Introduction

Neosporosis is one of important cause for abortion and reproductive abnormalities in vulnerable animal around the world, resulting in significant economic losses, notably among ruminants1,2,3. It is a worldwide parasitic disease caused by Neospora caninum, protozoan apicomplexan that includes canids as definitive hosts while birds and numerous species of mammals as intermediate hosts4,5. The main way of N. caninum infection in ruminants is vertical transmission or transplacental6. In addition, the horizontal transmission is possible via consumption of tissues harboring cysts and tachyzoites as well as the consumption of sporulated oocysts in contaminated feed or water7,8,9.

This parasite is a significant contributor to bovine foetal miscarriage, stillbirth, the birth of weak calves, and lower milk supply. Moreover, due to the absence of viable options for the prevention and treatment of this disease, culling of infected animals is frequently necessary10. Buffalo is regarded as natural intermediate hosts of N. caninum, despite the fact that water buffalo differ from cattle in behaviour and physiology and can act as reservoirs for infectious disease agents of cattle11. Experiments on buffaloes have demonstrated that pregnant animals are vulnerable. However, buffaloes are more resistant to abortion, and their inflammatory reactions to infection are weaker than those of cattle12.

Several studies have been detected antibodies against N. caninum in water buffaloes in many countries over the world. The average seroprevalence of N. caninum in water buffaloes in worldwide is 48%12, which is much greater than those in beef cattle (11.5%) and in dairy cattle (16.1%)13. Among these, N. caninum seroprevalence in water buffalo is 20% in Asia, 54.4% in South America, and 67.1% in Africa12. However, few studies have been conducted to investigate the risk factors for N. caninum infection in water buffaloes. The age of buffaloes and the presence of dogs on the property are considered as risk factors for incidence of neosporosis14,15.

In Egypt, N. caninum have been reported in various animal species, the seroprevalence rates were ranged between 6.97% to 42.8% in water buffaloes16, 12.21% to 20.43% in cattle16,17,18, 79.7% in sheep19, 93.4% in goats19 and 10.9% in camels3. Controlling of neosporosis requires the identification of risk factors for the presence of antibodies to N. caninum, particularly in the absence of efficient immunoprophylactic or treatment regimens since potential risk factors may change between geographical locations. However, few studies have been conducted to evaluate the associated risk factors for N. caninum infection.

Consequently, the purpose of this research was to estimate the seroprevalence of N. caninum in water buffaloes in some Egyptian governorates situated at North and Nile Delta of Egypt and to identify the risk factors associated for N. caninum infection.

Materials and methods

Ethical statement

The Benha University ethics committee for animal experiments approved all methods including the handling and collection of blood samples. In addition, all methods were performed in accordance with the relevant guidelines and regulations. Owners of the buffaloes gave their explicit consent for the collection of the samples. The ARRIVE criteria were adhered to throughout the study process.

Study area

For this study, the four governorates in northern Egypt with the greatest water buffalo populations were selected. The four governorates chosen for the study were Kafer ElSheikh, Qalyubia, Gharbia and Alexandria located at latitudes 31°06′42′′ N, 30.41° N, 30.867° N and 31°11′51″ N, respectively, and longitudes 30°56′45′′ E, 31.21° E, 31.028° E and 29°53′33″ E, respectively (Fig. 1).

Figure 1
figure 1

Map showed the governorates under the study (map generated by QGIS program).

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Kafr ElSheikh, Qalyubia and Gharbia situated at Nile Delta of Egypt, which characterized by moderate weather at summer and the average annual temperature is 28 °C (with a range of 17 °C to 42 °C), and its average annual precipitation is between 100 to 200 mm. In addition, the prevailing north wind, blowing across the Mediterranean, gives Alexandria a less harsh climate than the surrounding desert.

Study design and sample size

A cross-sectional study was performed during September 2020 to August 2021 to determine the prevalence of antibodies against N. caninum in water buffaloes in the four studied governorates. Using Epi Info™ 7, a representative sample size of 450 buffaloes was determined with a 95% confidence interval, a 5% statistical error, and a 42.8% expected seroprevalence for N. caninum16. A total of 450 water buffaloes of various ages and both sexes were chosen at simple random technique from individual animal raised by individual farmers. All of buffaloes included in the study were apparent healthy without in clinical signs and some of them had history of abortion.

Blood samples (5 mL) were obtained from the jugular vein, centrifuged at 3000 rpm for 10 min, serum separated, and kept at −20 °C. An epidemiology questionnaire was used at the time of sampling to collect data from each farmer on the study region, age, sex, breeding system (household or farming system), time of the abortion, previous abortions, and contact with dogs. The information about history and number of abortion or contact with dogs depends on data collected from farmers.

Serological analysis

The antibodies against N. caninum were detected in all sera using commercial indirect ELISA Kit (IDEXX Laboratories Inc., Westbrook, USA). The sensitivity of this kit is 100%, while the specificity 98.9%. the optical density (OD) was measured at 650 nm using microplate ELISA reader (AMR-100, AllSheng, China). Serum samples considered to be positive for N. caninum if their sample to positive (S/P) ratios were more than or equal to 0.50.

Data analysis

Data were collected and analyzed using SPSS ver. 24 program (IBM, USA). A univariate analysis of the variables of interest was carried out using the Pearson's chi-squared test to determine factors related to N. caninum infection. The result of P-value < 0.05 was regarded as statistically significant. The results were analysed through the use of univariable logistic regression to evaluate the association between each variable and prevalence of N. caninum in buffaloes. A logistic regression analysis was used to determine the relationship between the existence of anti-N. caninum antibodies and risk variables (sex, age, time and history of abortion, and contact with dogs). The multivariate regression model was used to assess odds ratios (ORs), and confidence intervals (CIs) of each significant variable20,21. Odds ratios more than one indicated an increased risk of N. caninum seroprevalence, whereas odds ratios less than one indicated a lower risk of N. caninum seroprevalence. The model's fit was evaluated using the Hosmer and Lemeshow goodness test.

Results

We examined 450 buffalo sera from four Egyptian governorates between September 2020 and August 2021, and the findings revealed that 31.1% (141/450) of examined animals had antibodies against N. caninum, with non-significant variation (P = 0.238) between studied governorates, Table 1.

Table 1 Seroprevalence of N. caninum in water buffaloes in relation to different studied factors.
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Several factors including sex, age, breeding system, time of abortion, history of abortion and contact with dogs were analyzed to determine the risk factors related with N. caninum infection in water buffalo (Table 2).

Table 2 Multivariate logistic regression analysis for variables associated with seroprevalence of N. caninum in water buffaloes.
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There was significant difference (P < 0.05) in seroprevalence in relation to sex and age of examined water buffaloes. The seroprevalence of N. caninum was greater in females (35.9%) than males (11.8%), and in elder buffaloes over 4 years old (53.2%) as compared to young buffaloes under 2 years (17.3%) or buffaloes aged 2–4 years (25.6%). Furthermore, the seroprevalence of N. caninum was significantly (P < 0.05) higher in buffaloes with a history of abortion (59.8%), particularly those aborted in the second trimester (81.5%). In the current investigation, the seroprevalence of N. caninum in buffaloes kept in touch with dogs was substantially greater (38.9%) than in other animals kept away from dogs, Table 1.

Multivariable logistic regression analysis was performed to assess the risk factors associated with N. caninum infection in water buffaloes, Table 2. The results revealed that females (OR = 1.96, 95%CI: 1.22−4.17), buffaloes more than 4 years age (OR = 5.80, 95%CI: 2.26−14.86), abortion in second trimester (OR = 16.48, 95%%CI: 2.99−34.03), history of abortion (OR = 3.45, 95%CI: 1.58−7.52) and contact with dogs (OR = 2.55, 95%CI: 1.51−4.32) were identified as risk factors for N. caninum infection in water buffaloes, Table 2.

Discussion

Infection with N. caninum causes retained fetal membranes and abortion, and subsequent infections raise the risk of reproductive disorders in water buffalo, which causes significant financial losses to the agricultural sector5,22,23. However, the available data on the prevalence of associated risk factors for N. caninum infections in Egyptian water buffaloes are scarce.

In the present study, the overall seroprevalence rate of N. caninum in water buffaloes was 31.3%, falling within the previously reported range (27.5% to 35.4%) in Brazil24,25. The reported seroprevalence rate for neosporosis in the current survey was higher than those reported in in Vietnam (1.05%)26, Thailand (9.1–16.7%)27, India (9.9%)28, Iran (19.3%)29 and Mexico (24.3–41.2%)30,31. Furthermore, the prevalence of the current findings appears to be lower when compared to research done in Brazil, where rates varied from 49 to 88%32,33,34, Argentina (64%)35 and Guangxi, China (50.9%)36.

The seroprevalence rates of N. caninum in buffaloes varied across countries due to a variety of factors such as climatic factors, where the mild temperature and humidity help in growth of oocyst of Neospora5,37,38. In addition, the rearing systems, geographic location, and farm management were varied in different countries15,37,39,40,41,42,43,44. Furthermore, comparing studies is challenging since researchers use varied sample criteria, serological procedures, and cut-off values, particularly for the immunofluorescence antibody test24.

In the line of previous results of Baltazar-Pérez, et al.31, the sex is significant risk factor for prevalence of N. caninum in buffaloes. Contrary to findings of Campero, et al.35 and Kengradomkij, et al.27, the results revealed strong association between N. caninum seroprevalence in water buffaloes and sex. According to our findings, vertical transmission is frequently thought to be the primary mode of infection in bovines45.

According to age group, adult animals in our study had a much greater prevalence than young animals. Similarly, several studies have been found significant association between age and the seropositivity for neosporosis in water buffaloes14,46,47,48,49. Indeed, elder age would suggest a possibly longer interaction with polluted water and/or feed, explaining the increased occurrence. It has been proposed that the age correlation merely reflects the annual increase in the likelihood of being exposed to parasite oocysts. In comparison to younger animals, older animals are predicted to have a larger likelihood of cumulative exposure to infectious agents. In contrast, the seroprevalence of N. caninum in water buffaloes from northern Brazil were not affected by age factor33,41,50,51,52. Interestingly, calves had a significant seroprevalence rate (17.3%), the persistence of maternal antibodies or trans-placental transmission47 might be to blame for this. According to Cardoso, et al.53, maternal antibodies against N. caninum can last up to 21 weeks after birth.

In contrast to the findings of Bărburaș, et al.46, we observed no significant relationship between breeding system and N. caninum seroprevalence. The seroprevalence rate was greater in farm animals than in house-hold animals, which was due to the chance of farm animals being exposed to sources of parasite oocyst contamination being higher.

There is no information in Egypt about buffalo reproductive losses caused by N. caninum infection. The findings of this study revealed strong association between N. caninum infection and abortion especially in second trimester stage. These findings were consistent with what have been found in previous studies of Romero-Salas, et al.30,54, Anderson, et al.55 and Wouda, et al.56. This provides circumstantial evidence that N. caninum may have a role in water buffaloes abortions in the research region.

Similarly, to the findings of Oliveira, et al.24, N. caninum prevalence rate was greater among water buffaloes kept with close contact with pet dogs. This could be attributed to dogs consuming aborted materials and consequently shedding infective oocyst which contaminate food and play a significant role in horizontal spreading of N. caninum infection to vulnerable animals57.

The limitation of this study is random sampling because it does truly represent about the prevalence of the disease in the studied areas.

Conclusion

The present findings confirmed that presence of antibodies against N. caninum in water buffaloes in studied governorates in Northern Egypt. In addition, some factors such as sex, age, time or history of abortion and contact with dogs considered as potential risk factors for N. caninum infection. These findings highlight the relevance of N. caninum control and prevention in Egypt, where water buffaloes are major domestic animals, as well as the need of risk factor analysis for efficient neosporosis control in various locations. The effects of Neospora infections in water buffalo should be further studied, but it's also important to look at the epidemiological significance of sick buffaloes and their connection to bovine neosporosis.