Prevalence of Neospora caninum and Toxoplasma gondii Antibodies and DNA in Raw Milk of Various Ruminants in Egypt

The prevalence of Neospora caninum and Toxoplasma gondii antibodies in raw milk samples was estimated in different ruminants and Egyptian governorates. Of 13 bulk milk samples tested by ELISA, five (38.5%) were positive for antibodies to N. caninum, and two samples were additionally positive for antibodies to T. gondii, resulting in a seroprevalence of 15.4% for both T. gondii and co-infection. In individual milk samples (n = 171) from the same bulks, antibodies to N. caninum were detected in 25.7%, to T. gondii in 14%, and 3.5% had antibodies to both parasites. A strong correlation between the OD values of the bulk samples and of the relevant individual milk samples was found for T. gondii (Pearson r = 0.9759) and moderately strong for N. caninum (Pearson r = 0.5801). Risk factor assessment for individual milk samples revealed that antibodies to T. gondii were significantly influenced by animal species, while no risk factors were detected for N. caninum antibodies. Additionally, DNA of N. caninum was detected in a bulk milk sample of cattle for the first time in Egypt, and DNA of T. gondii was found in bulk milk samples of cattle, sheep and goats. This is the first study in Egypt in which bulk milk samples of different ruminants were tested for the presence of N. caninum and T. gondii antibodies and DNA. Both individual and bulk milk samples are useful tools for monitoring antibody response to N. caninum and T. gondii infections in different ruminants in Egypt.


Introduction
Neospora caninum is a protozoan parasite that causes sporadic, endemic and epidemic abortions in cattle worldwide [1,2]. Endogenous transplancental transmission from dam to calf during pregnancy appears to be the main route of transmission [1,3]. Horizontal transmission of cattle by ingestion of sporulated oocysts shed by canids as final hosts can also occur [4,5]. Neospora caninum causes reproductive diseases which have a negative economic impact not only on milk yield but also on the replacement of dairy cows [6,7].
Milk has been demonstrated as a suitable sample for the detection of specific antibodies to N. caninum [8]. The antibodies found in milk, primarily IgG, are selectively transported from the serum into the mammary gland [9]. Milk samples from seropositive cows showed a significantly higher IgG level when compared with those from seronegative cows [10,11]. A high agreement, namely 95%, was recorded in antibody response between serum and milk [12]. Additionally, an excellent agreement among different diagnostic techniques in serum and milk samples was obtained [11,13]. Consistently, milk samples from sheep have been evaluated and validated for the detection of anti-N. caninum antibodies using bulk or individual samples [14][15][16].
Toxoplasmosis is an important and prevalent foodborne parasitic disease caused by Toxoplasma gondii. This protozoon can infect almost all warm-blooded animals, including human beings and farm animals [17]. The infection can occur in three ways: congenital transmission, organ transplant/blood transfusion, and contaminated food and water [18]. Human infection occurs frequently by consumption of raw or undercooked meat containing tissue cysts, unwashed fruit and vegetables contaminated with oocysts, and potentially also by tachyzoites shed in milk [19].
Topxoplasma gondii antibodies have been detected in the milk of various hosts like sheep, cattle, buffaloes, and camels [20,21], goats [21,22], and even in lactating women [23]. High agreement between ELISA results obtained with serum and milk samples of the same individuals was reported [22].
In cases of toxoplasmosis or neosporosis, substantial economic losses have been attributable directly to abortions in farm animals. Not only meat but also dairy sheep farms in Spain have suffered from economic losses resulting from T. gondii abortions [24]. Consistently, although neosporosis has been reported as a common cause of abortion in cattle, it has also been detected as a cause of abortion and perinatal mortality in sheep and goats [25][26][27]. Regarding public health, specific antibodies to N. caninum have been detected in women's sera using IgG [28] or IgM [29]. However, no evidence of a human clinical form of neosporosis has been detected yet. A recent study, however, discovered that two samples (1%) out of 201 examined human umbilical cord blood samples were Nc5 PCR-positive for N. caninum, while the placenta tested negative for this parasite [30]. On the contrary, approximately 25% of all people have experienced T. gondii infection globally. Congenital infections carry the risk of more serious results, and the growing foetus may exhibit symptoms that range from severe to moderate. Severe forms of congenital toxoplasmosis can cause congenital anomalies and even loss of life [31]. Severe infections in immunocompromised people often arise from the recurrence of a persistent illness [32].
In Egypt, antibodies to N. caninum and T. gondii, respectively, have been detected in numerous hosts using serum samples [33][34][35][36]. For milk samples, however, only one study looked for specific anti-N. caninum antibodies in cow's milk in Egypt so far [37]. Anti-T. gondii antibodies have been assessed in Egypt in human mothers' milk [23], donkey's milk [38], milk of sheep, cattle, buffaloes, and camels [21], and milk of sheep, goats and cows [39]. However, risk factors for antibodies in milk and the testing of bulk milk samples have not yet been assessed. Milk samples can represent a valuable, cheap, and non-invasive tool in monitoring N. caninum and T. gondii prevalence as alternatives to serum samples [40]. This current study aims at advancing the knowledge on surveillance of these important parasites using milk samples of ruminants.

Sample Collection and Preparation
Raw bulk milk samples (10 mL each) and individual raw milk samples (5 mL each) from 13 randomly chosen dairy farms and smallholders were collected from lactating cows, buffaloes, ewes and does between June to September 2022 (Table 1). Different regions were sampled in order to best represent Egypt: Dakahlia in the North, Cairo as the most densely populated governorate in the centre of Egypt, and Qena and Sohag in the South. Bulk milk samples have been collected directly at the farm (n = 7) or from shops (n = 6). During the direct visits at the farms, individual samples (n = 171) were randomly collected additionally (Table 1). Raw milk at shops usually was obtained freshly from a governmental or a private dairy farm and kept in bulk tanks for one-day use. Sometimes the milk was aliquoted in plastic bags of variable kilograms, and kept at +4 • C until selling out. No information on abortion history was available from these herds. The milk samples were centrifuged at 1000× g for 10 min. Lactoserum was collected from the layer below the cream layer and stored at −20 • C until used. * large farm: >200 animals; small farm: 50-200 animals; small holder: <50 animals; ** Shops sell the daily milk yield of one farm either from a large container or aliquoted in plastic bags.

Detection of Anti-N. caninum and Anti-T. gondii Antibodies in Milk
Samples were tested for specific antibodies to N. caninum using a commercially available ELISA (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France), according to the manufacturer's instructions. Briefly, undiluted milk samples and positive and negative controls were added to the microplate and incubated at 5 • C for 20 h. On the following day, the microplate was washed thrice using the washing buffer supplemented in the commercial kit. Washing out of any milk precipitates in all wells was visually confirmed. Then, 100 µL of diluted conjugate was added to the wells and incubated at room temperature (RT) for 30 min. After three washings, 100 µL substrate was added to each well and incubated at RT in a dark place for 15 min. Finally, 100 µL stop solution was added, and the optical density (OD) was measured. 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 50% were considered negative and considered positive if the S/N% was less than or equal to 50%.
Concerning T. gondii, the milk samples were analysed using an indirect multi-species ELISA for toxoplasmosis (ID.vet, Grabels, France) according to the manufacturer's instructions. Milk samples were added without dilution while the controls were diluted 1:10 and tested, as reported previously [22]. The OD obtained was used to calculate the percentage of sample (S) to positive (P) ratio (S/P%) for each of the test samples according 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 optical density of all ELISA results was measured at 450 nm with an Infinite ® F50/Robotic ELISA reader (Tecan Group Ltd., Männedorf, Switzerland).

DNA Extraction and Preparation
DNA extraction from bulk milk samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) as follows. After high-speed centrifugation (14,000 rpm/2 min) of 10 mL of milk samples, creamy and lactoserum layers were carefully discarded, and 25 Pathogens 2022, 11, 1305 4 of 13 mg of the pellet was mixed with 20 µL of proteinase K and 180 µL of ATL buffer and then incubated at 56 • C for 3 h. After incubation, 200 µL of AL buffer was added to the lysate, incubated for 10 min at 72 • C, and then 200 µL of 100% ethanol was added to the lysate. The lysate was then transferred to a silica column and centrifuged at 8000 rpm for 1 min. The sample was then washed and centrifuged following the manufacturer's recommendations. Nucleic acid was eluted with 100 µL of elution buffer provided in the kit.

PCR Application and Analysis
References, target genes (Nc5 for N. caninum and B1 for T. gondii), primer sequences and cycling conditions for the PCRs are given in Table 2 [41,42]. Primers were supplied from Metabion International (Planegg, Germany). PCR reaction was applied in a 25 µL reaction containing 12.5 µL of EmeraldAmp Max PCR Master Mix (Takara, Shiga, Japan), 1 µL of each primer (20 pmol concentration), 5.5 µL of water, and 5 µL of DNA template. The reaction was performed in an Applied Biosystem 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA). The PCR products were separated by electrophoresis on a 1.5% agarose gel (AppliChem GmbH, Darmstadt, Germany) with ethidium bromide in 1× TBE buffer at room temperature using gradients of 5 V/cm. For gel analysis, 20 µL of the products were loaded in each gel slot. Generuler 100 bp ladder (Fermentas, Hamburg, Germany) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra, San Leandro, CA, USA), and the data was analysed through computer software. The positive control DNAs were represented by field samples previously confirmed to be positive by PCR for the related genes in the Reference laboratory for veterinary quality control on poultry production, Animal health research institute, Giza, Egypt.

Statistical Analysis and Risk Factor Assessment
The significance of the differences in the prevalence rates and risk factor assessment was analysed with Fisher Exact Probability Test (two-tailed), 95% confidence intervals (including continuity correction) and odds ratios using an online statistical website www.vassarstats.net (accessed dates; 11-14 September 2022) as described previously [33]. p-values and odds ratio were also confirmed with GraphPad Prism version 5 (GraphPad Software Inc., La Jolla, CA, USA). The results were considered significant when the p-value was <0.05. Statistically significant differences in the OD values of the ELISA were estimated and interpreted using a t-test followed by the Mann-Whitney test for comparing different groups. Pearson's correlation coefficient was applied to test the correlation between the OD values of bulk samples and the mean OD of individual milk samples from the same group. Correlation coefficients were calculated using Pearson's correlation coefficient: |r| = 0.70, strong correlation; 0.5 < |r| < 0.7, moderately strong correlation; and |r| = 0.3-0.5 weak-to-moderate correlation [43].

Prevalence of N. caninum and T. gondii Antibodies in Bulk and Individual Milk Samples and Detection of DNA
In this study, we investigated the prevalence of N. caninum and T. gondii antibodies in bulk and individual raw milk samples. Samples were collected from cattle, buffaloes, sheep and goats from Qena, Sohag, Cairo, and Dakahlia governorates representing different Egyptian areas. Of 13 tested bulk milk samples representing 13 different farms, positive reactions to N. caninum were detected in five (38.5%) samples, three of which were from cattle farms. The other two positive samples were from a sheep and a goat smallholder farm, respectively. These two latter samples also tested positive for antibodies to T. gondii and thus for co-infection (seroprevalence for T. gondii and co-infection: 15.4%) ( Table 3). In the individual samples (n = 171) of the same bulk milk samples collected from three farms and four smallholders, antibodies to N. caninum were detected in 25.7%, to T. gondii in 14%, and 3.5% had antibodies to both parasites. In more detail, antibodies to N. caninum were detected in 26.2% (33/126) of the tested cattle, 18.8% (3/16) of the buffaloes, 33.3% (6/18) of the sheep, and 18.2% (2/11) of the goats, respectively (Table 4). Antibodies to T. gondii were detected in 81.8% (9/11) of the tested goats, 66.7% (12/18) of the sheep, 2.4% (3/126) of the cattle, and in none (0/16) of the buffaloes, respectively (Table 4). Detection of DNA was targeted in ELISA-positive bulk milk samples and in those containing a number of seropositive individual samples. From six bulk samples with the afore-mentioned criteria, bulk 1 from cattle showed a positive PCR result for the NC5 gene of N. caninum (337 bp), while bulk samples 7, 12, and 13 showed negative PCR results, although they were positive in ELISA (Figure 1). In the case of T. gondii, samples of bulk 12 from sheep and bulk 13 from goats that showed strong antibody reactivity using ELSA showed a positive PCR result for the B1 gene of T. gondii (196 bp). In addition, bulk sample 10 from cattle was also PCR-positive, despite being negative in ELISA (Figure 1). afore-mentioned criteria, bulk 1 from cattle showed a positive PCR result for the NC5 gene of N. caninum (337 bp), while bulk samples 7, 12, and 13 showed negative PCR results, although they were positive in ELISA (Figure 1). In the case of T. gondii, samples of bulk 12 from sheep and bulk 13 from goats that showed strong antibody reactivity using ELSA showed a positive PCR result for the B1 gene of T. gondii (196 bp). In addition, bulk sample 10 from cattle was also PCR-positive, despite being negative in ELISA (Figure 1).

Risk Factor Assessment for N. caninum and T. gondii Infection using Individual Milk Samples
Risk factor assessment was conducted for T. gondii and N. caninum antibodies in tested individual samples. Animal species was assessed as a risk factor for all tested samples (n = 171) collected from cattle, buffalo, sheep, and goat. Additional factors including region (Qena, Sohag, and Dakahlia), breed (native and Holstein Friesian), and management system (farm or small holder) could only be analyzed for cattle. Regarding N. caninum, no risk factors have been detected when analyzing any of the above-mentioned factors (Table 5).

Risk Factor Assessment for N. caninum and T. gondii Infection Using Individual Milk Samples
Risk factor assessment was conducted for T. gondii and N. caninum antibodies in tested individual samples. Animal species was assessed as a risk factor for all tested samples (n = 171) collected from cattle, buffalo, sheep, and goat. Additional factors including region (Qena, Sohag, and Dakahlia), breed (native and Holstein Friesian), and management system (farm or small holder) could only be analyzed for cattle. Regarding N. caninum, no risk factors have been detected when analyzing any of the above-mentioned factors (Table 5). Regarding T. gondii-associated risk factors utilizing individual milk samples, the tested sheep in this study were more susceptible to T. gondii infection (66.7%) than cattle (2.4%; OR = 82; p ≤ 0.0001) and buffaloes (0%; OR = 63.5; p ≤ 0.0001), while goats did not differ from sheep (81.1%; OR = 0.4; p = 0.67) ( Table 6). This was the only statistically significant association found.

Comparison of Antibody Responses to N. caninum and T. gondii in Individual and Bulk Milk Samples
Analysis of individual milk samples from bulk samples revealed that in negative bulk samples, positive individuals might be included, and vice versa. This effect was more pronounced in the analysis for N. caninum antibodies (Table 7). Optical densities of ELISA results were visualized to compare the reactivity of positive and negative samples for both parasites to provide further validation of our testing. Strong antigen-antibody reactivity was observed in positive bulk or individual samples for both parasites, particularly in the case of T. gondii (Figure 2A-D). The differences between OD values of both negative and positive individual samples within the relevant bulk milk sample were analysed for both antibodies to N. caninum ( Figure 2B) and T. gondii ( Figure 2D), respectively.
Optical densities of ELISA results were visualized to compare the reactivity of positive and negative samples for both parasites to provide further validation of our testing. Strong antigen-antibody reactivity was observed in positive bulk or individual samples for both parasites, particularly in the case of T. gondii (Figure 2A-D). The differences between OD values of both negative and positive individual samples within the relevant bulk milk sample were analysed for both antibodies to N. caninum ( Figure 2B) and T. gondii ( Figure 2D), respectively.  These results were confirmed by group comparisons of antibody levels of all individual samples from negative bulks versus those from positive bulks ( Figure 3A,B). Antibody levels of individual samples from bulks positive for T. gondii antibodies were significantly higher than that of negative bulks (p ≤ 0.0001) but not for N. caninum (p = 0.1810). In addition, Pearson's correlation coefficient was used to investigate the association between the OD values of bulk samples and the mean OD values of the individual samples from the bulk. A better correlation was identified for T. gondii (Strong; Pearson r = 0.9759) than for N. caninum (Moderately strong; Pearson r = 0.5801) ( Figure 3C,D). This data might be attributable to the number and distribution patterns of positive and negative individual samples among the bulk samples.
In case of N. caninum, the negative bulks contained a higher percentage of positive individual samples (13%; 5/38) than what was seen for T. gondii (2%; 3/142). Similarly, a lower percentage of positive individual samples in positive bulks (29%; 39/133) was seen for N. caninum, while 72% (21/29) of the individual samples in bulks positive for T. gondii were positive (Figure 4). tibody levels of individual samples from bulks positive for T. gondii antibodies were significantly higher than that of negative bulks (p = <0.0001) but not for N. caninum (p = 0.1810). In addition, Pearson's correlation coefficient was used to investigate the association between the OD values of bulk samples and the mean OD values of the individual samples from the bulk. A better correlation was identified for T. gondii (Strong; Pearson r = 0.9759) than for N. caninum (Moderately strong; Pearson r = 0.5801) ( Figure 3C,D). This data might be attributable to the number and distribution patterns of positive and negative individual samples among the bulk samples.

Discussion
The growing human population demands safe and high-quality food, including milk and dairy byproducts. The presence of N. caninum DNA in milk samples has been shown in a few studies for dairy cows [11,13,44] and in one study for lactating donkeys [45]. DNA of T. gondii was detected in milk samples from different animal species, including cattle [13], donkeys [45], as well as sheep and goats (including one report from Egypt) [21,46]. Therefore, milk might represent a potential risk of infection to suckling animals and facilitate parasite persistence in a flock. Another very important point is whether T. gondii in milk may be infective for humans. This risk seems to be higher in

Discussion
The growing human population demands safe and high-quality food, including milk and dairy byproducts. The presence of N. caninum DNA in milk samples has been shown in a few studies for dairy cows [11,13,44] and in one study for lactating donkeys [45]. DNA of T. gondii was detected in milk samples from different animal species, including cattle [13], donkeys [45], as well as sheep and goats (including one report from Egypt) [21,46]. Therefore, milk might represent a potential risk of infection to suckling animals and facilitate parasite persistence in a flock. Another very important point is whether T. gondii in milk may be infective for humans. This risk seems to be higher in the case of consuming raw milk from an individual animal than by consumption of bulk tank milk in which the parasites would be greatly diluted [47,48].
Our study demonstrated DNA of N. caninum in raw milk of cattle, and of T. gondii in raw milk of cattle, sheep and goats. Detection of DNA in milk is correlated to the stage of parasitemia which is short and transient in case of both tested parasites [49], oppositely to the long-lasting antibody response [50,51]. While in our tested sheep and goat samples, the results of the ELISA and the PCR for T. gondii were both positive, the agreement between the test methods was not as nice in the cattle samples. Only one of the two tested N. caninum seropositive bulk milk samples showed a positive Nc5-PCR result. The cattle bulk milk sample that was positive in the B1-PCR was seronegative for T. gondii.
Our findings demonstrate the potential hazards of raw milk of cows, ewes or does in the transmission of T. gondii or N. caninum to susceptible hosts. Indeed, the possibility of T. gondii transmission via milk was estimated using experimentally infected goats. Toxoplasma gondii viability in goat milk and cheese was demonstrated by bioassay in cats and mice [48]. However, similar experiments have yet to be reported for N. caninum [44].
The current control measures for bovine neosporosis primarily rely on serological testing and the replacement of infected animals [52]. Previous results indicated that the detection of antibodies in individual milk samples is a good, non-invasive alternative option to testing serum samples for detecting anti-N. caninum antibodies [52,53]. However, the use of milk samples also has some limitations, as only lactating cows can be tested, and young, diseased, and dry animals are excluded from the sample [54]. Detection of neosporosis in a herd by testing a single bulk milk sample has a significant financial and logistic advantage, but sensitivity is arguably lower than when testing individual milk samples due to the diluting effect of negative milk samples in a bulk sample. The use of milk samples to detect T. gondii has an additional public health aspect; indeed, T. gondii antibodies were detected in the milk of food animals, which may be considered a potential source for human infection [20][21][22].
For risk factor assessment, a comparison between animal species revealed a higher risk of T. gondii infection in sheep and goats than in buffaloes and cattle, but no influence of species was revealed on N. caninum infection. It is accepted that sheep and goats are more susceptible to T. gondii infection than cattle and buffaloes [1,2,17]. Only cattle samples were considered when analysing the influence of region, breed, and management system, as these factors did not vary for the other species. None of these factors was significantly influencing seropositivity neither for T. gondii nor for N. caninum.
The findings of the current study suggest the suitability of bulk milk samples of different ruminant species in monitoring N. caninum and T. gondii antibodies at the herd level in Egypt. This was also supported by the good agreement between the results of the individual samples compared to the overall bulk milk result. However, as only few bulk milk samples of goats, sheep and buffaloes were included, further studies testing combined individual and bulk milk samples are required to validate our results. Additionally, testing of individual samples is still necessary when aiming at detecting infected individuals, e.g., for replacing N. caninum-infected cows [54].

Conclusions
We provide novel data on the milk-based prevalence and risk factor assessment of N. caninum and T. gondii among cattle, buffaloes, sheep and goats from different areas in Egypt. The utility of bulk milk samples for detecting N. caninum and T. gondii antibodies was confirmed for the first time in Egypt. Furthermore, we report the first detection of N. caninum DNA in cow's milk in Egypt. The high prevalence of N. caninum antibodies in cattle suggests a high economic impact of this parasite in Egypt. Moreover, high seroprevalence and DNA detection of T. gondii in sheep's and goat's milk possibly threatens both human and animal health and should alert public health and veterinary authorities.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated and analyzed during this study are included in this published article. Raw data supporting the findings of this study are available from the corresponding author on request.