Seroprevalence of Specific Antibodies to Toxoplasma gondii, Neospora caninum, and Brucella spp. in Sheep and Goats in Egypt

Simple Summary Toxoplasma gondii, Neospora caninum and Brucella species are widely distributed infectious agents that induce severe clinical illness in numerous farm animals. In addition T. gondii and Brucella spp. are of public health concern because of their zoonotic potentials. In Egypt and other countries, toxoplasmosis, neosporosis, and brucellosis are three well-known etiological agents of infectious abortion in many farm animals including sheep and goats. Inaccurate diagnosis of the abortion cause results in great economic losses because of application of inappropriate control measures. In the current study, we provided the first report on the comparative seroprevalence of toxoplasmosis, neosporosis and brucellosis in sheep and goats in Egypt based on antibody detection. We found that the overall seroprevalence rate was the highest for T. gondii. Nevertheless, only antibodies to Brucella spp. were associated with recent abortion in sheep and goats. Our study provides solid data on the existence of abortifacient agents in the common small ruminants in Egypt. Accordingly, these data can be exploited in developing more efficient control policies against such serious infections. Abstract Toxoplasmosis, neosporosis, and brucellosis are devastating diseases causing infectious abortion and, therefore, substantial economic losses in farm animals. Toxoplasmosis and neosporosis are caused by the intracellular protozoan parasites Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum), respectively. Brucellosis is a bacterial disease caused by numerous Brucella species in multiple hosts. Toxoplasmosis and brucellosis are also considered foodborne zoonotic diseases. In the current study, specific antibodies to T. gondii and N. caninum, in addition to those to Brucella spp., were detected to gain a better understanding of the epidemiological situation for these three pathogens. Sheep and goat sera from Egypt (n = 360) of animals with and without a history of abortion were tested using commercial ELISAs. Seropositivity rates of 46.1%, 11.9%, and 8.6% for T. gondii, N. caninum, and Brucella spp., respectively, were revealed. Mixed infections with T. gondii and Brucella spp. (4.4%), T. gondii and N. caninum (4.2%), N. caninum and Brucella spp. (1.4%), and even some triple infections (0.6%) have been observed. Animals with a history of abortion had a significantly higher seroprevalence for Brucella spp. infection than those without abortion (12.6%; 28/222 vs. 2.2%; 3/138) (p = 0.0005; Odds ratio = 1.9–21.8), while none of the other pathogens showed a similar effect. This result suggests brucellosis as a possible cause of abortion in the study population. However, the high seroprevalence for T. gondii and N. caninum revealed in our study warrants further investigations.


Introduction
Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that affects almost all warm-blooded animals, including cats as definitive hosts as well as sheep, goats, pigs, and humans as intermediate hosts. The vertical transmission from dams to offspring, ingestion of food or water contaminated with oocysts excreted by infected cats, or ingestion of uncooked or undercooked meat containing tissue cysts are the major modes of transmission of T. gondii [1].
Neospora caninum (N. caninum) is a closely related protozoan parasite to T. gondii and affects dogs as final and intermediate hosts and numerous animal species, particularly cattle and sheep as intermediate ones. Neosporosis is characterized clinically by neurological disorders and reproductive problems in dogs and mainly abortion in other intermediate hosts. Transmission is remarkably similar to T. gondii and occurs either by horizontal ingestion of oocysts or tissue cysts or vertically from infected dams to their offspring [1,2]. Up-to-date, clinical neosporosis in humans has not been confirmed yet, despite the detection of specific antibodies in serum samples of human origin [3]. However, a recent study found N. caninum DNA in human umbilical cord blood samples but not in the respective placentas [4].
Brucella (B.) spp., Gram-negative coccobacillus bacteria, are also common aborting agents in multiple animal species, especially sheep, goats, cattle, buffaloes, and pigs. Brucella includes many species, of which B. abortus, B. melitensis, and B. suis are the most common among farm animals. Brucella melitensis is the most important one in sheep and goats, and abortion mainly occurs in case of infection of pregnant ewes or goats [5]. Transmission of Brucella organisms usually occurs through direct or indirect contact with infected animals or infected materials or ingestion of contaminated feed or water [6].
Although T. gondii and N. caninum are protozoan parasites and Brucella spp. are bacteria, they possess several similarities in aspects of infection and pathogenesis. All these pathogens are intracellular microorganisms that evade the killing by macrophages and even use them to reach their target tissues [7][8][9][10][11].
A placental and/or fetal infection caused by T. gondii, N. caninum, or B. melitensis in pregnant sheep and goats may result in fetal death, abortion, or stillbirth, as well as severe placentitis in the case of brucellosis [12][13][14][15][16][17]. In addition, Brucella spp. cause a decrease in progesterone production and an increase in estrogen levels, resulting in premature delivery [18]. Abortion induced by the three above-mentioned infectious agents is a major cause of substantial economic losses in livestock production. Financial losses have been estimated for abortions in sheep caused by T. gondii to amount to over EUR 100/abortion in Spain [19], and for abortions in sheep and goats caused by Brucella sp. to amount to over USD 120 million annually in India [20]. Brucellosis and toxoplasmosis not only cause enormous economic losses, but they also are of substantial public health concern [1, 21,22]. Brucellosis is mainly a serious occupational hazard (characterized by variable clinical signs ranging from fever to miscarriage [23][24][25]) for people in contact with infected animals or contaminated animal products, byproducts, or utensils, such as farm workers, animal owners or dealers, veterinarians, and abattoir or laboratory workers.
Numerous animal species, including sheep and goats, are susceptible to toxoplasmosis, neosporosis, and brucellosis in Egypt [26][27][28][29][30]. However, simultaneous investigation of antibodies to T. gondii, N. caninum, and Brucella spp. was not conducted before in Egypt, particularly in sheep and goats. Thus, this study aimed to estimate the prevalence of these infections in sheep and goats from various regions of Egypt and assess correlations with the abortion history of the sampled animals.

Ethical Statement
This study was conducted according to instructions established by the "Research Board" of the Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. The protocols were approved by the Research Code of Ethics at South Valley University number . Blood samples were collected by a group of highly trained veterinarians and staff after consultation with the officials and animal owners.

Description of the Animals and Regions of the Study
The present study was conducted in four governorates representing the northern part (Dakahlia), middle part (Beni Suef), southern part (Qena), and eastern part (Red Sea) of Egypt ( Figure 1). signs ranging from fever to miscarriage [23][24][25]) for people in contact with infected animals or contaminated animal products, byproducts, or utensils, such as farm workers, animal owners or dealers, veterinarians, and abattoir or laboratory workers.
Numerous animal species, including sheep and goats, are susceptible to toxoplasmosis, neosporosis, and brucellosis in Egypt [26][27][28][29][30]. However, simultaneous investigation of antibodies to T. gondii, N. caninum, and Brucella spp. was not conducted before in Egypt, particularly in sheep and goats. Thus, this study aimed to estimate the prevalence of these infections in sheep and goats from various regions of Egypt and assess correlations with the abortion history of the sampled animals.

Ethical Statement
This study was conducted according to instructions established by the "Research Board" of the Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. The protocols were approved by the Research Code of Ethics at South Valley University number 36 (RCOE-36). Blood samples were collected by a group of highly trained veterinarians and staff after consultation with the officials and animal owners.

Description of the Animals and Regions of the Study
The present study was conducted in four governorates representing the northern part (Dakahlia), middle part (Beni Suef), southern part (Qena), and eastern part (Red Sea) of Egypt ( Figure 1). The study animals were indigenous sheep and goats kept in farms (more than fifty animals) or small holdings (<50 animals/farm). Samples were collected at various time points from May 2016 to November 2021 (Table 1). All sheep and goats included in this study were older than 6 months and mostly females. None of the sampled animals was vaccinated against Brucella infection, as confirmed by animal owners. A total of 360 serum The study animals were indigenous sheep and goats kept in farms (more than fifty animals) or small holdings (<50 animals/farm). Samples were collected at various time points from May 2016 to November 2021 (Table 1). All sheep and goats included in this study were older than 6 months and mostly females. None of the sampled animals was vaccinated against Brucella infection, as confirmed by animal owners. A total of 360 serum samples were collected randomly from sheep (n = 239) and goats (n = 121) with or without a history of abortion. Sera collected from animals with a history of abortion were confined mostly to medium to large farms (50 to 200 heads/farm) and collected during the first few months after abortions occurred. The selection of samples for the study was designed to cover a large time span (5 years) and a wide geographical range (approximately 1000 kilometers apart) in order to give a reliable estimation of the epidemiological situation of the three pathogens. Furthermore, flocks with and without a history of abortion were included, while vaccination for Brucella was an exclusion criterium. Within the selected flocks, samples were collected randomly from apparently healthy animals.

Sample Collection and Serum Preparation
Approximately 5 mL of whole blood samples were collected by venipuncture from the jugular vein using sterile disposable plain vacutainer tubes and needles. The blood samples were kept in an upward position and allowed to clot at room temperature for 2-3 h. The samples were transported on ice in an icebox to our laboratory at the Faculty of Veterinary Medicine, South Valley University, Egypt. Sera were transferred into 1.5 mL Eppendorf tubes after centrifugation at 10,000× g for 15 min [30] and stored at −20 • C until use.

Serological Diagnosis of T. gondii, N. caninum, and Brucella Species
In order to detect antibodies to T. gondii, the serum samples were analyzed with the indirect multi-species ELISA for toxoplasmosis (ID.vet, Grabels, France) according to the manufacturer's instructions. Serum 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 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%.
For antibodies to N. caninum, serum samples were analyzed with the competitive multi-species ELISA for neosporosis (ID.vet, Grabels, France). Serum 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 it was considered positive if the S/N% was less than 50%.
Concerning Brucella spp., serum samples were analyzed with the indirect multi-species ELISA for brucellosis (ID.vet, Grabels, France). Serum samples and controls were diluted 1:20. The ODs obtained were 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 110% were considered negative; if the S/P% was between 110% and 120%, the result was considered doubtful, and it was considered positive if the S/P% was greater than or equal to 120%.
The ODs of all ELISA results were read at 450 nm and measured with an Infinite ® F50/Robotic ELISA reader (Tecan Group Ltd., Männedorf, Switzerland). More details for the commercial kits used are available in Table 2. * The sensitivity and specificity of the diagnostic kits were provided by the manufacturer of the kits. # CI 95%; 95% confidence interval.

Statistical Analysis
The significance of the differences in the prevalence rates and risk factors was analyzed with Chi-square (Pearson) test, 95% confidence intervals (including continuity correction), and odds ratios using an online statistical website www.vassarstats.net (accessed on 9 August 2022). 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 or highly significant when the p-value was <0.0001.

Discussion
Toxoplasma gondii, N. caninum, and Brucella spp. are professional abortifacient infectious agents in several animal species and different countries. Studies on the seroprevalence of all three pathogens and possible association with abortion history in sheep and goats have not yet been conducted in Egypt. Brucellosis is quite a common occupational disease among veterinarians and animal handlers in Egypt, and it is usually regarded as the first cause of infectious abortion in ruminants, especially in late gestation, and complicated by retention of the placenta [23]. These assumptions are supported by studies and official investigations, which diagnosed brucellosis as a cause of abortion in many abortion outbreaks [26,31,32]. However, our study of comparative seroprevalence of T. gondii, N. caninum and Brucella species in small ruminants had two main drivers: First, most of the reported brucellosis investigations were performed using screening tests, such as the Rose Bengal test and serum agglutination test, not confirmatory tests such as complement fixation test or ELISA. Second, numerous previous studies have recorded a high prevalence of T. gondii and N. caninum antibodies in many farm animals in Egypt, including in small ruminants, and therefore their contribution to abortions might currently be underestimated [27,28,30].
Our study indeed found the highest seroprevalence against T. gondii among all three tested pathogens, thus confirming previous studies from Egypt that found seroprevalences between 28.7% and 98.4% in small ruminants [33][34][35]. In our study, goats were significantly more affected (66.9%) than sheep (35.6%), and a region with a lower seroprevalence was also identified (Red Sea, eastern Egypt, compared to northern, middle, and southern Egypt). These findings confirmed earlier results from Egypt in that region and animal species have an effect on T. gondii seroprevalence [35], and also other studies found similar effects, namely Magalhães et al. [36] and Rêgo et al. [37], in Brazil; Dong et al. in China [38]; and Suazo-Cortez et al. in Mexico [39]. As for N. caninum, the seroprevalence in our study was 11.9% overall, 15.5% in sheep, and 5% in goats, respectively. Our results compared nicely with previous reports from sheep in Egypt: 8.6% reported by Selim et al. [30], and 36.1% reported by El-Ghaysh et al. [40]. Moreover, for this parasite, animal species and location had significant effects on the seroprevalence, with sheep being more affected than goats and northern and eastern Egypt being more affected than middle and southern Egypt. Other authors have also reported significant effects of location and animal species on N. caninum seroprevalence in small ruminants [30,[41][42][43].
Of all three pathogens tested, seroprevalence against Brucella spp. was the lowest, with 8.6% overall, 10.5% in sheep, and 5% in goats, respectively. Other authors found prevalences varying between 7.2% and 56.7% for sheep and goats in Egypt [29,32,44,45]. In our study, there was a trend of higher seropositivity in sheep compared to goats, but it was not significant. Northern Egypt, however, did have a significantly higher seroprevalence than the other regions tested. Similar factors were found before by other authors from Egypt [46][47][48] and elsewhere [49][50][51].
Arguably, the most interesting association revealed in this study was the seropositivity against Brucella spp. and a history of abortion. Interestingly, no such association was found between antibodies against T. gondii or N. caninum and recent abortions. A study conducted on pregnant ewes in Iraq found that ewes seroconverting to Brucella were 2.9 times more likely to lose their pregnancy than those remaining seronegative. In the same study, seroconverting to T. gondii had no significant impact on the loss of pregnancy [52]. Similarly, in another region of the world, only about 1.6% of lost lambs were attributed to T. gondii in a prospective cohort study in primiparous ewes from southern Australia, albeit at an overall much lower seroprevalence than in our study [53]. Moreover, in a study performed with breeding ewes from Mexico, seroprevalence for T. gondii (61.96%) and N. caninum (15.22%) was similar to our study, and T. gondii seroprevalence was not associated with a history of abortion, but N. caninum seroprevalence was [54]. However, although our results suggest an involvement of Brucella in the abortions, they do not negate the role of T. gondii or N. caninum as aborting agents. Numerous previous reports have documented toxoplasmosis [1,19,55] and neosporosis [56][57][58] as causes of abortion in sheep and goats.
The high prevalence of anti-Brucella antibodies in animals with an abortion history was correlated to the highest seropositivity in Dakahlia governorate in the Nile Delta region, northern Egypt. This area is endemic for different Brucella species, including B. abortus, the causative agent of bovine brucellosis, and B. melitensis, the main cause of ovine and caprine brucellosis [26,29]. In addition, the case history of abortion in that region demonstrated that Animals 2022, 12, 3327 9 of 12 some aborting ewes and does have experienced late abortion and retained placenta, both common signs of Brucella abortion [12,23]. In contrast, Neospora abortion can occur at any time with a special preference for mid-gestation, and usually, no clinical signs or lesions are evident in aborting dams or in the placenta or fetus [16]. In the case of toxoplasmosis, early abortion and fetal resorption are mostly noticed, and similarly to neosporosis, no abnormalities in the dams, fetus, or placenta are usually observed [21].
However, to obtain a better understanding of the causes of abortions of small ruminants in Egypt, fetuses, placentas, and serum samples of the mothers should be submitted for laboratory analyses. Moreover, our data provide insights into the current epidemiological situation for Brucella spp. and T. gondii, both important zoonotic species, and N. caninum, all three of which cause losses to the livestock industry. More efficient control programs will have to be established in the future based on a good understanding of the real situation, which can only be gained by investigations such as this one.
Due to the limitations in the number, content, and distribution of studies focusing on the relevant theme worldwide, including Egypt, we could not perform a comprehensive comparison with our study. Additionally, higher numbers of samples are required from sheep, goats, and other species and geographic locations to establish an accurate assessment of the main causes of abortions among our tested pathogens.

Conclusions
Abortion in farm animals, including sheep and goats, is a major and direct cause of enormous economic losses in animal production sectors. Toxoplasmosis, neosporosis, and brucellosis are well-known causes of infectious abortions in sheep and goats in Egypt and other countries. Misevaluation of the current situation of these infections as causes of abortion would jeopardize the livestock industry in Egypt. This study is the first to establish a comparison between toxoplasmosis, neosporosis, and brucellosis based on seroprevalence in sheep and goats in Egypt. Seroprevalence for T. gondii was highest, followed by N. caninum and Brucella spp. Animal species and location were considered as risk factors for T. gondii and N. caninum infections, while only the location was a significant factor for Brucella spp. infection. Importantly, antibodies to Brucella spp. were significantly higher in aborting than in non-aborting animals, a feature not observed for the other two pathogens.

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.