Seroprevalence and risk factors analysis of Neospora caninum and Toxoplasma gondii in cattle of Beheira, Egypt

Introduction Neospora caninum and Toxoplasma gondii are closely related obligate intracellular protozoan parasites. They are considered to be the major causes of infectious abortions and congenital abnormalities in livestock worldwide resulting in huge economic losses. Currently, there are no reports on the prevalence of neosporosis or toxoplasmosis in cattle in Beheira, Egypt's most important region for cattle industry. Methods The current study investigated the presence of anti-N. caninum and anti-T. gondii antibodies in apparent healthy cattle from eight localities representing the whole area of Beheira. A total of 358 plasma samples were randomly collected from 6 dairy and 10 beef farms and analyzed by commercially available ELISAs. Production type (dairy versus beef), sex (female vs male), age (< 3 years, 3–5, and > 5 years old), breed (mixed vs Holstein vs Colombian Zebu), and location (various locations) were assessed as risk factors for N. caninum and T. gondii infections. Results and discussion Of the samples, 88 (24.6%) and 19 (5.3%) were positive for anti-N. caninum and anti-T. gondii antibodies, respectively, and mixed infection was detected in 7. Of the 16 herds, 6 dairy and 7 beef herds were positive for antibodies to N. caninum. Antibodies to T. gondii were detected in 4, and 5 of dairy and beef herds, respectively. Production type (dairy) and, therewith, sex (female), age (aged over 5 years), and location were considered as risk factors for N. caninum infection. No factors statistically associated with T. gondii infection were identified. Overall, this study provided the first serological detection of N. caninum and T. gondii infections in cattle from Beheira, demonstrating the endemicity of both parasites in the main cattle rearing region of Egypt. This study also confirmed earlier reports of N. caninum being more present in dairy cattle than in beef cattle. Routine monitoring of N. caninum and T. gondii infections and the implementation of control strategies are urgently needed.


Introduction
Neospora caninum and Toxoplasma gondii are single-celled, obligate intracellular protozoan parasites causing neosporosis and toxoplasmosis, respectively (1). They affect a variety of warmblooded animals, including humans for T. gondii, and inflict serious clinical and economic losses, especially in the global cattle industry (2, 3). The reproductive stages of T. gondii are exclusively observed in members of the Felidae family (4), while canids serve as final hosts of N. caninum . /fvets. .
(5, 6). Neospora caninum is recognized as the most common infectious cause of abortion in cows globally (7). Early embryonic death and resorption, abortion, stillbirth, delivery of a calf with deformities, and the birth of healthy carrier offspring are all possible outcomes of N. caninum infection in pregnant cows (8). Early culling of seropositive cattle has been recorded (9) due to increased veterinary medical treatment expenditures and a drop in growth rates in these animals (10). Primary infection with T. gondii in cattle during gestation can cause abortion or congenital anomalies, including severe generalized toxoplasmosis with a possible fatal outcome (11). Infection in immunocompetent non-pregnant hosts, on the other hand, is frequently silent, resulting in mild or no clinical signs (12). The presence and spread of bovine neosporosis have been reported in more than 34 countries from Europe, Asia, America and Australia (13). However, few studies have investigated the prevalence of N. caninum infection in African countries (14). Toxoplasma gondii has been isolated from almost all geographical regions worldwide except Antarctica (15). Egypt has a cattle population of about 5.1 million cattle (16) with the highest density in Beheira province. Beheira province is located in the Nile Delta, the northern (lower) part of Egypt. It embraces the whole of the delta west of the Rosetta branch with a considerable desert region to the south. Economically, agriculture is the most important sector in Beheira province and produces wheat, rice, and corn as the major crops. Other cereals, potatoes, sugar beets, tomatoes, and sesame are also harvested (17). The unique location of Beheira province made it an attractive area for investment in cattle production, and currently, it has the highest number of cattle farms in Egypt with approximately 20% of the total cattle population, i.e., about 1 Mio heads (16). The majority of cattle herds are either mixed native breeds, raised in small to medium-sized herds by local farmers, or imported cattle breeds from Europe, Africa, and South America with improved meat and milk production characteristics, including Holstein Friesian, Zebu, Brown Swiss, and Simmental (16, 18). Cattle farming systems are either intensive (more than 200 cattle), semiintensive (20-200 cattle per farm), or smallholder (5-20 cattle per farm) systems (19).
Several investigations have been conducted in Egypt to determine the prevalence of neosporosis and toxoplasmosis in various animal species and people from different geographic locations (southern and northern) (3, 19-30). However, there has never been a thorough investigation of the prevalence of these parasites in cattle of the Beheira area. As reported in a literature review, only 15 blood samples from an abattoir in Beheira were analyzed in 1977 (31), and 7 (46.6%) of those samples proved positive for T. gondii antibodies. Therefore, this study aimed to investigate the presence of anti-N. caninum and anti-T. gondii antibodies in apparently healthy cattle from different localities and farms in Beheira province, and to provide insight into the risk factors related to such infections. We opted for commercial ELISAs in this study to obtain objective results that will allow for comparisons between different laboratories and countries.

Ethical approval
This research has obtained the approval of the Ethics of the Institutional Committee of the Faculty of Veterinary Medicine at Damanhour University, Egypt (DMU/VetINF-2019-/0145). The project and procedures were explained to the animal owners, and verbal consent was obtained, as requested by the Ethics Committee.

Blood sampling and plasma separation
Whole blood samples were collected from cattle using the tail vein puncture procedure and were stored in a glass tube with K 2 EDTA anticoagulant. Blood samples were transported immediately to the laboratory and kept at 4 • C. For serological investigation, plasma samples were separated by centrifugation at 3,000 rpm for 15 min at room temperature and kept at −20 • C prior to analysis.
Serological detection of N. caninum and T. gondii antibodies by ELISA All 358 collected cattle plasma samples were serologically investigated for infection by N. caninum or T. gondii or mixed infection via indirect ELISA assay, using commercial ELISA kits. For N. caninum, samples were analyzed with the competitive multispecies ELISA for neosporosis (ID.vet, Grabels, France). Such test has the potential to detect IgG and IgM through the use of purified extract of N. caninum as coated antigen and anti-N. caninum-HRP, with recorded sensitivity (100%; CI 95%: 98.8-100%) and specificity (100%; CI 95%: 99.63-100%) as provided by the manufacturer. Plasma samples and controls were diluted 1:2. The ODs obtained were used to calculate the percentage of sample (S) to negative (N) ratio (S/N%) for each of the test samples according to the following formula S/N (%) = OD sample/OD negative control × 100. Samples with an S/N% greater than 60% were considered negative; if the S/N% was between 50 and 60%, the result was considered doubtful, and considered positive if the S/N% was less than 50%.
Regarding T. gondii, the samples were analyzed with the indirect multi-species ELISA for toxoplasmosis (ID.vet, Grabels, France) according to the manufacturers' instructions. This tool was specified to detect IgG through the use of P30 as coated antigen and antimultispecies IgG-HRP, with recorded sensitivity (98.36%; CI 95%: 95.29-99.44%) and specificity (99.42%; CI 95%: 98.8-100%). Plasma samples and controls were diluted 1:10. The optical density (OD) obtained was used to calculate the percentage of sample (S) to positive (P) ratio (S/P%) for each of the test samples according .
/fvets. .  to the following formula: S/P (%) = (OD sample-OD negative control)/(OD positive control-OD negative control) × 100. Samples with an S/P% less than 40% were considered negative; if the S/P% was between 40 and 50%, the result was considered doubtful, and considered positive if the S/P% was greater than 50%.
The ODs of all ELISA results were read at 450 nm and measured with an Infinite R F50/Robotic ELISA reader (Tecan Group Ltd., Männedorf, Switzerland).

Statistical analysis
The significance of the differences in the infection rates of the different diseases and risk factors was determined by the Fisher Exact Probability Test (two-tailed) using online statistics software http:// vassarstats.net/ and GraphPad Prism version 5. A P value of < 0.05 was considered statistically significant. Odds ratios and the 95% confidence intervals were calculated using www.vassarstats.net and GraphPad Prism version 5.
The impact of several risk factors such as age, sex and production type, breed, and location of the tested cattle on the seroprevalence of N. caninum and T. gondii was investigated. For N. caninum infection, prevalence in cattle <3 years old was 24/142 (16.9%), and a non-significant increase [p = 0.23; odds ratio (OR) = 1.5] in seropositivity was observed in cattle aged 3-5 years (33/143; 23.1%). A significant increase in N. caninum seroprevalence (p < 0.0001; OR = 3.6) was demonstrated in older cattle aged >5 years (31/73, 42.5%; Table 4). Female cattle (dairy cows) showed a significantly higher seroprevalence for N. caninum (64/216, 29.6%; p = 0.008, OR = 2.1) than males that were used for beef production (24/142, 16.9%) ( For T. gondii, none of the tested factors (age, sex and production type, breed, and locality) had a significant effect on the seroprevalence. The lowest infection rate was found in cattle 3-5 years old (3.5%) followed by the youngest group (<3 years; 4.2%).
Noticeably, a complete absence of T. gondii infection was reported in tested cattle from Nubariyah but there was no significant difference to Damanhour (Table 5).

Discussion
This study provided the first report on the seroprevalence of N. caninum and T. gondii in cattle from Beheira province as well as a respective risk factor analysis (age, sex, and therewith production type, breed, and location). Our findings indicated a greater individual infection rate with N. caninum (24.6%) than with T. gondii (5.3%), which was also reflected on the herd level with 13 herds positive for N. caninum compared to 9 herds for T. gondii. Reportedly, cattle are naturally more susceptible to N. caninum than T. gondii infection and N. caninum can persist in a herd due to the highly efficient vertical transmission from cow to calf (32, 33). Indeed, in endemically infected dairy cattle herds up to 95% of calves of seropositive cows are born infected (13,34). In our study, the seroprevalence in the youngest age group (<3 years) was almost 17%, very close to the one reported before for this age group (17.5%) by Fereig  Indeed, the majority of cats in Beheira are now domesticated, and the number of stray cats has dropped considerably in past years. As an outcome, a progressive reduction in the number of new cattle infections was anticipated. The positive rate in female dairy cattle (29.6%) was significantly higher than in male beef cattle (16.9%). This supports earlier observations by Quintanilla-Gozalo et al. (38), who recorded higher seroprevalences to N. caninum in dairy cattle (83.2 and 36.8%) than those in beef cattle (55.1 and 17.9%) at the herd and individual level, respectively. Age might be an important factor in this equation, as in our study most of the males were younger than 3 years old and females were >3 years. This might also explain the contrast with an earlier study that did not identify sex as a risk factor for infection with N. caninum (24). Therefore, further investigation into the relationship between age, sex, and production type in cattle is necessary to determine the exact contribution of each variable to the infection risk. In accordance with an earlier study (19), the breed of cattle had no impact on the prevalence of N. caninum infection. However, a trend toward higher infection rates for both, N. caninum and T. gondii, was seen in mixed breed herds when compared to Holstein and Colombian zebu cattle in our study. Moreover, coinfection was reported only in 3 dairy mixed herds. These data could be linked to the management of these herds. Most of mixed cattle herds were not completely fenced against stray animals, suffered from shortage in veterinary services and regular screening of diseases, and were restocking with their own calves. The importance of the management system on the prevalence rate of N. caninum was also reflected by the significantly higher seroprevalence in Edku (only mixed breed analyzed), Dilinjat (all three breeds), and Kafr El-Dawar (mixed breed and zebu), respectively. This could be attributed to the nature of farming systems and the density of farms in these localities where most of farms are semi-intensive in comparison to the well-controlled intensive farms in other localities of the sampling area. These intensive herds mainly kept foreign breeds and the management was more professional. Furthermore, most of these cattle were imported as certified-free from diseases prior to stocking. Biosecurity, good veterinary practices, and use of artificial insemination in breeding system were also advantageous (16, 18). Neither age, sex and production type, nor breed were identified as risk factors for T. gondii infection in the examined cattle. This result was consistent with an investigation of the risk variables linked to T. gondii infection in cattle in southern Egypt (19). It's important to note that no Holstein cattle tested positive for anti-T. gondii antibodies, most probably for the management reasons discussed above.

Conclusion and recommendations
In conclusion, this study provided the first serological survey on subclinical infection with N. caninum and T. gondii in cattle in Beheira province. The findings indicated a higher prevalence of N. caninum compared to T. gondii among the cattle tested, as well as in comparison to earlier investigations carried out in Egypt. The most important risk factors for N. caninum infection in the cattle studied were age, sex, and therewith production type, and locality. However, the current investigation found no risk factors for T. gondii infection. Future research is needed to provide more detailed information about the prevalence of N. caninum and T. gondii in Egypt. The elaboration of effective control strategies and biosecurity measures in cattle farms has become an urgent need, because no vaccines or effective drugs against N. caninum or T. gondii are currently available.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
This research has obtained the approval of the Ethics of the Institutional Committee of the Faculty of Veterinary Medicine at Damanhour University, Egypt (DMU/VetINF-2019-/0145). Written informed consent for participation was not obtained from the owners because oral consent was obtained from each owner, as was agreed upon by the Ethics Committee (see respective letter). In Egypt, written informed consent is very hard to obtain because verbal explanations and consequent consent is the usual way of reaching agreements.

Author contributions
Conceptualization, design, resources, and shared materials: SM, RF, and CF. Experiments, formal analysis, and investigation: SM, RF, RH, KS, and CF. Writing-original draft: SM, RF, and RH. Writingreview and editing, project administration, and funding acquisition: RF and CF. All authors have read and agreed to the published version of the manuscript.