Introduction

Neosporosis is a parasitic disease, caused by Neospora caninum (Apicomplexa: Sarcocystidae), an intracellular protozoan and distributed worldwide1. N. caninum characterized by Heteroxenous biological cycles. The sexual phase of the N. caninum cycle only occurs in dogs, coyotes, Dingoes and grey wolves (Canis lupus), and is characterized by the release of oocysts in the faeces while bovines are the primary intermediate hosts of the parasite2,3. Transmission of N. caninum to cattle can be occur either horizontally by the oocyts ingestion or vertically through the placenta4. Because the pathogenesis of neosporosis in cattle is complicated, it is unclear why some animals abort while others do not5.

The infected cattle could be suffer from abortion, early foetal losses, neonatal deaths, stillbirths, and embryo reabsorption5,6. The dairy industry may suffer financial losses as a result of the infrequent, endemic, or epidemic abortion caused by N. caninum that frequently occur during the second trimester of pregnancy7. Moreover, there are no definite indicators of protective immunity in recovered animals, and the parasite may persist lifelong in the infected host5. Furthermore, there are currently no viable vaccines or treatment for Neospora infection8. In order to develop and apply strategies to control bovine neosporosis, it is crucial to understand risk factors related to N. caninum infection1.

In order to identify N. caninum infection in animals, a variety of laboratory techniques, including serology, histopathology and molecular techniques are now accessible9. Serologic tests, such as the indirect fluorescent antibody test (IFAT), direct agglutination test (DAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting (IB), were suggested as a diagnostic tool to investigate N. caninum antibodies9,10,11. ELISA is the most reliable serologic tool for determining specific antibody titers to N. caninum. Additionally, because of its relative quickness, it is more suitable for epidemiologic research1,12,13,14,15,16,17,18,19,20,21,22,23. There are some commercial ELISA assays available for identifying specific antibodies to N. caninum in bulk and bovine milk. Hence, bulk milk testing can be used to evaluate levels of N. caninum antibody in dairy herds24.

Studies have shown that frequent exposure to the sources of infection tends to increase the probability of animals to be seropositive for N. caninum25,26. The seroprevalence of N. caninum varies from 0 to 100% in various of animal species around the world. Egypt has reported a range of seroepidemiological data, including 4.3–8.6% in sheep27,28, 4.3% in goats28, 38.04% in cattle29 and 10.9% in camels14. However, there was little comprehensive information on the risk factors related to dairy cattle neosporosis in Egypt prior to our investigation.

Therefore, this study was conducted to assess the existence of antibodies against N. caninum and related risk factors in dairy cattle in some Egyptian governorates.

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.

Study area

A cross-sectional investigation was carried out to determine the relation between serological status of N. caninum in dairy cattle and possible risk factors. The study was performed in the governorates of Kafr ElSheikh, Menofia, Gharbia, and Alexandria, situated at northern Egypt at 31°06′42″ N 30°56′45″ E, 30.52° N 30.99° E, 30.867° N 31.028° E and 31°11′51″ N 29°53′33″ E, Fig. 1.

Figure 1
figure 1

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

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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 formula30, 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 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 determined19,21,31,32,33,34. The Hosmer–Lemeshow statistic was computed to assess the model's goodness-of-fit35.

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.

Table 1 Seroprevalence of Neospora caninum in cattle raising in the four governorates under the study.
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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.

Table 2 Multivariable logistic regression analysis for risk factors associated with Neospora caninum infection.
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Discussion

Neospora caninum is one important cause in cattle abortion36. 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 measures26,37,38.

In Egypt, the antibodies against N. caninum were detected in some species like sheep, cattle and camels14,27,29 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 Colombia39.

Neosporosis in globally distributed and the reported prevalence rates range from 10.7 to 19.6% in Africa40,41, 5.7–43% in Asia42,43, 7.6–76.9% in America44,45 and 0.5–27.9% in Europe46,47. However, the reported rate in the present study is not high, similar findings were reported in Brazil48 and Sengal49.

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 oocysts33,50,51,52,53,54,55,56,57,58,59,60.

Neosporosis in globally distributed and the reported prevalence rates range from 10.7 to 19.6% in Africa40,41, 5.7–43% in Asia42,43, 7.6–76.9% in America44,45 and 0.5–27.9% in Europe46,47. However, the reported rate in the present study is not high, similar findings were reported in Brazil48 and Sengal49.

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 factors17,18,23,59,61,62.

Studies have shown that frequent exposure to the sources of infection tends to increase the probability of animals to be seropositive for N. caninum25,26. 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 Brazil64,65, Croatia66, Jordan67, Romania46, and Venezuela68, 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 cattle15,69,70.

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 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 crossbreeding73. 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 milking75.

It is generally established that seropositive N. caninum cattle are more likely to prone abortion than seronegative N. caninum cattle1. 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 indications76.

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 and78. 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 oestrus77. 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 pregnancy49,79.

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 animals81,82. 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 population39.

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.