	14 (7.45)	0
Lymph nodes	n (%)	Seropositive (%)
Lymphadenopathy	69 (36.7)	8 (11.59)	0.740
Normal	101 (53.72)	9 (8.91)
Not recorded	18 (9.57)	1 (5.56)
Mucous membranes	n (%) ^a	Seropositive (%)
Pale (anemic)	32 (17.02)	3 (9.38)	0.570
Hyperemic	60 (31.91)	6 (10)
Icteric	4 (2.13)	1 (25)
Normal	78 (41.49)	8 (10.26)
Not recorded	14 (7.45)	0

The bold p-value indicates statistical significance.

Subgroups with very small sample sizes (n < 5) were considered descriptive only and were not used for inferential interpretation.

Blood samples (5 mL) were collected from the V. cephalica antebrachii of the dogs and transferred to sterile serum tubes and EDTA-containing tubes and then promptly transported to the laboratory. The blood samples were centrifuged at 1500 g for 10 min to obtain serum, and the serum samples were stored at −20°C until analysis. Complete blood counts were performed on EDTA-anticoagulated blood samples using an automated hematology analyzer (Auto Hematology Analyzer, Mindray BC-5000, China).

Serological analysis

Seropositivity was defined separately by IFAT and ELISA. Dogs were considered IFAT-seropositive if they tested positive by IFAT and ELISA-seropositive if they tested positive by ELISA. In addition, a dual-seropositive category was defined for dogs testing positive by both IFAT and ELISA.

IFAT

The analysis of serum samples for E. canis antibodies using the IFAT was performed with a commercial kit (MegaFLUO^® Vet, Megacor, Austria). Serum samples diluted 1:40 with phosphate-buffered saline (PBS) were analyzed alongside stock positive and negative controls. After adding 20 µL of diluted serum, positive control, or negative control to the appropriate wells of antigen-coated slides, the slides were incubated at 37°C for 30 min. Following PBS wash, 20 µL of conjugate was added to each well, and the slides were incubated for 30 min at 37°C in the dark. After a final wash, the slides were mounted with two drops of mounting medium and examined under a digital upright microscope (Olympus BX53, Japan) at ×400 magnification. Evaluation was carried out according to the manufacturer’s instructions, using the fluorescence intensity of the positive and negative controls as reference.

ELISA

Comparative analysis of serum samples for E. canis antibodies was performed using an indirect ELISA. For this purpose, a commercial indirect ELISA kit (MegaELISA^® EHRLICHIA, Megacor, Austria) was used. Five microliters of serum sample, positive control, or negative control were diluted with 500 µL of sample buffer and transferred into the corresponding wells of the ELISA plate, followed by incubation at 25°C for 60 min. After washing the ELISA plate with wash buffer, 100 µL of conjugate was added to each well, and the plate was incubated at 25°C for 45 min. The plate was then washed again with wash buffer, after which 100 µL of substrate was added to each well and incubated at room temperature in the dark for 15 min. At the end of the incubation period, 100 µL of stop solution was added to each well, and the absorbance was read within a short time (within 20 min) at a wavelength of 450 nm using a spectrophotometer (Epoch™ Microplate Spectrophotometer, BioTek, USA). Calculations were performed according to the manufacturer’s instructions using the optical density (OD) values obtained from the samples. Based on these OD values, the results were scored using the formula below provided by the kit manufacturer and expressed in Megacor Units (MU).

MU = \frac{Patient (mean) extinction value \times 10}{Cut - off}

Accordingly, samples with values <9 MU were classified as negative, 9–11 MU as equivocal (gray zone), and >11 MU as positive.

Sensitivity analysis

Equivocal (suspicious) ELISA results were handled according to a predefined approach. For the primary diagnostic performance analysis, equivocal results were excluded to allow binary classification. The number of samples included and excluded from this analysis is reported. In addition, sensitivity analyses were performed in which equivocal results were alternatively classified as positive and as negative to evaluate their impact on diagnostic performance estimates.

Statistical analysis

For the analysis of demographic, clinical, and hematological variables, only dogs that were positive by both IFAT and ELISA (dual-seropositive) were considered seropositive. This more stringent definition was selected to increase diagnostic specificity and reduce the likelihood of false-positive results. All analyses were performed using IBM SPSS Statistics 25.0. Associations between E. canis seropositivity and variables such as breed, sex, age, location, heart rate, respiratory rate, body temperature, coat condition, appetite, lethargy, and the status of lymph nodes and mucous membranes were evaluated using the Pearson chi-square test, Fisher–Freeman–Halton exact test, and Fisher’s exact test. To compare blood parameters between seropositive and nondual-seropositive groups, the distribution of each group was first assessed for normality using the Kolmogorov–Smirnov test. Variables showing normal distribution were analyzed with the Independent t-test, while those not showing normal distribution were analyzed using the Mann–Whitney U test. A p value <0.05 was considered statistically significant. Due to small subgroup sizes and missing data, the analysis of demographic and clinical variables was considered exploratory. No formal adjustment for multiple comparisons was applied.

Results

Results related to dogs

A total of 188 dogs were included in the study, consisting of 160 dogs representing 26 different breeds (24 purebred and 2 mixed-breed) and 28 dogs with no recorded breed. Among the dogs analyzed, the most common breeds were Kangal Shepherd Dog (51 dogs, 27.13%), small mixed-breed dog (24 dogs, 12.77%), and large mixed-breed dog (23 dogs, 12.23%), while the rarest breeds were Border Collie, Bull Terrier, Chihuahua, Doberman, English Cocker Spaniel, French Bulldog, Labrador Retriever, Pekingese, Pomeranian, Poodle, Presa Canario, Pug, and Rottweiler, each represented by a single dog. The number of male dogs (109 dogs, 57.98%) was higher than that of female dogs (61 dogs, 32.45%). Most of the dogs in the study were young, aged between 0 and 24 months (103 dogs, 54.79%), while 62 dogs (32.98%) were between 25 and 96 months and 6 dogs (3.19%) were between 97 and 120 months of age. Most dogs (106 dogs, 56.38%) had a pulse rate between 101–120 and ≥121, while the respiratory rate was within the normal range (12–40/min) in approximately half of the dogs (47.87%). Body temperature was within the normal range (37.5–39.3°C) in most dogs (52.66%). Additionally, a considerable proportion of the dogs (46.81%) exhibited loss of appetite, which may indicate a general health problem (Table 1).

Hematological results

For hematological comparisons, dogs were classified as seropositive based on dual IFAT/ELISA positivity, and values are presented as mean ± standard deviation depending on distribution; the number of animals included in each comparison is reported in Table 2. These findings should be interpreted with caution due to the limited number of seropositive animals. When hematological findings were compared between E. canis seropositive (n = 18) and nondual-seropositive group (n = 170), no statistically significant differences were observed in any parameter (p values ranged from 0.065 to 0.343). Although the mean WBC, granulocyte, and monocyte counts in the seropositive group were relatively higher than in the non-dual-seropositive group, and the mean lymphocyte, RBC, hemoglobin, and platelet counts were lower, these differences did not reach statistical significance (p values ranged from 0.079 to 0.343). Similarly, no significant differences were found between the groups in erythrocyte indices (MCV, MCH, and MCHC) or in RDW, MPV, and PDW values (p values ranged from 0.065 to 0.341). In conclusion, these data indicate that E. canis seropositivity does not have a marked or statistically significant effect on hematological parameters in the studied population (Table 2).

Table 2.

Hematological Parameters in Seropositive and NonDual-Seropositive Dogs

Parameters	Reference range	Seropositive (n = 18)		Nondual-seropositive (n = 170)		p Value
Parameters	Reference range	Mean ± SD	Range	Mean ± SD	Range	p Value
WBC (×10³/µL)	6–17	16.89 ± 11.92	1.86–42.64	14.48 ± 9.3	0.81–53.97	0.223
Lym (×10³/µL)	1–4.8	2.02 ± 1.23	0.17–3.74	2.36 ± 2.73	0.12–17.36	0.343
Mon (×10³/µL)	0.15–1.35	1.05 ± 1.04	0.07–3.32	0.81 ± 0.86	0.03–6.44	0.247
Gran (×10³/µL)	3–12.5	13.77 ± 10.65	1.62–35.58	11.21 ± 8.19	0.26–43.51	0.231
RBC (×10⁶/µL)	5.5–8.5	5.81 ± 2.09	1.29–8.86	6.34 ± 1.49	1.99–10.49	0.161
MCV (fL)	60–77	66.01 ± 5.64	60.03–78.2	64.93 ± 7.71	22.6–80.6	0.341
MCH (pg)	19–24	23.42 ± 1.55	21.2–25.1	24.39 ± 20.43	11.6–233.3	0.066
MCHC (g/dL)	32–36	35.57 ± 1.8	32–38.9	34.37 ± 2.42	27–42.4	0.076
RDW (%)	11–15	37.88 ± 13.63	9.9–62.7	29.83 ± 13.42	8.3–57.5	0.065
HB (g/dL)	12–18	13.53 ± 4.81	3.2–20.5	14.3 ± 3.92	4.8–24.8	0.289
PLT (10³/µL)	200–500	230.44 ± 95.64	80–333	325.71 ± 198.5	20–1150	0.079
MPV (fL)	7–11	9.58 ± 2.02	6.8–13.4	8.73 ± 1.46	5.7–15.8	0.081
PDW (%)	15–17	15.03 ± 3.2	7–18	13.98 ± 5.52	0–55	0.209

IFAT and ELISA results

In this hospital-based sample, 42 (22.34%) of the 188 dog serum samples were found to be positive for E. canis by IFAT, while 20 (10.64%) were positive by ELISA. Among IFAT-positive samples, 6 (3.19%) were classified as equivocal and 18 (9.57%) as negative by ELISA. Among IFAT-equivocal samples, 2 (1.06%) were positive, 4 (2.13%) equivocal, and 26 (13.83%) negative by ELISA. Among IFAT-negative samples, 10 (5.32%) were equivocal, and 104 (55.32%) were negative by ELISA, while no samples were ELISA-positive and IFAT-negative (Table 3).

Table 3.

Diagnostic Performance of Tests for Ehrlichia canis Detection

Tests	IFAT			Diagnostic values
Tests	Positive (n = 42) (22.34%)	Equivocal (n = 32) (17.02%)	Negative (n = 114) (60.64%)	Se (%) (95% CI)	Sp (%) (95% CI)	PPV (%) (95% CI)	NPV (%) (95% CI)	DA (%) (95% CI)	Kappa (95% CI) (SE)
ELISA
Positive (n = 20) (10.64%)	18 (9.57%)	2 (1.06%)	0 (%0)	50 (32.92–67.08)	100 (96.52–100)	100 (81.47–100)	85.25 (80.65–88.9)	87.14 (80.44–92.2)	0.598 (0.439–0.757) (0.081)
Equivocal (n = 20) (10.64%)	6 (3.19%)	4 (2.13%)	10 (5.32%)
Negative (n = 148) (78.72%)	18 (9.57%)	26 (13.83%)	104 (55.32%)

CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; NPV, negative predictive value; PPV, positive predictive value; Se, sensitivity; SE, standard error; Sp, specificity.

IFAT was used as a reference method for the serological assessment of E. canis exposure. When IFAT was considered the reference method, ELISA showed a sensitivity of 50% and a specificity of 100% relative to IFAT under the conditions and assumptions of this study, with a positive predictive value of 100%, a negative predictive value of 85.25%, and a diagnostic accuracy of 87.14%. A moderate-to-good agreement was observed between IFAT and ELISA (κ = 0.598) (Table 3).

Sensitivity analyses demonstrated that the classification of equivocal results influenced diagnostic performance estimates. When equivocal results were considered positive, sensitivity, specificity, PPV, and NPV were 57.14%, 91.23%, 70.59%, and 85.25%%, respectively. When considered negative, these values were 42.86%, 100%, 100%, and 82.61%, respectively. These findings indicate that the handling of equivocal results has a substantial impact on diagnostic performance estimates (Table 4).

Table 4.

Sensitivity Analysis of ELISA Diagnostic Performance Under Different Classifications of Equivocal Results

Scenario	TP	FP	TN	FN	Se (%) (95% CI)	Sp (%) (95% CI)	PPV (%) (95% CI)	NPV (%) (95% CI)
Excluding equivocal results	18	0	104	18	50 (32.92–67.08)	100 (96.52–100)	100 (81.47–100)	85.25 (80.65–88.9)
Equivocal considered positive	24	10	104	18	57.14 (41.0–72.3)	91.23 (84.6–95.5)	70.59 (52.5–83.9)	85.25 (77.8–91.0)
Equivocal considered negative	18	0	114	24	42.86 (29.1–57.8)	100 (96.7–100)	100 (82.5–100)	82.61 (75.5–88.1)

ELISA, enzyme-linked immunosorbent assay; FN, false negative; FP, false positive; NPV, negative predictive value; PPV, positive predictive value; Se, sensitivity; Sp, specificity; TN, true negative; TP, true positive.

Distribution of E. canis seropositivity according to various determinants

The distribution of E. canis seropositivity according to various determinants is presented in Table 1. When evaluating the relationship between the determinants and E. canis positivity, only the seropositive samples (n = 18) simultaneously detected by both IFAT and ELISA were considered; hereafter, this definition is referred to as dual-seropositivity. For most variables, some data could not be recorded during the sampling period (labeled as “no record”), and these data were excluded during statistical analysis. However, several of the observed percentages should be interpreted with caution due to very small subgroup sizes, and values such as 100% seropositivity may reflect single-animal observations rather than meaningful epidemiological patterns. In particular, categories represented by only one animal (1/1) are not epidemiologically informative (Table 1).

The relationship between locations, dog breed, pulse rate, respiration rate, body temperature, coat structure, appetite status, and mucous membrane condition with E. canis seropositivity was evaluated using the Fisher–Freeman–Halton exact test. No statistically significant differences in seropositivity were observed for any of these variables (p values ranged from 0.170 to 0.580). The E. canis seropositivity rates for these variables are presented only as percentages. Higher seropositivity was observed in dogs from the Sarıkamış District (66.67%), in Bull Terrier and French Bulldog (each 100%, based on n = 1), in dogs with normal (12–40/min) respiration rates (11.11%), and in dogs with alopecia (100%). Additionally, higher seropositivity was noted in dogs with normal (70–89/min) pulse rates (20%), in dogs with low body temperature (<37.4°C) (18.18%), in dogs with normal appetite (14.58%), and in dogs with icteric mucous membranes (25%) (Table 1).

The relationship between dogs’ age groups and E. canis seropositivity was evaluated using the Fisher–Freeman–Halton exact test, and a statistically significant difference in seropositivity was observed between age groups (p = 0.008). E. canis seropositivity was found to be higher in the older age group (97–120 months) at 33.33%; however, this finding should be interpreted with caution due to the small number of dogs in the oldest age group (n = 6), and no clear linear trend was observed across age categories (Table 1).

The relationship between E. canis seropositivity and the sex of the dogs was evaluated using the Pearson chi-square test. Seropositivity was higher in male dogs (11.01%); however, no statistically significant difference in seropositivity was observed between male and female dogs (p = 0.338) (Table 1).

The relationship between lethargy status and E. canis seropositivity was evaluated using Fisher’s exact test. Although seropositivity was higher in nonlethargic dogs (11.18%), no statistically significant difference in seropositivity was observed between lethargic and nonlethargic dogs (p = 0.361) (Table 1).

The relationship between lymph node status and E. canis seropositivity was evaluated using Fisher’s exact test. Although seropositivity was higher in dogs with lymphadenopathy (11.59%), no statistically significant difference in seropositivity was observed between dogs with lymphadenopathy and those with normal lymph node status (p = 0.740) (Table 1).

Discussion

Canine monocytic ehrlichiosis is often overlooked in diagnosis due to the nonspecific nature of its clinical signs, the potential for the infection to remain subclinical for long periods, and the fact that hematological changes do not manifest prominently in all cases. Especially in endemic regions, the infection can persist even in clinically healthy dogs, making it difficult to assess the true prevalence of E. canis based solely on clinical cases (Sainz et al., 2015). Therefore, seroepidemiological studies are of great importance for accurately determining the prevalence of the agent. Serological diagnostic methods are widely preferred in field studies because they allow the evaluation of acute, chronic, and subclinical stages of infection. In this context, the IFAT, long considered the reference method, and ELISA, due to its practical applicability and suitability for screening purposes, are among the most commonly used diagnostic methods for determining E. canis seroprevalence. Evaluating IFAT and ELISA together reduces the risk of missing infections in diagnosis and contributes to a more accurate estimation of the disease’s true prevalence at the population level (Erdeğer et al., 2003; Beall et al., 2012; Aziz et al., 2022). In the present study, E. canis seropositivity was assessed in a total of 188 dogs, including clinically healthy individuals as well as dogs of different ages, breeds, and sexes with various clinical and paraclinical findings, using IFAT and ELISA. The study revealed the seroprevalence of the agent and its relationships with hematological profiles as well as various demographic and clinical determinants.

Monocytic ehrlichiosis in dogs is of global significance, but it is more commonly observed in subtropical and tropical regions. Reported seropositivity rates in different geographic regions range widely from 0.33% to 92.1% (Gomes dos Santos et al., 2020; Di Bella et al., 2024), indicating that diagnostic methods, vector density, and climatic conditions play a determining role in the epidemiology of the disease. In this study, E. canis seropositivity was found to be 22.34% by IFAT and 10.64% by ELISA. These results are not only within the range of global seroprevalence values but are also consistent with seroprevalence rates reported in various regions of Türkiye (0–69.4%) (Cihan et al., 2010; Demir and Aktaş, 2020). However, as this hospital-based sample may not represent the general dog population, the findings should not be interpreted as true regional seroprevalence. Within this context, the results suggest that E. canis seropositivity is common in the Kars region, as in other parts of the country, and that dogs in areas with a high prevalence of Rhipicephalus sanguineus are at an increased lifetime risk of exposure to the infection (Potkonjak et al., 2013; Vieira et al., 2013; Checa et al., 2024).

IFAT is an established and widely used reference method for the serological diagnosis of E. canis exposure, particularly due to its ability to detect low antibody titers in subclinical or chronic infections (Waner et al., 2000; Bélanger et al., 2002; Ferrero et al., 2025). Although IFAT is widely used as a reference method, it has inherent limitations, including subjective interpretation of fluorescence, potential cross-reactivity, interobserver variability, and the inability to distinguish between current and past infections. Therefore, the diagnostic performance metrics of ELISA reported in this study should be interpreted as relative to IFAT rather than as absolute measures of sensitivity and specificity. In contrast, recently developed recombinant antigen-based ELISA tests can achieve high sensitivity (over 92%) and specificity (over 95%) compared with the IFAT reference, making them practical and reliable serological screening tools in the field (Ferrero et al., 2025; Shepherdson et al., 2026). The lower sensitivity of ELISA observed in our study (50%) may be explained by factors such as the antigen composition of the commercial kit used, the cutoff value of the test, and differing antibody levels depending on the stage of infection. This is further supported by the moderate agreement between IFAT and ELISA (κ = 0.598) and the marked variability in sensitivity estimates depending on the classification of equivocal results (42.86–57.14%). This variability has also been reported in previous studies demonstrating the effect of antigen selection on ELISA performance (Oliveira et al., 2015; Ferrero et al., 2025). These findings support the literature consensus that evaluating IFAT and ELISA together increases diagnostic accuracy compared with using a single serological test (Erdeğer et al., 2003; Waner., 2022; Ferrero et al., 2025). Therefore, in our study, when assessing the relationship between E. canis seropositivity and hematological, demographic, and clinical findings, a combined approach was adopted in which only samples simultaneously positive by both IFAT and ELISA (n = 18, 9.57%) were considered. This dual-seropositivity criterion likely increased the specificity of case classification and reduced the likelihood of false-positive cases; however, it may have reduced sensitivity by excluding animals positive by only one assay. Moreover, this stringent approach substantially reduced the number of positive cases included in the analysis, which may have limited statistical power and led to potential underestimation of associations between E. canis seropositivity and the evaluated determinants. Similarly, the exclusion of equivocal ELISA results in the primary analysis may have further contributed to an overestimation of diagnostic performance, particularly specificity and predictive values. Sensitivity analyses confirmed that different classifications of equivocal results influenced these estimates, highlighting the importance of transparent reporting and cautious interpretation.

When evaluated in terms of hematological parameters, no statistically significant differences were observed between E. canis seropositive and non-dual-seropositive group, and the hematological values obtained in our study generally remained within normal reference ranges (Table 2). These findings are consistent with the fact that most seropositive dogs did not exhibit acute-phase disease. In the acute stage of E. canis infection, commonly observed changes such as thrombocytopenia, leukopenia, and anemia are typically absent in subclinical or chronic stages. This supports previous studies, in both experimental and natural infection models, reporting that hematological alterations in subclinical ehrlichiosis may be mild or virtually absent (Nakaghi et al., 2008; Yağcı et al., 2010; Fonseca et al., 2017; Singh et al., 2022). In these studies, it is emphasized that hematological parameters alone are not diagnostic in clinically healthy seropositive dogs, serving only as epidemiological indicators and highlighting the risk of overlooking the infection. The clinical stability of most seropositive dogs in our study may explain the lack of pronounced hematological differences. Furthermore, in such infections, the presence of concurrent parasitic or bacterial infections cannot be excluded, which may increase variability in hematological parameters and result in individual differences. Therefore, interpreting hematological findings alone as indicative of E. canis infection is not recommended and should be approached with caution.

Analyzing demographic and clinical variables together in tick-borne diseases is important for identifying potential risk groups and supporting preventive veterinary strategies. In the present study, a proportion of seropositive dogs were clinically apparently healthy, which may suggest subclinical exposure to E. canis; however, in the absence of a clear clinical case definition and molecular confirmation, this finding should be interpreted with caution. Regarding breed, it is known that all dog breeds are susceptible to monocytic ehrlichiosis. Nevertheless, some studies have reported that, likely due to immunological factors, German Shepherd Dogs, Labrador Retrievers, and mixed-breed dogs may exhibit a higher prevalence of infection and may be at a disadvantage in terms of clinical severity and prognosis (Risheen et al., 2022; Kumar et al., 2023; Gurung et al., 2025; Topal et al., 2025). In the present study, E. canis seropositivity was observed at 100% in Bull Terrier and French Bulldog breeds; however, as only one individual from each of these breeds was included in the study, these high rates cannot be generalized statistically or epidemiologically. One of the important findings of our study is the statistically significant association between age groups and E. canis seropositivity. Higher seropositivity in older dogs aligns with increased lifetime exposure to ticks and the tendency of the infection to progress to subclinical or chronic forms. Current epidemiological studies indicate that seropositivity to tick-borne pathogens, including E. canis, significantly increases in older dogs, reflecting cumulative exposure (Singh et al., 2022; Kumar et al., 2023). Although a higher seropositivity rate was observed in male dogs (11.01%), no statistically significant association between sex and seropositivity was found, which is largely consistent with the literature (Bogićević et al., 2017; Van Hai and Khuong, 2021; Shepherdson et al., 2026). While some studies have reported higher seropositivity rates in males (Risheen et al., 2022; Singh et al., 2022; Kumar et al., 2023), this is more likely explained by behavioral factors, such as greater roaming and increased likelihood of tick exposure in male dogs, rather than a biological predisposition. Overall, the analysis of demographic and clinical determinants is limited by small subgroup sizes, missing data, and multiple comparisons. Some categories included very few animals, and proportions such as 100% seropositivity in single-animal groups are not epidemiologically meaningful. Although a statistically significant association with age was observed, the oldest age group included only six dogs and no clear linear trend was evident. Therefore, these findings should be interpreted with caution.

In our study, the lack of a significant association between seropositivity and clinical variables such as vital signs, mucous membrane status, lethargy, and lymphadenopathy supports the notion that E. canis infection exhibits a heterogeneous clinical course. The literature reports that a substantial proportion of infected dogs may be completely asymptomatic, and that clinical signs can vary widely depending on the stage of infection, the host’s immune response, and the presence of concurrent infections (Fonseca et al., 2017; Cabrera-Jaramillo et al., 2022; Shepherdson et al., 2026). Furthermore, the inability to achieve statistical significance in some subgroups despite high seropositivity rates may be related to the limited sample size. Nevertheless, this finding highlights the importance of not overlooking dogs that are clinically healthy but at risk, from a diagnostic perspective.

In conclusion, this study demonstrates that E. canis seroprevalence is notable (22.34% by IFAT and 10.64% by ELISA) in a hospital-based sample of dogs from the Kars region, and that seroreactivity cannot be excluded based solely on clinical or hematological findings. Therefore, the results should be interpreted as evidence of seroreactivity rather than definitive proof of ongoing infection. The combined use of IFAT and ELISA represents a more conservative approach to defining seropositivity rather than evidence of improved diagnostic accuracy, particularly in identifying seroreactive animals (9.57% in the present study). The identification of age as an important risk factor underscores the need for regular serological screening in older dogs. Future long-term studies with larger sample sizes, including molecular diagnostic methods, are expected to provide more comprehensive insights into the epidemiology, pathogenesis, and clinical course of E. canis exposure.

Authors’ Contributions

F.B., E.E.E., and Y.E.: Conceptualization, methodology, and writing—original draft. Y.E., G.Ö., E.B., Y.U.B., and S.G.: Data collection, investigation, and formal analysis. F.B., Y.E., G.Ö., E.E.E., E.B., Ö.Ç., Y.U.B., S.G., and A.G.S.: Visualization and writing—review and editing. F.B., E.E.E., Ö.Ç., and A.G.S.: Supervision, project administration, and funding acquisition.

Study Limitations

This study has several limitations, including the lack of information on important epidemiological variables such as tick infestation status, ectoparasiticide use, ownership conditions, outdoor exposure, previous treatment, and potential coinfections. These unmeasured factors may have influenced the observed seroprevalence and associated risk factors. Therefore, the findings should be interpreted with caution, as residual confounding cannot be excluded.

Ethical Approval

Ethical approval for the study was obtained from the Kafkas University Animal Experiments Local Ethics Committee (KAÜ-HADYEK) with decision number 009, dated January 15, 2023.

Footnotes

Author Disclosure Statement

The authors do not have any conflicts of interest to declare.

Funding Information

This study was supported by the Scientific Research Projects Coordination Unit of Kafkas University (Türkiye) under the project code “KAÜ-BAP 2023-TS-50.”

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