Neospora spp. Seroprevalence and Risk Factors for Seropositivity in Apparently Healthy Horses and Pregnant Mares

Simple Summary Neosporosis, caused by the parasite Neospora species, is recognized as one of the major causes of abortion in cattle worldwide causing large economic losses. Over the past few years, Neospora infection and parasite transmission from the mare to the fetus has been documented in horses and was associated with reproduction failure. In the present study, we investigated parasite prevalence and associated risk factors in the general equine population and in a group of pregnant mares during pregnancy and after parturition. Our findings revealed high exposure of horses to Neospora spp. parasites, with significantly higher prevalence in pregnant mares. Abstract Equine Neospora infection has been linked to neurological disorders and infertility in horses. This study looked into the risk factors for infection and the exposure to Neospora spp. in horses. The study was performed in two independent populations in Israel. The first consisted of apparently healthy horses, and the second consisted of mares examined during pregnancy and after parturition. Sera samples collected from horses and mares were tested for Neospora exposure by the indirect fluorescent antibody test (IFAT). The study revealed seroprevalence of 24% in apparently healthy horses and 66.4% and 48.6% in mares during gestation and after parturition, respectively. Among the investigated risk factors, older age (p = 0.026) and housing in both stalls and paddocks (p = 0.033) in apparently healthy horses, and Arabian breeds (p = 0.005) in pregnant mares, were found to be significantly associated with Neospora spp. seropositivity in univariable, but not multivariable, statistical analysis. This study revealed high exposure of equines to Neospora parasites, especially mares. Horse farm management, in combination with active surveillance, including serological testing and follow up, could help reduce the spread of the parasite among horses in endemic areas.


Introduction
Neosporosis is considered a serious disease of cattle and dogs worldwide. The disease, caused by the protozoan parasite Neospora caninum, is recognized as one of the major causes of abortion in cattle, leading to large economic losses in the livestock industry. Neospora is an obligatory intracellular parasite belonging to the phylum Apicomplexa, with dogs and related canids as definitive hosts [1]. Neospora caninum have been reported to infect various host species [2], while N. hughesi has only been reported in horses (and its definitive host has yet to be recognized). In the past, both N. caninum and N. hughesi have been reported in horses, and were associated with abortions and neurological disorders, respectively [3,4].
Neospora infection in intermediate hosts is frequently asymptomatic, and exposure is confirmed by the presence of antibodies against Neospora. Detection of anti-Neospora Animals 2022, 12, 2699 2 of 10 antibodies in serum samples of mammals is the most frequently used method in the study of the epidemiology and prevalence of neosporosis in live animals [5]. However, the potential of cross-reaction between N. caninum and N. hughesi in serological tests complicates the interpretation of the results of serological surveys, as serological differentiation between these two organisms is probably not possible [6]. Therefore, previous studies in horses based solely on serology cannot be considered species-specific.
The seroprevalence of Neospora spp. in horses has been reported in many regions. Antibodies against Neospora were found in 2% of equine serum samples in South Korea [7], 2.5% in South America [8], 11.5% in North America [9], 23% in France [10] and 23.8% in New Zealand [11]. A serological survey performed on horses in Israel (2007) showed a seroprevalence for Neospora spp. of 11.9% among 800 apparently healthy horses. A significantly higher seropositivity was found in horses with neurological signs (21.2%) and in aborting mares (37.5%) [3]. A recent study from Israel identified Neospora infection in aborting mares and aborted fetuses. All Neospora infections found in aborted fetuses were classified as N. caninum by molecular analysis [12]. Neospora hughesi has not been identified in horses in Israel, so far.
Neospora caninum is one of the most efficiently transplacentally transmitted parasites [13]. Transplacental transmission occurs when tachyzoites from the mare cross the placenta and infect the fetus. Fetal damage may occur due to tissue damage caused by the multiplication of N. caninum in the fetus or in the placenta [14]. A survey conducted in Brazil demonstrated the importance of transplacental transmission in horses. Among 129 seropositive mares, 34.8% gave birth to positive pre-colostral foals. The seroprevalence in mares was higher than in foals and seropositive mares were likely to transmit the parasite to their offspring, demonstrating that Neospora can be disseminated via the placenta [15]. A study performed in California over a two-year period, which followed 74 paired samples of mares and foals, showed that Neospora spp. persists in the equine population via endogenous transplacental infection [16]. In this study, three foals had pre-colostral antibody titers ranging from 2560 to 20,480 for Neospora spp. An additional study confirmed neosporosis as the cause of abortion in an equine fetus aborted after 280 days of gestation by histology, immunohistochemistry, ultrastructure and molecular analysis [17]. A recent study demonstrated high seroprevalence for Neospora in a group of aborting mares in Israel (70.9% among 31 mares). Positive titers varied from 1:50 to 1:6400, and 13 (out of 31) fetuses were found to be positive for N. caninum by polymerase chain reaction (PCR) [12].
In this study, we investigated the exposure to Neospora spp. in two horse populations. The first population consisted of apparently healthy horses from different areas of Israel. The second population was comprised of a group of mares examined during pregnancy and after parturition. Seroprevalence of Neospora spp. was examined in both groups to better understand the risk factors associated with seropositivity and the dynamics of antibodies in mares during and following pregnancy.

Experiment Design
The survey was conducted on two independent study populations. The first study population consisted of a group of apparently healthy horses selected to represent the geographical distribution of the Israeli equine population. In this group, horses were sampled once. The second study population was comprised of pregnant mares, sampled on two occasions, during gestation (mainly in the first trimester) and after parturition. In some instances, serum collection postpartum could not be performed due to lack of owner consent, change in ownership, or mare death. Horse details, including age, sex (for asymptomatic horses' group), breed, reproduction history (for the mares' group), geographic location, housing management (stalls, paddocks, pasture) and presence of dogs in the farm, were recorded and used for further analyses of possible risk factors for seropositivity. Sample collections were performed with the horse owners' consent, and the study was approved by the Internal Research Committee of the Koret School of Veterinary Medicine-Veterinary Teaching Hospital (KSVM-VTH/02_2018, HU-NER-2020-055-A).

Study Population
Samples were obtained from two independent study populations: First population: Blood samples from 334 horses were collected during 2018-2019 from 30 farms, representing the geographical distribution of the Israeli equine population. Four to thirty-three horses were sampled at each farm. The farms were mainly riding schools, therapeutic riding schools and provided trail riding for inexperience riders. Few of the horses were sport horses (reining, jumping or dressage), and one farm kept endurance horses. Most of the horses were born in Israel; several had been imported, but not recently. This population was comprised of 161 mares (48.2%), 165 geldings (49.4%) and 8 stallions (2.4%) of various breeds. Horses' age ranged from six months up to 47 years (mean (M) = 11.7; standard deviation (SD) = 6.04).
Second population: Blood samples from 152 pregnant mares were collected at 36 farms located in different geographical locations in Israel during 2019. One to twenty sera were sampled at each farm. A total of 107 serum samples after parturition (in some cases after abortion or fetal absorption) were collected. The mares' age ranged from two to twenty years old (M = 7.6; SD= 3.4).

Anti-Neospora spp. Antibodies Detection by Indirect Fluorescent Antibody Test (IFAT)
Blood samples were collected from each horse's jugular vein into sterile vacuum tubes without anticoagulant. Blood samples were centrifuged at 2500 × rpm for six min. Sera were collected and samples were kept frozen at −20 • C until being processed. All samples were examined for the presence of antibodies against Neospora spp. by IFAT, using culture-derived NC-1 tachyzoites prepared in-house, as previously described [3]. In brief, serum samples from the general equine population were tested at 1:50 and 1:200 dilutions. Serum samples from the pregnant mares were tested at an initial screening dilution of 1:50. All samples that showed fluorescence at the initial dilution were considered positive, and were further diluted at a 1:2 ratio to the endpoint titer. Positive and negative controls were included on each slide. The highest dilution of serum exhibiting fluorescence of the whole Neospora organism was considered as the endpoint titer.

Statistical Analysis
The data were analyzed for association between demographic, environmental and husbandry parameters, and Neospora seropositivity. Fisher's exact or Chi-squared tests were used for categorical variables, as appropriate, and odds ratios (OR) were calculated. Student's t test and Spearman's correlation were used for continuous parameters (age). All factors that were found to be significantly associated with Neospora seropositivity were included in a multivariable generalized estimating equation (GEE) using the logit link function, with the horse defined as subject and the farm as within-subject effect. Statistical significance was set at p < 0.05. The analysis was performed using the SPSS v. 25  The overall seroprevalence of Neospora spp. found in the general equine population was 24% (80 of 334). Of them, 6.6% (22 of 334) presented the titer of 1:200. Of the 30 farms examined, 24 (80%) were positive for Neospora spp., with an intra-farm seroprevalence ranging from 10% to 86% (Figure 1). High seroprevalence was found in the Golan Heights and Western Negev areas (57% and 86% respectively). tus (rho = 0.134, p = 0.015). Neither age (p = 0.073) nor housing (p = 0.061) significant in the multivariable model. Neospora spp. infection was not fou icantly associated with horse's sex, breed or geographical area. None of th was found to be significantly associated with seropositivity, when usin titer.

Risks Factors Associated with Neospora spp. Infection
Associations between environmental and demographic factors and exposure to Neospora spp. using the 1:50 cut-off titer are summarized in Table 1. Significant difference in seropositivity was found between horses with different housing management. Horses kept in both stalls and paddocks had significantly higher seroprevalence than horses kept in either stalls, paddocks, or pasture (OR = 2.12, 95% confidence interval (CI): 1.18-3.76, p = 0.008). Age was also associated with Neospora spp. seropositivity. Mean age of seropositive horses was significantly higher than that of seronegative horses (p = 0.026, Table 2), and a weak but significant correlation was found between horses' age and serological status (rho = 0.134, p = 0.015). Neither age (p = 0.073) nor housing (p = 0.061) was found to be significant in the multivariable model. Neospora spp. infection was not found to be significantly associated with horse's sex, breed or geographical area. None of the factors tested was found to be significantly associated with seropositivity, when using a 1:200 cutoff titer. Among the 152 sampled pregnant mares, 66.4% (95% CI: 58.3-73.9%) were found seropositive for Neospora spp., with titers ranging from 1:50 to 1:400. Following parturition, the seroprevalence was significantly reduced to 48.6% (95% CI: 38.8-58.5%). The odds ratio for being seropositive was twice higher in mares during gestation than in mares after parturition (OR = 2.09; 95% CI: 1.22-3.59%, p = 0.005).
Data regarding the pregnancy outcome was available for 135 mares. Of them, 125 had a healthy foal and ten experienced problems during pregnancy or parturition. Three mares aborted between the sixth and seventh month, three absorbed and four had dystocia (two of them died during parturition). The three aborting mares were seropositive for Neospora spp. during and after parturition (Table 3). Of the mares that absorbed their fetuses, one was seropositive, and one was seronegative for Neospora spp. during gestation, however, post-partum serum could not be collected. The third mare which absorbed its fetus was negative during and after pregnancy. Of the mares with healthy foals, 68% (85 out of 125) were seropositive for Neospora spp. No abortions were observed among seronegative mares, apart for one absorption. Table 3. Anti-Neospora antibody titer variation detected by IFAT between mare's pregnancy period and after parturition.

Mare Serum Antibody Titers Number of Mares Pregnancy Outcome * During Pregnancy
After Parturition

Variations in Anti-Neospora Antibody Titers during and after Gestation
Paired data from mares during pregnancy and after parturition were obtained from 107 mares (Table 3). Antibody levels remained the same after parturition in 34.6% of the pregnant mares. Antibody levels increased in 22.4% and decreased in 43% of them. There were significantly higher rates of mares with decreased antibody titers postpartum than with increased antibody titer (OR = 2.61, 95% CI: 1.39-4.96%, p = 0.002). Seroconversion from negative to positive after parturition was found in 42.4% of the pregnant mares and seroconversion from positive to negative was found in 48.6% of them. These rates were not significantly different (p = 0.411).

Risk Factors Associated with Neospora spp. Infection in Pregnant Mares
The differences in seropositivity based on housing, pregnancies in the past, abortion history, proximity to dogs or cattle, food conditions, food access by canids and geographical location were not significant in pregnant mares and mares after parturition. Seropositivity was found to be associated with Arabian breeds both during pregnancy and following parturition. The proportion of seropositive Arabian mares was significantly higher than in the other breeds (during pregnancy: OR = 3.51, 95% CI: 1.28-9.51%, p = 0.008; after parturition: OR = 4.32, 95% CI:1.24-17.05%, p = 0.016, Table 4). The mean age of seropositive mares after parturition was significantly lower than that of seronegative mares (p = 0.006). A significant negative correlation was found between age and seropositivity after parturition (rho = −0.285, p = 0.006). Neither breed (p = 0.062) nor age (p = 0.164) was found to be associated with Neospora spp. seropositivity after parturition in the multivariable analysis. A positive tendency was found for mares after parturition living in proximity to dogs in the farm (Table 4). * Not all data was available for every mare. ** The term "paddock" includes mare living only in paddocks or both in stalls and paddocks. *** The abortion history also includes cases of absorption.

Discussion
The seroprevalence of Neospora spp. was higher in both study populations compared to the seroprevalence found in horses in 2007 in Israel [3]. The present study revealed seroprevalences of 24% and 66.4% in apparently healthy horses and in pregnant mares, respectively, compared to 11.9% and 37.5% in the survey conducted on apparently healthy horses and aborting mares in 2007. The increase in seroprevalence of Neospora spp. parasites in horses from 2007 to 2020 may suggest an expanded exposure to Neospora spp. over those years in Israel.
The risk factors associated with Neospora spp. infection in the univariable analysis were housing management and age for apparently healthy horses and breed and age for pregnant mares. However, none of these factors remained significant in the multivariable model. Previous studies from other geographical areas, including neighboring Jordan, which evaluated risk factors associated with neosporosis in horses also did not reveal any significant factors in the multivariable analyses [18][19][20][21].
A seropositive tendency was found for post-partum mares living near dogs where there is a possibility of oocyst contamination and horizontal transmission. Exposure to dogs was found to be a significant risk factor for Neospora spp. infection in cattle and other intermediate hosts in other studies [18,19,22,23]. Since canids are defined as the definitive hosts of N. caninum [13,[24][25][26], we assume that horses living not only in stalls but also in paddocks or pasture are more likely to be in contact with oocysts shed by the definitive host and to be infected by horizontal transmission than horses living only in stalls.
The geographical area was not found to be associated with seropositivity in both study populations. In apparently healthy horses, the seroprevalence varied between farms, Animals 2022, 12, 2699 8 of 10 and the number of horses sampled at each location varied between four and 33 horses. Higher seroprevalence was found in two farms located in northern and southern regions in Israel (57% and 86%, respectively). However, the number of horses sampled at each farm was relatively low (between four and ten horses) compared to the other farms (between 11 to 33 horses). Overall, it was impossible to conclude whether some farms had greater risk of infection. It is interesting to note that, in a previous study performed in Israel, the geographic area was found to be a risk factor to Neospora spp. infection as mares from the north of Israel had lower exposure than mares from central and southern Israel (46.1% versus 88.9%, p = 0.017) [12]. However, the sample size in that study was relatively small.
Most of the previous studies which evaluated the association between various risk factors and seropositivity only performed univariable statistical analysis [3,18,20,26]. Several factors were identified as significantly associated with Neospora seropositivity, most of them related to the management of the farm and the horses. These include routine use of anthelmintics [21], proximity to other animal species [19,20], feeding management [19,20], the use of horses [19] and quarantine [19]. In the few studies which also performed multivariable analysis [19] and the current study none of these factors remained significantly associated with exposure. This may suggest that neosporosis is a multifactorial infection and that the risk of infection is related to different environmental and management parameters, while antibody titers of chronic carriers may vary according to the animal's general health and reproductive status. Also, many of the management parameters are linked when horses are sampled at the same farm and the contribution of each individual factor is difficult to assess. Therefore, better understanding of the pathogenesis and host-parasite interactions is needed to comprehend the complex epidemiology of neosporosis.
The present study demonstrated that Neospora spp. seropositivity is higher in mares, with rates of 66.4% during pregnancy and 48.6% after parturition, than in the general equine population (24%). This difference cannot be attributed to differences in sex, as seropositivity did not differ between geldings, stallions, and mares. Most mares sampled as a part of the general horse population were not pregnant, but the reproduction status was not recorded in all cases. Nonetheless, when comparing the seroprevalence only in mares in the general population (24.2%) and pregnant mares (66.4%, OR = 6.2, 95% CI: 3.68-10.47, p < 0.001) or mares after parturition (48.6%, OR = 2.96, 95% CI: 1.7-5.16, p < 0.001), both these differences are statistically significant. In addition, it is relevant to point out that all three aborting mares in the pregnant group were found seropositive during and after gestation. These findings are in accordance with a study revealing high seropositivity of 70.9% (22 of 31) in mares after abortion using the same serological cut-off value 1:50 [12]. Mares during gestation are in a stressful period, which may affect the life cycle of Neospora and generate modifications to the immune system response, thus providing an opportunity for parasite recrudescence and multiplication [27].
Anti-Neospora antibody titer fluctuations were found in paired samples of mares. Only 34.6% of these mares examined had the same antibody titers during pregnancy and after parturition. A decline in antibody titer after parturition occurred more frequently than augmentation. Seropositivity in pregnant mares was significantly higher than in mares after parturition (p = 0.005). Bradyzoite cysts are the chronic stage of infection which can escape recognition by antibodies [27]. In pregnant or stressed animals, bradyzoites can reconvert into tachyzoite, emerge out of the cysts, and then be recognized by the immune system, leading to a potential fetal loss due to transplacental infection [27,28]. We assume that recognition of the parasite by the immune system leads to an augmentation of anti-Neospora antibodies in the mare's blood, explaining the increased antibody levels founded during pregnancy.
The pregnancy outcome was not found to be significantly associated either with an increase or a decrease in antibody titer.