Introduction

Toxoplasmosis, due to an intracellular obligatory protozoan parasite, Toxoplasma gondii (T. gondii), is one of the most import- ant parasitic infections which is widespread throughout the world and overwhelms near- ly all warm-blooded animals as intermediate hosts and it is prevalent globally even in the arctic (Dubey, 2016). The Center for Disease Control and Prevention (CDC) has assigned toxoplasmosis as one of the most neglected parasitic infections (NPIs) with high preva- lence and chronic nature (Foroutan-Rad et al., 2016). On average, 25-30% of the world’s population has acquired the infection at least once; the highest seroprevalence of the dis- ease among human belongs to Latin Amer- ica, Central and Eastern Europe, and South- east Asia (75-85%), while the prevalence rate in the Middle East ranges 30-50% (Daryani et al., 2014). The infection dominance also differs in different geographical regions and among various populations of a country. Ac- cording to some meta-analysis studies, the mean prevalence of toxoplasmosis in the general population of Iran is  approximate- ly 40% (Daryani et al., 2014), whereas it is higher among Iranian immunocompromised patients with 50% (Ahmadpour et al., 2014). Furthermore, the prevalence rate of infection in cat, sheep, goat and cattle populations in Iran reaches 33.6%, 31%, 27% and 18%, re- spectively (Rahimi et al., 2015; Sarvi et al., 2015; Sharif et al., 2015). Among domestic animals, sheep and goats are easily infected with T.  gondii (Sharif et al., 2015). Owing  to their milk and meat products, sheep and goats are crucial for livestock industry, par- ticularly in developing countries (Cenci-Go- ga et al., 2011; Dubey, 2009). Toxoplasmo- sis was first described in sheep and goats by Hartley and Feldman (Garcia et al., 2012).

Oocyst-infected water and food supplies serve as the major sources of infection for sheep and goats. According to seroprevalence studies, antibodies against Toxoplasma are commonly detectable among sheep and goat populations worldwide (Sharif et al., 2015).

Toxoplasmosis  is   frequently   prevailed in lowlands with warm and humid weath-  er; these climatic conditions are necessary for oocyst sporulation (Dabritz and Con-  rad, 2010). The primary routes of human infection include: 1) ingesting raw or un- dercooked meat containing bradyzoites, 2) consuming oocyst-contaminated vegetables and water, 3) vertical transmission, 4) tissue grafts, and 5) blood transfusion (Khadem- vatan et al., 2017). The infection has two clinical phases: the parasite tachyzoites (rap- id, invading form) are predominant in acute phase, while bradyzoites (slow, non-invad- ing form) are developed in tissue cysts most- ly in muscle and brain during chronic phase (Dubey, 1998). Pregnant women are one of the susceptible individuals to toxoplasmosis, as the infection may render serious sequel in their offspring (Jones et al., 2001). The time of toxoplasmosis establishment in gestation course is central to the severity of conse- quences in the fetus. As the end of pregnan- cy approaches, more parasites are capable of trans-placental transmission, although harsh outcomes often emerge in first trimester of conception (Kravetz and Federman, 2005). Congenital infection in the fetus  may lead  to mental retardation, deafness, microceph- aly, hydrocephaly as well as retinochoroid- itis. Also, toxoplasmosis yields some serious consequences in sheep and goats such as mummification, abortion, stillbirth, fetal and neonatal death, inflicting considerable finan- cial burden annually (Foroutan-Rad et al.,

2016; Sharif et al., 2015).

Based on seroepidemiologic evidence, one-third of the world population are sero- positive, though asymptomatic in terms of toxoplasmosis (Torgerson and Mastroiaco- vo, 2013). Regarding the fact that Iranians are the major consumers of the milk and meat of sheep and goats, they may be at risk of toxoplasmosis infection. With respect to unknown aspects of toxoplasmosis epide- miology in Sistan region, we decided to in- vestigate the seroprevalence of this infection among pregnant women and the population of sheep and goat herds in Zabol city, Sistan & Baluchestan Province, Southeast Iran.

Materials and methods

Study area

This cross-sectional study was done in Zabol region, Sistan and Balushestan Prov- ince, from December 2016 to February 2017. Bordering Afghanistan, this city  possesses two major water reservoirs, i.e. Chahnimeh and Hamoon lakes. However, the drying up  of the lake Hamoon on the one hand, in addi- tion to the hot and dry climate in the last few decades on the other hand, have led to harsher environments in this territory (https://en.wiki- pedia.org/wiki/Zabol).

Sample collection

In order to assess the role of some possi- ble risk factors, socio-demographic informa- tion of each pregnant woman, enclosing age, month of gestation, history of abortion, con- tact with dog, cat and other domestic animals, consuming raw foods, level of education and place of residence were obtained,  according to a pre-designed questionnaire. Subsequent- ly, 90 blood samples were collected. Then, the serum of each sample was separated by means of centrifugation at 3000 rpm for 6 min and stored at -20 oC for further use. Additionally,

blood samples were collected from 184 sheep and goats by venipuncture. Briefly, after shav- ing and disinfecting the skin next to the jugu- lar vein, a volume of 10 ml blood was taken in specific tubes without anticoagulant agents. All taken blood samples were transferred to the Parasitology laboratory, College of Veter- inary Medicine, Zabol University, observing the cold chain. Similar to human samples, se- rum was separated and kept at -20 oC.

ELISA assay

A single test was carried out on human blood samples to evaluate the anti-Toxoplasma IgM and IgG antibodies using a commercial ELISA kit (Pishtaz Teb Zaman, Tehran, Iran).

The serological assay was performed based on the manufacturer’s protocols. Once com- pleted, the results were read at 540 nm by an ELISA reader device (Hiperion microplate reader, England). The experiment was ac- complished as single test. Also, three standard solutions with various concentrations were used in the experiment for kit validity.

On animal blood samples, a single test was used to evaluate the anti-Toxoplasma IgG anti- bodies in animal serum samples using a com- mercial ELISA test (ID screen® Toxoplasmo- sis Indirect Multi-species ELISA kit, IDVET, Montpellier, France). The P30 (SAG1) anti- gen of Toxoplasma gondii is used as coated antigen in this assay. The serological assay was performed based on the manufacturer’s protocols. Briefly, a volume of 100 µl of 1:10 diluted sera and control samples were embed- ded in P30-coated microtiter plates. Then, the plate was maintained in ambient temperature (21 ± 5 oC) for 45 min. After a phase of three times washing, 100 µl of prepared conjugate 1X was appended to each well and the plate was incubated at room temperature for 30 min. Subsequently, each well was washed three times with 300 µl of the wash solution. Next,

100 µl of the substrate solution was added to the wells. After incubation of the plate in dark room for 15 min, 100 µl of stop solution was incorporated into each well in order to stop the reaction. Finally, the optical density (OD) of each well was read by an ELISA reader de- vice at 450 nm. The interpretation of obtained results was accomplished using the following formula:

Positive samples were those with an S/P percentage equal to or greater than 50%.

Statistical analysis

Obtained data were analyzed by Chi-Squared and Fisher’s exact tests. Also, 95% confidence interval for serum prevalence was calculated by

binomial distribution. The level of statistically significant results was considered as P<0.05.

Results

Pregnant women

The seroprevalence of IgM and IgG an- ti-Toxoplasma antibodies in blood serum of pregnant women referred to the reference laboratory of Zabol was 0% (0/90) and 14% (13/90), respectively (Table 1). Moreover, correlation between dependent and indepen- dent variables is shown in Table 2. Toxo- plasmosis  seroprevalence  in  women   with a previous history of abortion was signifi- cantly higher than women without abortion (P<0.05). There was no statistically signifi- cant relationship between T. gondii infection and other risk factors.

Table 1. Anti-Toxoplasma IgG and IgM prevalence in pregnant women of Sistan region

Anti-Toxoplasma antibody           No. of examined samples              No. of infected animals           Prevalence of serum infection

(95% Confidence interval)

IgM

90

0                                       0 % (0% - 4%)

         
IgG                                                                                                    13                                     14% (8% - 23%)

Sheep and goat

In this study, 37/184 cases (20.1%, CI: 14.6% to 26.6%) were previously  exposed to the organism and possessed specific an- ti-Toxoplasma antibodies.  Additionally, Table 2 represents the correlation between independent variables and the presence of anti-T. gondii antibodies in serum of exam-

ined sheep and goats. Accordingly, the prev- alence of serum infection was significantly associated with animal species (P<0.05), history of abortion (P<0.05) and frequency of parturition (P<0.05). There was no statis- tically significant correlation between other risk factors and serum prevalence of toxo- plasmosis (Table 3).

Table 2. The seroprevalence status of toxoplasmosis in Sistan pregnant women, based on risk factors

Variable                        Level                       No. of exam- ined women

No. of infected women Prevalence of serum

infection (%)

P value

Age

>30                                38                                     3                                     8

0.131

Gestation Period

First trimester                         39                                     6                                    15

Second trimester                       51                                     7                                    14

0.824

Contact with domestic animals

Raw vegetable consumption

Yes                                19                                      6                                    32

No                                 80                                     10                                   13

Yes                                10                                      3                                    30

No                                 48                                      8                                    17

Yes                                42                                      5                                    12

0.155

0.521

Illiterate                              5                                      2                                    40

Level of education

Primary school                        14                                     2                                    14

Secondary school                      28                                     4                                    14

Academic                            43                                     5                                    12

0.215

*significantly different between groups

Table 3. The seroprevalence status of toxoplasmosis in small ruminants of Sistan, based on risk factors

*significantly different between groups

Discussion

Toxoplasmosis is a zoonotic protozoal dis- ease with probable congenital manifestations in the fetus of pregnant women and small ru- minants such as still-birth, sudden abortion and brain calcifications (Foroutan-Rad et al., 2016; Sharif et al., 2015); hence, it is rec- ommended to clarify the seroepidemiology status of this infection for better preventive measures. This serological research was conducted to elucidate the seroprevalence of anti-Toxoplasma gondii antibodies in serum samples of pregnant women and small rumi- nant populations of Sistan region, Southeast Iran. Based on the results of human samples, the prevalence rate of anti-parasite IgM and IgG antibodies was 0% and 14%, respective- ly. Positive IgG and negative IgM titers often represent a previous (more than a year ago) exposure to the parasite (Robert-Gangneux and Dardé, 2012). During the last decade, several studies have investigated the T. gon- dii seroprevalence in pregnant women glob- ally. Toxoplasmosis infection is more domi- nant in the Americas and Africa continents, where seroprevalence in pregnant women ranges from 6.1-77.5% and 25.3-75.2%, re- spectively, whereas serological rates in Asia and Europe are confined to 0.8-60.4% and 9.1-63.2%, respectively (Pappas et al, 2009). In Iran the overall prevalence rates have been as follows: Mazandaran 75% (Panah et al., 2013), East Azerbaijan 71.61% (Rajaii et al., 2015), Kurdistan 55.22% (Parvizpour et al.,

2010), Khuzestan 45.45% (Saki et al., 2017),

Golestan 41.8% (Sharbatkhori et al., 2014), Ghazvin 31% (Tabatabaie et al., 2015), Al- borz 30% (Akhlaghi et al., 2014), Khuzestan 29.35% (Yad et al., 2014), Hamedan 29.01% (Hamidi et al., 2015) and Sistan and Baluch- istan 10.3% (Mousavi et al., 2014). Accord-

ingly, a lower seroprevalence was achieved in our study, in comparison to other parts of the country and similar to a serological in- vestigation in Nikshahr (10.3%) (Mousavi et al., 2014). Climate is a substantial parameter for the development of parasite oocysts. In contrast with the southern and northern parts of Iran, where there is sufficient humidity for parasite sporulation and higher prevalence rates have been documented in pregnant women, there exists low prevalence in Sistan and Baluchistan province due to hot and dry climate, particularly in Sistan district (Dary- ani et al., 2014; Foroutan-Rad et al., 2016). Pertinent to our findings, only one out of six evaluated risk factors, i.e. history of abor- tion, was significantly correlated (P<0.05) to toxoplasmosis seroprevalence in pregnant women, which is consistent with the results of Sarkar et al (Sarkar et al., 2012). Besides, there was no statistically significant asso- ciation between Toxoplasma seropositivity and other risk factors such as age, concep- tion course, contact with domestic animals, level of education and consumption of raw vegetables.

Based on the results of the  present  study,  it seems that there is a direct relationship be- tween the history of abortion and seropos- itivity against T.gondii infection in pregnant women of Sistan region. Alteration in im- mune system responses and presence of some hormones such as 17β-estradiol and low lev- els of progesterone and estrogen are the likely reasons of abortion in acute toxoplasmic in- fection, particularly in first trimester of preg- nancy (Galván-Ramírez et al., 2014).

Regarding Toxoplasmosis seroprevalence among small ruminants, 34 sheep (24.6%) and 3 goats (6.5%) out of 184 (138 sheep and 46 goats) serum samples were posi-

tive for parasite-specific IgG. Up to now, there were multiple studies that reported the prevalence  of  anti-Toxoplasma  antibodies in sheep and goats. During a cross-section- al study in Spain, parasite-specific antibod- ies were found  in 49% of sheep and 25%  of goats (Garcia-Bocanegra et  al.,  2013).  In a research conducted in Greece, 48.6% and 30.7% of sheep and goats, respectively, were positive for toxoplasmosis (Tzanida- kis et al., 2012). In Kerman province it was determined that 1.7% of goats and 3.3% of sheep were positive for Toxoplasma-spe- cific antibodies, using fluorescent antibody test (Derakhshan and Mousavi, 2014). Ak- houndi and Youssefi, (2017) demonstrated that 28.2% of sheep in Golestan province have antibodies against T.gondii using IFA method. In a study performed in the center of Iran, Kashan, two separate tests were used to evaluate sheep and goats, resulting in 12.2% and 17.8% of sheep and 4.4% and 8.9% of goats testing positive by ELISA and PCR, respectively (Rasti et al., 2018). These stud- ies represent similar results with our study (P<0.05). Some authors also reported con- trary results; for instance, agglutination latex test was used to examine 200 serum samples, showing 25.4% and 11.2% of goat and sheep infection, respectively (Ramzan et al., 2009). Higher infection seroprevalence in  sheep and goats were also reported (Wang et al., 2011). Regarding open husbandry system in the area, small ruminant exposure to contam- inated pastures is inevitable. Meanwhile, se- roprevalence rate was higher among sheep, which may be due to higher susceptibility of these animals to toxoplasmosis (Sharif et al., 2015). On the other hand, lower goat infec- tion may be due to their eating habits, as they feed on upper plant parts, hence they are less exposed to lower, and probably more-con-

taminated plant parts (Lu, 1988).

Based on our findings, 36% of seropreva- lence was in animals with a previous history of abortion, which was statistically significant (P<0.05). In Italy, following 31 cases of toxo- plasmic abortion in a farm, anti-parasite IgG prevalence ranged from 31.5% in first sam- pling to 62.6% in fourth sampling (Zedda et al., 2010). In a study on 48000 sheep in Bra- zil, the prevalence of anti-Toxoplasmaanti- bodies in animals with abortion history was reported to be 67.8% (Cosendey-KezenLeite et al., 2014). In Pakistan, the seroprevalence rate of infection in sheep populations with abortion history was 51.66% (Ramzan et al., 2009). Also, in separate studies the seroprev- alence rate among sheep herds with previous abortion was 17.35% and 97.4% in Iraq, and 32.3% in Jordan (Abu-Dalbou et al., 2011; Issa and Omer, 2011; Khadi et al., 2009). Ac- cording to studies, the seroprevalence rate is lower in current research, contrary to neigh- boring countries. This may be dependent on the climate condition in Sistan region, which probably leads to decreased sporulation  rate of Toxoplasma oocysts in the environment. Then, lesser animal infection would occur. Furthermore, the mechanism of toxoplasmic abortion in small ruminants is not completely understood as yet, but it was substantiated that the blood levels of progesterone in sheep de- clines following Toxoplasma infection. This hormone is vital for maintaining pregnancy and its reduction exposes the risk of abortion (Galván-Ramírez et al., 2014).

Moreover, in our study we found that fre- quency of parturition is relatively directed to the rate of seroprevalence infection of toxo- plasmosis in examined animals (P<0.05). This relationship could be justified by the fact that seroprevalence increases with age (Dubey, 2016), although the correlation be-