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Epidemiological Study on Toxoplasmosis of Human and Animals at Dhamar Governorate, Yemen

ISSN: 2319-7706 Volume 7 Number 12 (2018) Journal homepage: http://www.ijcmas.com This cross sectional study was carried out in Dhamar governorate from December 2015 to November, 2016 with main objective to determine the seroprevalence of toxoplasmosis in humans and animals. A total of 323 individuals and 323 animals (241 sheep, 69 cattle and 13 goats) from different districts of Dhamar governorate were selected and tested using ELISA and PCR techniques for detection antibodies against Toxoplasma gondii in human and animals. The results revealed that, the overall seroprevalence of toxoplasmosis in human was 20.43%. The seroprevalence rates of IgG, IgM and both IgG +IgM were 13.62%, 4.33% and 2.48% respectively. The higher rate of infection was recorded (29.50 %) in age group of 1120years old; whereas, the lower rate (11.11%) was in age group of less than 10 years old. Females were more susceptible to infection (23.19 %) compared to males (2.02 %). The higher seroprevalence rate was recorded in summer (28.41%); whereas, the lower rate in spring (13.15%). The higher prevalence of rate was recorded in Utoma district (32.00%); whereas, lower rate in Alhada district (3.85%). Statistically, significant differences (P<0.05) were observed between seroprevalence rate and age group, sex; while none with season and area factors. In animals, the results of ELIZA revealed that, the overall seroprevalence of toxoplasmosis was 23.84 percent, the seroprevalence of IgG, IgM and both IgG +IgM were 15.5%, 1.5% and 6.8% respectively. Out of 241 sheep, 69 cattle and 13 goats tested, 21.16%, 31.88%, 30.77% respectively were seropositive for toxoplasmosis. The infection rate of toxoplasmosis was high in age group of 5 years old and above (33.33%); whereas, low (21.55%) in age group of two years. The seroprevalence rate in males was (28.28%) higher compared to females (21.88 %). The higher seroprevalence rate was recorded in spring season (33.33%); whereas, the lower rate in winter (18.02.67%). The higher of seroprevalence was recorded in Utoma (35.71%); whereas, the lower rate in Mifa`a ans (10.715%) and none in Jabal asharq. Statistically, significant differences (P<0.05) were observed between seroprevalence rate and animal species, season, area factors; while, none with other factors studied. In the present study, all ELISA seropositive samples of human and animals were retested by PCR as confirmatory tool. Results of PCR revealed that, out of 66 and 77 samples of human and animals tested 30 (45.45%) and 18 (23.38%) were found positive respectively for toxoplasmosis. The results of the present study revealed that toxoplasmosis is prevalent in human and animals in study area. Combinations of serological and molecular techniques are recommended for accurate diagnosis of Toxoplasmosis. K e y w o r d s Animals, ELIZA, Human, PCR, Prevalence, Toxoplasmosis, Dhamar, Yemen Accepted: 12 November 2018 Available Online: 10 December 2018 Article Info Int.J.Curr.Microbiol.App.Sci (2018) 7(12): 1480-1495


Introduction
Toxoplasmosis is one of the most worldwide parasitic zoonoses caused by the protozoan Toxoplasma gondii (Muqbil and Alqubatii, 2014;Zhou et al., 2018). It is a major public health concern for human and domestic animals (Montoya et al., 2009;Gebremedhin et al., 2013;Luo et al., 2017;Tonouhewa et al., 2017). Domestic cats and wild felids play a crucial role in the epidemiology of toxoplasmosis as the definitive hosts, through the shedding of millions of oocysts when infected (Dubey, 2010;Gebremedhin et al., 2013). Human beings and other warm-blooded animals become infected primarily by ingesting food or water contaminated with sporulated oocysts or by ingesting meat that contain tissue cysts of T. gondii (Hamzavi et al., 2007;Dubey and Jones, 2008;Gasior et al., 2013;Smit, et al., 2017;Sroka et al., 2018).
T. gondii has a worldwide distribution in human populations infecting up to one third of the global population and a wide range of other mammalian and avian species (Sukthana, 2006;Gharavi et al., 2018). Toxoplasmosis is a major public health problem, with a high socioeconomic impact in terms of human suffering including the cost of caring for sick, mentally retarded and blind children (Roberts et al., 1994;Mahdi et al., 2008;Zhou et al., 2018). The parasite is an extremely successful pathogen, responsible for significant morbidity and mortality, especially in congenitally infected and immunocompromised individuals (Tenter et al., 2000;Luft and Remington, 1992;Elsheikha, 2008;Mahdi et al., 2008). In animals, toxoplasmosis leads to great economic losses in food animals by causing embryonic death, fetal death, abortion and infertility (West, 2002;Tonouhewa et al., 2017). The risk of infection can be reduced by proper food preparation. Meats thoroughly cooked, avoid cross contamination of foods and utensils with oocysts of cats (Cook et al., 2000).
The diagnosis of Toxoplasma gondii infection is conventionally made by the direct demonstration or isolation of the parasite from biopsy or autopsy material (Jula et al., 2013), but such techniques are unsuitable for use in large-scale surveys. Therefore, recourse has been made to immunoserological tests for specific host antibody, and a variety of tests such as PCR has been used (El_Ghany et al., 2012;Jula et al., 2013;Tonouhewa et al., 2017).
An increasing prevalence of toxoplasmosis in humans and animals has been drawn the attention of the researchers and workers to address toxoplasmosis as serious public health problem and many studies have been conducted in different geographical regions of the world and Yemen (Nimri et al., 2004;Sharif et al., 2006;Hamzavi et al., 2007;Saleh et al., 2010;Shahiduzzaman et al., 2011;Subasinghe et al., 2011;Tasawar et al., 2011;Gebremedhin et al., 2013;El-Madawy and Metawea, 2013;Kadle, 2014;Saif et al., 2014;Bakre, 2016;Tonouhewa et al., 2017;Luo et al., 2017;Gharavi et al., 2018). However, the information on toxoplasmosis of human and food animals are limited at Dhamar governorate, Yemen. Therefore, the present study was designed to investigate the toxoplasmosis prevalence and associated risk factors in human and animals at Dhamar Governorate, Yemen.

Study areas
The cross sectional study was conducted in different districts of Dhamar governorate, namely, Alhada, Jahran, Anis, Jabal Alshaq, Mifa'a ans, Manar, Wusab, Utoma and Dhamar city during the period from December, 2015 to Novmber, 2016. Geographically, Dhamar is located at 14°.58′N latitude, 44° 43′E longitude and at an altitude of 2425 mater above sea level. The average rainfall, temperature and relative humidity is 40.5mm, 17.3ᴼc and 57.8% respectively during the year of 2016.

Study population and size of sample
The study subjects were individuals attending to public and private hospitals for routine examination and farmers from different districts of Dhmar governorate. Animal population investigated were sheep, goats and cattle from various district areas and central slaughterhouses of Dhamar governorate. A total of 323 individuals and 323 animal heads were selected by systematic random sampling method. The sample size was calculated according to Thrusfield (2007) technique considering 30 % expected prevalence and 95% confidence interval with a 5% desired absolute precision using the following formula: N= (Z) 2 P(1-P)/d 2 , where, (p)expected prevalence and (Z) 95% confidence interval (Z= 1.96) and (d) a 5% desired absolute precision.

In humans
Blood samples were obtained from individuals/subjects referred to public and private hospitals for clinical and routine examinations. A 5 ml venous blood sample was collected from each screened subject. Serum was separated from half of each sample and kept at -20 °C until used, while the other half of the sample was placed in a sterilized EDTA tube and stored at-80 °C for amplification by PCR. Information related to subject i. e age, sex, area, season were recorded and maintained confidentiality. The blood samples were then brought to the Veterinary parasitology laboratory, Faculty of Agriculture and Veterinary Medicine, Thamar University, Dhamar for processing and testing.

In animals
Blood samples were collected from animals. Approximately a 5 ml of whole blood sample was collected from each animal by venipuncture from the jugular vein using disposable plain vacutainer tubes and needles. Animal species include sheep, cattle, and goats. The blood samples were kept in two tubes, one with anticoagulant for PCR analysis; while another with none for serological testing. The blood samples were allowed to clot and then centrifuged to separate the serum. The serum was collected into 1.5 ml Eppendorf tubes and brought to the Veterinary parasitology laboratory, Faculty of Agriculture and Veterinary Medicine, Thamar University, Dhamar for processing and testing. All samples were labeled properly with necessary information.

Testing of samples (Sera)
The entire sera of human and animals were screened for Toxoplasma gondii infection with serological technique (ELISA) and then, the sero-positive samples of serological technique were retested by molecular technique(PCR) as confirmatory tool.

Serological technique
In laboratory, All the collected sera samples of human and animals were tested for the presence of IgG, IgM and both antibodies against T. gondii by indirect enzyme linked immunosorbent assay (ELISA) following the protocol of the manufacturers.

Molecular technique
Extraction of DNA from whole blood DNA was extracted from whole blood for both human and animals using a commercial purification system (AccuPrep® Genomic DNA Extraction Kit, Bioneer, Korea) according to manufacturer instructions.

Nested PCR Assay (Simplex Polymerase Chain Reaction)
Conventional PCR was used on all DNA samples to amplify a fragment of DNA Toxoplasma gondii. The specific primers for used for amplification of the sequence of T. gondii DNA were: forward ITS1-F 5`ACA CGT CCT TAT TCT TTA TTA ACC A3` and revers primers ITS1-R 5`ATC CCA ACA GAG ACA CGA ATT 3` as described by (Rahumatullah et al., 2012).
These primer amplify the 234bp from ITS1 gene of Toxoplasma gondii were obtained from AccuPower ® HotStart PCR PreMix (bioneer Korea). The PCR reaction was contain 1µl of each primer, 1µl of DNA and 18µl ddH2O until the total volume of mixture becomes 20 µl.
Thermal PCR conditions consisted 94°C for five minutes, followed by 35 cycles at 94°C for 45 seconds, 57°C for 45 seconds and 72°C for 45 and a final extension at 72°C for 5 minutes. A 10 μL aliquot of the amplified product was analyzed on 1% agarose gel and stained with ethidium bromide. Every PCR run included positive and negative controls. After electrophoresis, generated bands were screened and digitally photographed under UV light.

Ethical consideration
Verbal consent from all subjects involved in this study was obtained after provided them detailed explanation on objective of the study. Subjects also were assured that the obtained information should be kept confidentially and used only for research purposes.

Statistical analysis
The data obtained from this study were entered in Microsoft excel spreadsheet (Microsoft Corporation). All statistical analyses were performed using the statistical package for the social sciences SPSS (version 17). The frequencies and percentages were presented and Chi square test was used to analyze the T. gondii seroprevalence in respect of species, age, season, gender, and area. The differences were considered to be statistically significant when the P value was less than 0.05.

Results of human study
Out of 323 sera samples of human examined, 66(20.43%) were found seropositive for toxoplasmosis according to ELIZA results. In addition, Out of 66 samples seropositive for toxoplasmosis, 44(13.62%), 14(4.33%) and 8(2.48%) were seropositive for IgG, IgM and both IgG and gM respectively (Table 1). There were significant differences (P<0.05) among seroprevalence rate of antitoxoplasma antibodies in human.
The effects of risk factors age, sex, season and area on distribution of toxoplasmosis in human were investigated and the results were presented in Table 2. The results revealed that, the higher seroprevalence rate (29.50%) was recorded in age group of 11-20 year; whereas, the lower rate (11.11%) in age group of 10≤ years old. The seroprevalence rate of toxoplasmosis in males was 2.02 %; whereas, the rate in female was 23.19%. The higher seroprevalence rate was recorded in summer season (28.41%); whereas, the lower rate in spring season (13.15%). The higher of seroprevalence of toxoplasmosis was recorded in Utoma district (32.00%); whereas, the lower rate in Mifa`a ans district (15.00%). Statistically, significant differences (P<0.05) were observed between seroprevalence rate and age group, sex; while none with season and area factors. The results of the correlation analysis revealed that, the association between seroprevalence and metrological such as temperature (r = -0.327, P< 0.01), rainfall(r = -0.357, P< 0.01) and humidity (r =-0.201, P< 0.01).

Results of animals study
A total of 323 animals were examined, 77(23.84%) animals were found infected with toxoplasmosis according to ELIZA technique results. In addition, Out of 77 seropositive samples of animals for toxoplasmosis, 50 (15.5%), 5(1.5%) and 22(6.8%) were seropositive for IgG, IgM and IgG +IgM respectively (Table 3). There are significant differences (P<0.05) among Seroprevalence of antitoxoplasma antibodies in animals.
The association between the seroprevalence of toxoplsmosis in animals and risk factors also was investigated in this study and the results are presented in Table 4. As shown, the seroprevalence rates recorded in Sheep, cattle and goats were 21.16%.31.88 and 30.77% respectively. The higher seroprevalence rate was recorded (33.33%) in age group of 5 year and above; While, the lower (21.55%) in animals group of two years old. The seroprevalence of toxoplasmosis in males (28.28%) were higher compared to females (21.88 %). The higher seroprevalence rate was recorded in spring season (33.33%) and the lower rate in winter (18.02%).
The higher seroprevalence rate of toxoplasmosis in animals was recorded in Utoma district (35.71%); and the lower rate in Mifa`a ans (10.715%) and none in Jabal asharq district. Significant differences (P<0.05) were observed between the seroprevalence rate and animal species, season, area factors; while none in with other factors investigated.
In the present study, all ELISA seropositive samples of human and animals were retested by PCR as confirmatory technique tool. Results of PCR revealed that, Out of 66 and 77 samples, 30(45.45%) and 18(23.38%) were found positive for toxoplasmosis in human and animals respectively (Table 5 and Fig. 1). Significant differences were (P<0.05) observed between the prevalence rates of human and animals.     (Jones et al., 2001;Xiao et al., 2010). The epidemiology of toxoplasmosis in human in many countries has been investigated, but at Dhamar Yemen has not been clear. In this study, results revealed the overall seroprevalence of toxoplasmosis in human in Dhamar governorate was 20.43% which was generally lower than that reported in some governorates of Yemen (Qotinah, 2009;Al-Nahari, and Al-Tamimi, 2010;Saleh et al., 2010;Saif et al.,014;Muqbil and Alqubatii, 2014) and other countries of the world (AlKalaby et al., 2002;Chaudhary et al., 2006;Madi et al., 2008;Hassanain et al., 2013;Gasior et al., 2013;Almasian et al., 2014;Gharavi et al., 2018) the prevalence rate reported by above workers was ranged from 29.8%-83.1%. However, our result was higher than seroprevalence rate (12.3%) reported by Xiao et al., (2010) in China. The contrary between the results of current study and findings of above workers could be attributed to difference in environmental conditions, immune status of the individuals, socio-cultural differences, habit of consuming meat, abundance of definitive as well as intermediate as suggested by Yamaoka and Konishi (1993) and Xiao et al., (2010). In this study the results revealed out 66 samples positive for toxoplasmosis, 44(13.62%), 14(4.33%) and 8(2.48%) were seropositive for IgG, IgM and IgG7 and gM. These results are lower than findings of other workers in Yemen (Al-Nahari, and Al-Tamimi, 2010;Saif et al., 2014) and different part of the world (Mahdi et al., 2008;in Qatar;Xiao1 et al., 2010 in China;Hassanain et al., 2013 in Egypt), who reported the seroprevalence rates of IgG, IgM and both IgG and IgM between (11.88-60%), (1.5-19.6%) and (3.5-22.6%) respectively. The discrepancy between the results of this study and findings of above may be due to the size samples, human errors, efficiency of equipment used and immunological status of patients.
In the present study, the higher seroprevalence rate of toxoplasmosis was recorded in age groups of10-20 years' old and lower rate in age group of less than 10 years old. These results are in consistent with findings of Madi et al., (2008) in Qatar, Saleh et al., (2010); Muqbil and Alqubatii (2014) in Yemen, who studies the effect of age in distribution seroprevalence rate of toxoplasmosis in humans. However; it`s lower rate than findings of Mohamad et al., (2013). The reason behind, the higher infection rate in age group of 10-20 years old and above might be due to that elder individuals are exposed to infection more than younger. Furthermore, Dupouy-Camet et al., (1993) and Remington et al., (2001) suggested that seroprevalence of Toxoplasma is well known to increase with age.
The results of the current study displayed that, the seroprevalence of toxoplasmosis was higher in female compared to male. These results are in agreement with findings with others workers (Xiao et al., 2010 in china;Saleh et al., 2010 andMuqbil andAlqubatii, 2014 in Yemen), who studied the relationship between the seroprevalence of toxoplasmosis and gender factor in human. The higher infection rate in females could be attributed to the females are more associated to animals either in the home or in field including cats the final host of the toxoplasmosis, thus may be facilitating the transmission of infectious diseases to them. Moreover, in Yemen, traditionally, women undertaking the care of animals including feeding, breeding, milking of animals etc either in field or home (Al-Nahari and al-Tamimi, 2010).
Regarding the effect of season (month variation) on seroprevalence rate of toxoplasmosis in human, the results revealed that, the higher rate of infection was recorded in summer; whereas, lower rate in spring. The higher infection rate in human recorded in summer may be due to the environmental conditions in summer are suitable for propagation and survival of the causative agent of the infection.
On area basis, the results displayed that, the higher seroprevalence rate of toxoplasmosis was recorded in individuals tested from Utoma district; while, the lower rate from Alhada district. This could be explained in view of Muqbil and Alqubatii (2014) who stated that, educational status of the people, society manner, number of stray cats available in study areas and hygienic conditions are influencing significantly in distribution prevalence of toxoplasmosis among different areas of study,

In animals
The results revealed that an overall seroprevalence of toxoplasosis in animals was 23.84 % according to ELIZA results. This seroprevalence rate is lower than rate reported by other workers in different regions of world (Moreno et al., 1991, in Spain;Kritsepi, 1992, in Serbia;Klun et al., 2006, in Greece;Santos et al., 2009, in Brazil;Iovu et al., 2009 in Romaia;Shahiduzzaman et al., 2011, in Bangladesh;Rahman, 2012, in Iraq;Hassanian et al., 2013, in Egypt;Kadle, 2014, in Somalia;Ahmed and Taswar, 2015, in Pakistan;Zhou et al., 2018;Tilahun et al., 2018, in Ethiopia). The seroprevalence rates reported by them were ranged between 27.75-92.0%. However, the rate of our study was higher than that seroprevalence rate recorded by Gondum et al., (1999) in Brazil;Song et al., (2011), in Korea;Jula et al., (2013) in Iran; Gąsior et al., (2013) in Poland. The contrary between current results and findings of previous studies may be explained in view of Gąsior et al., (2013) who suggested the differences in sero-prevalence of toxoplasmosis in animals between various countries depend on many factors such as type of breeding and management practices, zoo-hygienic status, age of examined animals, serological techniques and geographical regions.
In current study, the results revealed that, out of 77 animals positive for toxoplasmosis, 15.5%, 1.5 % and 6.8 % were seropositive for IgG, IgM and both IgG and IgM respectively. These results are lower than findings of Prelezov et al., (2008) in Bulgaria; Zakaria (2011) in Iraq, Shahiduzzaman et al., (2011 in Bangladesh; Jula et al., (2013) in Iran; Gebremedhin et al., (2013) in Ethiopia; who reported the seropositive rates ranged between (18.3-83.8%), (17.9-82.1%) and for IgG, IgM and both respectively. The detection and presence of IgG and IgM could be explained as high levels of specific IgG antibodies indicate that the individual has been previously infected. However, these antibodies do not distinguish a recent infection from one acquired a long time before. Detection of specific IgM antibodies can help to determine if infection recently occurred; although, these antibodies can persist for months or even years after acute infection (Liesenfeld et al., 2001).
The risk factors such as animal species, age, sex, season and areas were studied and considered potentially influencing in seroprevalence rate of toxoplasmosis in animals. Regarding to animal species investigated. The results raveled that the seroprevalence rate of toxoplasmosis in sheep was 21.16%. These results are in agreement of findings of Savio and Nieto (1995) in Uruguay; whereas, lower than findings of Asgari et al., (2013) in Iran;Klum et al., (2006) in Serbia; Iovu et al., (2009) in Romania;Kadle(2014) in Somalia, who reported the seroprevalence rates ranging between 34%-84.5%. In goats the seroprevalence recorded was 30.77%. These results are in consistent with findings of Shahiduzzaman et al., (2011) in Bangladesh and lower than rate reported by kadle (2014) in Somalia. However, Its higher than findings of Asari et al., (2013) in Iran;Gondum et al., (1999) in brazil, who recorded the seroprevalence rate as 18.8% and 28.93% respectively. In cattle, the seroprevalence rate was 31.88%. This result is lower than that recorded by Asgari et al., (2013) in Iran and klum et al., (2006) in Serbia, who reports the prevalence as 55% and 76.3 % respectively. However its higher than seroprevalence rate recorded by Shahiduzzaman et al., (2011) in Bangladesh;kadle (2014) in Somalia who, reported the seroprevalence rate ranging between 7.1-27.85%. The contrary between the seroprevalence rate recorded in present study and findings of above workers in animal species could be attributed to number of animals examined, farming management, hygiene condition, grazing habit, and methods used for detection of specific antibodies against T. gondi.
In present study, the higher seroprevalence rate of infection was recorded in elder animal groups; whereas, the lower rate in younger animal groups. These results are in line with findings Savio and Nieto (1995) in Uruguay; Oncel and Vural (2006) in Turkey; Hassanain et al., (2013) in Egypt; Elfahal et al., (2013) in Sudan who observed that elder animals are more susceptible to toxoplasmosis infection compared to younger animals. The higher seroprevalence rate infection in elder animal groups could be attributed to that aged animals have longer exposure time to oocysts of T. gondii compared to younger, higher likelihood on ingestion of oocysts by elder animals and immunological factors. Moreover, Gebremedhin et al., (2013) suggested that the rate of infection is increasing with age. The results revealed that the seroprevalence rate was higher in male compared to females. These results are in agreement with findings of Asgari et al., (2013) in Iran. Moreover; the impact of gender/sex on the frequency of toxoplasmosis has been previously studied by many researchers (Oncel and Vural, 2006, in Turkey;Rahman, 2012, in Iraq;Jula et al., 2013, in Iran;Elfahal et al., 2013, in Suden;Gebremedhin et al., 2013, in Ethiopia) who observed the prevalence of toxoplasmosis was high in females compared to males. The differences between the results of current study and finding of above workers could be explained in view of Gebremedhin et al., (2013) who suggested the females are retained in the farm for longer periods for breeding purpose than males this may increase the probability of infection; whereas, Few males are retained for mating while the majority are culled and sold for cash purpose. In addition, the hormonal difference in relation to stress of lactation and pregnancy leading to immunosuppressant may also increase susceptibility to toxoplasmosis in females.
Results of season wise revealed that, the higher of seroprevalence of infection was recorded in summer which coinciding with rain season; whereas, the lower rate in winter. These results are in consistent with findings of Oncel and Vural (2006) who studied the effect of seasons on distribution of seroprevalence rate of toxoplasmosis in sheep in Turkey. The higher seroprevalence rate during summer could be attributed to the longer viability of T. gondii oocysts in warm and humid environments which is suitable for propagation of parasite as suggested by Puije et al., (2000).
PCR has been used as confirmatory tool for serological tests results (Nimri et al., 2004;Hassanain et al., 2013;AL-Dujaily and Amir, 2014;Hashoosh and Majeed, 2014;Sroka et al., 2018). In the present study, all ELISA seropositive samples of human and animals were retested by PCR technique. The results of PCR revealed that, out of 66 and 77 samples of ELIZA, 45.45 % and 23.3% were positive for toxoplasmosis. This result could be explained in view of El-Madawy and Metawea (2013) who stated that, Although serological testing is considered the main diagnostic technique for toxoplasmosis, it has some limitations because of the false positive results which have been reported by many investigators, in addition, to the false negative results that may happen during the active phase of T. gondii. The need for more sensitive techniques as PCR assay is essential to demonstrate the actual infection in human and animal, to obtain more data on prevalence, frequencies and phases of the parasite. Moreover, Hassanain et al., (2014) also, suggested that, the presence of toxoplasma-specific antibodies in an insufficient criterion for identifying toxoplasma infection.
The results of this study, based on serological and molecular assays, confirmed our expectation that toxoplasmosis is indeed prevalent in Dhamar governorate. Combinations of serological and molecular techniques are recommended for accurate diagnosis of toxoplasmosis.