Introduction

Babesia bigemina and B. bovis are tick-borne hemoprotozoan parasites causing bovine babesiosis, a disease negatively affecting livestock industry worldwide, especially in subtropical and tropical regions [1]. Economic losses caused by babesiosis are ascribed to poor feed conversion, reduced milk production, and increased mortality [2]. Babesia sporozoites invade the bovine red blood cells (RBCs) and transform into merozoites, which egress and cause destruction of RBCs, resulting in anemia and anemia-related clinical signs, and in some cases, may lead to neurological and respiratory syndromes [1, 3].

Proper diagnosis of bovine babesiosis plays a critical role in the management, monitoring, and control of the disease [4, 5]. Traditionally, microscopic techniques for blood examination using thick or thin smears stained with Giemsa can confirm the presence of Babesia, and remain the most convenient tool to diagnose acute babesiosis [6]. However, this technique has limited value in chronic babesiosis due to the low parasitemia. Moreover, the main challenge of using this method is how to differentiate different Babesia species. Serological diagnostic tests, including the enzyme-linked immunosorbent assay (ELISA) based on specific recombinant proteins, have been established to assess the exposure to different Babesia species, monitor the immune status of the host animals, and obtain critical data that can be used to control the disease [7,8,9,10,11]. The recombinant proteins including the C-terminal region of rhoptry-associated protein 1 (BbigRAP-1a) and spherical body protein-4 (BbSBP-4) are commonly used to diagnose and specifically differentiate between B. bigemina and B. bovis [12,13,14,15,16].

In Egypt, B. bigemina is transmitted by Rhipicephalus annulatus and R. microplus ticks, while B. bovis is transmitted to bovines exclusively by R. microplus ticks [17, 18]. Several studies previously detected these protozoan parasites in Egyptian cattle and water buffaloes bred in different geographical regions of Egypt [12, 19,20,21,22], including Menoufia province, where cattle and water buffaloes are widely distributed. Menoufia ranks second in bovine population in Lower Egypt [23]. However, information on the seroprevalence of bovine babesiosis in Menoufia has not been updated in recent years. The present study aimed to determine the seroprevalence of B. bigemina and B. bovis in cattle and water buffaloes in Menoufia province, Egypt through ELISA based on recombinant Babesia antigens.

Materials and Methods

Calculation of Sample Size

The minimum sample size required for this study was calculated in the online platform Epitools [24] by the formula: \(N\, = \,\left\lfloor {\frac{{Z^{2} \, \times \,P\, \times \,\left( {1\, - \,P} \right)}}{{e^{2} }}} \right\rfloor \, \div \,\left\lfloor {1\, + \,\frac{{(Z^{2} \, \times \,P\, \times \,\left( {1\, - \,P} \right)}}{{\left( {e^{2} \, \times \,n} \right)}}} \right\rfloor\), where Z is the confidence level (95%), P is the expected true proportion (0.5) and e is the margin of error (± 0.05). The population size (n) inputted was 7.8 million, the total population of cattle and water buffaloes in Egypt in 2017 [25]. The minimum sample size required is N = 385.

Animal Sampling

A total of 506 blood samples of cattle (N = 262) and water buffaloes (N = 244) were collected from open public markets in Qewaisna and Birket Al Saba, Menoufia province, Egypt (Fig. 1) during the different seasons (spring, summer, autumn and winter) of June 2017–May 2018. The markets were chosen based on convenience while sampled animals were randomly chosen. The examined animals are kept in farmers’ stables in the evening and used in the fields in daytime. Cattle ages 1 month to 8 years old were classified into two groups: young (2 years or less) and adult (more than 2 years). Likewise, water buffaloes with the age range of 1 month to 10 years old were classified into two groups: young (3 years or less) and adult (more than 3 years). Blood samples were collected from the jugular or caudal vein of the animals. Then, the samples were incubated at room temperature for 1 h and centrifuged at 3000 rpm for 15 min. Sera were collected and stored at − 20 °C until use.

Fig. 1
figure 1

Map of sampling areas in Menoufia province, Egypt

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Recombinant B. bigemina RAP-1a and B. bovis SBP-4 Preparation

The Argentina strain of B. bigemina and Texas strain of B. bovis were continuously cultured in bovine erythrocytes (RBCs) using a micro-aerophilous stationary-phase culturing system [26]. Total RNA was isolated and cDNA was synthesized from each parasite culture. The DNA fragments encoding the B. bigemina C-terminal region of rhoptry-associated protein 1 (BbigRAP-1a/CT: 390–480 aa, GenBank accession number M60878) and B. bovis spherical body protein-4 (BbSBP-4, GenBank accession number AB594813) were amplified by PCR from B. bigemina and B. bovis cDNA, respectively, subcloned into a pGEX-4T1 plasmid vector (Amersham Pharmacia Biotech, Inc., USA) using suitable restriction enzyme sites, then, expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli BL21 strain (Amersham Pharmacia Biotech, Inc.). The recombinant proteins were purified from the soluble fractions of E. coli lysates using Glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech, Inc.) and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis [13]. The purified recombinant proteins were used for the subsequent assays.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was done in accordance to the modified procedure described previously [12]. Antigen-coated wells were incubated with 50 μl of cattle or water buffalo serum samples diluted to 1:100. Either 50 μl of horseradish peroxidase (HRP)-conjugated rabbit anti-bovine IgG (Bethyl Laboratories, USA) diluted at 1:10,000 or 50 μl of HRP-conjugated rabbit anti-bovine IgM (Chongqing Biospes Co. Ltd., China) diluted at 1:10,000 was used as secondary antibody. Absorbance was detected at 405 nm using an ELISA reader (Radim Diagnostics, Italy). The results were calculated by obtaining the differences in mean optical densities between the recombinant antigens (BbigRAP-1a/CT or BbSBP-4) and those of the GST protein. The cutoff points were calculated as the OD405 mean value for B. bigemina- and B. bovis-negative sera plus 3 standard deviations (n = 20): for IgG, BbigRAP-1a/CT: 0.019 and BbSBP-4: 0.018 in cattle, BbigRAP-1a/CT: 0.018 and BbSBP-4: 0.018 in water buffaloes; for IgM, BbigRAP-1a/CT: 0.019 and BbSBP-4: 0.018 in cattle, BbigRAP-1a/CT: 0.02 and BbSBP-4: 0.019 in water buffaloes. The negative sera from our standard sera stock were tested and confirmed negative by PCR [12].

Statistical Analysis

Association of the seropositivity of animals with sampling region, age, sex and season of sample collection was evaluated using the Pearson’s chi-squared or Fisher’s exact test in Microsoft Excel and GraphPad Prism 7. The logistic regression model was used to evaluate the differences in seroprevalence rates of animals based on age, sex and season of sample collection in IBM SPSS® version 11. The dependent variable used was the outcome of the test (seropositive or seronegative) while risk factors, i.e., age, sex and season of sample collection, were the independent variables. A p value < 0.05 was considered significant.

Results

Overall, 41.60% (109/262) and 38.17% (100/262) of cattle showed seropositivity (positive for IgM, IgG or both) for B. bigemina and B. bovis, respectively. Seroprevalence of B. bigemina was 37.40 and 9.54% for acute infection (IgM) and chronic infection (IgG), respectively. The number of cattle with positive reactivity against B. bigemina IgM was significantly higher (p = 0.0004) in the area of Birket Al Saba (49.56%) compared to Qewaisna (28.19%) (Table 1). Seroprevalence of B. bovis for IgM and IgG antibodies was 35.88 and 6.11%, respectively. B. bovis IgM-seroreactive cattle serum samples from Qewaisna is notably higher than in Birket Al Saba while no significant differences were observed in B. bovis IgG-positive cattle between the two areas. Mixed infection with B. bigemina and B. bovis was detected in 24.05% (63/262) of the cattle serum samples.

Table 1 Seroprevalence of Babesia bigemina and Babesia bovis infections in cattle from Menoufia province
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In water buffaloes (N = 244), the overall seroprevalence of B. bigemina was 35.66% (87/244; 27.87% for IgM and 15.16% for IgG), while the overall seroprevalence of B. bovis was 31.97% (78/244; 21.72% for IgM and 15.16% for IgG). B. bigemina seropositivity for IgM antibodies (p = 0.007) and B. bovis seropositivity for IgG antibodies (p = 0.00000018) were significantly increased in the area of Qewaisna compared to Birket Al Saba area. Mixed infection rate with B. bigemina and B. bovis was 15.16% (Table 2).

Table 2 Seroprevalence of Babesia bigemina and Babesia bovis infections in water buffaloes from Menoufia province
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The seroprevalences of B. bigemina and B. bovis based on age, sex and season are summarized in Table 3 for cattle and Table 4 for water buffaloes. During acute infection, the seroprevalence rates for both parasites in adult cattle (> 2 years) and water buffaloes (> 3 years) were higher than those in young animals, although not significant. Moreover, during chronic infection, the B. bigemina seroprevalence rates in adult animals were notably higher than that of the infection rates in the young ones. In female water buffaloes, higher seropositivity rates for all infections were observed compared to male animals (p < 0.001) (Table 4). Moreover, the seroprevalence was higher in female cattle during the chronic infection of B. bigemina and the acute infection of B. bovis in both animals (Tables 3 and 4). No significant differences were recorded between infection percentages of both parasites in cattle according to sex (Table 3).

Table 3 Seroprevalence of Babesia bigemina and Babesia bovis in cattle according to age, sex and season
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Table 4 Seroprevalence of Babesia bigemina and Babesia bovis in water buffaloes according to age, sex and season
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In acutely B. bigemina-infected cattle, binary logistic regression analysis indicated that the differences in seropositivity rates in serum samples collected between winter season and spring/autumn were significant (Table 3). In water buffaloes, during acute infection, statistical analysis indicated that the seroprevalences of B. bigemina and B. bovis were significantly higher in the summer season compared to the spring and the autumn season, respectively. No significant changes were noted during chronic infection of B. bigemina in cattle and water buffaloes. Meanwhile, the seroprevalence of B. bovis chronic infection in water buffaloes was significantly (p < 0.001) increased to 30.0% in the summer season compared to 2.86% seropositivity rate in the winter season (Table 4).

Discussion

In this study, we determined the seroprevalences for B. bigemina and B. bovis in cattle (41.60 and 38.17%, respectively) and water buffaloes (35.66 and 31.97%, respectively) in Menoufia province. These are higher compared to previous serological surveys conducted in other provinces of Egypt which recorded seroprevalences of 10.6–33.20% for B. bigemina and 9–20.43% for B. bovis in cattle, and 15.63–22.20% for B. bigemina and 11.46–22.20% for B. bovis in water buffaloes using ELISA [12, 22, 27]. In addition, a study in Menoufia employing PCR assays demonstrated that the Babesia spp. infection rate was 12.66% in cattle [20] while another study in Menoufia revealed molecular detection rates of 7.62 and 2.54% for B. bigemina and B. bovis in cattle, respectively, and 3.03% for B. bovis in water buffaloes [21]. Several molecular investigations in other Egypt provinces also recorded infection rates of 5.3–19.33% for B. bigemina and 3.97–5.88% for B. bovis in cattle, and 10.42 and 4.17% in water buffaloes, respectively [12, 21, 28]. Higher positivity rates reported in the current study using ELISA compared to studies which employed PCR assays indicate differences in the exposure of animals to the parasites and the presence of the parasites during the sampling [22]. Same observation has been documented from previous studies in Egypt [12, 22].

The differences in prevalences of B. bigemina and B. bovis in cattle and water buffaloes among different regions in Egypt may be attributed to the geographic distribution of tick vector [1, 29], climate condition, farm management and the time of sampling [30]. Adham et al. [18] demonstrated the presence of infected tick vector of bovine babesiosis in Egypt wherein B. bigemina was more predominant than B. bovis infection in Rhipicephalus (Boophilus) ticks [18]. In Mediterranean countries such as Egypt, R. (Boophilus) annulatus is considered as the most prevalent and main tick vector transmitting B. bigemina and B. bovis [30, 31]. Although this mirrors the higher seroprevalences of B. bigemina compared to that of B. bovis in both animals in the current study, the infection status of the tick vector of these parasites in Menoufia remains to be elucidated as this study did not evaluate tick infections.

Higher seropositivity rates were observed in adult animals with the exception during the chronic infection of B. bovis. This was consistent with previous studies that showed increased seroprevalence in older cattle [32,33,34]. Both young and adults are susceptible to babesiosis, but the stronger innate immune response in younger animals affords them with higher tolerance against Babesia infections [34]. In the present study, no significant changes were observed among male and female cattle. These results were consistent with previous studies carried out in cattle [35, 36].

Although seroprevalence of B. bigemina showed significant increase during the winter season compared to the spring and the autumn seasons during acute infection in cattle, the seroprevalences of B. bigemina and B. bovis were significantly increased during the summer season compared to those of the spring and the autumn season, respectively, during acute infection in water buffaloes. Moreover, during chronic infection of B. bovis in water buffaloes, the summer season prevalence was significantly higher than that of the winter season. Vieira et al. [37] reported that B. bigemina infection rate reduced significantly during the winter compared to those of spring, summer, or autumn. A previous study from Egypt revealed a significant increase in the tick infestation during summer months among cattle [38]. The increase in the tick infestation during summer months might be attributed to the increase in temperature and humidity [39]. Seasonal differences of tick infestation may lead to the varying degrees of season-related infection.

We found higher levels of seropositivity for both B. bigemina and B. bovis IgM antibodies, which indicate high frequency of recent acute infections among cattle and water buffaloes in Menoufia. This finding is particularly important and calls for intensive implementation of control measures for bovine babesiosis in the area.

In conclusion, the current study indicated that cattle and water buffalo babesiosis is frequent and presents a threat of an epidemic in Menoufia province. The current study provides additional information on the bovine B. bigemina and B. bovis infections in Egypt and will assist in developing strategies for controlling the disease. Further investigations are needed to understand the reasons behinds the high rates of these parasitic infections in Menoufia province, Egypt.