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Seasonal and Age Distribution of Toxoplasma gondii in Milk of Naturally Infected Animal Species and Dairy Samples

(Received 14/12/2019, accepted 29/01/2020) DOI. 10.21608/ejvs.2020.20965.1143 ©2020 National Information and Documentation Centre (NIDOC) PRIMARY infection by Toxoplasma gondii is habitually food borne and ingestion of foods with animal origins is measured as a risk factor. The current survey was performed to assess the seasonal and age distribution of T. gondii in diverse kinds of raw milk and traditional dairy products. Three-hundred and seventy raw milk and traditional dairy samples were collected and subjected to DNA extraction. B1 specific gene of T. gondii was perceived by means of the molecular technique. Eighteen out of 370 (4.86%) studied samples were positive for T. gondii. Molecular incidence of T. gondii in raw milk was 6.08%. T. gondii was also perceived in 2.85% of studied traditional dairy samples, respectively. Sheep milk (10%) and cheese (6.66%) had the uppermost molecular incidence of parasite. Molecular incidence of T. gondii in raw camel milk and cream and butter samples were 3.33%, 5% and 5%, respectively. Samples which were collected through autumn (15.55%) and summer (9.37%) seasons had the maximum molecular incidence of T. gondii. Raw milk samples of older than 4 years animal species (11.39%) had the maximum molecular incidence of T. gondii. The existingsurvey is the first report of seasonal and age distribution of T. gondii in diverse kinds of dairy samples. Raw sheep, goat, buffalo, bovine and camel milk and also traditional cheese, cream and butter dairy samples may harbor T. gondii infection. Seasonal and senile distribution should be measured for molecular incidence of T. gondii in milk of primary infected animals and also traditional dairy products.


Introduction
Milk and dairies have an imperative importance in the human regime. They provide a set of nutrient materials such as fats, proteins, calcium, vitamins, potassium and phosphorus [1,2]. Milk and traditional dairies are not essentially safe. It is because of numerous surveys demonstrated their consumption as a risk factor for occurrence of diverse kinds of diseases such as toxoplasmosis [3][4][5][6].
Toxoplasma gondii (T. gondii) is an intracellular cyst-forming Apicomplexan Sarcocystidae protozoan parasite responsible for occurrence of infectious toxoplasmosis in humans and animals [7][8][9][10]. T. gondii is also responsible for biologically diverse diseases such as abortion in livestock and predominantly sheep, encephalitis in immunocompromised individuals and abortion or congenital flaws in fetuses [7]. Additionally, consumption of contaminated dairies is a significant risk factor for occurrence of toxoplasmosis in human population [7,8,11].
Consumption of contaminated milk and other high risks food stuffs is the most substantial route of transmission of T. gondii to intermediate hosts [3,5,[7][8][9][10][12][13][14]. Gaps in present information about the risk assessment of T. gondii by dairy consumption are renowned. Inconsistent information is perceived inside risk assessment of dairy product consumption and occurrence of toxoplasmosis. While some reports described positive relationships amid dairy consumption and transmission of T. gondii infection to human [8,11], others specified irrelevant effects of dairy consumption [15,16]. Thus, it is important to find the portion of raw milk and traditional dairies in transmission of T. gondii infections to human. Furthermore, there is no strict data about the role of season and age of animals in the incidence of T. gondii. Some scarce reports disclosed that season and age may affect the incidence of toxoplasmosis [17,18].
Affording to uncertain role of raw milk and traditional dairies in transmission of T. gondii, the current survey was performed to evaluate the seasonal and age distribution of T. gondii in raw cow, buffalo, sheep, goat, camel and donkey milk and traditional yoghurt, cheese, butter, cream, doogh, kashk, and ice-cream dairy product samples by means of the nested-Polymerase Chain Reaction (nested-PCR).

Moral questions
The survey was permitted by the ethical team of the author's institute.Licenses of sampling were taken from the Head of the Faculty.

Samples
From and ice-cream (n=20) samples were arbitrarily obtained through simple random sampling procedure from the retail centers. Samples were collected from diverse provinces located at the center and south-west of Iran. All samples were stored at refrigerator in the retail centers. Sheep, donkey and goat raw milk samples were only collected through the spring and summer seasons. Dairy samples all were derived from cow's milk. Age of targeted livestock was determined by an expert professor of the field of Animal Sciences. Briefly, Samples (10 mL) were collected through hygienic circumstances by means of sterile glass tubes. First few squirts were overlooked during milk collection. Samples were directly transferred to the laboratory by means of sterile cool boxes.

DNA extraction
DNA was extracted from a 200-μL aliquot of samples by means ofthe DNA extraction and purification kit (Thermo Fisher Scientific, Germany) rendering the factory's guidelines. For this purpose, 900 QL of cell lysis solution was added to 300 QL of sample in a cryotube. Samples were subjected to enzymatic pre-digestion by means of proteinase K (20 QL, 20 mg/ml) and incubated at 56°C overnight. Extracted DNA samples were subjected to quantification by NanoDrop device (NanoDrop, Thermo Scientific, Waltham, USA), qualification (2% agarose gel) and purity checking (A260/A280).

Detection of T. gondii B1 gene
DNA thermo-cycler (Eppendorf Master cycler 5330, Hamburg, Germany) was applied in PCR reactions. T. gondii B1 gene was perceived in DNA samples by means of the method conveyed formerly [19]. Table 1 signifies the PCR circumstances applied for detection of T. gondii B1 gene. Visualization was performed by means of electrophoresis on agarose gel (2%in 1× TBE buffer stained with SYBR Green (Thermo Fisher Scientific, Germany). Negative control (PCR grade water (Thermo Fisher Scientific, Germany) and positive control (positive DNA for the B1 gene obtained from the Faculty of Veterinary Medicine, University of Tehran) were applied in PCR reactions.

Statistical analysis
Data obtained from the survey were numerically examined by SPSS 21.0 software. Noteworthy relations amid variables were determined by chi-square and Fisher's exact twotailed tests. P value <0.05 was determined as level of significance.

Results
Three-hundred and seventy dairies were tested for presence of T. gondii B1 gene by the PCR method. Figure 1 signifies the amplification of T. gondii B1 gene in first and second (nested) PCR reactions. Table 2 signifies the molecular incidence of T. gondii in diverse kinds of dairies. Eighteen out of 370 (4.86%) raw milk and dairy samples were positive for T. gondii B1 gene. Four out of 140 (2.85%) traditional dairy product and 14 out of 230 (6.08%) raw milk samples were also positive for T. gondii B1 gene. Sheep milk (10%) was the most commonly contaminated sample amongst the raw milk samples. Additionally, traditional cheese (6.66%) was the most commonly contaminated sample amongst the traditional dairy product samples. Reversely, camel milk (3.33%) and cream (5%) and butter (5%) had the lowermost molecular incidence of T. gondii. There were    Table 3 signifies the seasonal distribution of T. gondii in diverse kinds of dairies. Molecular incidence of T. gondii in spring, summer, autumn and winter seasons were 2.10%, 9.37%, 15.55% and 0%, respectively. Moreover, molecular  Table 4 signifies the age distribution of T. gondii in diverse kinds of raw milk. Molecular incidence of T. gondii in raw milk samples of < 2 years old, 2-4 years old and > 4 years old livestock species was 1.81%, 5.26% and 11.39%, respectively. Only 7.14% of raw milk samples of younger than 2 years old ovine were positive for T. gondii. There were no detectable T. gondii in the raw milk samples of younger than 2 years old buffalo, bovine, caprine and camel. Statistically noteworthy variances were gotten amid age of animal species and molecular incidence of T. gondii (P<0.05).
Though, preceding work disclosed that T. gondii's tachyzoites may sporadically survive in acidic solutions and their ingestion may cause infection in cats and mice [53]. Moreover, T. gondii's tachyzoites may penetrate to mucosal tissue and reach to the blood and lymphatic systems of hosts before reaching the stomach [42,43,54]. Furthermore, T. gondii's tachyzoites can survive in milk for about three to seven days at 4°C [55] and in cheese for a about 8 to 10 days [32]. Thus, presence of T. gondii in dairies should be measured as a dangerous concern. Serologic surveys conducted in Iran [56], Czech Republic [57], Romani [58], Brazil [59], Greece [60], and finally Thailand [61].
Our findings also disclosed that dairies collected through autumn and summer seasons had the higher molecular incidence of T. gondii. Moreover, raw milk samples collected from older animals (>4 years old) had the higher molecular incidence of T. gondii. Higher molecular incidence of T. gondii in samples collected through autumn and summer seasons, signifying that fresh grasses may harbor T. gondii oocysts. Moreover, humid environment exist in these seasons may favor the survival of T. gondii oocysts. Our findings were similar to results of previously published works [53,62,63]. Though, some surveys have exposed negative portion of season for incidence of T. gondii [64,65].Previous work [66] described that 85 out of 3531 (2.41%) samples collected through autumn seasons were positive for T. gondii. Additionally, higher seroprevalence of this parasite was conveyed on samples collected through autumn (OR 3.462; P = 0.039) [66]. Dubey [67] conveyed that access of livestock to outdoors and grazing pasture (in autumn and summer seasons) are imperative risk factors for incidence of T. gondii. Role of age of animals has also been investigated [31,68].Recent survey [69] also disclosed that > 8 months old pigs had the higher seroprevalence of T. gondii than young pigs. Previous investigation [70] described that animals and especially sheep and goat can be exposed to T. gondii at any stage of their life, while incidence increased in higher ages. Recent report [71] described that incidence of T. gondii increased from 37.70% in young sheep to 73.80% in >6 years ewes. Another survey [72] specified the role of age as an substantial risk factor for incidence of T. gondii. Totally, portion of food-borne microbes, particularly bacteria, in occurrence of food-borne diseases has been measured in Iran and diverse surveys have been conducted in this field [20,[73][74][75][76][77][78][79][80][81][82].

Conclusions
We examined the molecular incidence of T. gondii in raw milk and traditional dairy product samples collected from different parts of Iran. Nested-PCR reaction was introduced as a safe and accurate diagnostic method for detection of the T. gondii B1 gene. T. gondii molecular incidence in raw milk and traditional dairies was 6.08% and 2.85%, respectively. Sheep milk and traditional cheese were the most commonly contaminated samples. Camel milk and cream and butter had the lowermost molecular incidence of T. gondii. There were no detectable T. gondii in donkey raw milk and yoghurt, kashk and doogh samples. Higher molecular incidence of T. gondii was found in dairies collected through the autumn and summer seasons. Additionally, higher molecular incidence of T. gondii was seen in raw milk samples of older than 4 years old animal species. Our outcomes may disclose that the raw buffalo, cow, goat, sheep, and camel milk samples and traditional cheese, butter and dairies are likely sources of T. gondii infection in the community.