Seroprevalence of Canine Leishmaniasis and the Public Health Risk of Asymptomatic Dogs in Baringo County, Kenya: Implication for an Integrated One Health Surveillance

1. Introduction

Leishmaniasis is a neglected tropical disease (NTD) caused by protozoan parasites of the genus Leishmania. It is transmitted by the bites of infected female phlebotomine sand flies, specifically Phlebotomus species in the Old World and Lutzomyia species in the New World [1,2]. Transmission involves complex zoonotic and anthroponotic cycles with mammalian hosts and sand fly vectors [3,4,5,6]. Leishmaniasis continues to pose a significant global public health challenge in Mediterranean, tropical, and subtropical regions.

Clinical manifestations of leishmaniasis include cutaneous, mucocutaneous, and visceral forms. Visceral leishmaniasis (VL), also known as Kala-azar, represents the most severe form and may progress to post-kala-azar dermal leishmaniasis (PKDL). Kala-azar is characterized by systemic dissemination of the parasite to vital organs such as the spleen, liver, and bone marrow, and can be fatal if left untreated [7,8,9].

The epidemiology of visceral leishmaniasis (VL) is largely determined by the reservoir host involved in transmission. Anthroponotic visceral leishmaniasis (AVL), primarily caused by L. donovani, has humans as the primary reservoir. In contrast, zoonotic visceral leishmaniasis is associated with domestic and wild canids, which serve as the principal reservoirs for L. infantum infection. Domestic dogs (Canis familiaris) are recognized globally as the primary reservoirs due to their close association with humans and frequent exposure to infected sand flies. Stray and free-roaming dogs play a particularly significant role in sustaining transmission cycles because they often lack regular veterinary care and preventive interventions [2,10].

Canine leishmaniasis (CanL) is a chronic systemic disease characterized by a spectrum of clinical manifestations, ranging from asymptomatic infection to severe illness. Common clinical signs include weight loss, lymphadenopathy, splenomegaly, anemia, epistaxis, alopecia, and various dermatological lesions [5,7,11]. Notably, a substantial proportion of infected dogs may remain asymptomatic while harboring significant parasite loads in the skin, thereby serving as sources of infection for sand fly vectors and facilitating ongoing transmission. This situation poses a significant epidemiological challenge, especially in endemic regions where asymptomatic dogs function as silent reservoirs for human infection [12,13,14]. Although other animals, including cats, horses, and wild canids, may also harbor Leishmania parasites, their definitive role in transmission remains uncertain [9,15].

CanL has been documented in over 50 countries and is increasingly recognized as a significant veterinary and public health issue. Disease transmission is influenced by factors such as vector abundance, environmental conditions, host susceptibility, canine behavior, and preventive measures. Climate change, urbanization, and increased movement of humans and animals have contributed to the spread of leishmaniasis into new regions by altering the distribution of sand fly vectors [5,6,11]. This geographic expansion presents a substantial threat to public health systems, particularly affecting vulnerable groups including children, the elderly, and immunocompromised individuals, who are at the greatest risk for severe disease progression [7,16].

The seroprevalence of canine leishmaniasis varies substantially across endemic regions, with reported rates ranging from 2% to over 30%, influenced by ecological and climatic conditions [17]. Identified risk factors for canine infection include the extent of exposure to sand flies, housing conditions, the dog’s age, and owners’ implementation of preventive measures [6,17,18].

Effective control of canine leishmaniasis remains challenging despite interventions such as insecticide-treated collars, vaccination, reservoir management, and dog culling. These difficulties are primarily attributed to vector adaptability, limitations in available treatments, and the persistence of asymptomatic infections. As many infected dogs do not exhibit clinical signs, laboratory-based diagnosis is critical [5]. ELISA demonstrates high sensitivity and is appropriate for large-scale surveillance. In contrast, rapid immunochromatographic tests offer prompt field-based results but exhibit lower sensitivity, particularly in asymptomatic cases. Given that no diagnostic test achieves perfect accuracy, Bayesian Latent Class Analysis is recommended to enhance diagnostic evaluation and reduce bias [19].

In East Africa, particularly in Sudan, Ethiopia, and Kenya, VL has traditionally been attributed to L. donovani. However, recent evidence indicates that dogs may also serve as significant reservoir hosts in disease transmission [20]. In Kenya, particularly within Baringo County, the prevalence and epidemiological significance of L. infantum infection in dogs are not well characterized. Climatic changes, alterations in vector ecology, and increasing dog populations may further affect transmission dynamics. The present study aims to address key knowledge gaps concerning the role of dogs as reservoir hosts in endemic, resource-limited pastoral communities of Baringo South [1,21].

This study aimed to determine the seroprevalence of CanL, the potential risk factors associated with infection and to evaluate the potential role of domestic dogs as reservoirs of Leishmania infection in Baringo County, Kenya. The findings are expected to provide epidemiological data to inform integrated One Health strategies for the prevention and control of human and canine leishmaniasis.

2. Materials and Methods

2.1. Description of the Study Area

The study was conducted in Baringo County, Kenya, which was purposively selected because it is a major endemic area for both VL and CL [1]. Baringo is situated approximately 250 km northwest of Nairobi, at an altitude of 1,067 meters above sea level, and is characterized by a semi-arid climate. The region experiences high annual temperatures averaging 32.8 ± 1.6 °C and receives an average annual rainfall of 512 mm (ranging from 300 to 700 mm), distributed across two distinct rainy seasons. Baringo County covers approximately 11,075 km² with an estimated human popu-lation of 555,561 (KNBS census 2009)[22].

The county contains diverse ecological niches that support the breeding and survival of sand fly vectors, particularly P. martini and P. duboscqi. These habitats include termite mounds, animal burrows, rock crevices, and dense vegetation such as Acacia and Prosopis juliflora [23]. Traditional dwellings constructed with mud-plastered or stick walls and grass-thatched roofs are also prevalent. Cracks and crevices within these structures provide suitable resting sites for vectors and have been significantly associated with increased leishmaniasis transmission [21,23,24].

The population primarily engages in pastoralism, mixed farming, and livestock keeping, activities that frequently involve extended periods outdoors and close contact with animals [21]. Pastoralism has been identified as a significant socioeconomic risk factor for leishmaniasis in the region. Additionally, cultural practices such as sleeping outdoors and limited community awareness of vector-borne transmission further increase vulnerability to infection [1,21].

Baringo County share boundaries with the following counties: Turkana and West Pokot to the north, Samburu and Laikipia to the east, Nakuru and Kericho to the south, Uasin Gishu to the southeast, and Elgeyo Marakwet to the west. Administratively, the county is divided into seven sub-counties: Baringo South, Baringo Central, Baringo North, Mogotio, Eldama Ravine and Tiaty. (Figure 1).

2.2. Study Design and Selection of Study Sites

A cross-sectional study was conducted in Baringo South and Tiaty Sub-counties to provide a snapshot of the prevalence of visceral leishmaniasis prevalence and to assess the distribution of infection in dogs. This methodology is commonly employed in endemic regions to estimate disease prevalence and identify potential reservoirs [25]. The elevated prevalence in the human population within the study area establishes a critical baseline for investigating the role of domestic dogs as reservoirs that maintain transmission cycles in this region [26].

Baringo South and Tiaty sub-counties were purposively selected due to their high documented human leishmaniasis seroprevalence. Site selection further considered logistical feasibility, including accessibility and the need to maintain adequate security considering regional tribal conflicts and cattle rustling, to ensure safe access to affected populations [27]. Within Baringo South sub-county, four locations—Marigat, Kimalel, Sandai, and Kapkuikui—were purposively selected based on their epidemiological significance. Marigat was identified as a high burden leishmaniasis hotspot [23], while Kimalel was selected from earlier fining of the established center that acted as a primary regional treatment and research center [27]. Sandai and Kapkuikui were chosen due to their endemic lowland ecology and proximity to villages with high documented human seroprevalence [25].

In Tiaty sub-county, Chemolingot has been recognized as a well-documented endemic focus of human VL. Epidemiological surveillance and laboratory records from Chemolingot Sub-County Hospital consistently demonstrate a substantial disease burden in this area [25,26]. Additionally, a study of Chemolingot residents found a high positivity rate among symptomatic individuals, with 39.6% testing positive using rK39 antigen kits.

2.2.1. Sampling Strategy

A multistage random sampling technique was employed to select study participants. Initially, sub-locations within Baringo South were identified and stratified into four geographical zones according to ecological and epidemiological characteristics relevant to Leishmania transmission. Villages were then selected through a simple randomization procedure. The choice of specific study sites was guided by human leishmaniasis case records from Kimalel Health Centre, Marigat Sub-County Hospital, and Chemolingot Sub-County Hospital. One sub-location from each target area—Rabai, Sabor, Mbechot, Kapkuikui, and Chemolingot—was included based on the reported incidence of leishmaniasis.

2.2.2. Household Selection

A sampling frame was constructed using household registers obtained from village elders, local authorities, and the Kenya National Bureau of Statistics. Eligible households were selected through simple random sampling with computer-generated random numbers. Due to challenges in obtaining an accurate list of all dogs, a multi-stage cluster sampling approach was implemented to ensure effective representation of dog samples by randomly selecting villages, followed by households or dogs within those villages [28]. Community sensitization and mobilization activities were conducted by the local administrators and community health workers. A written informed consent was obtained from all participants and dog owners before enrollment.

2.3. Inclusion Criteria

Households were eligible for inclusion only if both residents and their dogs had resided in the study area for at least 1 year, thereby ensuring that detected infections reflected local transmission. All available dogs in selected households were enrolled, underwent clinical examination for signs of canine leishmaniasis, including lymphadenopathy, skin lesions, and weight loss, and had blood samples aseptically collected for serological testing.

2.4. Sample Size Determination

The sample size (N) for canine sampling was determined using the formula for estimating prevalence in cross-sectional study described by Dohoo et al. (2003) based on seroprevalence of 21.3% from a similar study in Egypt[29].

$n={\displaystyle \frac{Z^{2}pq}{e^2}}$A 7% margin of error was selected instead of the standard 5% to accommodate logistical challenges, low population density, and the mobile nature of pastoralist populations in Baringo South sub-county [21,25].

Where:

(n) = required sample size

(Z) = 1.96, standard normal deviate at 95% confidence interval

(p) = 0.213 was the baseline prevalence

(q = 1-p)

(L) = desired precision (0.07)

Using these parameters, the calculated sample size was 131 dogs, but we ended up collecting samples from 140 dogs.

2.5. Ethical Considerations

Ethical approval for this study was obtained from the Ethical Committee for Animal Experimentation of the Faculty of Veterinary Medicine, University of Nairobi (REF: FVM BAUEC/2024/582). All animal handling procedures, clinical examination, and biological sample collection were conducted in accordance with relevant institutional guidelines and regulations. Informed consent was obtained from dog owners prior to sample collection and questionnaire data collection.

2.6. Data Collection and Variables

A structured questionnaire was also administered to dog owners to collect data on relevant epidemiological risk factors associated with canine leishmaniasis. Data collected included demographics characteristic of dogs, such as geographical location, age, sex, and breed. In the absence of birth records for most dogs, age was estimated using dental formula, ocular, and musculoskeletal indicators. Information on management and environmental factors including housing conditions (e.g., indoor housing, outdoor confinement, or earthen-floor shelters), the veterinary care such as use of protective measures against vector-borne diseases like insecticides, and the primary reason for keeping the dog (e.g., hunting, livestock herding, companion or security). Additionally, the dogs were also examined for clinical signs suggestive of canine visceral leishmaniasis such as lymphadenopathy, dermatology lesions, alopecia, weight loss and other relevant clinical manifestations and data of each dog were recorded.

2.7. Blood Sample Collection and Processing

Blood samples were collected from restrained dogs using aseptic techniques and appropriate personal protective equipment (PPE). Approximately 5 mL of blood was drawn from the cephalic or lateral saphenous vein with sterile Vacutainer needles following disinfection with 70% isopropyl alcohol by a qualified veterinarian. Samples were labeled and transported under refrigerated conditions to the Baringo South Veterinary Laboratory. Blood samples were allowed to clot at room temperature to ensure complete coagulation prior to centrifugation, thereby preventing latent fibrin formation in the serum. Following clotting, samples were centrifuged for 10 minutes at 2,000-3,000 rpm to obtain serum for rapid tests and ELISA. Serum aliquots were transferred into cryovials and stored at −20 °C. Subsequently, serum samples were transported to the Kenya Medical Research Institute (KEMRI) for serological testing. All procedures for handling, storage, and transportation adhered to established quality assurance and biosafety protocols.

2.7.1. Rapid Diagnostic Testing (rK39 RDT)

Canine serum samples were initially screened for the presence of anti-Leishmania antibodies using the rapid diagnostic test (RDT) for IgG or IgM, following the manufacturer’s instructions Speed Leish K®. The rK39 immunochromatographic assay is commonly used for the serodiagnosis of VL due to its simplicity, rapid turnaround time, and high diagnostic accuracy. The test used in this study demonstrated a reported diagnostic sensitivity of 82.4% and specificity of 99%.

In brief, the required volume of serum was applied to the sample well cassette, followed by the addition of assay buffer. Results were interpreted within the manufacturer-specified timeframe. Samples displaying both control and test lines were classified as positive for anti-Leishmania antibodies, while those showing only the control line were classified as negative. Tests without a visible control line were deemed invalid and repeated. All samples that tested positive on the rK39 RDT were aliquoted and stored at −80 °C for subsequent confirmatory analysis by ELISA.

2.7.2. Serological Analysis Using Indirect ELISA

Anti-Leishmania antibodies in canine serum samples were detected using the VetLine Leishmania, Ref. LEIVT0310, Gold Standard Diagnostics, Germany following the manufacturer’s instructions. This indirect enzyme-linked immunosorbent assay enables qualitative detection of antibodies against Leishmania spp. in canine serum samples. The samples, along with positive, negative, and cut-off controls supplied with the kit, were added to a 96-well microtiter plate pre-coated with Leishmania antigen. The plates were incubated under manufacturer-specified conditions to facilitate binding of Leishmania-specific antibodies in the samples to the immobilized antigens.

After incubation, wells were washed to remove unbound material, and enzyme-conjugated anti-canine immunoglobulin was added. Following a second incubation and wash, tetramethylbenzidine (TMB) substrate solution was introduced, producing a colorimetric reaction proportional to the amount of antibody bound. The reaction was terminated with the stop solution provided in the kit, and optical density (OD) values were measured at 450 nm using a microplate ELISA reader, as recommended by the manufacturer.

Results were interpreted according to the manufacturer’s criteria, utilizing the calculated cut-off value and control performance parameters. Samples with OD values exceeding the established cut-off were classified as positive, while those below the threshold were classified as negative. Quality control was confirmed by verifying that all positive, negative, and cut-off controls met the manufacturer’s acceptance criteria prior to interpreting sample results.

The VetLine Leishmania ELISA demonstrates a reported diagnostic sensitivity of 95.8% (95% CI: 90.47–98.62%) and a diagnostic specificity of 95.43% (95% CI: 91.19–98.01%) for anti-Leishmania antibodies in canine samples. Positive control sera were sourced from dogs previously confirmed to be infected with Leishmania spp. through parasitological, serological, molecular, and clinical diagnostic methods. Sera from dogs originating from non-endemic areas served as negative controls.

2.8. Data Management and Statistical Analysis

Data were entered, cleaned, coded, and validated prior to analysis. Statistical analyses were performed using Stata version 14 (Stata Corp LLC, College Station, TX, USA). Data completeness was assessed through descriptive checks and missing-value analysis. Descriptive statistics were used to summarize the characteristics of the study population. Continuous variables, including age, were summarized using measures of central tendency and dispersion, while categorical variables were presented as frequencies and percentages.[30] Dog age was subsequently categorized into three groups: young (≤1 year), mature (>1 to <5 years), and old (≥5 years) [10].

The prevalence of Leishmania infection was estimated separately for the rK39 rapid diagnostic test (RDT) and ELISA by calculating the proportion of positive dogs among the total number of animals tested. Corresponding 95% confidence intervals (95% CI) were computed [29,30]. Associations between infection status and potential risk factors, including sex, age category, housing system, purpose of keeping dogs, breed, clinical status, veterinary care such as use of ecto-parasite use, and geographical origin, were initially assessed using contingency table analyses [33]. Pearson’s chi-square test or Fisher’s exact test, where appropriate, was used to evaluate differences in prevalence between categories [34].

A stepwise logistic regression procedure with a significance level for entry of p < 0.20 was employed to identify the most parsimonious model and determine independent predictors of ELISA positivity [35]. Model performance was evaluated using the likelihood ratio test, and goodness-of-fit was assessed using the Hosmer Lemeshow test [36]. The discriminatory ability of the final model was evaluated through receiver operating characteristic (ROC) curve analysis, and classification accuracy was assessed using classification tables [37]. Agreement between the rK39 RDT and ELISA results was evaluated using Cohen’s kappa (κ) statistic, complemented by cross-tabulation analyses [31,36]. All statistical tests were two-sided, and statistical significance was defined at p < 0.05.

3. Results

3.1. Potential Risk Factors for Leishmania Infection.

The descriptive characteristic of the study population including origin, age, sex, housing, reason for keeping dogs, clinical status, veterinary care, use of insecticide, breed, are summarized in Table 1. A total of 140 dogs were enrolled and sampled during the study. Most of the dogs originated from Sabor (32.14%), followed by Mbechot (28.57%), Rabai (19.29%), Chemolingot (11.43%) and Kapkuikui 12(6.57%).

3.2. Prevalence and Risk Factors Associated with Leishmania Seroprevalence

The serological tests confirmed active exposure of dogs to L. infantum infection in Baringo County. The result from both rK39 RDT and ELISA tests demonstrated that there was an ongoing exposure of dogs to Leishmania spp. In Baringo County. In fact, the overall seroprevalence of canine leishmaniasis determined using the rK39 RDT was 7.79%; [95% CI: 4.38–13.70]. Using the ELISA the estimated seroprevalence was 12.14% [95% CI: 7.65–18.73].

Bivariate analysis using Fisher’s exact with ELISA test results identified several factors associated with Leishmania seropositivity among dogs in Baringo South Sub-county (Table 2). Among the variables examined, geographical origin, clinical status, breed, and housing conditions were significantly associated with seropositivity, whereas sex, age, vaccination status, insecticide use, and the primary reason for keeping dogs were not. Although a greater proportion of seropositive dogs were males (82.4%) than females (17.6%), the association between sex and seropositivity was not statistically significant (p = 0.560).

Similarly, seropositivity tended to increase with age, with older dogs accounting for 41.2% of positive cases, followed by mature (35.2%) and young dogs (23.5%); however, these differences were not statistically significant (p = 0.760). A significant association was observed between geographical origin and seropositivity (p = 0.005). The highest proportion of seropositive dogs originated from Mbechot (41.2%), followed by Rabai (29.4%) and Kapkuikui (23.5%), whereas only one positive dog was detected in Sabor (5.9%) and none in Chemolingot. Clinical status was strongly associated with infection (p < 0.001). Dogs presenting with emaciation accounted for nearly half (47.1%) of all seropositive animals, while alopecia and diarrhea were observed in 11.8% and 5.9% of positive dogs, respectively. In contrast, 35.3% of seropositive dogs did not exhibit any apparent clinical signs, indicating the occurrence of asymptomatic infections within the canine population. Breed was also significantly associated with seropositivity (p = 0.010). Local breeds accounted for 76.5% of seropositive dogs, compared with 23.5% among mixed breeds of dogs. Housing conditions significantly influenced seropositivity (p = 0.042). Most seropositive dogs were free roaming (88.2%), whereas only 5.9% of positive dogs were housed in kennels and 5.9% were kept within household compounds. No seropositive dogs were detected among those housed in animal sheds. The higher infection frequency among roaming dogs may reflect increased exposure to sand fly vectors and contact with infected reservoir hosts. Although most seropositive dogs lacked veterinary care such as use of insecticide treatment (94.1%), neither the dogs with proper veterinary care nor without were significantly associated with seropositivity (p = 1.00 for both variables).

Likewise, dogs kept for hunting (58.8%) and grazing (29.4%) appeared to have higher seropositivity than those kept as pets or for security purposes (5.9% each), but these differences were not statistically significant (p = 0.397). Overall, geographical origin, clinical status, breed, and housing system emerged as important factors associated with Leishmania seropositivity at the bivariate level and were therefore considered candidates for inclusion in subsequent multivariable analyses. (Table 2)

3.3. Multivariable Logistic Regression for Risk Factors Associated with Leishmania Infection in Dogs

A stepwise multivariable logistic regression model was fitted to identify independent predictors of Leishmania seropositivity among dogs. The final model included clinical status, geographical origin, housing conditions, breed, and vaccination status. Overall, the model was statistically significant (Likelihood Ratio χ² = 28.12, df = 5, p < 0.001), indicating that the included variables collectively contributed to explaining variation in ELISA seropositivity. The model yielded a pseudo-R² of 0.294, suggesting a moderate fit and indicating that approximately 29.4% of the variation in infection status was accounted for by the variables included in the model.

There was a close association between clinical disease and Leishmania seropositivity. In fact, clinical status emerged as a strong independent predictor of infection. Dogs without clinical signs suggestive of canine leishmaniasis had significantly lower odds of ELISA seropositivity compared with dogs presenting clinical manifestations (OR = 0.06, 95% CI: 0.014–0.289, p < 0.001). Specifically, asymptomatic dogs had approximately 94% lower odds of infection than clinically affected dogs.

Breed was also significantly associated with infection status. Mongrel dogs exhibited substantially higher odds of seropositivity compared with local breeds (OR = 11.92, 95% CI: 1.79–79.37, p = 0.010). This finding indicated that mongrel dogs were nearly 12 times more likely to be ELISA-positive than local breeds. However, the relatively wide confidence interval suggests limited precision of the estimate, likely due to the small number of seropositive animals within some breed categories.

Geographical origin showed a tendency toward association with infection risk. Dogs originating from Rabai had approximately 4.6-fold higher odds of seropositivity compared with those from the reference location (Kapkuikui) (OR = 4.60, 95% CI: 0.78–27.15, p = 0.092). Although suggestive of increased risk, the association did not reach statistical significance because the confidence interval included unity.

Similarly, dogs housed within household compounds had lower odds of seropositivity than free-roaming dogs (OR = 0.13, 95% CI: 0.01–1.51, p = 0.103), indicating a possible protective effect of confinement. Nevertheless, this association was not statistically significant after adjustment for other covariates.

Veterinary care was not significantly associated with Leishmania seropositivity. Dogs with proper veterinary care showed lower odds of infection than those without (OR = 0.12, 95% CI: 0.005–2.69, p = 0.181), but the wide confidence interval and non-significant p-value indicate insufficient evidence to support a protective effect in this study population. (Table 3)

3.4. Area Under a Curve After Logistic Model with ELISA Positive

The logistic regression model demonstrated excellent discriminatory performance, with an area under the ROC curve (AUC) of 0.85, indicating good ability to distinguish ELISA-positive from ELISA-negative dogs.

3.5. Agreement Between RDT Positive and ELISA Positive

There was strong concordance observed between the two diagnostic methods (κ = 0.763, p < 0.001. The overall observed agreement between the tests was 95.7%. Cross-tabulation of the results revealed that 123 dogs were negative by both RDT and ELISA, while 11 dogs were positive by both methods. Six dogs tested negative by RDT but positive by ELISA, whereas no dogs were positive by RDT and negative by ELISA. These findings indicated that the RDT produced no false-positive results but failed to identify a proportion of ELISA-positive animals. Using ELISA as the reference test, the RDT demonstrated a sensitivity of 64.7% and a specificity of 100%. The positive predictive value was 100%, showing that all dogs testing positive by RDT were confirmed positive by ELISA. The negative predictive value was 95.3%, indicating that most RDT-negative dogs were truly negative. However, the occurrence of six false-negative results suggests that the RDT failed to detect approximately one-third of ELISA-positive dogs.

Overall, the rK39 RDT exhibited excellent specificity and positive predictive value, together with substantial agreement with ELISA. These findings suggest that a positive RDT result is highly reliable for identifying Leishmania-seropositive dogs. However, because of its moderate sensitivity, a negative RDT result should be interpreted cautiously, and confirmatory testing using more sensitive serological assays such as ELISA may be warranted in epidemiological surveys and surveillance programs

4. Discussion

This research presents an epidemiological assessment of canine leishmaniasis and its associated risk factors in Baringo County, Kenya, emphasizing the potential role of dogs in sustaining Leishmania transmission within this endemic region. The study identified an overall seroprevalence of 7.79% using Rapid Diagnostic Test and 12.14% using ELISA. These findings highlight that seropositive dogs serve as primary reservoirs, maintaining parasite circulation and facilitating sand fly infection, irrespective of the presence of clinical disease [6].

The seroprevalence observed in this study is consistent with findings from several endemic regions worldwide. For example, a serological survey in Zambia reported seropositivity rates of up to 17% using L. donovani Soluble Antigen ELISA and rK39 ELISA [9].Comparable results have been documented in other studies employing serological methods such as the Direct Agglutination Test (DAT), Indirect Fluorescent Antibody Test (IFAT), or ELISA. The ELISA technique detects specific antibodies, primarily Immunoglobulin G and its subclasses, produced by the canine immune system in response to Leishmania antigens [12,15,17].Comparable seroprevalence rates have been reported in several regions, including 12.5% in Portugal using the Direct Agglutination Test [17], 12% in Bangladesh with DAT[39], 14.2% in Turkey via the Indirect Fluorescent Antibody Test [40], 15.4% in Sardinia, Italy, using IFAT [41], and 16.7% in Palestine using ELISA [42]. Other studies indicate varying endemicity, such as 9.9% in northeastern Portugal by ELISA and IFAT [8], an unspecified rate in France using an in-house ELISA[12], 21.3% among asymptomatic dogs in Northern Egypt using ELISA [29], up to 36.79% in China using ELISA [15] and 84% in Ethiopia using ELISA [10]. These differences in seropositivity across geographical regions are primarily attributed to ecological factors, vector abundance, and climatic conditions that affect vector breeding and dog exposure [41].

The variation in seroprevalence observed across the five study areas—Mbechot (41.2%), Rabai (29.4%), Kapkuikui (23%), Sabor (5.9%), and Chemolingot (0%)—indicates an uneven distribution of transmission intensity within Baringo County. This pattern aligns with recent findings from Ethiopia, where seroprevalence varied significantly among Weliso (79.82%), Ambo (76.11%), and Ejaji (93.84%), suggesting the influence of localized environmental factors on infection rates [10]. Evidence from Zambia also identifies specific areas as active transmission hotspots, likely due to environmental suitability for sand fly breeding and increased vector-dog contact [9]. In Palestine, domestic dogs in districts such as Hebron and Jenin serve as primary reservoirs for human VL, further underscoring the importance of geographical hotspots in disease persistence [42]. Spatial heterogeneity in transmission is frequently associated with rural or peri-domestic conditions, including the presence of sheep pens and accumulation of organic waste, which promote sand fly proliferation [17].

A principal finding of this study was that, although seroprevalence was significantly higher among dogs exhibiting clinical signs, a substantial proportion of the infected population remained asymptomatic. In canine leishmaniasis, IgG2 is typically associated with a protective or asymptomatic immune response, while IgG1 is linked to increased susceptibility and clinical disease. Elevated IgG1 levels are frequently observed in dogs presenting with weight loss, alopecia, and lymphadenopathy. In chronic infections, both IgG1 and IgG2 levels may remain elevated, which supports their utility as biomarkers for monitoring disease progression [43]. These findings align with studies from Tunisia [44], Italy [41]where the majority of infected local dogs (up to 80%) exhibited no overt symptoms. Comparable results have been reported in Palestine [42], and Brazil [45], where only a minority of infected dogs displayed clinical signs and most remained asymptomatic. Conversely, a study in Zambia reported that many seropositive dogs did not show obvious clinical signs of leishmaniasis, indicating the presence of hidden or subclinical infections [9]. These subclinical infections are epidemiologically important because asymptomatic dogs can transmit Leishmania to sand flies, thereby enabling the silent circulation of the parasite within the canine population [6,34]. Furthermore, subclinical cases may progress to clinical disease if the animal becomes immunosuppressed or develops concurrent infections [13]. This is critical for managing reservoir populations, as asymptomatic but persistently seropositive dogs still serve as sources of infection for sand flies.

Higher seropositivity was linked to dogs that were not housed or lived in shelters with earthen floors. This was in agreement with a study in Egypt [29], where earthen floors were associated with increased exposure because they provide a conducive environment for sand fly breeding through the accumulation of organic matter. Similarly, in Sardinia, Italy, the lack of nocturnal refuges (dogs spending the night outdoors) was identified as a risk factor for exposure [41].

Male dogs in this study exhibited higher seroprevalence compared to females, although this association was not statistically significant. This finding aligns with previous reports from Egypt [29], Italy [46], and Brazil [30,37]. The observed pattern may be attributed to behavioral differences, particularly increased roaming among males due to territorial and reproductive behaviors, which could elevate exposure to infected vectors.

Older dogs exhibited higher seropositivity compared to younger animals, consistent with findings from studies in Portugal [8], Bangladesh [4], Egypt [29], Palestine [42], and Italy [41]. This increased seropositivity in older dogs is likely attributable to cumulative exposure to infected sandflies over time, resulting from longer periods spent outdoors. Furthermore, older dogs may develop more robust immune responses due to prolonged exposure, and asymptomatic individuals can remain infectious for years [46]. Higher cumulative exposure in older dogs leads to more persistent and elevated antibody titers, with a significant positive correlation between age and anti-Leishmania antibody levels [48]. Additionally, older dogs are more likely to seroconvert and remain seropositive for extended periods, even if their clinical status changes [49]. However, a study conducted in Turkey reported higher seropositivity among younger dogs than older dogs [40]. These findings suggest that susceptibility may vary across epidemiological settings, and dogs of all age groups remain at risk of exposure to infection.

Dogs used for hunting and herding animals in this study were at higher risk of infection than those kept primarily for security or companionship. This occupational risk is consistent with findings from Portugal [8], and Sardinia, Italy [41], where dogs involved in herding or outdoor hunting experienced significantly greater exposure to infected sand flies in rural habitats The elevated risk among these working dogs is primarily due to their prolonged exposure to sylvatic or peridomestic environments, where vector activity is most intense.

Local dog breeds exhibited higher seropositivity than mixed breeds, which may be attributed to their predominance in the study population and due to increased participation in outdoor activities such as grazing and hunting. Comparable findings have been documented in Turkey and Italy [29,30]. Conversely, research from Egypt identified greater susceptibility among pure breeds, including German Shepherds and Rottweilers [29], indicating that host genetics, management practices, and environmental exposures may influence breed-related susceptibility.

Dogs that did not receive veterinary care, such as vaccination and ectoparasite control, exhibited higher seropositivity rates than those that did. Studies conducted in Egypt [29], and Portugal [17], similarly reported that dogs lacking access to repellents, insecticides, or vaccination programs faced a significantly increased risk of infection; Inadequate veterinary management may increase vector exposure and contribute to sustained parasite transmission. Evidence from Brazil also indicated that enhanced veterinary care and animal health management were linked to a reduced risk of infection [14].

Several factors were identified as major drivers of seropositivity in Baringo County, including housing and environmental conditions. The study found that roaming dogs had higher odds of seropositivity than those housed within a compound, although the association was not statistically significant. This trend is consistent with findings from Ethiopia, where a mixed indoor-and-outdoor living environment was an independent predictor of infection (OR = 2.19). Outdoor housing has been shown to increase exposure to leishmaniasis by facilitating greater contact with sandfly vectors [10]. Furthermore, roaming dogs may experience increased exposure to sandfly vectors due to sedentary outdoor lifestyles, rural environments, and inconsistent preventive treatment [8].

A key finding of this study was the significant association between breed and seropositivity, with Mongrel dogs exhibiting nearly 12 times higher odds of ELISA positivity compared to local breeds. This difference likely results from increased exposure to outdoor activities, including hunting, for these dogs. Research conducted in Egypt has also identified breed as a significant risk factor, indicating that breed-related variations in immune response may affect susceptibility to Leishmania infection [29]. Although local breeds demonstrated high seropositivity in the present study area, global evidence indicates that management practices have a greater impact than breed alone. The absence of veterinary care, particularly the lack of insecticides, repellents, and vaccinations, substantially increases the risk of infection. Studies from Portugal demonstrate that dogs receiving regular veterinary care, such as those in managed shelters employing preventive measures with anti-feeding effects, often exhibit lower seroprevalence than domestic dogs with less oversight [8].

Dogs without clinical signs exhibited significantly lower odds of ELISA positivity (OR = 0.06). The observed correlation between symptomatic disease and increased seropositivity aligns with findings from studies conducted in Portugal [8], Italy [41], and Brazil [47]. In Brazil, symptomatic dogs had a significantly higher risk of testing positive (OR = 1.8) than asymptomatic dogs [47]. These results indicate that clinical manifestations are indicative of advanced infection and enhance the likelihood of serological detection [3].

Dogs from Rabai had approximately 4.6 times higher odds of ELISA positivity than those from Kapkuikui; however, this was not associated with infection. Comparable geographical clustering has been documented in Palestine and Portugal, where specific endemic regions, such as Hebron and Jenin, were identified as major areas linking domestic dogs to human visceral leishmaniasis (VL) risk [42]. Similarly, a study in Portugal reported higher seroprevalence in certain geographical areas, with dogs residing in interior regions nearly twice as likely to be infected due to rural and peridomestic conditions favorable for sand fly breeding, including sheep pens, cattle sheds, and organic waste accumulation [17].

Reduced seropositivity in dogs receiving veterinary care, particularly vaccination and ectoparasite control, highlights the critical role of management strategies. In Egypt, dogs lacking regular veterinary care or insecticide use were 2.7 to 3.09 times more likely to be infected [29]. Vaccination has also been demonstrated to significantly decrease the progression to clinical disease (OR = 3.8) in naïve dogs exposed during transmission seasons [50]. Collectively, these findings indicate that although biological factors such as age and sex may vary regionally, consistent veterinary management serves as a universal deterrent to the spread of canine leishmaniasis [17].

Age and sex were not significant risk factors in our Baringo County cohort. Our results do align with a study in Bingöl, Turkey, which also found that age and sex were not definitive predictors of seroprevalence [40]. This is in contrast with findings in Egypt [29], and Portugal [17], where older dogs and males often show significantly higher likelihoods of infection (up to 12 times higher in some age groups.

The RDT demonstrated high specificity (100%) but only moderate sensitivity (64.7%) in this study. All RDT-positive dogs were confirmed positive by ELISA, resulting in a 100% positive predictive value. However, the RDT failed to detect approximately 35% of animals identified as seropositive by ELISA. This diagnostic discrepancy highlights a significant limitation: although a positive RDT result is highly reliable for identifying infected dogs in the field, a negative result does not definitively exclude infection [51]. Similar findings have been reported in other endemic regions, including Zambia [9] and Italy [41], where differences in sensitivity among diagnostic methods often lead to undetected infections in seronegative animals. Therefore, while the RDT is useful for rapid field assessment, its limited sensitivity reduces its suitability as a standalone tool for comprehensive screening or surveillance programs targeting elimination [17,42]. Seronegative dogs by RDT may still harbor infection, indicating hidden infection.

In contrast, ELISA demonstrated superior diagnostic sensitivity by detecting significantly more infected dogs than the rapid diagnostic test (RDT). The data indicated that ELISA could detect low antibody levels, which are frequently observed in asymptomatic carriers. These findings are consistent with previous studies conducted in Brazil [53], Turkey [40], Portugal [8], and China [15]. This evidence reinforces the role of ELISA as a “gold standard” for mass screening in field epidemiological studies, particularly in high-transmission areas such as China and Portugal [17,42]. Additionally, because many dogs in endemic regions such as Baringo possess antibodies from prior exposure or subclinical infection rather than active disease, ELISA offers an effective means to accurately assess the true prevalence of the parasite in the population [8,19].

The logistic regression model provided additional validation for these findings, demonstrating strong discriminatory performance with an area under the ROC curve of 85%. This result indicates a robust capacity to differentiate between ELISA-positive and ELISA-negative dogs, aligning with diagnostic models from other endemic regions, including Brazil and Portugal, which reported comparable AUC values of approximately 84% [19,43]. Nevertheless, the model’s high false negative rate (76.5%) underscores the persistent challenge of identifying the entire infected population, implying that a significant proportion of positive animals may remain undetected during routine screening [9,42].

This study demonstrates that the combination of rapid diagnostic tests and ELISA is a highly effective strategy for detecting Leishmania exposure in canine populations. By utilizing a multi-test approach, we were able to capture a broader spectrum of infection status, particularly in asymptomatic dogs. Our findings align with recent research in Zambia, where the use of both rK39-based RDTs and ELISA provided strong evidence of true exposure, even when individual test results varied [9]. Such synergistic diagnostic protocols are increasingly recommended to improve the detection of subclinical infections that single-method screenings often miss [15,42].

The identification of phlebotomine sandfly species, particularly those from the genera Phlebotomus and Sergentomyia, provides critical entomological evidence of local vector-borne transmission in Baringo County [54]. The presence of these vectors, together with the observed significant canine seroprevalence, demonstrates that the Leishmania transmission cycle is firmly established in the region. Integrating entomological data with canine surveillance is vital for developing a comprehensive One Health strategy to control the spread of zoonotic visceral leishmaniasis.

5. Conclusions

This study revealed a high seroprevalence of L. infantum among dogs in Baringo County, highlighting a significant zoonotic risk for visceral leishmaniasis transmission. Asymptomatic dogs were identified as important reservoir hosts, complicating diagnosis and disease control due to the absence of obvious clinical signs. Sustained transmission is likely driven by untreated or stray dogs and inadequate vector control measures. The findings showed that all dogs are susceptible to infection regardless of age, sex, breed, or origin when exposed to infected sand flies. These results emphasize the need for routine canine screening, strengthened surveillance, and an integrated One Health approach incorporating reservoir management, treatment of infected animals, and effective vector control to reduce the risk of human infection.