Seroprevalence of Toxoplasma gondii and Neospora caninum in Dromedary camels ( Camelus dromedarius from Saudi Arabia

http://doi.org/10.1590/S1984-29612020008 Abstract Serological screening of 199 serum samples from Dromedary camels—from different cities in Saudi Arabia—was performed using enzyme-linked immunosorbent assay for detecting antibodies against two cyst-forming coccidian parasites, namely Toxoplasma gondii and Neospora caninum. Antibodies against T. gondii were detected in 68 (34.2%) samples, while those against N. caninum were present in 33 (16.6%) samples. The highest seroprevalence of T. gondii antibodies was reported in samples from Taif (51.2%), while the lowest seroprevalence was reported in samples from Riyadh and Hofuf (15.1%). The highest seroprevalence of N. caninum antibodies was reported in samples from Jizan (35.9%) while the lowest was reported in samples from Taif (2.4%). A total of 47 male and 21 female camels exhibited antibodies against T. gondii , while 19 male and 14 female camels showed antibodies against N. caninum . Concurrent detection of both T. gondii and N. caninum antibodies was observed in 18 camels. It has been demonstrated that T. gondii and N. caninum antibodies are prevalent in camels from different cities of the Kingdom of Saudi


Introduction
Neospora caninum and Toxoplasma gondii are obligate intracellular protozoan parasites that infect a wide range of domestic and non-domestic animals, as well as humans (Dubey, 2010;Donahoe et al., 2015). The zoonotic nature of toxoplasmosis has been well established, whereas despite serological evidence in humans, the disease caused by N. caninum is not considered zoonotic (Tranas et al., 1999;Lobato et al., 2006). T. gondii (a cyst-forming coccidium) was first discovered in 1908 and named a year later; however, its life cycle was fully elucidated in the 1970s (Dubey & Frenkel, 1972). The other cyst-forming coccidium, N. caninum, was first identified in 1984 (Bjerkas et al., 1984) and its detailed life cycle was elucidated in 1998 (McAllister et al., 1998).
Both parasites are believed to be transmitted to intermediate hosts via consumption of food contaminated with the sporulated oocysts shed by the definitive hosts. Domestic cat and other members of the family Felidae are the definitive hosts for T. gondii (Dubey, 2010), while dogs and coyotes are the definitive hosts for N. caninum (McAllister et al., 1998;Gondim et al., 2004). Vertical transmission from mother to fetus through the placenta is an alternative route for transmission of both parasites (McAllister et al., 2000;Dubey, 2010).
N. caninum is a bovine pathogen and abortion is the principal clinical manifestation of infected pregnant animals (Dubey & Schares, 2011). Intrauterine fetal death may occur during late gestation; the dead fetuses are usually expelled and show moderate autolysis. Fetuses succumbing in less than five months or during early gestation may be mummified and retained in the uterus for several months. Repeat breeding may be a consequence of fetal death during early stage of gestation in infected cows (Moore et al., 2002). One of the clinical features of toxoplasmosis is abortion, along with neurological symptoms in its intermediate hosts (Dubey & Beattie, 1988;Dubey, 2010).
Only two studies dealing with the seroprevalence of N. caninum from camels (C. dromedarius) were conducted in Saudi Arabia; the first one was limited to Riyadh Province (Alanazi, 2011). The second study was by Aljumaah et al. (2018) from different regions of Saudi Arabia who reported high prevalence of N. caninum antibodies. There was a contrast between the findings of the two studies where seroprevalence was found 5.6% in the first study while it was 21.99% in the second study. Furthermore, both studies dealt only with N. caninum and did not show the cities from where the samples were collected. Cities are important for the availability of camels slaughtered for human consumption. Studies on T. gondii in camels were only few and restricted to Riyadh and Hofuf, the study from Hofuf was not even on seroprevalence and it was on investigating the role of camels as an intermediate host for the parasite, no study has covered different cities in Saudi Arabia (Hussein et al., 1988;Hilali et al., 1995;Alanazi, 2011Alanazi, , 2013. In the present study, we reported the antibody prevalence of both T. gondii and N. caninum in the Dromedary camel (Camelus dromedarius) from blood samples collected from different cities of Saudi Arabia in order to understand the potential role of this animal species in the epidemiology of these parasites and the possible risk of zoonosis.

Materials and Methods:
Blood samples were obtained from apparently healthy camels by drawing 5 ml of jugular blood into plain vacutainer tubes (Becton, Dickinson and Company 1 Becton Drive, Franklin Lakes, NJ, USA) and then left to clot. Serum was collected after subjecting the clotted blood to centrifugation at 900 g for 10 minutes and stored at -20 °C until further use. A total of 199 blood samples were collected from camels from different cities in Saudi Arabia. Hofuf (n=38) represented the eastern region, Riyadh (n=38) represented the central region, Tabuk (n=43) represented the northern region, Jizan (n=39) represented the southern region and Taif (n=41) represented the western region. None of the sampled camels showed apparent signs of any disease and none of the females appeared to be pregnant.
An Enzyme-Linked Immuno Sorbent Assay (ELISA)-utilizing a kit available from Idexx (HerdCheck ® Anti-Neospora; and Idexx Toxo-test IDEXX Laboratories; Bommeli Diagnostics, AG, Bern, Switzerland)-was used to detect anti-Neospora and anti-Toxoplasma IgG-antibodies in the camel serum samples. The kits were used as per the manufacturers' instructions. In the laboratory, the detection of the primary antibodies to T. gondii and N. caninum in the camel serum samples was brought about by using the secondary antibody anti-llama IgG-horseradish peroxidase conjugate (Bethyl Laboratories, Montgomery, TX) at a dilution of 1:15,000. This conjugate was previously used at the laboratory for the detection of antibodies to the Middle East Respiratory Syndrome corona virus (MERS CoV) in camel serum samples (Alagaili et al., 2014). The presence or absence of antibodies was determined via reading the reaction result on the ELISA reader at 450 nm. The presence or absence of specific antibodies for each test sample was determined by comparing the optical density percentage of the test samples with those of the control samples provided with the kit. Samples showing values of ≥ 40% were considered positive using the following formula: Statistical analysis of data obtained for the seroprevalence of N. caninum and T. gondii were computed using the Chi square test in the GraphPad statistical software (Prism 6.0). Chi square test values were considered significant if p ≤ 0.05. The relative risk (RR) and 95% confidence interval were calculated according to Altman (1991).

Results
The overall antibody prevalence of T. gondii was found to be 34.2%, with the highest prevalence being reported in serum samples from Taif (51.2%), while the lowest was recorded from camels from Riyadh and Hofuf (15.8%), as shown in Table 1 and Figure 1. Antibodies against T. gondii were detected in 47 (35.1%) females and 21 (32.1%) males. T. gondii was more prevalent in males than in females in Taif (56.8%) and Jizan (54.2%), while the highest female seroprevalence of T. gondii was reported in samples from Tabuk (58.8%) ( Table 2). The risk ratio of T. gondii prevalence is significantly higher in Taif, Jizan, and Tabuk than in Riyadh and Hofuf (Table 1).   The overall antibody prevalence of N. caninum was 16.6%, with the highest prevalence being reported in serum samples from Jizan (35.9%); the lowest prevalence was found in samples from Taif (2.4%) (Table 1; Figure 1). Antibodies against N. caninum were reported in 19 males and 14 females ( Table 2). The risk ratio of N. caninum prevalence is significantly higher in Jizan and Hofuf than in Riyadh, Tabuk, and Taif (Table 1).
Males have significantly higher prevalence of T. gondii antibodies compared to those against N. caninum (p=0.0001), while the difference between the prevalence of antibodies against these two parasites in females was not significant (p=0.24). There was no significant difference between the prevalence of T. gondii and N. caninum antibodies among males and females (p=0.75 and p=0.22, respectively).
Concurrent detection of both T. gondii and N. caninum antibodies was also reported during the present study. Toxoplasma gondii antibodies were detected in 18 camels which tested positive for antibodies against N. caninum too. Of those 12 were from Jizan, 3 from Tabuk, 2 from Hofuf and 1 from Taif.

Discussion
In the present study, a seroprevalence analysis of two cyst-forming coccidia (T. gondii and N. caninum) in the Dromedary camel (Camelus dromedarius) in different cities covering different regions in Saudi Arabia was performed. The seroprevalence of T. gondii was found to be 34.2%, while that of N. caninum was found to be 16.6%.
The seroprevalence of T. gondii was comparable to that reported in previous studies on camels from Saudi Arabia and its neighboring countries (Hussein et al., 1988;Hilali et al., 1998;Wernery et al., 2008;Hosseininejad et al., 2010;Alanazi, 2011;2013;Hamidinejat et al., 2013;Mentaberre et al., 2013). Extremely high prevalence-as high as 90.9% (from Turkey) (Utuk et al., 2012), 67% (from Butana plains, eastern Sudan) (Elamin et al., 1992) and 40.5% (from Fentale district, central Ethiopia) (Gebremedhin et al., 2014)-of T. gondii antibodies in camels have been reported. In our study, the difference in the prevalence of T. gondii antibodies between male and female camels was not significant and was in agreement with that reported in previous studies (Elamin et al., 1992;Wang et al., 2013;Gebremedhin et al., 2014). However, this is in contrast with what has been reported earlier by Hussein et al. (1988), who detected significantly higher prevalence of T. gondii antibodies in female camels than in male camels; they attributed this difference to the husbandry practices. In most studies carried out on the prevalence of T. gondii antibodies in camels, it was found that old camels generally had a higher prevalence compared to younger camels (Hussein et al., 1988;Gebremedhin et al., 2014). Calves are also affected by the infection, but it was not certain whether they acquired the infection as a result of innocuous consumption of cysts arising from Felidae family members or as a result of vertical transmission from mothers such as that in the case of N. caninum in camels from the Canary Islands (Mentaberre et al., 2013).
There are three reports on the prevalence of N. caninum in animals in Saudi Arabia and two of them are about camels (Alanazi, 2011;Alanazi et al., 2014;Aljumaah et al., 2018). In the present study, a prevalence of 16.6% was reported, while Alanazi (2011) and Aljumaah et al. (2018) reported a prevalence of 5.6% and 21.99% from camels, respectively. Different studies on the seroprevalence of N. caninum in camels from other countries reported different rates and the highest (86%) was reported in camels from the Canary Islands, Spain (Hilali et al., 1998;Sadrebazzaz et al., 2006;Wernery et al., 2008;Hosseininejad et al., 2010;Hamidinejat et al., 2013;Mentaberre et al., 2013). Mentaberre et al. (2013) attributed the high prevalence of antibodies against N. caninum in camels from the Canary Islands to the absence of the definitive host in the surveyed farms, resulting in the vertical transmission of this parasite in camels, as has been reported earlier in cattle (López-Gatius et al., 2004).
In the present study, camels from Taif city showed the highest prevalence of T. gondii antibodies and the lowest N. caninum antibodies. This could be attributed to the fact that cats, which are the definitive host for T. gondii, are more abundant than dogs in Taif. It is quite possible that the transmission of infection occurs via contamination of animal feed or water. The definitive hosts of both T. gondii and N. caninum were common in the areas where the camels were sampled. Dogs are kept as sentinel animals by shepherds while cats may contaminate camel feed and the infective oocysts may stay viable until the feed is eaten by the camels. Particularly in the Tabuk area, it was noticed that several dogs were in the vicinity where the samples were collected and some of the camel owners complained that they have had some animals aborting for no obvious reason.
Detection of antibodies to T. gondii or N. caninum from camels is dependent of the level of infection and the availability of the definitive hosts and obviously on the test utilized in the investigation. Most of the laboratory tests employed in serological surveys dealing with N. caninum were based on serological tests such as indirect fluorescent antibody test (IFAT) or modified agglutination test (MAT). Only a few studies employed ELISA for the detection of antibodies, such as Wernery et al. (2008) and Aljumaah et al. (2018). All three investigations, including the present study, where ELISA was used, have detected a high percentage of seropositive animals; this is likely due to the efficiency of the secondary antibodies used in the test. Andreotti et al. (2009) had reported that the specificity of the ELISA was 98.3% compared to that of IFAT when testing sheep serum samples. In the present study, detection of antibodies for both parasites in the same individual was not surprising, as the concurrent detection of both T. gondii and N. caninum antibodies has previously been reported in goats from the Czech Republic (Bartova & Sedlak, 2012). It is possible that cross reactivity might have occurred in the samples positive for both parasites; however, the serological method used in the present study is highly specific and sensitive.
Consuming camel meat is common in Saudi Arabia and it is likely that undercooked meat might be a potential source of both, T. gondii and N. caninum infections in humans as a result of consuming camel meat, especially that of young camels. This inference was validated through the presence of viable cysts of T. gondii in non-infected cats, which were fed infected camel meat (Hilali et al., 1995). Dogs that were fed camel meat shed Hammondia heydorni and Sarcocystis cameli. However, the identification of H. heydorni based on the morphology of the oocysts was questionable and it could probably be mistaken for N. caninum, as the dimensions of the cysts of both parasites are quite similar (Schares et al., 2005).
Transmission of T. gondii by male goats through semen has been experimentally demonstrated (Dubey & Sharma, 1980;Santana et al., 2010). It is unknown whether a similar transmission can occur in camels. Artificial insemination is not practiced in camels and only the traditional method of mating is practiced; therefore, any infected camel could be a potential hazard to the females it mates with.
Antibodies against both N. caninum and T. gondii have been detected in camels from different cities in different regions of Saudi Arabia. There was no information regarding the disease situation of both T. gondii and N. caninum in cattle in Saudi Arabia. The role which may be played by camels and equine in the epidemiology of these diseases in cattle in Saudi Arabia cannot be underestimated (Alanazi et al., 2014).