Dairy cattle farming constitutes a major component of national milk production in Türkiye, where raw milk production increased by 4.7% in 2024 to 22,487,757 tons, of which 93.6% was derived from cows, placing Türkiye among the leading milk-producing countries in Europe and worldwide (1). Sustaining both the quantity and quality of milk production requires effective herd health management, as parasitic infections can reduce productivity, impair reproductive performance, and result in significant economic losses (39). Neospora caninum, an obligate intracellular protozoan parasite, is widely recognized as one of the leading infectious causes of bovine abortion worldwide, with substantial adverse effects on reproductive efficiency and herd profitability (2, 3). Current evidence indicates that, while human exposure has been reported, N. caninum is not regarded as a confirmed zoonotic pathogen.

Dogs and wild canids serve as the definitive hosts of N. caninum, while cattle and various other mammals act as intermediate hosts (2). Transmission occurs primarily through two routes. Horizontal transmission takes place when cattle ingest sporulated oocysts shed by infected canids, contaminating feed, water, or the environment. However, vertical (transplacental) transmission from dam to fetus is the dominant mechanism sustaining infection within herds and allows the parasite to persist across generations (4). Infected cows may abort, produce weak or stillborn calves, or give birth to clinically healthy but congenitally infected calves that remain infected for life and show an increased likelihood of abortion in subsequent pregnancies (5, 6).

Diagnosis in live animals relies primarily on serological detection of N. caninum-specific antibodies in serum or milk (7, 8). ELISA is widely used for herd-level surveillance because of its practicality and scalability. IgG1 antibodies are transferred into milk, and ELISA values obtained from milk and serum samples show strong linear correlation (9). Compared with individual serum sampling, bulk-tank milk ELISA enables efficient assessment of herd-level infection status using a single sample per farm, without animal handling, allowing faster and more cost-effective sample collection and feasibility at the national scale. Bulk-tank milk ELISA (BTM) therefore offers a minimally invasive and cost-effective tool for monitoring herd exposure, particularly suitable for large-scale epidemiological studies and routine herd-level screening (10–12).

Prevalence estimates for N. caninum vary widely between regions and production systems, influenced by environmental factors, herd management, and dog contact patterns (2, 3). In Europe, seroprevalence ranges from 16 to 76% in dairy herds and 41 to 61% in beef herds (13). In Türkiye, the mean animal level seroprevalence has been estimated at 14.7% based on individual cattle serum samples, similar to levels reported in several Asian and African countries (14). However, herd-level prevalence based on bulk-tank milk ELISA has not previously been reported in Türkiye.

Neosporosis also represents a substantial economic burden. Annual losses associated with N. caninum have been estimated at 546.3 million USD in the U. S. dairy industry and 9.7 million EUR in Swiss dairy herds (14, 15). For Türkiye, the economic impact has been estimated at approximately 710 USD per infected dairy cow, corresponding to national annual losses of 40.5 million USD (14). These estimates underscore the need for effective surveillance and management strategies.

Therefore, the objective of this study was to determine the nationwide herd level prevalence and spatial distribution of N. caninum in dairy cattle in Türkiye using bulk tank milk ELISA, and to identify management and environment related risk factors associated with infection. The findings aim to support the development of targeted control strategies to improve reproductive performance and reduce economic losses in the Turkish dairy sector.

Neospora caninum is widely recognized as a major infectious cause of reproductive loss in dairy cattle worldwide, with substantial economic consequences for the cattle industry (6). In the present study, a herd-level seroprevalence of 27.47% was detected across 466 dairy farms in Türkiye, demonstrating that exposure to N. caninum is widespread at the national level. Previous serological studies conducted in Türkiye, primarily based on individual serum samples, have reported highly variable prevalence estimates across regions (16–19). Such variability may partly reflect Türkiye’s pronounced climatic heterogeneity, ranging from humid and temperate coastal areas to continental and semi-arid inland regions, which can influence oocyst survival, environmental contamination, and exposure risk. In this context, bulk-tank milk (BTM) ELISA represents a practical and scalable herd-level surveillance tool, enabling assessment of infection status with reduced sampling effort and cost and allowing repeated monitoring across large production systems (10, 20). Importantly, to date, no nationwide herd-level evaluation of N. caninum exposure based on BTM ELISA had been conducted in Türkiye. The prevalence observed in this study aligns with global estimates, being comparable to reports from Italy (30.7%) (21), higher than those from Sweden (8.3%) (22) and Alberta, Canada (7.4–18.2%) (23), and lower than values reported from Iran (55%) (24), situating Türkiye within the broader international epidemiological context.

Marked regional variation in herd-level seropositivity was observed, with the highest prevalence recorded in the Marmara Region (39.2%), followed by Southeastern Anatolia (37.5%) and the Aegean Region (31.8%), while the lowest prevalence was observed in the Mediterranean Region (16.7%). These regional differences likely reflect variation in herd management practices, the density and behavior of domestic and stray dogs, grazing intensity, and local environmental conditions that influence oocyst persistence and transmission. Comparable geographic patterns have been documented in European and Asian production systems, where dog contact and grazing management are recognized as key determinants of exposure risk (25–27). Although overall regional heterogeneity was statistically significant, the absence of significant pairwise contrasts after adjustment suggests that these differences reflect broad regional trends rather than discrete, isolated hotspots of infection.

A strong association was identified between herd seropositivity and abortion history. N. caninum positive herds had 29.4 times higher odds of reporting abortion than seronegative herds, reinforcing the well-established role of N. caninum as a leading cause of infectious abortion in dairy cattle (28–30). Similarly, a large-scale study involving over 3,200 herds in Rhineland-Palatinate, Germany, reported that bulk tank milk–positive herds experienced an annual abortion rate approximately 3% higher than negative herds, further confirming the herd-level reproductive impact of N. caninum (10). Previous research has also demonstrated that herds with high BTM antibody levels may incur productivity losses of approximately 1.6 kg of milk per cow per day (9). Collectively, these findings emphasize that N. caninum infection contributes not only to reproductive loss but also to reduced herd-level performance and economic efficiency.

Risk factor analysis further underscored the importance of environmental and management-related transmission pathways. In particular, the use of groundwater was associated with a significantly higher likelihood of herd seropositivity compared with farms relying on municipal water supplies. This association supports the hypothesis that untreated or naturally sourced water may serve as an important environmental transmission route, potentially through contamination with oocysts shed in dog feces (31, 32). Although dogs were present on all surveyed farms, notable regional differences were observed in dog feeding practices. The majority of farms (95.9%) reported feeding dogs leftover household food, raw meat, or allowing scavenging and hunting behavior, whereas supplementation with commercial dog feed was confined to a limited number of farms in western Türkiye. Feeding practices involving raw or scavenged animal tissues increase the likelihood of exposure to infective tissue cysts and may therefore contribute to sustained parasite circulation on farms (33). Comparable associations between N. caninum seropositivity and environmental or management-related factors, including water source and production system, have also been reported in recent studies of small ruminants in Mexico and southeastern Türkiye, underscoring the broader relevance of these transmission pathways across livestock systems (34, 35).

In addition to horizontal environmental exposure, vertical (transplacental) transmission represents the dominant mechanism sustaining N. caninum infection within cattle populations (2). Through this route, infection can persist across multiple generations even when dog-mediated environmental contamination is reduced. In the absence of effective treatment options to prevent abortion or congenital infection, control strategies must therefore prioritize interruption of vertical transmission, particularly through selective breeding practices such as avoiding the use of seropositive cows as replacement breeders. Nevertheless, horizontal transmission remains epidemiologically relevant. Dog presence and local dog density have been identified as important drivers of elevated BTM seroprevalence (12), and although the present study did not capture variability in dog presence, it confirmed a consistent presence of dogs on dairy farms, often with unrestricted access to pastures and feed storage areas. Accordingly, complementary control measures should include restricting canine access to placental tissues and feed materials and ensuring appropriate disposal of aborted tissues (36). Over time, the combined implementation of these measures may contribute to a gradual reduction in herd antibody levels and overall infection pressure (37).

The relevance of dog-mediated transmission is further amplified in the Turkish context by the widespread presence and high density of stray dogs across both rural and peri-urban landscapes. Recent ecological evidence from Türkiye has demonstrated extensive spatial and temporal overlap between stray dogs and native wildlife species, highlighting the capacity of free-ranging dogs to move across fragmented ecosystems and interact with multiple hosts (38). Such unrestricted movement increases the likelihood of environmental contamination with N. caninum oocysts, particularly in shared grazing areas, water catchments, and feed-access zones used by livestock. Given the scale and persistence of stray dog populations in Türkiye, their role in sustaining environmental transmission cycles should be considered a critical component of neosporosis epidemiology and control strategies at the national level.

Pasture use varied significantly between regions; however, the proportion of pasture in the diet did not show a statistically significant association with seropositivity after multivariable evaluation. This may partly reflect differences in seasonal grazing practices, as pasture access in many regions is limited to specific periods of the year, potentially reducing sustained exposure to contaminated environments. In contrast, drinking water sources are used continuously throughout the year and therefore appear to represent a more consistent risk factor for N. caninum exposure.

The findings of this study align with national economic estimates indicating that neosporosis represents a substantial financial burden in Türkiye, with an estimated cost of approximately 710 USD per infected dairy cow and total annual industry losses of 40.5 million USD (14). Identifying region-specific risk profiles and modifiable management practices is therefore essential for designing cost-effective control strategies.

The present findings indicate that N. caninum exposure is widespread among dairy herds in Türkiye and that herd-level seropositivity is associated with specific management- and environment-related factors.

Based on the risk factors identified, control strategies should prioritize preventing dog access to feed storage and calving areas, improving disposal of placental tissues and aborted materials, minimizing the use of untreated water sources where feasible, and implementing routine bulk-tank milk monitoring to guide herd-level intervention. Importantly, because vertical transmission is the primary mechanism maintaining infection within herds, management programs should also consider avoiding the selection of seropositive cows as replacement breeders to prevent perpetuation of infection across generations. Future work should include longitudinal follow-up to clarify transmission dynamics and integrate molecular genotyping to support the development of targeted and regionally adapted control strategies.