Neospora caninum infection in dairy cattle in Egypt: a serosurvey and associated risk factors

Neospora caninum (N. caninum) is one of the causative agents that causing cattle abortion, and severe economic losses. Due to the scarcity of data on N. caninum infection in Egyptian cattle, the purpose of this study was to estimate the seroprevalence and determine the risk factors for parasite infection. In four governorates in northern Egypt, 540 blood samples from cattle were taken, and tested using a commercial ELISA kit. The overall seroprevalence of N. caninum in examined cattle was 28.89%. A multivariate logistic regression model determined that age (OR = 2.63, P < 0.001), manual milking (OR = 1.39, P = 0.14), abortion history (OR = 2.78, P < 0.0001), repetition of estrus (OR = 2.31, P < 0.0001), and contact with dogs (OR = 2.57, P < 0.0001) were significant risk factors. The findings proved that N. caninum infection was one of the factors contributing to abortion and financial losses in dairy cattle in Egypt. Therefore, the application of sanitary security and control programs is very important in dairy farms.


Materials and methods
Ethical statement.The Benha University ethical committee for animal studies approved all methods including the handling and collection of blood samples.The cattle owners informally consented for the collection of samples.The Faculty of Veterinary Medicine's ethical committee ensured that all procedures followed the rules and regulations.The ARRIVE criteria were adhered to throughout the research process.
The climate of these areas are usually warm with an average annual temperature of 25 °C, relative humidity between 40 and 60% with moderate rainfall 100-mm per year.Because of this, the study region is suitable for farming and raising animals, especially dairy cows.
Animals and sampling.The study was performed during 2021 to determine the presence of anti-N.caninum antibodies among cattle raising in four Egyptian governorates.The sample size needed to determine seroprevalence was estimated according to the Thrusfield formula 30 , based on previous prevalence rate in cattle 38.04% reported by Gaber et al. 29 , precision 10% and level of confidence 95%.A total of 540 blood samples were randomly collected from dairy cattle using simple random sampling.For each examined cattle, the veterinarian www.nature.com/scientificreports/and owner filled out a brief questionnaire that was used to gather data at the time of sampling.According to collected data, animals were classified based on location (Kafr ElSheikh, Gharbia, Menofia and Alexandria), age (< 2, 2-4 and > 4 years old), sex (female and male), breeding service (natural or artificial insemination), milking (automated or manual), stage of pregnancy (1-3, 4-6 and 7-9 months), parity (primiparus and pluriparus), gestation (yes or no), abortion history (yes or no), repeat of estrus (yes or no), retention of placenta (yes or no) and dog contact (yes or no).Using vacutainer tubes, five mL of blood were drawn from the jugular vein.The blood was then allowed to coagulate before being centrifuged at 3000×g for 10 min to separate the serum, then it was stored until use at − 20 °C.

Serological analysis.
All animals' sera were examined for anti-IgG to N. caninum using an ELISA kit (ID Screen Neosporosis indirect multi-species; ID-Vet, France) in accordance with the manufacturer's instructions..Each sample's optical density (OD) was determined using microplate reader at 450 nm, and seropositive animals (Sp) were identified using the computation of the S/P%, whereas serum samples were regarded as positive if the S/P% was more than 50%.

Statistical analysis.
The statistical software SPSS version 24 (IBM, SPSS Inc., Chicago, IL, USA) was used for all calculations.The relation of neosporosis with various risk factors was assessed using the non-parametric Chi-square test.Significant results were defined with P value < 0.05.All variables that had a P < 0.25 in the univariate analysis were subjected to the multivariate logistic regression model to evaluate the independent risk factors of each variable.Using multivariate logistic regression, the odds ratio (OR) and the corresponding 95% CI were determined 19,21,[31][32][33][34] .The Hosmer-Lemeshow statistic was computed to assess the model's goodness-of-fit 35 .

Results
Out of 540 examined cattle, 156 had N. caninum antibodies, with overall seroprevalence rate of 28.89%.The seroprevalence rate in Kafer ElSheikh was not substantially greater than that in the other locations under study, Table 1.
The statistical findings regarding the risk variables for seroprevalence showed no significant relationship between breeding service, parity, gestation, retained placenta and stage of gestation and seroprevalence of N. caninum (P > 0.05), Table 1.
Regarding of cattle age, there was a highly significant (p < 0.05) correlation between age and seroprevalence.Young cattle (< 2 years) had a significantly lower seroprevalence (15.71%) than older cattle (32.38%) in cattle of 2-4 years and 34.74% in cattle of > 4 years.For milking, the higher seroprevalence was found in cattle subjected to manual milking (32.22%) when comparing to those subjected to automated milking (22.22%;P < 0.05), Table 1.
The prevalence of N. caninum was significantly correlated with the history of abortion in females.The prevalence in this group was higher (48.33%) than in cows without a history of abortions (23.33%;P < 0.0001).Of the 310 females with a history of recurrent estrus, 110 (35.48%;P < 0.0001) were seropositive for N. caninum, whereas only 46 (20%) of the 230 females without a history of repeated estrus were seropositive, Table 1.
Considering contact of examined cattle with dogs, strong significant association was found between cattle contact with and seroprevalence, the highest seroprevalence rate was reported in cattle contact with dogs (34.29%) when comparted to other animals, Table 1.
The multivariable logistic regression model was applied to the variables in the univariable analysis that had a P < 0.25.The findings showed that the odds of contracting N. caninum infection were three times higher in adult cattle older than four years (OR = 2.63, 95% CI: 1.47-4.71),one time higher in manual milking (OR = 1.39, 95% CI: 0.89-2.17),three times higher in cattle with a history of abortion (OR = 2.78, 95% CI: 1.76-4.41),two times higher in cattle suffering from repetition of estrus (OR = 2.31, 95% CI: 1.52-3.53)and three times higher in cattle contact with dogs (OR = 2.57, 95% CI: 1.63-4.05),Table 2.

Discussion
Neospora caninum is one important cause in cattle abortion 36 .Analyzing seroprevalence, and consequently the exposure of dairy cattle populations to N. caninum, is crucial for determining populations that may be susceptible to neosporosis and for looking into the probable modes of the parasite's transmission.The prevalence and risk factors of cow neosporosis must be understood in order to develop and implement control programme measures 26,37,38 .
In Egypt, the antibodies against N. caninum were detected in some species like sheep, cattle and camels 14,27,29 but the epidemiological data about the disease in cattle is very limited and restricted in some areas in Egypt.Thus, this study aimed to investigate the seroprevalence of N. caninum in dairy cattle and assess the potential risk factors associated to infection.
In the present study, there were 28.89% of animals that tested positive for N. caninum, which come in agreement with prevalence rate 28.3% reported in Colombia 39 .
Neosporosis in globally distributed and the reported prevalence rates range from 10.7 to 19.6% in Africa 40,41 , 5.7-43% in Asia 42,43 , 7.6-76.9% in America 44,45 and 0.5-27.9% in Europe 46,47 .However, the reported rate in the present study is not high, similar findings were reported in Brazil 48 and Sengal 49 .
Among the study's governorates, the seroprevalence rate of N. caninum varied non-significantly and Kafr ElSheikh had the highest rate in comparison with other areas.These results concurred with those of Gaber et al. 29 , they reported that Kafr ElSheikh had a high incidence of N. caninum.This could be explained by the fact that this governorate's management, climate and environmental factors play a significant effect in the survivability of N. caninum oocysts 33,[50][51][52][53][54][55][56][57][58][59][60] .
Neosporosis in globally distributed and the reported prevalence rates range from 10.7 to 19.6% in Africa 40,41 , 5.7-43% in Asia 42,43 , 7.6-76.9% in America 44,45 and 0.5-27.9% in Europe 46,47 .However, the reported rate in the present study is not high, similar findings were reported in Brazil 48 and Sengal 49 .
These variances could be brought about by alterations in the climate, study design, detection techniques, farm management, sample size, and varying degrees of exposure to risk factors 17,18,23,59,61,62 .
Studies have shown that frequent exposure to the sources of infection tends to increase the probability of animals to be seropositive for N. caninum 25,26 .According to Moore et al. 63 , the risk of seropositivity increased 3.5% for every year that bovine and buffalo ages increased.Our findings are in line with earlier research and demonstrate that elder cattle > 4 years were more likely than younger to have infection with sporulated oocysts of N. caninum.Contrarily, other studies from various countries, including Brazil 64,65 , Croatia 66 , Jordan 67 , Romania 46 , and Venezuela 68 , found no correlation between age and N. caninum infection, indicating that transplacental transmission is likely more significant for these herds.Our findings suggest that horizontal transmission is also a significant factor in the epidemiology of N. caninum in cattle, despite the fact that vertical transmission is typically prove to be the main route of transmission in cattle 15,69,70 .
In cow neosporosis, the semen plays a significant role in disease transmission.Compared with cattle bred naturally from Iran (17.1%) and Spain (7.4%) 71,72 , pregnant heifers undergoing artificial insemination (AI) had www.nature.com/scientificreports/higher levels of IgG against N. caninum.The artificial insemination of seropositive dairy cows with beef bull semen may affect the role of the placenta as a result of crossbreeding 73 .Okumu et al. 74 found that abortion was considerably higher in pregnant cows with AI when there was no quick testing for cow neosporosis on the semen donors.Interestingly, cattle were subjected to manual milking showed significant higher seroprevalence than those subjected to automated milking, which come in accordance with findings of Llano et al. 39 .This attributed to poor hygienic condition and contamination of milker's hand by feces contain sporulated oocyst have significant role in horizontal infection transmission during milking 75 .
It is generally established that seropositive N. caninum cattle are more likely to prone abortion than seronegative N. caninum cattle 1 .We found that the proportion of seropositive cows that had a history of abortion (48.33%) was substantially higher than the proportion of seronegative cows (23.33%) in a group of cows with the same clinical symptoms.This gives circumstantial evidence that N. caninum may contribute to cow abortions in the area under study.These fundamental conclusions concur with those made by Llano et al. 39 in Colombia.
Furthermore, a considerable percentage of recurrent estrus cattle (35.48%;P < 0.0001) had anti-N.caninum antibodies.Similar results were found in a study carried out in the southeast of Brazil, where animals with repeat oestrus and transient anoestrus were 3.8 and 3.4 times, respectively, greater likelihood of seropositivity than those without the same clinical indications 76 .
In the present study, cattle suffered from repeat breading and early embryonic death had high seropositivity for N. caninum infection which come in agreement with prior findings of Buxton et al. 77 and 78 .This could be as a result of the fetus's immature immune system and lesions induced by parasites in the placental tissues, which result in early embryonic mortality and the return to oestrus 77 .This theory is consistent with research from Australia and Senegal that found that seropositive animals for N. caninum needed more inseminations to conceive, which is related to embryonic loss in the early stage of pregnancy 49,79 .
Similar to the findings of Barling et al. 80 , N. caninum infection in dairy cattle had a substantial correlation with close contact with dogs.This might be due to eating of aborted materials by dogs, which play an important role in horizontal transmission of infection to susceptible animals 81,82 .In Canada, Vanleeuwen et al. 83 verified that there is a higher risk of infection on properties with dogs who have access to placentas and foetuses than on properties where dogs are not permitted to come into contact with these materials.In this area, preventive actions are advised to reduce the likelihood of dogs consuming contaminated bovine tissues.Since the dogs on one property frequently visited the neighbors' properties, the close proximity of the farms also made it impossible to get reliable information about the canine population 39 .

Conclusion
N. caninum seroprevalence and distribution throughout all examined areas confirm that the parasite is common in Northern Egypt.Concerning to risk factors associated with N. caninum infection, the higher seroprevalence was observed in elder cattle, subjected to manual milking, with history of abortion or repetition of estrous and close contact with dogs.Further studies are necessary to examine sanitary application in dairy farms and to implement an efficient control program.

Figure 1 .
Figure 1.MAP showed the studied areas and prevalence of N. caninum (QGIS 3.18.3software used to generate the MAP).

Table 1 .
Seroprevalence of Neospora caninum in cattle raising in the four governorates under the study.*The result is significant at P < 0.05.

Table 2 .
Multivariable logistic regression analysis for risk factors associated with Neospora caninum infection.B: Logistic regression coefficient, SE: standard error, OR: odds ratio, CI: confidence interval.