Sero-Prevalence of Toxoplasma Gondii in Different Horses Groups from Khartoum State, Sudan

Research Article 152 Sero-Prevalence of Toxoplasma Gondii in Different Horses Groups from Khartoum State, Sudan Abdalla Mohamed Ibrahim 1* ; Osman Mukhtar Osman 2 ; Rabab Haroun Mohamed Ali 1 ; Ahmed Ali Ismail 3 ; Tamador Elkhansa Elnour Angara 4 1 College of Veterinary Medicine University of Bahri, Khartoum, Sudan. 2 Veterinary research Institute, Khartoum, Sudan. 3 College of Veterinary Medicine, Sudan University of Science and Technology (SUST), Khartoum, Sudan. 4 College of Science and Technology of Animal Production, SUST, Khartoum, Sudan. (Received: June 30, 2014; Accepted: August 19, 2014) Abstract: A serological survey of Toxoplasma gondii was conducted using Latex Agglutination Test (LAT) on 223 sera collected from horses during dry (107 horses) and wet season (116 heads). Horses kept in equestrian, masts and markets in the Khartoum State, Sudan were included in the present study. The overall sero-prevalence of these horses was 30.04%(67/223). The seropositivity was significantly (P<0.05) higher in dry season (37.4%), compared to wet season (23.3%). Antibodies to T. gondii were detected in 27.3%, 30% and 33.3% of equestrian, Masts and market horses respectively. The antibody titers were 1:2 (18 heads), 1:4 (14 heads), 1:8 (12heads), 1:16 (19 heads), 1:32 (3 heads), and 1:64 (1 mare). The seroprevalence in the Khartoum district was insignificantly (P>0.05) higher (32.7%) compared to that of Khartoum North (31.1%) and Omdurman district (27.3%). In locality base, Khartoum locality is superior among the six investigated localities followed by Ombadda and Bahri localities with 37.8%, 33.3% and 27.5% seropositivity of antibodies against T. gondii respectively. Area, horse group, age and sex showed insignificant (P>0.05) differences between the investigated groups. Season was reported to show significant (P<0.05) effect on horse toxoplasmosis in the Sudan in the present study. It was concluded that the environment is highly contaminated with T. gondii oocyst which reach horses stable. The results of the present study revealed the high risk of human exposure to T. gondii in the investigated area and may be the whole country, where, more research on human toxoplasmosis is recommended. Index terms: Toxoplasma gondii; Horses; Seroprevalence; Latex Agglutination Test; Sudan. I. INTRODUCTION Toxoplasmosis is an infection distributed worldwide and affecting almost all warm-blooded animal species, and specially humans [1]. The zoonotic protozoan *Corresponding author: Abdalla Mohamed Ibrahim, M.Sc. Lecturer of Veterinary Parasitology (Protozoology) College of Veterinary Medicine University of Bahri, Khartoum11111, Sudan. E. Mail: abdallami79772@gmail.com parasite, Toxoplasma gondii are widely prevalent in humans and animals [1, 2] including equines [3, 4] on all continents. However, very few data is available about the prevalence of human [5, 6, 7] and animal toxoplasmosis in the Sudan including camel [8, 9], sheep [10, 11] and other food animals [12, 13, 14]. Equines are not included in the work of these authors. The present paper is a part of our research project on animal toxoplasmosis in the Sudan as the main source of human toxoplasmosis [14]. Equines in the Sudan are mainly horses and donkeys. Horse s accounts to 784.5 thousand head [15]. Horses in the Sudan are kept for working, masts, individual riding or racing. Infections due to Toxoplasma gondii in equines have been reported worldwide [3, 16, 17, 18, 19]. Last year we published the first report on the presence of antibodies against T. gondii in working equines [20] in the Sudan. The main goal of the present work is to study the seroprevalence of Toxoplasma gondii antibodies in horses kept in equestrian, masts and market in the Khartoum State, Sudan. This may increase the importance of scientific research for more epidemiological data on human toxoplasmosis in the Sudan. Moreover, this work is to raise up the awareness of Sudanese on the level of the environment contamination with T. gondii parasite, and to encourage in-depth research in the socio-economic, public health and the epidemiology of human toxoplasmosis in the country. II. MATERIALS AND METHODS The Study Area The study area, Khartoum State is divided into three districts (Table 1) with seven localities including, Khartoum district (two localities), Omdurman (three localities) and Khartoum North (two localities). Horses of six out of these seven localities were investigated in this study (Table 2). As shown in table 3, eleven Areas (Herds) where equestrian, masts horses located as well as one market were visited twice (dry and wet season) for sample collection (Table 5).

153 Animals The study population encompassed 223 horses (107 during dry season and 116 during wet season) including 11 heads of equestrian horses, 9 heads from market and 203 heads of masts horses (Table 4). All of the investigated horses were adult ranging from 2 to 26 years old (Table 6). These horses are used for racing champions, police (masts) and pulling cart in the market. Both available sexes (212 male and 11 female) were tested (Table 7) for antibodies against T. gondii using Latex Agglutination Test (LAT). Samples Collection Blood samples were obtained from the jugular vein of 223 horses in the Khartoum State, Sudan during April, 2013 (dry season) and December, 2013 (wet season). Blood was transported to Parasitology Laboratory, College of Veterinary Medicine, Sudan University of Science and Technology. For each sample, serum was removed from clotted blood and stored at -20 ºC until tested. Latex agglutination Test (LAT) The Toxo-Latex diagnostic kits (Spinreact, S.A./S.A.U, Spain) were purchased from Shifak Company, Khartoum, Sudan. Initially sera were examined for anti- T. gondii qualitatively in the LAT based on the manufacturer instructions (25µl of antigen in 50 µl of tested serum). Sera with positive reactions were then diluted twofold serially (1:2 up to 1:128) and tested for detection of level antibody titration. Data Analysis Data was analyzed using the statistical package for social sciences (SPSS) version 17.0. A p-value less than 0.05 were considered statistically significant. III. RESULTS AND DISCUSSION The overall sero-prevalence of T. gondii in horses Antibodies to T. gondii were found in 67 heads out of 223 (30%) horses with titration ranging from 1:2 (18 heads), 1:4 (14 heads), 1:8 (12heads), 1:16 (19 heads), 1:32 (3 heads), and 1:64 (1 mare). Most of the positive samples (52.2%) revealed high T. gondii antibody (>1:8) titration (table 1). Table 1. Sero-prevalence of T. gondii in horses in the three districts of the Khartoum State, Sudan. *Distribution of specific antibody titres to T. gondii positive reaction (%) District Tested *Positive (%) 1:2 1:4 1:8 1:16 1:32 1:64 Negative Khartoum 49 16(32.7) 4(25) 1(6.3) 3(18.8) 6(37.5) 1(6.3) 1(6.3) 33 Kh. North 90 28 (31.1) 9(32.1) 9(32.1) 6(21.4) 4(14.3) 0(0.0) 0(0.0) 62 Omdurman 84 23 (27.4) 5(21.7) 4(17.4) 3(13) 9(39.1) 2(8.7) 0(0.0) 61 *Not significant at P 0.05. The effect of area on horse toxoplasmosis in the Khartoum State, Sudan: The higher seropositivity was reported in Khartoum district (32.7%), followed by Khartoum North (31.1%) and Omdurman (27.4%), without any statistically significant (P>0.05) differences (table1). Khartoum locality was also the superior (37.8%) among the six localities investigated (table 2) followed by Ombadda (33.3%) and Bahri (31.5%) localities. Antibody against T. gondii was detected in all (100%) investigated herds (1 market heard and 10 Stables) without statistically significant differences between areas (table 3). The highest prevalence rate (47.1%) was reported in mast stable from Khartoum area, followed by North Ombadda mast stable (46.7%). Table 2. Sero-prevalence of T. gondii in the investigated horses from six localities in the Khartoum State, Sudan. District Locality Tested samples Positive (%) Negative Khartoum Khartoum 37 14 (37.8) 23 Jabal Awlia 12 2 (16.7) 10 Khartoum North Bahri 54 17 (31.5) 37 Eastern Nile 36 11 (30.6) 25 Omdurman Omdurman 39 8 (20.5) 31 Ombadda 45 15(33.3) 30 Total 223 67(30) 156 *Not significant at P 0.05.

154 Table 3. Sero-prevalence of T. gondii in the investigated herds (stables) in the Khartoum State, Sudan. District Locality Area (Stables) Tested samples Positive (%) Negative Khartoum Jabra 11 3(27.3) 8 Khartoum 17 8(47.1) 9 Khartoum Sajana* 9 3(33.3) 6 Jabal Awlia Kalakla 12 2(16.7) 10 Khartoum North Elmazad 31 12(38.7) 19 Bahri Halfaya 23 5(21.7) 18 Eastern Nile Haj Yosif 36 11(30.6) 25 Omdurman Omdurman 23 3(13.0) 20 Omdurman Ombadda Molazmeen 16 5(31.3) 11 South Ombadda 30 8(26.7) 22 North Ombadda 15 7(46.7) 8 223 67(30.0) 156 Not significant at P 0.05. Total *Market Horse groups There was no statistically significant difference (P>0.05) in the seroprevalence of T. gondii between the equestrian (27.3%), masts (30%) and market (33.3%) horses (Table 4). However, statistically significant differences (p<0.05) was reported in the antibody titration between these groups. Although there is no statistically significant difference, but the effect of management system appear when horses from market (extensive system) showed higher seropositivity compared to horses kept in stables (semi-intensive system). Table 4. Prevalence of T. gondii antibodies and latex agglutination test titers in three different groups of horses in the Khartoum State, Sudan *Distribution of specific antibody titers to Toxoplasma gondii positive reaction (%) group Tested Positive (%) 1:2 1:4 1:8 1:16 1:32 1:64 Negative Masts 203 61(30) 15(24.6) 13(21.3) 12(19.7) 18(29.5) 3(4.9) 0(0.0) 142 Equestrian 11 3(27.3) 0(0.0) 1(33.3) 0(0.0) 1(33.3) 0(0.0) 1(33.3) 8 Market 9 3(33.3) 3(100) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 6 Insignificant differences in the prevalence (P>0.05), but significant (P<0.05) in antibody titration. Seasonal effect on horse toxoplasmosis in the Khartoum State, Sudan The seroprevalence of T. gondii was significantly (p=0.16) higher (37.4%) in dry season than that reported in wet season (23.3%). Moreover, a highly (p<0.01) statistically significant difference was also reported in the antibody titration between the two seasons, when most of positive 72.5% (29/40) samples in the dry season has more than 1:8 titers. However, only 22.2% (6/27) has titers more than 1:8 in the wet season (table 5). Table 5. The effects of Season on horses toxoplasmosis in the Khartoum State, Sudan *Distribution of specific antibody titers to Toxoplasma gondii positive reaction (%) Season Tested *Positive (%) 1:2 1:4 1:8 1:16 1:32 1:64 Negative Dry 107 40(37.4) 3(7.5) 8(20) 9(22.5) 16(40) 3(7.5) 1(2.5) 67 wet 116 27(23.3) 15(55.6) 6(22.2) 3(11.1) 3(11.1) 0(0.0) 0(0.0) 89 *significant (P<0.05) differences in both, the seropositivity and the antibody titration. Prevalence of T. gondii infection in different age groups The prevalence of toxoplasmosis was found to be higher in the older (>5 years) horses compare to younger group (2-5 year old). However, there were no significant differences (P>0.05) between the investigated three age groups (table 6).

155 Table 6. The effect of age on horse s toxoplasmosis in the Khartoum State, Sudan Distribution of specific antibody titers to Toxoplasma gondii positive reaction (%) *Age /Year Tested Positive (%) 1:2 1:4 1:8 1:16 1:32 1:64 Negative 2-5 18 4(22.2) 1(25) 1(25) 0(0.0) 2(50) 0(0.0) 0(0.0) 14 6-10 151 42(27.8) 8(19) 10(23.8) 9(21.4) 12(28.6) 3(7.1) 0(0.0) 109 >10 54 21(38.9) 9(42.9) 3(14.3) 3(14.3) 5(23.8) 0(0.0) 1(4.8) 33 *Insignificant differences in both the prevalence and in antibody titration (P>0.05). The effect of Sex on horse toxoplasmosis in the Khartoum State, Sudan As shown in the following table (7), although the difference was statistically insignificant, male horses reported higher prevalence on anti-t. gondii antibodies (30.2%) compared to the investigated mares (27.3%). There were statistically significant differences in the antibody titration, when the highest antibody titration was reported in mare with history of abortion. Table 7. The effect of sex on horse s toxoplasmosis in the Khartoum State, Sudan *Distribution of specific antibody titers to Toxoplasma gondii positive reaction (%) Sex Tested Positive (%) 1:2 1:4 1:8 1:16 1:32 1:64 Negative Male 212 64(30.2) 18(28.1) 13(20.3) 12(18.8) 18(28.1) 3(4.7) 0(0.0) 148 Female 11 3(27.3) 0(0.0) 1(33.3) 0(0.0) 1(33.3) 0(0.0) 1(33.3) 8 * Insignificant differences in the prevalence (P>0.05), but significant (P<0.05) in antibody titration. There is meager data on animal and human toxoplasmosis in the Sudan. In order to encourage more research on human toxoplasmosis in the country, our ongoing research project aim to avail data on animal toxoplasmosis [14, 20] in the Sudan. In the present study Latex agglutination test was used to detect the seroprevalence of T. gondii in different groups of horses kept in stable and market. The uses of different serological tests for Toxoplasma gondii in horses have been made in several countries with prevalence rate ranging from 6.9% to 65.6% [16, 17]. The prevalence rate of T. gondii antibodies in horses in this study was 30%. Nationally, this result is lower than that reported in our last work [20] in working horses (38%). That is may be because of the large number of investigated horses in this study. Additionally, most of these horses (Masts and equestrian group) are kept in stables (semi-intensive management system). Internationally, the present result is lower than the findings of Egyptian (38.1%) horses [17]. However, it is higher than the American (6.9%) findings [16]. That is may be due to different serological tools used for anti-t. gondii antibody detection. In the present study, variables of area (districts, localities and sites) have no any significant effect in the horse toxoplasmosis prevalence as well as the level of antibody titers. This may highlights the similar contamination of all the Khartoum State environments by this parasite. Although there was significant variation in antibody titration, the different groups of horses showed no significant differences in seropositivity. Most of these horses (Masts and equestrian group) are kept in similar management system. That is clear when the market group (extensive management system) reported the highest seroprevalence (33.3%) among the investigated horse groups. In this study, where most of the investigated animals were male (95%) and aged (>5 years old), age has no significant effect in horse toxoplasmosis, but sex affect the antibody titration significantly. The increase of antibody titration in aged horses could be explained by the possibility of neonatal infection rather than congenital infection. Generally, age and sex effect were rarely reported in seroprevalence of human and other animal toxoplasmosis [21, 22, 23]. The highest titration (1:64) was reported in mare kept in equestrian stable with history of abortion and neonatal death. It worth mentioning that, horses are considered to be more resistant to clinical T. gondii infection [18, 19]. Highly statistically significant differences in both, the prevalence and the antibody titration were reported between the dry and wet season in the present study. This study is not enough to explain the effect of seasonality in T. gondii infection. However, the significant differences may be due to the high resistance of oocysts [24] as a source of infection for several months, passing the wet season to dry season where the wave of seropositivity increased. IV. CONCLUSION & RECOMMENDATIONS It was concluded that, similar to other livestock [8-14], horses in the Sudan are widely infected with T. gondii. Additional study is recommended for accurate assessment of equine clinical toxoplasmosis in the Sudan. Moreover, the role of animals in the

156 epidemiology of human toxoplasmosis need more care in the country. REFERENCES [1] Tenter, A. M.; Hekeroth, A. R. and Weis, L. M. Toxoplasma gondii from animal to humans. Int J Parasitol. 2000; 30: 1217-1258. [2] Innes, E. A.. Neosporosis and toxoplasmosis in farmed livestock: Options for control. Proceedings of European Buiatrics Forum, Palias de Faro, Marseille, France from November 16 th to 18 th, 2011, 7-15. [3] El-Ghaysh A. Seroprevalence of Toxoplasma gondii in Egyptian donkeys using ELISA. Veterinary Parasitology 1998; 80 : 71-73. [4] Shaapan R. M. and Fathia Khalil A. M. Evaluation of different Toxoplasma gondii Isolates as antigens used in modified agglutination test for detection of toxoplasmosis in camels and donkeys. American Eoropean J. agric. & environ. Sci., 2008; 3(6): 837-841. [5] Siddig. A. B.. Study on Toxoplasmosis in Humans and Cats in the Red Sea State, Sudan M.Sc. Thesis SUST 2010. [6] Musa H.O. Detection of Toxoplasma gondii antibodies in females in Port Sudan City. B.Sc. thesis, University of Ahlia, PortSudan 2008. [7] Anon. Annual report 2006-2010: Private Medical laboratories. 2010 Port Sudan, Red Sea State, Sudan. [8] Elamin,E.A, Elias S., Daugschies, A. and Rommel, M. Prevalence of Toxoplasma gondii antibodies in pastoral camels (Camelus dromedarius) in the Butana plains, mid-eastern Sudan Veterinary Parasitology. 1992; 43 (3-4): 171-175. [9] Manal Y.I. Study on Toxoplasma and Sarcocystosis from camel (Camelus dromedaries) in the Sudan. PhD. Thesis. University of Khartoum, Sudan 2003. [10] Abdel Hafez, A. A. Epidemiological study on Toxoplasma gondii infection in Beja sheep in The Red Sea State, Sudan. PhD thesis, Sudan academy of Science, 2013. [11] El Badawi K.S.; Micheal S.A.; Morsy T.A. Serological demonstration of toxoplasmosis among sheep in Sudan. J Egypt.Soc. Parasitol, 1984; 14(1): 289-293. [12] Khalil, M. K. and Elrayah, I. E. Seroprevalence of Toxoplasma gondii antibodies in farm animals (camels, cattle, and sheep) in Sudan. Journal of Medicine and Animal Health, 2011; Vol. 3(3), 36-39. [13] Zain Eldin, E. A.; Elkhawad, S. E. and Kheir, H. S. M. A serological Survey for Toxoplasma antibodies in cattle, sheep, goats and camels (Camelus Acknowledgements We would like to thank Miss Reem Moaz for laboratory work assistance. dromedaries) in the Sudan. Rev. Elv. Med. Vet. Pays trop., 1985; 38(3): 247-249. [14] Ibrahim A. M.; Isamail A. A.; Tamador E.E. Angara 3, Osman M. O. Sero- prevalence of Toxoplasma gondii in Farm Animals in Khartoum State, Sudan. Proceedings of ICOPHAI Conference August 14-17, Brazil, 2013, pp. 233. [15] Ministry of Animal Resources, Range lands and Fisheries (MARF). Annual report, 2012. Khartoum, Sudan. [16] Dubey J. P., Thulliez P., Romand S., Kwok O. C., Shen S. K., and Gamble H. R.. Serological prevalence of Toxoplasma gondii in horses slaughtered for food in North America. Veterinary parasitology. 1999; 86(4): 235-238. [17] Ghazy A.A.; Shaapan R.M. and Eman H. Abdel-Rahman. Comparative serological diagnosis of toxoplasmosis in horses using locally isolated Toxoplasma gondii. Veterinary Parasitology 2007; 145, 31 36. [18] Dubey J. P., Kerber C. E. and Granstrom D. E. Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil. Journal of the American Veterinary medical Association. 1999; 215(7): 970-972. [19] Dubey J. P., Venturini M. C. Venturini L., McKinney J. and Pecoraro M. Prevalence of antibodies to Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses from Argentina. Veterinary Parasitology. 1999; 86(1): 59-62. [20] Shadia Mohamed Ahmed, Abdalla Mohamed Ibrahim, Nabaa Kamal Alshafie, Rabab Haroun Mohammed, Ahmed Ali Ismail and Tamador Elkhansa Elnour Angara. The first Report on Sero-prevalence of Toxoplasma gondii in working Horses and Donkeys in The Sudan. Journal of life sciences, 2013; vol.7, No 12 pp. 1284-1287. [21] Petersen E. and Eaton R. B. Control of congenital infection with Toxoplasma gondii by neonatal screening based on detection of specific immunoglobulin M antibodies eluted from phenylketonuria filter-paper blood spot samples. Acta Paediatrica, supplement. 1999; 88(432): 36-39. [22] Baril L., Ancelle T., Goulet V., Thulliez P., Tirard-Fleury V., and Carme B. Risk factors for Toxoplasma infection in pregnancy: a case- control study in France. Scandinavian Journal of Infectious diseases. 1999; 31(3): 305-309. [23] Gorman T., Arancibia J. P., Lorca M., Hird D. and Alcaino H. Seroprevalence of Toxoplasma

157 gondii in sheep and alpacas (Llama pacos) in Chile. Preventive Veterinary Medicine. 1999; 40(3-4): 143-149. [24] Frenkel J.K.. Biology of Toxoplasma gondii: in Ambroise-Thomas P.; Peterson E. editors. Congenital Toxoplasmosis. Scientific background, clinical signs and Control. Paris: Springer-Verlag, France, 2000, pp 9-25.