1. Background
Nearly all warm-blooded animals worldwide, including humans, are susceptible to infection by the food-borne intracellular parasite Toxoplasma gondii (1). The human population has been widely exposed to T. gondii to some extent. In particular, parasite infection can result in death or severe damage to the brain and eyes in patients with immune deficiencies (2-4). Humans primarily become infected with T. gondii through the consumption of raw meat, water contaminated with oocysts from the environment, or through vertical transmission (5, 6). Toxoplasma gondii can also negatively affect animal growth, development, and reproduction, leading to significant financial losses in livestock husbandry. Livestock often become infected by consuming food and water contaminated with sporulated oocysts (7, 8). Sheep are significant intermediate hosts for T. gondii, as the parasite cysts remain dormant within their cardiac and skeletal muscles. Consequently, consuming raw or partially cooked meat is one of the main risk factors for T. gondii infection (9). T. gondii infection in sheep can produce many non-specific symptoms, such as fever, dyspnea, depression, lethargy, vomiting, diarrhea, chorioretinitis, lymphadenopathy, and even miscarriages and stillbirths (10).
Iran is a country in Western Asia with four distinct climate zones: Hot and dry in the central plateau, cold and mountainous in the west and northwest, warm and humid on the southern coast of the Persian Gulf, and temperate and humid on the Caspian Sea coast in the north. The frequency of T. gondii infection in ruminants varies significantly across the country due to these climate differences (11). While T. gondii seroprevalence in sheep has been previously reported from Iran (11-13), we provide comprehensive epidemiological information here, including infection risk variables.
2. Objectives
Therefore, the goal of the current investigation is to conduct a seroepidemiological survey for T. gondii infection in sheep bred in Iran's Kohgiluyeh and Boyer-Ahmad province. This study is significant because it identifies the key factors that predispose sheep to T. gondii infection, and the findings can be used to prevent and control this parasite in sheep.
3. Methods
3.1. Study Area
The study was conducted in the Kohgiluyeh and Boyer-Ahmad province, located in the southwest of Iran. This province is situated between latitudes 30° and 31° N and longitudes 51° and 52° E. With an average annual temperature of 15°C and an annual rainfall of 900 millimeters, Kohgiluyeh and Boyer-Ahmad experience cold weather conditions (Figure 1).
Kohgiluyeh and Boyer-Ahmad province
3.2. Sampling
Over the course of four seasons, blood samples were taken from 247 sheep in 20 selected flocks. Using sterile Venoject tubes, the samples were taken directly from each animal's jugular vein and brought to the laboratory. Priority was given to pregnant female sheep from herds that had previously experienced abortions. Serum samples were separated from 5 mL blood samples using centrifugation at 3000 g for 150 minutes before testing. The samples were then stored at -20°C.
3.3. Determination of Antibodies Against T. gondii
Specific IgG antibodies to T. gondii were examined using the Modified Agglutination Test (MAT) as described by Desmonts and Remington (14). The Department of Parasitology at Ahvaz University of Medical Sciences in Iran provided the T. gondii RH strain tachyzoites, which were grown in mice in cell culture before being separated and preserved in formaldehyde solution (15). In addition to negative and positive controls, sera were tested on 96-well plates with U-shaped bottoms (25 µL of prepared mix antigen + 25 µL of serum) using two-fold serial dilutions from 1: 10 to 1: 160. An antibody titer of ≥ 1: 20 was deemed positive (13). Every test included both positive and negative controls, which were examined using the identical serum sample dilutions.
3.4. Risk Factors
Two categories of risk factors were examined to correlate the prevalence of T. gondii infection with potential risk factors: Those related to the biological traits of the animals (age and sex) and those related to environmental conditions (access of cats to feeding facilities, access of cats to water supplies, season, and source of drinking water) (16).
3.5. Statistical Analysis
The statistical analyses were conducted using SPSS software version 22 in Chicago, IL, USA. The researchers utilized the chi-square test and logistic regression to assess the association between several variables: Age (sub-adult and adult), sex (male vs. female), season (wet vs. dry), and abortion with infection. Differences were considered statistically significant when P < 0.05.
4. Results
4.1. Seroprevalence
Sixty of the 247 sera examined (24.3%) contained T. gondii antibodies. Among the 247 samples, there were 201 (81.38%) ewes and 46 (18.62%) rams. Table 1 reveals that 51 (25.4%) of the ewes and 9 (19.6%) of the rams had antibodies against T. gondii.
| Variables | Positive | Negative | Seroprevalence (%) | P-Value | 95% CI | OR |
|---|---|---|---|---|---|---|
| Age | ||||||
| ≤ 1 year | 13 | 62 | 17.3 | 0.001 | 0.17- 0.64 | 0.33 |
| > 1 years | 47 | 125 | 27.3 | 1.76- 5.11 | 3.15 | |
| Gender | ||||||
| Female | 51 | 150 | 25.4 | 0.052 | 0.63- 3.09 | 1.40 |
| Male | 9 | 37 | 19.6 | 0.32- 1.58 | 0.72 | |
| Source of drinking water | ||||||
| Surface | 47 | 111 | 29.7 | 0.0001 | 1.25- 4.89 | 2.48 |
| Underground | 13 | 76 | 14.6 | 0.21- 0.79 | 0.40 | |
| Access of cats to water supplies | ||||||
| Yes | 43 | 78 | 35.5 | 0.0001 | 2.16- 7.60 | 4.05 |
| No | 17 | 125 | 11.9 | 0.13- 0.46 | 0.25 | |
| Access of cats to feeding facilities | ||||||
| Yes | 27 | 78 | 25.7 | 0.758 | 0.64- 2.05 | 1.43 |
| No | 33 | 109 | 23.2 | 0.49- 1.57 | 0.87 | |
| Season | ||||||
| Wet | 36 | 81 | 30.7 | 0.035 | 1.09- 3.55 | 1.96 |
| Dry | 24 | 106 | 18.4 | 0.28- 0.92 | 0.51 |
Risk Factors Associated with Biological and Environmental Characteristics of Animals
4.2. Environmental and Biological Risk Factors
Age, cats' access to water sources, source of drinking water, and season were all associated with risk factors for T. gondii seroprevalence (Table 1). Adult sheep had a greater seroprevalence (27.3%) compared to younger sheep (17.3%). Additionally, females had a higher seroprevalence than males. Seasonal variations were also significant, with wet seasons showing higher susceptibility. On 25.7% of sheep farms, cats were seen even in the feed store. The area's cats' freedom of movement also impacted the contamination of animal drinking water, with farms using surface water to water sheep showing a higher seroprevalence (29.7%).
5. Discussion
Toxoplasmosis is a prevalent infectious disease affecting both humans and animals worldwide. It is one of the main reasons why sheep abort and give birth to stillborn offspring (17, 18). Humans can become infected after birth by eating meat containing tissue cysts, drinking water or eating food contaminated with oocysts, or accidentally ingesting oocysts dispersed in the environment. Contaminated lambs are one of the main sources of T. gondii infection in humans and carnivorous animals (1, 19). Additionally, T. gondii uses these species as intermediate hosts (9).
This study showed that 24.3% of the sheep in Kohgiluyeh and Boyer-Ahmad were positive for T. gondii. Several studies on T. gondii infection in sheep have been conducted in many countries, and the prevalence in sheep was reported as 51.8% in Egypt (20), 26.2% in Pakistan (21), 36.2% in Nepal (22), 85% in Brazil (23), 59% in Senegal (24), and 53.5% in Romania (25). According to published research, the frequency of T. gondii infection among sheep in various regions of Iran ranges from 3.3% to 38.3% (26). Comparing the results of the present study to earlier studies in Iran revealed that the prevalence of our seropositive sheep was similar to other parts (27-29). Variations in prevalence can be explained by factors such as climate, age, animal breeding conditions, and different parasite detection techniques (30). It is widely known that sporulated oocysts can endure for up to 12 months in a favorable environment (humidity and temperature) (31).
Research linking T. gondii seropositivity to the biological and environmental traits of animals indicates that age, cats' access to water sources, the source of drinking water, and season can all affect an animal's susceptibility to T. gondii infection. High seroprevalence levels have been found in sheep that are female and older than one year. An increased prevalence of T. gondii infection in older animals compared to younger ones has been repeatedly documented in earlier research, including studies on Northern Italian sheep, Scottish sheep, sheep in Argentina, and sheep in Iran (30, 32-34). These findings imply that T. gondii contamination in sheep occurs primarily after birth and that horizontal contamination is the primary method of infection transmission in herds (19, 34). Additionally, seroprevalence rises with increasing age, which is related to an elevated cumulative risk of exposure to environmental infectious agents (32).
The findings revealed a strong positive association between the prevalence of T. gondii antibodies in sheep and the access of cats to water provided for livestock on farms. This suggests that interaction with feline species, specifically through exposure to water contaminated with oocysts from cats, plays a crucial role in the epidemiology of T. gondii infection. The presence of cats, both domestic and stray, on farms appears to be a significant factor contributing to the high incidence of T. gondii-specific antibodies in sheep observed in the study. Cats can shed oocysts in their feces, contaminating the environment, including water sources. Therefore, contaminated water may be just as important as contaminated feed in transmitting T. gondii infection. The findings align with a recent review paper, which suggests that the transmission of T. gondii through water may be more widespread than previously recognized, emphasizing the potential role of waterborne transmission in the spread of toxoplasmosis (35). The "use of surface water sources for drinking" was noted as a possible risk factor for T. gondii seropositivity in sheep in an earlier study (36). Static water use was linked to seroprevalence, which could be explained by the fact that cats were more likely to have access to it than to rushing water. Any feces from infected cats will be dangerous because oocyst survival in soil has been observed to last for up to two years (37). Unfiltered water infected with T. gondii can cause illness since T. gondii oocysts can live in water for up to 54 months in cold water (38, 39).
Sheep sampled during wet seasons had a twofold higher likelihood of testing seropositive compared to those sampled during dry seasons. Our findings are consistent with earlier data from Iran, where it was discovered that sheep had significantly greater T. gondii seropositivity in humid and mild climates, which are thought to be the best environments for the growth and survival of T. gondii oocysts (30). In contrast, a study conducted in Egypt found that the seropositivity rate of T. gondii was not significantly associated with climate (40).
Environmental and climatic factors can affect the spread and survival of the oocysts, increasing the likelihood that susceptible animals will be exposed to the parasite (41, 42). The oocysts can disperse in freshwater after heavy rains due to their hydrophilic and hardly sticky surface. In addition, heavy rains may influence the flow of rivers and streams, facilitating the spread of oocysts in the environment (43). According to Afonso et al., cats that have endured rainy winters for several years have a greater seroprevalence (43). Analysis of bioclimatic variables found that places with higher levels of precipitation had a higher chance of instances, supporting this idea. A moist environment helps the oocysts survive and makes food available to the insects that serve as T. gondii transport hosts (42).
5.1. Conclusions
This study provides new data about the epidemiology of T. gondii infection in sheep. Age, the rainy season, cats' access to water sources, and surface water sources all significantly contribute to the dissemination of oocysts and the rise in the rate of oral infection in farm animals. The emphasis on the high seroprevalence and risk factors for T. gondii transmission in sheep may serve as a springboard for educating Iranian farmers about the significance of the issue and the necessity of implementing control measures to stop the spread of T. gondii.
