Serological Determination of Toxoplasma gondii among Sheep (Ovis aries) in Guilan Province, Iran

Introduction

Toxoplasma gondii is one of the most common zoonotic diseases that can be transmitted through meat around the world ( Schluter et al., 2014 ). Toxoplasmosis is a major contributor to abortion, congenital infection, and stillbirth in humans and animals ( Schluter et al., 2014 ). Toxoplasmosis in patients with intact immune systems is usually asymptomatic; however, it can be life-threatening in patients with a weak immune system (for example, patients with the human immunodeficiency viruses/acquired immunodeficiency syndrome or cancer and transplant recipients) ( Abdoli et al., 2016 ). The cat is the final host, and a wide range of warm-blooded animals, including humans and ruminants, are the intermediate hosts for T. gondii ( Schluter et al., 2014 ).

The sexual cycle of T. gondii occurs only within the feline intestine, and the produced oocysts are excreted through the host’s feces. Sporulated oocysts can resist environmental conditions for 12-18 months ( Schluter et al., 2014 ). Eating foodstuffs, water, vegetables, or even soils contaminated with oocysts is the major source of transmission of the parasite to humans and animals ( Schluter et al., 2014 ). The formation of tissue cysts is also the most important stage of T. gondii infection.

Humans and felines are infected through the consumption of raw or half-cooked meat containing tissue cysts ( Schluter et al., 2014 ).

Sheep are one of the most important intermediate hosts for T. gondii since the parasite cysts lie dormant within the cardiac and skeletal muscles. Therefore, the consumption of raw and half-cooked meat is one of the major risk factors for T. gondii infection ( Schluter et al., 2014 ). Iran is located in Western Asia with four main climates, including temperate and humid on the coast of the Caspian sea located in the north of Iran, hot and dry in the Central Plateau of Iran, cold and mountainous in the west and northwest of Iran, and warm and humid on the southern coast of the Persian Gulf. These climatic changes play a major role in changing the prevalence of T. gondii infection in ruminants in different regions of the country.

In previous serological studies since 2007 to 2019, due to climate changes in different provinces of Iran, Guilan and Mazandaran provinces had a temperate and humid climate on the coast of the Caspian sea ( Sharif et al., 2007 ; Havakhah et al., 2014 ). Tabriz, Urmia, Kurdistan, Kermanshah, Markazi, and Qazvin provinces, Iran, had a cold and mountainous climate ( Khezri et al., 2012 ). Kerman, Jahrom, and Kashan provinces, Iran, had a hot and dry climate in the Central Plateau of Iran ( Ahmed et al., 2016 ; Rasti et al., 2018 ). Furthermore, Khuzestan, Iran, had a warm and humid climate. According to the published reports, the seroprevalence of T. gondii among sheep in Iran is reported within the range of 3.3-36.8%. It is noteworthy that the prevalence of T. gondii is associated with climatic conditions; accordingly, in areas with a moderate and humid climate, the prevalence is higher than in hot and dry areas ( Sharif et al., 2015 ).

However, in a study carried out by Havakhah et al. in Iran (2014), the prevalence of toxoplasmosis in sheep was reported only in three cities of Guilan province, and there has been no information about the prevalence of toxoplasmosis across the province. Since sheep are among the major sources of meat production in Guilan province, the current study used the serological investigation of T. gondii infection among sheep in Guilan within 2018 to 2019 to identify the prevalence rates of the parasite.

Material and Methods

Study area. Guilan, one of the northern provinces of Iran with a population of about 2,500,000, is located at 37° 27' North latitude and 49° 58' East longitude from the meridian, and its altitude from the sea level varies in different regions (Figure 1). Its area is ​​about 14,711 km2, and the average annual rainfall is 1,275 mm. Guilan is divided into three regions of plains with a temperate climatic condition, hillsides with a semi-humid climate, and heights with a cold mountainous climate.

Figure 1. Geographic map of Guilan province, Iran

Sample collection. In the current study, a total of 400 sheep blood samples obtained from the jugular vein were collected according to the geographical distribution of the sheep population in Guilan province. The samples of pregnant female sheep of the herds with a history of abortion were prioritized in the study. The blood samples were centrifuged at 3,000 rpm for 10 min, and the isolated sera were stored at -20˚C until analysis. According to the climate variations in Guilan province, the samples were divided into three groups of plains, hillsides, and heights based on the geographical location. The sheep were also divided into three age groups of < 2, 3-4, and > 4 years. The study protocol was approved by the Ethics Committee of the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

Serological detection. The indirect enzyme-linked immunosorbent assay (ELISA) was used for the detection of immunoglobulin G (IgG) antibodies against T. gondii in the sera (multi-species ID Screen® Toxoplasmosis Indirect, IDVet, Montpellier, France) according to the manufacturer's instructions. The optical density (OD) was measured at 450 nm with an ELISA automated plate reader (BioTek, USA). The Sample to Positive (S/P) ratio was calculated according to the following formula:

S/P% = OD sample-OD negative control OD positive control-OD negative control × 100

The samples presenting an S/P% ≤ 40% were considered negative for T. gondii; the samples with S/P% within 40% to 50% were considered doubtful for T. gondii; the samples with S/P% ≥ 50% were considered positive for T. gondii.

Statistical analysis. The data were statistically analyzed using the Chi-square test by SPSS software (version 24; SPSS, Inc., Chicago, IL).

Results

A total of 400 female sheep with a mean age of 3.4±1.84 years (age range: 1-13 years) were evaluated in the current study. T. gondii-IgG antibody was detected in 166 cases (41.5%; n=166). Age was significantly associated with IgG seropositivity with the highest frequency of positive samples (72.7%; n=56) observed in the age group of > 4 years (P=0.0001). The geographical location and IgG seropositivity were also reported with a significant relationship (P=0.0001) with the highest frequency of the positive samples (53.9%; n=103) observed in the sheep of the plains (Table 1).

With increasing the age from 3 to 4 years and over, the frequency of IgG seropositivity decreased from the plains to heights (Table 2).

Discussion

Toxoplasmosis is a common disease among animals and humans around the world and one of the major causes of abortion and stillbirth in sheep ( Edwards and Dubey, 2013 ). Humans, after birth, are infected via the reception of the tissue cysts observed in half-cooked meat, consumption of water and food contaminated with oocysts, or accidentally ingestion of oocysts spread in the environment. Contaminated lambs are among the major sources of T. gondii infection in humans and carnivorous animals ( Dubey, 2009 ). These animals are also the intermediate hosts for T. gondii ( Schluter et al., 2014 ).

Iran has a wide variety of climates; in the northern regions of Iran, with a moderate and humid climate, the oocysts excreted through felines feces can remain for months or even years in the environment ( Zhang et al., 2016 , Subedi et al., 2018 ). Therefore, the highest prevalence of T. gondii infection in humans and animals in Iran is reported for the northern region ( Sharif et al., 2015 ; Izadyar et al., 2019 ) indicating the high importance of this issue in Northern Iran. In previous studies conducted on pregnant women in Northern Iran, the prevalence of T. gondii infection was reported within 41.8-75.02% ( Foroutan-Rad et al., 2016 ). In addition, in a serological investigation on rural individuals in Amol, Northern Iran, the prevalence of this infection was 75.7%. This high prevalence demonstrates that Northern Iran is an endemic area for toxoplasmosis. There was also a significant relationship in the current study between the lamb meat (P=0.015) and raw or half-cooked meat (P <0.001) consumers and presence of T. gondii-IgG in their serum ( Rostami et al., 2016 ).

According to previous studies conducted in different parts of the world, the serological prevalence rates of T. gondii among sheep are reported as 33.6% in Portugal ( Lopes et al., 2013 ), 15.1-84% in Mexico ( Hernández-Cortazar et al., 2015 ), 8% in South Africa ( Hammond-Aryee et al., 2015 ), 47.8% in Brazil ( Rêgo et al., 2016 ), 26.2% in Pakistan ( Ahmed et al., 2016 ), 22.0% in the USA ( Guo et al., 2016 ), 20.71% in China ( Yang et al., 2017 ), and 42.1% in Northern Iraq ( Al Hamada et al., 2019 ). The prevalence of T. gondii infection among sheep in different regions of Iran is within the range of 3.3-38.3% according to the published reports ( Rasti et al., 2018 ). These differences can be attributed to the difference in climates, ages, livestock breeding conditions, and various methods for the detection of the parasite ( Izadyar et al., 2019 ).

In the present study, the serological prevalence of T. gondii in sheep in Guilan province was detected at 41.5%. In another study carried out in Mazandaran province, Northern Iran, with a temperate and humid climate, the seroprevalence of T. gondii was 35% ( Sharif et al., 2007 ) which was lower than that reported for the present study. In a study conducted by Havakhah et al. (2014) in Guilan province, the prevalence of T. gondii infection in sheep was 36.8%, which was lower than that of the present study; however, Havakhah et al. used Sabin-Feldman serologic dye test only in three counties. This could account for the differences reported in the present study, owing to the population distribution of sheep in Guilan province, as the first attempt investigating most regions of the province by the serological method of ELISA, which has a higher sensitivity than the dye test ( Balsari et al., 1980 ).

In addition to the temperate and humid climate, the extensive grazing system of sheep breeding, presence of wild felines, and crop storage facilities that felines have access to are other causes of the high prevalence of T. gondii in Guilan province. These findings are similar to the results of a study carried out by Subedi et al. on sheep in Nepal ( Subedi et al., 2018 ). Guilan province was divided into three regions of plains, hillsides, and heights according to the geographical location. In the current study, the highest (53.9%) and lowest (10%) frequency rates of T. gondii infection were observed in the plain and height samples, respectively. Furthermore, a significant relationship was observed between the geographical location and frequency of T. gondii infection (P=0.0001).

With increasing the altitude from the sea level, the amount of humidity and temperature decreases, and a dry and cold winter dominates the heights of the province reducing the survival rate of oocysts in such climatic conditions; however, in the plains, due to the lower altitude from the sea level and proximity to the sea, the conditions are suitable for the survival of oocytes during winter. Oocytes can survive in moderate and humid climates for up to 18 months ( Katzer et al., 2011 , Gazzonis et al., 2015 ).

In a study carried out by Khezri et al. (2012) on sheep in Kurdistan province, Iran, the prevalence of T. gondii infection in the southern regions due to hot and humid climate was higher than that of the western regions due to cool and dry climate dominating these regions. In another study performed by Sharif et al. (2007) on the sheep in Mazandaran province, the high prevalence of T. gondii infection in the western region was due to the difference in moisture content increasing the number of oocysts, compared to that reported for the eastern and central regions. The aforementioned results are consistent with the findings of the current study.

In the present study, there was a significant correlation between mean age and seropositive (4.2±33.17) and seronegative (2.1±7.2) samples (P=0.0001). Moreover, the highest (72.7%) and lowest (16%) frequency rates of the infection were observed in the age group of > 4 and < 2 years, respectively. The relationship between the age group and seropositivity was statistically significant (P=0.0001). In previous studies, including Gazzonis et al. (2015) on Northern Italian sheep, Katzer et al. (2011) on Scottish sheep, Hecker et al. (2018) on sheep in Argentina, and Izadyar et al. (2019) on sheep in Qazvin, an increased prevalence of T. gondii infection in older animals in comparison to younger ones was consistently reported as observed in the current study ( Rahman et al., 2014 , Sharif et al., 2015 , Izadyar et al., 2019 ). These results suggest that contamination with T. gondii in sheep mostly occur after birth, and horizontal contamination is the main route of infection transmission in herds ( Dubey, 2009 , Hecker et al., 2018 ).

Conclusion

A high prevalence of T. gondii infection in Guilan province was observed in the present study which was related to the temperate climate and high humidity of the region. Due to climate variations in Guilan province, the prevalence of contamination in the plains of the province was higher than that reported for other regions. It was also shown that as the sheep age increased, the prevalence of T. gondii infection also increased, confirming the horizontal transmission of contamination. A high prevalence of T. gondii infection was reported in sheep; therefore, in order to improve the management of sheep in this region, it is recommended to develop a comprehensive educational, preventive, and continuous treatment plan in this regard.

Authors' Contribution

Study concept and design: M. R. Ch. N. and B. Sh.

Acquisition of data: M. R. Ch. N. and P. Sh.

Analysis and interpretation of data: M. R. Ch. N.

Drafting of the manuscript: M. R. Ch. N.

Critical revision of the manuscript for important intellectual content: M. R. Ch. N.

Statistical analysis: M. R. Ch. N.

Administrative, technical, and material support: M. R. Ch. N.

Ethics

The authors declare that all the ethical standards were respected in the preparation of the submitted article.

Grant Support

This research did not receive any specific grant from the university.

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