SEROLOGICAL STUDIES OF TOXOPLASMA GONDII INFECTION IN CAMELS (CAMELUS DROMEDARIUS)

Veterinary Serum and Vaccine Research Institute,

Abbasia, Cairo, Egypt.

Central Veterinary Diagnostic Lab,

Ministry of Agriculture, Riyadh. KSA.

Serological studies of Toxoplasma gondii infection in Camels

(Camelus dromedarius)

(With One Table and One Figure)

By

R.M. Al-Khatib

Received at 13/6/2011)

دراسات مصلية لإصابة الجمال (وحيدة السنام) بطفيل التکسوبلازما جوندى

رجب محمد الخطيب

طفيل التکسوبلازما جوندى واحد من أهم الطفيليات المشترکة واسعة الانتشار الجغرافي على مستوى العالم. جميع الحيوانات تعتبر عائل وسيط ماعدا العائلة  القطيه فهي تعتبر العائل الأساسي للطفيل. ما بين عامي 2009/2010 تم جمع عدد 482 عينة سيرم (182 ذکر – 300 أنثى) من الجمال (وحيدة السنام) وکانت أعمارها من 3-6 سنوات من خمس محافظات مختلفة من المملکة العربية السعودية, حيث تم إجراء اختبار الاليزا الغير مباشر وذلک للکشف عن الأجسام المضادة لطفيل التکسوبلازما جوندى في عينات السيرم التي تم جمعها. أظهرت نتائج هذه الدراسة إن عدد  219 (من جملة 482) عينه سيرم کانت ايجابية لوجود الأجسام المناعية المضادة الدالة على الإصابة بطفيل التکسوبلازما جوندى وذلک بنسبة (45.44%) وکانت نسبة الإصابة في الإناث (47%) اعلي منها في الذکور (42.86%) ولوحظ أن نسبة الإصابة في محافظة الرياض والخرج (50.68%- 47.15%) کانت اعلي من المحافظات الثلاث الأخرى وهي وادي الدواسر (41.84%) وضر ماء (40.58%) والسليل (38.46%) علي الترتيب. هذه الدراسة النوعية توضح مدى إصابة الجمال بطفيل التکسوبلازما جوندى على نطاق واسع بالمملکة السعودية وذلک مما يعکس أثرها الاقتصادي و أهميتها على الصحة العامة للإنسان لا سيما وان البدو يستهلکون الحليب والکبد الغير ناضج من هذه الحيوانات وهذا مما يستحق نظرة فاحصة إلى کيفية إصابة الجمال وطريقة اکتسابها العدوى بطفيل التکسوبلازما جوندى  خصوصا في بيئتها القاحلة.

SUMMARY

Toxoplasma gondii is one of the important zoonotic parasites of worl-dwide zoological and geographical distribution. All animal species act as intermediate hosts to the Toxoplasma gondii, except feline species which act as a definitive host. A total of482 serum samples (182 male and 300 female) were collected during 2009/2010 from native camels (3-6 years), represented five different provinces of Saudi Arabia to monitoring serologically for Toxoplasma gondii antibodies by the indirect enzyme linked immuno-sorbant assay (ELISA). A total of 219 (45.44%) camel sera were serologically positive for toxoplasmosis. The prevalence was higher in female (47%) than male (42.86%).The prevalence was high in Al-Riyadhand Alharig (50.68% and 47.15%) comparing to other areas: Wady Al-Dawaser (41.84%), Dar-maa (40.58%) and Al-Solyel (38.64%) respectively. This study suggests widespread infection with Toxoplasma gondii among camels, a finding that warrants a closer look into the possible ways infection are acquired by camels in their arid environment, its economic impact, as well as its public health significance, especially among the nomads who consume cameline milk and raw liver.

Key words: ELISA, Camel, Sero-prevalence, Toxoplasma gondii.

Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite that is probably capable of infecting all species of mammals, including man and cause one of the important zoonotic parasite diseases; Toxoplasmosis of world-wide distribution (Shaapan et al., 2010). It can also cause abortion or neonatal mortality in other animal species, such as sheep, goats, horses, camels and deer (Dubey, 2003). Approximately one-third of humanity has been exposed to the parasite world wide (Evengard et al., 1999; Sukthana, 2006). The parasite induces high levels of gamma interferon (IFN-γ) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. During chronic infection, parasite-specific T lymphocytes release high levels of IFN-γ, which is required to prevent cyst reactivation (Denkers and Gazinelli, 1998). One attenuated vaccine has been successfully used to reduce abortions in sheep (Buxton, 1993); this live vaccine is unlikely to be used in humans. In addition; many inactivated vaccines developed in the past have produced only little to moderate protective efficacy against infections with a lethal dose of the virulent strain of T. gondii, therefore; effective and reliable vaccines comprising subunit or recombinant antigens are likely to be approved for use (Waldeland and Frenkel, 1986). Cats, including all felines; are definitive hosts for T. gondii and excrete environmentally-resistant oocysts in their feces (Dubey, 2006). Domestic cats are probably the major source of contamination, because they are common and produce large numbers of T. gondii oocysts (Dubey and Frenkel, 1972; Dubey, 2001). T. gondii infection results in significant reproductive losses and economic losses (Faria et al., 2007), Hosts became infected by ingestion of food or drink contaminated with oocysts or by ingesting undercooked meat from infected animals with   T. gondii which causes mental retardation and loss of vision in congenitally- infected children and abortion in pregnant women and animals (Dubey et al., 2005). Cats are essential in perpetuation of the life cycle, because T. gondii infection is rare or absent in areas devoid of cats (Wallace, 1969; Dubey et al., 1997).

Seroprevalence data for cats are more useful than results of fecal examination, because cats with antibodies have probably already shed oocysts and are an indicator of environmental contamination (Dubey and Frenkel, 1972). Sporulated oocysts of Toxoplasma gondii are very resistant to environmental conditions. They remain infectious in moist soil or sand for up to 18 months (Frenkel, 2000). Serologic testing is an important method for detecting these parasitic infections, and includes immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), competitive-inhibition ELISA, Western blotting, and direct agglutination test (DAT) using intact tachyzoite or tachyzoitederived antigens (Jenkins et al., 2002; Huang et al., 2004), however; seroprevalence rates do vary depending on the serologic test and the initial serum dilution tested (Dubey, 2003).

Camels constitute an important economical activity in the desert population in east and central regions of Saudia Arabia. The camel is used as a load animal, which is an important role in transportation in rural areas; in addition, it is also a source of meat, skin, and leathers (Sadrebazzaz et al., 2006).

Since there is a little information on the role of toxoplasmosis in camel in KSA; the presented study was aimed to estimate the sero- prevalence of the T. gondii antibodies among camel (Camelus dromedarius) population in five provinces of  Saudi, and subsequently to estimate the role of this parasite in the abortion of camel (Camelus dromedarius) as preliminary step in long lasting program to prevent transmit the infection to animal or man population due to its medical importance of T.gondii as a major source of parasitic zoonosis

MATERIALS and METHODS

1. Blood samples

Random blood samples were collected from different farms with previous history of abortion and reproductive disorders from five provinces of KSA (Al-Riyadh, Alharig, Al- Solyl, Dar- maa and Wady Al- Dawaser): a total of 482 samples were collected (182 male and 300 female) from camels aged 3-6 years, by jugular vein-puncture and removed from the clotted blood by centrifugation at 3000 rpm for 15 minutes. All the sera were aspirated and kept in micro-tubes and stored at −20^°C until tested for antibodies to T. gondii.

2. Enzyme linked immunosorbent assay (ELISA):

The commercial ELISA kits were supplied by Institute of Pourquier, France [Lot batch No., 2-TXRM- 003]. The test was carried out according to the manufacture's instructions. Each sample; individually tested in single well. The results were monitoring using ELISA reader with 450nm filter and expressed as the percentage of the mean absorbance value of the samples (S) to the mean absorbance value of the positive (P) control sample provided with the diagnostic kit. The resultant S/P ratio was expressed as a percentage (S/P %) according to manufacture's recommendation where; sera with S/P% less than 40% were regarded as negative, between 40% - 50% as suspicious, and more than 50% as positive.

Results

As shown in Table 1 the prevalence of T. gondii antibodies in examined camel sera revealed that 219 (45.44%) {42.86%male, 47% female} serum samples from totally examined 482 camels tested by ELISA were positive. Among which; 75(50.68%) {46.16% male,53.13% female}, 58(47.15%){44.19% male and 48.75%} and 41(41.84%) {34.09% male and female 48.15%} had antibodies against T gondii inAl_Riyadh., Alharig and Wady Al Dawaser respectively. But the prevalence is 28(40.58%) {38.71% male and 42.11% female}, 17(38.64%){33.33% male and 40.63% female} in Dar-maa and Al Solyel.

Table 1: The prevalence (antibodies) of Toxoplasma gondii infection from five Province in Saudi Arabia.

Fig. 1: Percentage of positive cases in different provinces

Discussion

T.gondii is a protozoan parasite that infects mainly the cat as a definitive host and sheep as a very important intermediate host. In ewes, this parasite is now considered to be the major cause of abortion worldwide (Dubey and Beattie, 1988); it has been found worldwide from Alaska to Australia so public health organizations repeatedly encourage the collection of accurate data about T. gondii in animals and humans due to its medical importance as a major source of parasitic zoonosis (Rinaldi and Scala, 2008). In KSA, there role of toxoplasmosis were recorded as one of the most important causative agent of abortion and infertility in both sheep and goat (Al-Khatib, 2010).

Toxoplasmosis usually results from the ingestion of food contaminated with oocysts or cysts of the parasite. Oocysts are shed by cats -the definitive hosts of Toxoplasma gondii - while; cysts are present in tissues from chronically infected animals (Dubey and Beattie, 1988). The prevalence data for the determination of T. gondii infection in camels at the different localities of the world is extremely variable. In Egypt, the infection rates among slaughtered camels were 46% using DAT (Rifaat et al., 1979), 54.2%, 18% using IHAT and IFAT, respectively (Ibrahim et al., 1997; Derbala et al., 1993), 17.4% using MAT (Hilali et al., 1998) and recently by bioassay in mice and cats a total of 23 out of 90 camel meat samples were found to be infected with an incidence rate of 25.6% (Shaapan and Fathia, 2005). The infection rate was 16% (IHAT) in Saudi Arabia, (Hussein et al., 1998), 31.4% (ELISA) (Abu-Zeid, 2002) and 22.4% (MAT) (Abu-Zeid et al., 2006) in United Arab Emirates; in Sudan 67% (latex agglutination) (Elamin et al., 1992) and 51.3% (latex agglutination) (Manal and Maijd, 2008). Seroprevalence data on T. gondii that have been recorded in livestock from different regions in Saudia, showing the high level of exposure of small ruminants (76.84% for sheep and goats) (Al-Khatib, 2010).

In current work; the total prevalence of 45.44 % Toxoplasma gondii antibodies in the examined camel sera were similar to some what that prevalence in Egypt (48.8%) (Ibrahim et al., 1997), and lower than that recorded in Sudan (67%) (Elamin et al., 1992), but it was higher comparing to that reported in Emarates (36.4%) (Afzal and Sakkir, 1994), in Saudi Arabia (16%) (Hussein et al., 1988), and Egypt (17.4%) (Hilalia et al., 1998) among camel population. The variety of  prevalence of toxoplasmosis in the current and previous studies could emanate from difference in the serological tests employed and the initial serum dilution tested (Dubey, 2003), geographical variability or continuous exposure of camels to infection due to heavy environmental contamination with oocysts shed from the observed stray cats in the farms,  frequency of felines on the farms, age of the animals, and the climatic variations from one region to another (Dubey, 1990; Sawadogo et al., 2005). Variation in prevalence of T. gondii occurs not only among different continents or countries but also within a given country (Teshale et al., 2007), camels free range livestock associated with T. gondii infection, acquisition of Toxoplasma infection by camels is through ingestion or inhalation of sporulated oocysts that are shed by cats in the environment, they are kept on pastures with an increased pressure of infection due to contamination of environment with oocysts and the frequency of stray cats in a humid rainy climate. The high ratio of Toxoplasma gondii obtained in these work may be attributed also to the fact that, the stray cats are widely spread specially in El-Rhyaid which is in favor of a higher prevalence of oocysts within hot and humid environments compared to cold and dry ones is attributed to the longer viability of T. gondii oocysts under humid conditions (Fleck, 1972; Fayer, 1981; Elamin et al., 1992; Remington   et al., 2001). The effect of sex on the prevalence of T.gondii in camel obtained in our study indicated that infection rates were higher in female than male; in this regard; (Duby and Beattie 1988; Randall et al., 2000) proved that antibody prevalence of T. gondii was directly related to sex and they ascribed this fact to opportunity stresses (pregnancy and lactation) exposed by females. This study suggests widespread infection with Toxoplasma gondii among camels, a finding that warrants a closer look into the possible ways infection are acquired by camels in their arid environment, its economic impact, as well as its public health significance, especially among the nomads who consume cameline milk and raw liver.    

References

Abu-Zeid, Y.A. (2002): Protein G ELISA for detection of antibodies against Toxoplasma SAG1 in dromedaries. J. Egyptian Soc. Parasitol., 32: 247-257.

Abu-Zeid, Y.A.M.; Enan, R.; Amina, M.; Hassan, Huda; Shaheen, M.G.; Ramadan, A.; Al Tayarri and Wafaa, A.R.S.K. (2006): Genotyping of Toxoplasma gondii Isolates from Camels from Abu Dhabi. The SixthAnnualU.A.E.University Research Conference.

Afzal, M. and Sakkir, M. (1994): Survey of antibodies against various infectious disease agents in racing camels in Abu Dhabi, United Arab Emirates. Rev. Sci. Tech.13 (3): 787-92.

Al-Khatib, R.M. (2010): Sero-prevalance of Toxoplasma gondii in small Ruminants Assiut Vet. Med. J. (56): 271-278.

Buxton, D. (1993): Toxoplasmosis: the first commercial vaccine. Parasitol. Today 9: 335–337.

Denkers, E.Y. and Gazinelli, R.T. (1998): Regulation and Function of T-Cell-Mediated Immunity during Toxoplasma gondii Infection. Clinical Microbiol. Rev.11(4): 569-588.

Derbala, A.A.; Zayed, A.A. and Tawfik, M.A. (1993): Prevalence of Toxoplasma antibodies in serum of cattle and camels. J. Egyptian Vet. Med. Assoc., 53: 319-324.

Dubey, J.P. and Beattie, C.P. (1988): Toxoplasmosis of animals and man, p. 1-220. CRC Press, Boca Raton, Fla.

Dubey, J.P. (2001): Oocyst shedding by cats fed isolated bradyzoites and comparison of infectivity of bradyzoites of the VEG strain Toxoplasma gondii to cats and mice. J. Parasitol, 87: 215–9.

Dubey, J.P. (2003): Review of Neospora caninum and neosporosis in animals. Korean J. Parasitol 41: 1–16.

Dubey, J.P. (1990): Status of toxoplasmosis in sheep and goats in the United States. J. Am. Vet. Med. Assoc., 196: 259–262.

Dubey, J.P. (2006): Comparative infectivity of oocysts and bradyzoites of Toxoplasma gondii for intermediate (mice) and definitive (cats) hosts. Vet. Parasitol., 140: 69–75.

Dubey, J.P.; Hill, D.E. and Sreekumar, C. (2005): Toxoplasmosis, In Kahn, C.M. Eds. The Merck Veterinary Manual Ninth Edition, Publication Services, Merck and CO., White House Station, New Jersey, pp: 549.

Dubey, J.P. and Frenkel, J.K. (1972): Cyst-induced toxoplasmosis in cats. J. Protozool, 19: 155–77.

Dubey, J.P.; Rollor, E.A. and Smith, K. (1997): Low seroprevalence of Toxoplasma gondii in feral pigs from a remote island lacking cats J. Parasitol., 83: 839–41.

Elamin, E.A.; Elias, S.; Daugschies, A. and Rommel, M. (1992): Prevalence of Toxoplasma gondii antibodies in pastoral camels (Camelus dromedarius) in the Butana plains, mid-Eastern Sudan. Vet. Parasitol. 43: 171-175.

Evengard, B.; Lilja, G.; Capraru, T.; Malm, G., Kussofsky, E.; Oman, H. and Forsgren, M.A. (1999): Retrospective study of seroconversion against Toxoplasma gondii during 3,000 pregnancies in Stockholm. Scand. J. Infect. Dis., 31: 127-1.

Faria, E.B.; Gennari, S.M.; Pena, H.F.; Athayde, A.C.; Silva, M.L. and Azevedo, S.S. (2007): Prevalence of anti- Toxoplasma gondii and anti- Neospora caninum antibodies in goats slaughtered in the public slaughterhouse of Patos city, Paraíba State, Northeast region of Brazil. Vet. Parasitol. 149: 126–129.

Fayer, R. (1981): Toxoplasmosis update and public health implications. Can Vet. J., 22: 344–352.

Fleck, D.G. (1972): The seroepidemiology of Toxoplasma infection in man. Proc. Royal Soc. Med., 65: 50.

Frenkel, J.K. (2000): Biology of Toxoplasma gondii. In: Ambroise-Thomas P, Peterse E, editors. Congenital toxoplasmosis: Scientific background, clinical management and control. Paris: Springer-Verlag. P 9-25.

Hilali, M.; Romand, S.; Thulliez, P.; Kwok, O.C. and Dubey, J.P. (1998): Prevalence of Neospora caninum and Toxoplasma gondii antibodies in sera from camels from Egypt. Vet. Parasitol., 75: 269-271.

Huang, X.; Xuan, X.; Hirata, H.; Yokoyama, N.; Xu, L.; Suzuki, N. and Igarashi, I. (2004): Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats. J. Clin. Microbiol. 42: 351–353.

Hussein, M.F.; Bakkar, M.N.; Basmaeil, S.M. and Gar el Nabi, A.R. (1988): Prevalence of toxoplasmosis in Saudi Arabian camels (Camelus dromedarius).Vet. Parasitol. 28(1–2): 175–178.

Ibrahim, B.B.; Salama, M.M.; Gawish, N.I. and Haridy, F.M. (1997): Serological and histopathological studies on toxoplasma Gondii among the workers and the slaughtered animals in Tanta Abattoir, Gharbia Governorate. J. Egypt Soc. Parasitol. Apr., 27(1): 273-8.

Jenkins, M.; Baszler, T.; Bjorkman, C.; Schares, G. and Williams, D. (2002): Diagnosis and seroepidemiology of Neospora caninum –associated bovine abortion. Int. J. Parasitol. 32: 631–636.

Manal, Y.I. and Maijd, A.M. (2008): Association of diarrhea with congenital Toxoplasmosis in calf- camel (Camels dromedaries). International J. of Tropical Medicine. 3(1): 10-11.

Randall, L.; Zarnke, S.; Dubey, J.P.; Kwok, O.C.H. and VerHoef, J.M. (2000): Seroprevalence for Toxoplasma gondii in selected wild species from Alaska. J. Wildlife Dis., 36: 219-224.

Remington, J.S.; McLeod, R.; Thulliez, P. and Desmonts, G. (2001): Toxoplasmosis, Infectious diseases of the fetus and newborn infant; In: J.S Remington and J. Klein, (5thEd). W.B. Saunders Philadelphia, Pa pp: 205.

Rifaat, M.A.; Morsy, T.A.; Sadek, M.E.; Azab, M.S.; Khaled, M.L.M. and Safar, E.H. (1979): Incidence of toxoplasmosis among farm and slaughtered animals in costal zone of Egypt. J. Egyptian Soci. Parasitol., 9: 193-197.

Rinaldi, L. and Scala, A. (2008): Toxoplasmosis in livestock in Italy: an epidemiological update. Parassitologia 50(1-2): 59-61.

Sadrebazzaz, A.; Haddadzadeh, H. and Shayan, P. (2006): Seroprevalence of Neospora caninum and Toxoplasma gondii in camels (Camelus dromedarius) in Mashhad, Iran.Parasitol Res. 98: 600–601.

Sawadogo, P.; Hafid, J.; Bellete, B.; Sung, R.T.M.; Chakdi, M.; Flori, P.; Raberin, H.; Hamouni, I.B.; Chait, A. and Dalal, A. (2005): Seroprevalence of T. gondii in sheep from Marrakech, Morocco. Vet Parasitol, 130: 89–92.

Shaapan, R.M. and Fathia Khalil, A.M. (2005): Isolation of Toxoplasma gondii from camel meat in Egypt. J. Egyptian Vet. Med. Assoc., 65: 187-195.

Shaapan, R.M.; Hassanain, M.A. and FathiaKhalil, A.M. (2010): Modified agglutination test for serologic survey of Toxoplasma gondii infection in goat and water buffalo in Egypt. Res. J. Parasitol., Issn 1816-4943.

Sukthana, Y. (2006): Toxoplasmosis beyond animals to humans. Trends Parasitol., 3: 173-142.

Teshale, S.; Dumetre, A.; Darde, ML.; Merga, B. and Dorchies, P. (2007): Serological survey of caprine toxoplasmosis in Ethiopia: Prevalence and risk factors. Parasite-Journal de la Societe Française de Parasitologie, 14, 2: 155-159.

Waldeland, H. and Frenkel, J.K. (1986): Live and killed vaccines against toxoplasmosis in mice. J. Parasitol 69: 60–65.

Wallace, G.D. (1969): Serologic and epidemiologic observations on toxoplasmosis on three Pacific atolls. Am. J. Epidemiol., 90: 1011.