Neospora caninum serostatus in dairy cattle of the Northern plains of Antioquia, Colombia

Estudio serológico de Neospora caninum en ganado de leche del altiplano Norte de Antioquia, Colombia

Neosporosis is one of the most important causes of infectious abortion in bovines across the world. Transmission can occur horizontally from ingestion of Neospora caninum oocyst shed by definitive hosts (i.e, canines), or through parasite migration during pregnancy to the fetus (vertical transmission) which is generally accepted to be the primary method of transmission in cattle (1,2). In Colombia, the presence of seropositive animals in the absence of a vaccination program can provide information on the status of infection in a herd. Reports from various countries have shown that the risk for abortion increases not only with the percent of seropositive cows in a herd, but also with the level of seroconversion, such that the odd ratios for aborting can vary from 3 to 12 times for animals with low to high antibody titers, respectively (3,4). However, the majority of seropositive cows still give birth to clinically normal but infected calves that are prone to have subsequent abortions (2,5). The clinical outcome of such transplacental fetal infection depends largely on the maternal and fetal immune responses which are always unpredictable (5).

A definitive diagnosis on the cause of any abortion requires the assistance of a veterinary diagnostic laboratory capable to perform a thorough diagnostic examination of the aborted fetuses and placentas. In this regard, limited diagnostic services in Colombia are still hampering investigations on the main agents causing abortions in livestock. In spite of this limitation, the use of serological tests to monitor natural infections by pathogens like N. caninum, can yield important diagnostic information at a herd level (6). For example, when seroprevalence is similar across different age groups it may indicate that the infection has been likely perpetuated by vertical transmission (1). In addition, high titers are known to be a significant risk factor for abortion compared to low positive animals, so it is a factor that could also be considered to design control programs (3). Notwithstanding that serology has been the main technique to diagnose N. caninum infection, it has limited application to diagnose whether an individual cow has aborted due to neosporosis. Most of N. caninum seropositive cattle do not abort and seronegative dams can give birth to seropositive calves (7). In Colombia, the only reports documenting the presence of N. caninum in bovines are based on serological surveys, but associations with abortions or other reproductive problems have not been explored (8, 9, 10).

The aim of this study was to estimate the seroprevalence to N. caninum in a major dairy area of Colombia. It was part of a larger study that included serologic viral pathogens and parasitic infections (data not shown).

Type of study and location. A cross-sectional study was conducted in the municipality of San Pedro de los Milagros, with an estimated population of 65.000 dairy cattle. A total of n=1003 animals were selected from N=29 herds as described in the next section.

Population and statistical model. The sample size for each farm, number of farms, and ages within each farm, was chosen to be proportional to the municipality’s bovine population, with an expected prevalence of 50%, margin of error of 3.1% (≤5%), and a 95% confidence interval. The breeds of the cows were Holstein, Holstein-Jersey, Jersey and others. The number of farms with land area of <50, 50-100, and >100 “fanegas” (1 fanega = 0.66 hectares) were 19, 7 and 1, respectively. Six herds (20.6%) had less than 50 cows, nine herds (31%) had between 50-100 cows and fourteen herds (49%) had more than 100 cows. Data was entered into Excel worksheets and then exported to Stata 12.0 (StataCorp, 2011) for analysis. Descriptive statistics were computed and an univariate logistic regression model was constructed to evaluate the relationship between the prevalence of infections and age as an independent variable. Serological status (positive/negative) was the dependent variable.

Husbandry and management practices. The management system in the area is primarily an intensive rotational grazing system based on Kikuyo grass (Penisetum clandestinum) monoculture pastures with no confinement of adult cows, and calves were usually kept in separate paddocks between the ages of 3-9 months and then moved with replacement heifers until they were ≥ 15 months old. Pastures are rested for approximately 30-40 days, and occupation days vary with density of animals. No vaccines against neosporosis had ever been used in the area.

Sample collection and laboratory analysis. Blood samples were collected between May and June 2014, and centrifuged to obtain serum within 24 hours. Serum samples were stored at -20°C until analyzed using an indirect ELISA immunoenzymatic assay for the detection of specific antibodies to N. caninum in bovine serum (Ingezim, 1.2. NC. L1, Ingenasa S.A, Spain). The cutoff for a positive result was a sample-to-positive control ratio of ≥0.45, although according to the manufacturer samples with optical density (OD) between 0.45-0.60 should be considered suspicious. Data of correspondence with the reference ELISA (HerdChek® Neospora caninum Antibody Test Kit) used in Canada is summarized as follows. Considering doubtful sera as positive, the relative sensitivity of the Ingezim kit was 99,4% (95% CI=96-100%) and the relative specificity 100% (95% CI= 99.7-100%). Considering doubtful sera as negative, the relative sensitivity is 88,2% (95% CI=81.8-92.7%) and the relative specificity 99.1% (95% CI= 98.1-99.6%).

The prevalence of N. caninum seropositive animals by age categories is presented in Table 1. All N=29 herds were positive with a prevalence range between 7-97%. Older age groups tended to have higher prevalence with values of about 40% for cows of ≥2 years compared to 25-30% for animals in the first 2 years of life. The slightly higher seroprevalence in older animals suggests that postnatal infection, in addition to congenital transmission, is also occurring. Farm dogs were present in all sampled herds and are a potential source for horizontal transmission. Unfortunately, data for the existence of abortion during the year was unreliable as many farmers do not record fetal loses following a positive diagnosis of pregnancy.

Table 1.
Age distribution of the prevalence to Neospora caninum in sera from dairy cattle in the Northern plains of Antioquia.

Neosporosis is one of the major causes of abortion in cattle and serologic surveys indicate it is worldwide distributed (11). The present study, aimed to describe the seroprevalence of N. caninum in a dairy cattle area of Antioquia, observed a within-herd prevalence ranging between 7-97% in 29 herds, with an overall mean of 37.1% (n=1003). However, the implications of such a high prevalence of infection for a disease that has only been proved to cause abortions, are difficult to assess without establishing the rate of abortions due to neosporosis.

In a recent study where the overall seroprevalence of N. caninum in a large dairy herd was 35.5% (4), the percentage of abortions was 3 times higher in seropositive dams compared to seronegative mates (21.6 and 7.3%, respectively). They also observed that the risk of repeated abortion among seropositive cows was 5-fold greater than seronegative ones. However, the factors that promote the likelihood of a seropositive cow to abort are usually unknown; several stress factors related to management practices, weather conditions, co-infections, age, and others, have been reviewed by Dubey et al (11) and are likely to play an important role at reactivating latent infections. Consequently, and considering the different management and husbandry practices of dairy cows in our area, making extrapolation on the incidence of abortions from studies in different places would be impossible.

Serological test for N. caninum antibodies have been the most common method to diagnose infection status in whole herds. However, because titers fluctuate considerably and chronically infected animals may become negative, a single whole herd screening could always misinterpret the real status of some animals. In this respect, some studies have conducted serial testing for various lengths of time to evaluate the sensitivity and specificity of one-time sampling (6,12, 13,14).

In a Dutch study that also used an ELISA method, a total of 1676 animals were sampled 3 times at 11 month intervals (12). They showed that one-time testing for N. caninum had a 95.3% agreement (kappa = 0.90) to the results of three consecutive samplings. Interestingly, their values for prevalence were very similar to the ones in this study, with an overall mean prevalence of 36%, ranging between 4.8-80.3% within individual herds. The positive predictive value of their test, that is, the probability that a seropositive animal was truly infected was 92.4%. Their studies also showed that it was important to differentiate between low and high positive titers, as most fluctuations that resulted in false positives or negatives values occurred in the low positive results (12,13). However, when a cow had aborted with a confirmed diagnosis of neosporosis in the fetus done by histopathology, it would be expected that sera will have 2 to 7-fold greater titers than sera from cows that abort due to other causes (6), and typically there will be a rise in antibody titers between the third and seventh month of gestation (14). In this respect, the kinetics of antibody responses during gestation has also been proposed as a tool to predict abortion (14).

The high fidelity of vertical transmission typically reported in chronically infected dams (about 80% heifers and 66% older cows) is an effective way for the parasite to sustain prevalence within a herd (11,12). In this respect, the presence of seropositive dam-calf pairs and seropositive family lines is a strong indication for congenital transmission (1). In the present study the higher prevalence in older animals would suggest that horizontal transmission was likely contributing to the prevalence of infected animals, and since dogs lived on every farm sampled, it is possible they may be serving as a continuous source of oocyst exposure. An increased probability of postnatal infection with age has also been described as a factor associated with seropositive status in other studies (1,15).

In herds with an epidemic pattern of abortion, a point source exposure should be suspected, as opposed to being endemic in chronically infected animals. It could be speculated that a N. caninum-induced abortion storm will be followed by elevated endemic and sporadic abortions in subsequent years. However, long-term studies have shown that herds with N. caninum abortion outbreaks will not necessarily have elevated abortions in the following years (13), clearly revealing the importance that risk or protective factors play in the occurrence of endemic abortions.

In conclusion, this study showed a high prevalence of N. caninum infection in a main dairy area of Colombia. However, the large variation between herds suggests the presence of mayor risk factors whose identification may be crucial to future control strategies. The results warrant future investigations on the epidemiology of N. caninum in the area.

This work was supported by Vecol, Zoolab and the Facultad de Ciencias Agrarias de La Universidad de Antioquia. The authors thank the Centauro Sustainability Project 2013-2014 (Estrategia de Sostenibilidad CODI 2013-2014) and the Universidad de Antioquia for the economic support.

The authors declare that there is no conflict of interest.