Spatial analysis of toxoplasmosis through EcoHealth approaches using GRA-1 recombinant: case in Sleman, Yogyakarta

Toxoplasmosis is an obligate intracellular zoonotic parasite caused by Toxoplasma gondii that can infect all warm‐blooded animals including humans. Prevalence of toxoplasmosis varies depending on climate, geography, and the presence of cats in an area. This study aimed to identify the prevalence and distribution of toxoplasmosis in Sleman, Yogyakarta through EcoHealth approaches. A total of 385 blood samples were collected from residents in the district of Sleman. Seven people from 55 villages were selected for blood sampling using a cluster method. The collected serums were tested by ELISA using recombinant Granule 1 protein (GRA‐1) as coated antigen. Data on altitude and coordinates of sampling sites were collected using GPS instruments, soil surface temperature in Sleman was obtained by satellite imagery, and cat population in residential areas was determined by questionnaire. The prevalence of toxoplasmosis in Sleman was 58%, of which distributed around rivers and in cattle pens. Based on altitude and temperature, toxoplasmsosis cases were found the highest at 0–150 m (66.3%) and at temperatures of 26–30 °C (66.4%). Areas with large numbers of cats had toxoplasmosis prevalence of 75.8% while areas with moderate and few cats were 56.5% and 49.0%, respectively. Thus, differences in the prevalence of toxoplasmosis at settlement were found based on altitude, soil surface temperature and cat


Introduction
Toxoplasmosis is an important parasitic zoonosis caused by Toxoplasma gondii which is distributed worldwide, ex cept in polar regions and desert areas. Zoonoses is a disease transmitted between human and animals. Warm blooded animals, including humans and other mammals are the intermediate hosts, while cats and other of Feli dae are definitive hosts (Tenter et al. 2000; Lopes et al. 2011. The prevalence of cats exposed to toxoplasmo sis in Indonesia was 3573%, while in Yogyakarta is 40% (Agustin and Mukono 2016). The serological studies in farm animals around the world showed that toxoplasmo sis is commonly found in cattle (14%), goats (27%), pigs (25%), sheep (66%), and horses (14%). The prevalence of toxoplasmosis in goats and cattle in Yogyakarta was 78% and 21%, respectively (Sujono 2010; Retmanasari et al. 2017. The worldwide prevalence of toxoplasmo sis varies, depending on climate, geography, and the pres ence of cats in an area. The seropositive of T. gondii us ing ELISA test in adults human worldwide is 1359%, in the USA is 1059%, while Europe and Brazil reported ap proximately 5080% were infected (Jones et al. 2001). At several places in Indonesia, the prevalence of toxoplasmo sis varies between 4388% (Subekti and Arrasyid 2006). Toxoplasmosis in humans and animals often shows sub clinical symptomatic or asymptomatics, so that they can be unwittingly infected by toxoplasmosis. Humans can be infected by T. gondii through food, undercooked meat, and vegetables contaminated by oocysts or congenital trans mission from pregnant mother to fetus in the womb. The congenital transmission does occur and may lead to se vere problems in unborn children and babies, including abortion, hydrocephalus, and retinochoroiditis (Chacin Bonilla et al. 2001; Sukthana 2006; Sadiqui et al. 2019). many centuries. This close relationship between human and animal can induce antropozoonosis diseases such as toxoplasmosis. The occurrence of zoonotic diseases can be affected by changes in weather, climate, and envi ronmental conditions. The environment includes every thing outside the relevance of organisms such as sunlight, temperature, humidity, topology, vegetation, rainfall and competitors. Therefore, to handle the zoonotic disease, it should be controlled in human, animal, and environ mental health aspects. Culture, social, economic and eco logical situations has an interrelated with health and dis eases. Lifestyle, sociodemographic and environment are risk factors for toxoplasmosis. EcoHealth is one of several integrative approaches that consider interactions between human health, animal health and the environment. Inter disciplinary collaborative research is essential to reduce zoonosis such as toxoplasmosis (Retmanasari et al. 2017). Ecosystem approach to human health is the study of envi ronmental change in biological, physical, social and eco nomic factors, and linking these changes to their impact on human health. Ecosystem is the habitat of a particular species when it possesses the ability to support the life of the species. The relationship of T. gondii with humans, animals and the environment has an interdependent rela tionship to maintain their life sustainability. Toxoplasma gondii requires catlike animals to keep its viability (Rap port 2007; Meerburg andKijlstra 2009). As the defini tive host, cats play an important role in transmitting tox oplasmosis to cattle, wildlife and humans. Toxoplasmo sis occurs when the oocysts are released along with cats stool, which can contaminate the surrounding environ ment. These oocysts will survive in the environment for a long time, depend on soil conditions (moisture, sunlight, temperature and humidity) and stability of the surrounding ecology, including the presence of definitive hosts and in termediate hosts of T. gondii (Jones et al. 2001; Chahaya 2003.
The study about the prevalence of toxoplasmosis com paring various risk factors has been conducted in several countries and showed highly variable results. The most dominant risk factor for toxoplasmosis infection can not be determined exactly to date. Nevertheless, there is no study about the relationship toxoplasmosis incidence with ge ographical dissemination of toxoplasmosis, environmen tal changes, especially in Sleman regions, Yogyakarta, In donesia. The changes in environmental conditions includ ing temperature, topography region, the presence of farm animals and distribution of the population can negatively affect the prevalence of toxoplasmosis. Thus it is neces sary to study the prevalence of toxoplasmosis in Sleman through EcoHealth approach. Analysis of the prevalence of toxoplasmosis in Sleman through EcoHealth approach can be performed using remote sensing technologies and mapping by Geographic Information Systems (GIS) for the detection of vulnerable populations and environmen tal diseases. Utilization of GIS to provide spatial data that describes the distribution or pattern of disease spread, to understand prevalence and populations at risk for a dis ease, that was observed based on the concept of EcoHealth (Chivian 2001; Sujono 2010; Dong et al. 2018.
Nowadays, the diagnosis of toxoplasmosis carried out using serological and molecular tests. Several serologi cal tests have been developed to detect specific antibodies against T. gondii in humans and animals. Immunodetermi nant antigens of the toxoplasma protein can stimulate the host immune response. ELISA is one of the tests used to detect existing of toxoplasmosis in humans and animals. The basic principle of this method is to detect the pres ence of immunoglobulin in the serum with T. gondii spe cific antigens. Granule 1 (GRA1) protein is an immuno genic granule protein of T.gondii and is most expressed compared to other GRA proteins. GRA1 protein can be found on the parasitophorous vacuole membrane, the vac uole matrix and tubular structures in the T. gondii par asitophorous vacuole (Subekti 2014; Muflikhah andAr tama 2017). GRA1 protein from local isolates showed equivalent virulence with T. gondii RH strain in experi mental mice (Subekti et al. 2008). These proteins can be used as a vaccine candidate and diagnostic tool because GRA1 has the capability to induce humoral and cellular immune response in both human and mice (Wihadmady atami et al. 2011). One way to get pure GRA1 protein is recombinant protein through the cloning approach and expression of GRA1 proteincoding gene (Subekti 2014; Muflikhah and. The aims of this study are to measure the prevalence and distribution of toxoplasmo sis in the Sleman region through EcoHealth approaches and the effect of altitude, soil surface temperature and the presence of cats in the surrounding human residential ar eas against the prevalence of toxoplasmosis using GRA1 recombinant proteins.

Design and sampling
This study was an analytical survey with a crosssectional design. Blood samples were obtained from 385 respon dents in Sleman district, Yogyakarta and then serum was kept at 20°C. The choice of respondents performed with two cluster sample design stage, probability proportion ate to size (PPS) and simple random sampling. Research clusters were grouped into 55 sections, and 7 people at each cluster were selected for blood sampling. The sam ples were examined by ELISA method using GRA1 pro tein tachyzoite locally isolated in the Laboratory of Bio chemistry, Center for Biotechnology, Universitas Gadjah Mada. Optical density was measured by a microplate reader (Zenix, ZN320, China) at 405 nm of wavelength. Ethical clearance was obtained from the Ethical Clearance Commission of the Faculty of Medicine, Universitas Gad jah Mada (approval number: KE/FK/318/EC). The sample size was calculated by the following formula: Whereas n is the sample size of the research, p is the ex pected proportion in populationbased on previous studies, z1p is standard normal variation [α= 5% (1,96)], and d is the absolute error (0.05).

Expression and purification of GRA-1 proteins
Recombinant plasmid (pET32+) that contain gene encod ing for GRA1 protein were transformed to E. coli BL21 (DE3). Two milliliters of starter colony containing recom binant plasmids (pET32a (+)/R1) were cultured in LB liq uid medium (25 mL) supplemented with 25 mg/mL ampi cillin at 37°C overnight. Furthermore, 10 mL bacterial suspension inoculated to 100 mL LB medium at 37°C to reached optical density 0.600 (OD=0.600), and added 1 mM isopropyl1thioβDgalactopyranoside (IPTG) to culture and incubate for 4 h. A total of 20 mL of bacterial suspension was removed to a new tube and centrifuged at 4°C 3,000 rpm for 20 min. The supernatant was removed, and the pellets were washed by phosphate buffer saline (PBS). Centrifugation at 4°C 3,000 rpm for 10 min and washing pellets using PBS three times were carried out. The pellets were dissolved with 500 μL PBS solution and destroyed by sonication. Finally, the supernatants were collected by centrifugation at 4°C, 10,000 rpm for 5 min.
The GRA1 recombinant proteins were purified using nickel column chromatography and the purified lysate was loaded into a 10 mL NiNTA column (NiNTA Purifica tion System, NOVEX by Lab Technologies Cat No. K950 01, USA). Initially, the column was washed with wash buffer (250 mMNaH 2 PO 4 , 2.5 M NaCl and 100 µL 3 M imidazole). The proteins were eluted with elution buffer (250 mMNaH 2 PO 4 , 2.5 M NaCl, and 1.25 mL 3 M im idazole). The purified recombinant proteins and super natant of cell pellets were analyzed using 15% sodium do decyl sulfatepolyacrylamide gels (SDSPAGE). The pro tein samples were taken and then added with loading dye with a ratio of 1:4 (10 μL loading dye:40 μL sample), then heated at 80°C for 5 min. Electrophoresis was performed at 120 V for 2 h. The proteins were visualized by stain ing with Coomassie blue for 1 h using a shaker. The gel was washed with aquabidest, then washed with a staining washing solution (50% methanol, 10% acetic acid glacial, and 40% aquabidest) for 30 min until the band was clearly visible.

ELISA assay
Initially, ELISA plates (Nickel Chelated Plates, Nunc. USA) were washed three times with 300 mL PBST [PBS (pH 7.3) containing 0.05% (v/v) Tween 20], and the puri fied recombinant proteins (GRA1) were coated overnight at 4°C in 100 mL PBS (concentration of each recombinant proteins was 5 µg/mL). Plates were washed and blocked (5% serum bovine albumin in 0.05% PBST) at 37°C for 1 h. After washing the plate three times with washing solu tion, 50 µL serum diluted one in blocking solution (1:100) was added to duplicate wells for each sample and kept at 37°C for 1 h. The plates were then washed five times and in cubated with 50 µL of horseradish peroxidaseconjugated goat antihuman IgG (Sigma, St.Louis, USA) diluted in blocking solution (1:4,000) per well at 37°C for 1 h. Af ter washing five times, the 96 well plates were incubated with 100 µL pNitroPhenyl Phosphate (pNPP) substrate at 37°C for 15 s. The absorbance was detected at 405 nm using a microplate reader.

Data analysis
The ELISA data were collected and calculated based on the mean optical densities at a value of 405. Data on al titude and coordinates of sampling sites were taken us ing a GPS instrument (GPS Garmin ETREX 10, Russian). Land surface temperature in Sleman was obtained by satel lite imagery (CITRA ASTER), while cats population data in residential areas were taken through charging question naire by respondents. Data analysis was performed by Chi Square and Logistic Binary Regression with 95% level of confidence and the geographic distribution of toxoplas mosis is made spatially and analyzed by plotting ordinate points of toxoplasmosis, buffering and overlay of a map.

Result
The expression of GRA1 T. gondii locally isolates in E. coli BL21 (DE3) using pET32a (+)/R1 plasmid is shown in Figure 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) of the unpurified GRA1 re combinant proteins in Figure 1 showed that the molecular weights were 24 kDa, accordance with Western blotting assay. The pure of GRA1 proteins used for ELISA test to evaluating the prevalence and distribution of toxoplas   mosis in the Sleman district. The prevalence of toxoplas mosis in Sleman after the volcano eruption of Merapi was 58% (Table 1). The seroprevalence difference of toxoplas mosis in each subdistrict in Sleman was related to the be havior, environmental conditions and demographics of the community in each district. The risk factors that greatly influence the prevalence of toxoplasmosis incidences in an area were behavioral, geographic environment and the presence of cats. The toxoplasmosis distribution in sub district of Sleman, Yogyakarta are presented in Table 1, and the case of toxoplasmosis based on behavioral, geo graphic environment and the presence of cats are presented in Table 2 (bivariate analysis). The significant result from bivariate analysis contin ued to multivariate analysis and the result of odds ratio are shown in (Table 3). Surface temperature and population of cats had Odds ratio more than one. Among that, the pop ulation of cats was the highest risk factor. Results of the Multivariate test by Binary Logistic Regression showed that the Odds Ratio of the cat population in the settlements has the most effective when compared to other environ mental factors (1.729). These different results are associ ated with population density.
The distance between river shed and cattle pens is one of risk factors that supports toxoplasmosis accurance in Sleman District (Table 4). The results of disease map ping showed that the most incidence of toxoplasmosis are around the river (Figure 2). Inhabitant settlement close to the cage farms were positive for toxoplasmosis ( Figure 3). The purple color is the nearest settlement from the farm and the green color is furthest from farms. More than 20% of respondents live around the farms.
Toxoplasmosis was higher in lowland (0500 m) than highland (>500 m) (Figure 4). The dark brown location (sepia and brown) showed highland whle the light brown (russet) shows lowland. Another risk factor observed in this study was temperature. The variation temperature in this study is shown in Figure 5. The results of satellite imagery, and ground surface temperature in Sleman, Yo gyakarta are ranged between 23°C-36°C. The results of IgM and IgG anti toxoplasma tests are based on differences of temperature, which showed significant results among several soil surface temperatures in the value was 0.023 (P value <0.05). The orange color is the highest cases of toxoplasmosis in the Sleman District (26°C-30°C). Pop ulation cats in Sleman District are one of the risk factors in this study (Figure 6). The brown color showed that many cats could be found in that location.

Discussion
Granule protein is a major component of excretory and secretory antigen (ESA) secreted by organelles T. gondii, and these proteins play an essential role during the inva sion of the parasites into the host cells (Subekti 2014). The GRA1 proteininduced humoral and cellular immune responses of the host. The availability of GRA1 pro tein for the purposed diagnosis is very limited. There fore, overexpression of GRA1 by subclone of the cor responding gene in the pET 32a will be very beneficial for diagnostic development. The results showed that re combinant GRA1 protein could be isolated and charac terized by electrophoresis 24 kDa and detected by its spe cific antibody anti T. gondii through immunoblotting. In this study, recombinant proteins of GRA1 T. gondii from local isolate were used to determine the distribution and the prevalence of toxoplasmosis in Sleman, Yogyakarta  The study conducted by Utami (2009) showed that the serology assay used GRA1 ELISA was sensitive and spe cific. Characteristic performance of IgM GRA1 ELISA showed a sensitivity of 94.12% and specificity of 94.12%, while the test of IgG GRA1 ELISA showed a sensitiv ity of 94.12% and specificity of 100%. This result sim ilar to previously reported research showed the sensitiv ity, the specificity, and the positive predictive values of GRA1 as an antigen are 100%, 86.36%, and 94.11%, re spectively (Muflikhah and Artama 2017). Based on these data, the recombinant protein of GRA1 is known to have high immunogenic activity in patients with toxoplasmosis compared with other excretory proteins of T. gondii. Gen erally, antigens commonly used for ELISA assay derived from surface and membrane proteins of T. gondii. Mem brane antigens can be recognized by the immune system after T. gondii is destroyed by the host immune system dur ing an ongoing infection, while the GRA1 proteins are re leased and early recognized by host immune system when the parasites infecting host cells. (Utami 2009; Muflikhah and. Toxoplasma gondii can infect the in termediate host in a very wide range of animals. Toxoplas mosis in cats, livestock and wild animals can be a source of transmission to humans. Multi Center Study in Europe re ported that eating cystcontained meat are estimated to be infected by T. gondii in approximately 3063% of cases, while contact with soil 6-17% and 14-49% of unknown risk factor (Fromont et al. 2009). The differences preva lence of toxoplasmosis depends on conditions in regions, climates, farming methods, customs, meat consumption habits, and contact with cats (Elmore et al. 2010; Subedi et al. 2018. The eruption of Merapi has changed many environ mental conditions, that affect the ecology of existing species, including the vegetation inside. Vegetation is sig nificant for breeding Annelida and arthropods such as flies and cockroaches, which acts as a vector to move oocysts in the environment to livestock or humans (Meerburg and Kijlstra 2009). The eruption also affected environmental damage and the animal population especially, those are the definitive and intermediate host of toxoplasmosis. Meat from animals such as goats, sheep, pigs, cattle, poultry and other livestock plays an essential role as a source transmis sion of T. gondii to humans (Iskandar 2008). Data from the local authority of farm in Sleman district showed about 1,900 castles die due to eruption; goat 2,400; and many other animals such as rabbits, chickens, and poultry. The intermediate hosts such as animal farms and the environ ment, are factors that can break the chain of disease caused by T. gondii. The population of cats around the area of eruption did affect to the number of oocysts that contami nated the environment (Dubey 1998; Fromont et al. 2009).
Cats have a significant role in the epidemiology of tox oplasmosis through shedding oocysts in feces during their prepatent period. Cats can be an intermediate host for the parasite with the occurrence of clinical signs; however, in fection of T. gondii in cats is usually asymptomatic. Cats can become infected by eating the carcasses of small mam mals (rats, mice, and birds) or ingesting oocysts from the environment (Sroka et al. 2018). Rats are considered the most important as intermediate hosts of T. gondii. Do mestic cats are obligate carnivores and annually consume birds and small mammal as prey. Feral cats are poten tial reservoirs for the parasite of T. gondii because they have easy access to food resources, such as farms garbage bins (Salman et al. 2018). The study has carried out by Salman et al. (2018) showed that the prevalence of toxo plasmosis in cats aged 1-5 years, 6-10 years, and older than 11 years were 26.3%, 17.6%, and 8.2%, respectively. Cats that lived outdoors or had access to the outside have a higher risk of infection than indoor cats. The contam ination of the environment by cat feces is a serious risk factor of spreading toxoplasmosis to human beings or an imals. The existence of many wild cats also increases the possibility of the environmental contamination by the cat's feces. A study conducted in Egypt described that 97.4% wild cats T. gondii oocysts positive in the feces contam inated their surroundings (AlKappany et al. 2010). The large population of the wild cats would increase the con tamination of the environment caused by cat's feces, which is correlated with widespread toxoplasmosis infection to human beings or other animals (Subrata et al. 2015).
The toxoplasmosis disease transmission depends on the hydrologic characteristics of the river because the river characteristics related to water conditions in the cross section of the river. Based on indications of proximity to cross the river, toxoplasmosis is found around the river. Oocyst contaminates the environment through the surface of the water as a river following the flow of rainwater. Res idents who live around the river were possibly infected by oocyst caused by using river water as daily necessary, wa tering the yard or other needs. Therefore, existing oocysts in these watersheds will potentially transmit toxoplasmo sis to humans or surrounding pets. Oocystcontaminated water is a carrier that can spread toxoplasmosis in humans and animals. Oocysts are very resistant in an aqueous envi ronment and are resistant to many disinfectants, including chemicals like chlorine (Aubert and Villena 2009).
Toxoplasmosis transmission in the residents who live around the cattle shed is due to contamination of food or drinking water by the oocysts that are derived from cat tle pens carried by flies or cockroaches. Cats seek food around the cage that can defecate in the corral. Cattle sheds are a suitable place for the sporulation of oocysts due to the conditions and moisture, which are optimum for the oocyst sporulation process. Oocysts sheds in the environment can contaminate water wells, which are located around the FIGURE 6 Distribution of toxoplasmosis based on cat populations in settlement in Sleman district. The areas with rare cat populations (green), a small cat populations (yellow), and large cat populations (brown) showed prevalence of toxoplasmosis were 49%, 56.5%, and 75.8% respectively.
neighborhood. Oocysts that contaminate the environment can be distributed through the air, surface water, rainwa ter, and crops, as it also can spread through worms soil and compost (De Camps et al. 2008; Tenter 2009). The flies and mice have a very important role against T. gondii oocysts transferring to farm animals or pets. The research about seroepidemiology of T. gondii from China has found increasing seroprevalence of T. gondii in food animals those life at river run area (Dong et al. 2018). Results sug gested that T. gondii oocysts may be transmitted through water, and annual precipitation possibly help the oocyst spread and retain accessible for the potential host.
Weather and geographic location significantly affect to the spread of T. gondii in the environment. T. gondii does not undergo sporulation, survive and become infec tious without the influence of climatic conditions (Subedi et al. 2018). Sleman district is a wet tropical climate with an average temperature of 26.1°C to 27.1°C and the hu midity is 70% to 95% (Meteorological, Climatological and Geophysics Agency, Yogyakarta). Temperature and hu midity in Sleman are ideal conditions for the sporulation of T. gondii oocysts. The variation of temperature in each region in Sleman is due to differences in land usage. Land usage for building leads to temperature increasing in the region compared to areas with lots of green vegetation or crop rice fields so that the temperature differences can af fect the process of sporulation oocysts to infective form. There is a difference in the temperature range at present with the temperature in the previous year. Ninety five per cent of the area in Sleman shows the temperature condi tions are suitable for the process of sporulation oocysts to become infective form. The sporulation process of Tox oplasma gondii oocyst occurs at 20°C-28°C (Sasmita 2006). This range is in accordance with the sporulation process proposed by Dubey who conducted the study in moist soil with temperatures ranging between 23°C-29°C (Dubey 1998). The sporulation process usually ranges from 1 to 5 days, depending on the humidity and temperature of the surrounding environment (Tenter et al. 2000). At a tem perature of 35°C the sporulation process takes about 32 days (Meerburg and Kijlstra 2009). Increased sporulation oocysts in the environment will result in an increased hu man risk factor toxoplasmosis. Surface temperature in Sle man after the eruption of Merapi and the effect of global warming was ranging between 23°C-36°C. Temperatures above 30°C cause the sporulation process of oocyst to be in the infective form becomes longer. As a result there will be an effect on the incidence of toxoplasmosis. Sporu lated oocysts form are very resistant in environment and infective for a year or more at a temperature of 24°C, but if it's at a temperature of 30°C it is only lasting for 20 days and at 50°C or more then will quickly die (Iskan dar 2008; Meerburg andKijlstra 2009). The prevalence of toxoplasmosis in Sleman district was 54.4% (20-25°C), 66.4% (2630°C) and 48% (>30°C) respectively. These results indicate that an environment with a temperature above 30°C led to the oocyst sporulation process being longer, so that influences against the prevalence of tox oplasmosis in humans. The prevalence of toxoplasmosis in the district of Depok, Sleman, Yogyakarta (temperature >30°C) was 85%, the factors that cause high incidence of toxoplasmosis in this area because of the habit of eating undercooked meat or consuming fresh vegetables which are not washed adequately. According to the European Multi Center Case Control Study, a comparison between the transmission through the existing cysts in meat and oocysts in cat feces to humans result in 510 times higher risk in people who consume meat (Dubey 1998).
Based on altitude, the prevalence of toxoplasmosis in Sleman, Yogyakarta can be influenced by several factors including the presence of oocysts in the environment and public behavior. The number of oocysts at an altitude of more than 500 m, less than the height 0-150 m due to num ber of oocysts in each height is influenced by the environ mental damage caused by the eruption of Merapi. Popu lation of cats is as a source of oocyst contamination into the surrounding environment at an altitude of 500 m has decrease, so that oocyst contaminate the environment is reduced. Besides that, the oocysts in high areas can be carried by rain water through the river to areas of lower altitude, so the number of oocysts in these areas will in crease. The areas that showed a high prevalence toxoplas mosis (71-85%) as Berbah, Gamping, Depok, Godean, and Moyudan, Sleman have a slope area of 0-2%, while the regions that have a slope area of 8-15% such as Tem pel, Sleman, Turi, Ngemplak, Pakem, and Cangkringan showed a lower prevalence of toxoplasmosis that is 32-57%.
Research conducted by ChacinBonilla et al. (2001) reported that residents living in the mountains with an al titude of 800 m from the sea surface with a temperature of 24°C showed a lower prevalence of toxoplasmosis than population living in low lying areas which have a temper ature of 30°C and altitude below 100 m from sea level. At an altitude of 500 m, the area showed a high prevalence of toxoplasmosis is Cangkringan (60%), where the area is most severely affected by the eruption of Merapi. The high prevalence of toxoplasmosis in this area is probably due to poor hygiene in the consumption of food or drink. Com munity in this area is living in shelters. Sources of toxo plasmosis transmission come from vegetables or drinking water sources, where the vegetables are not washed clean because of limited water. Decline in public health condi tions also resulted in the emergence of reinfection cases of toxoplasmosis. The presence of cats is found in areas close to urban areas. Urban areas are usually very densely popu lated and there are many sources of food for cats (Meireles et al. 2004; Salman et al. 2018. The area in low altitude, and having a subtropical cli mate (hot and humid), had a higher prevalence of toxo plasmosis. The probable reasons for high seroprevalence are semiintensive sheep farming, humid conditions in the study area at low altitude, the presence of feral cats, and open feed storage with access to cats. Similarly, higher prevalence of T. gondii in Pokhara district, Nepal, can be attributed to the high rainfall and semi intensive sheep farming (Subedi et al. 2018). The study by (Caballero Ortega et al. 2008) who revealed that the highest preva lence of T. gondii is presented at low altitude. A 10 years study in France revealed the relationship of T. gondii prevalence with temperature and rain (CaballeroOrtega et al. 2008). The risk of acquiring infection was enhanced when the weather was moist and warm. Oocyst survival increases in moist conditions during longer periods of hot weather (Meerburg and Kijlstra 2009). The study carry ing out by Subedi et al. (2018) reveals that prevalence of toxoplasmosis in sheep at low altitude and has subtropi cal climate (hot and humid) is higher than areas with high altitude and the arid environment.
The amount of domestic waste by poor management in dense populated areas is one of the causes of an increase cat and mouse populations. Seroprevalence of toxoplas mosis infected cats is estimated from 0.23 to 0.39 per cat per year. The average soil contaminated with oocysts from infected cats are estimated about 94 to 4671/m 2 each year, it depends on the cat population which is infected by T. gondii and the oocysts which are excreted (Afonso et al. 2010). The cat population will affect the high oocyst that will pollute the environment if the cat is infected by T. gondii. Latent toxoplasmosis is common among domestic cats, corresponding to a high prevlence in adult cats. En vironments that are contaminated by oocysts of T. gondii could potentially spread the infection to an uninfected cats, livestock and humans (Meireles et al. 2004; Salman et al. 2018).

Conclusions
The prevalence of toxoplasmosis in Sleman, Yogyakarta after eruption of Merapi was 58% and the distance of rivers and cattle pens had affected against toxoplasmosis. There are differences seroprevalence of toxoplasmosis based on altitude, soil surface temperature and presence of cats in human settlements checked by ELISA using recombinant proteins GRA1 T.gondii local isolates.