Toxoplasma gondii Antibodies in Raw Milk and Sera of Cows in China

Toxoplasma gondii is a worldwide food-borne protozoa that has harmful influences on animal and human health. Raw milk containing T. gondii has been considered as one of the possible infectious sources for humans. Although China is one of the world’s leading milk consumers, there is still no study to investigate the seroprevalence of T. gondii in raw cow milk in China; especially for cows in rural areas. Thus, we conducted this study to examine the specific anti-T. gondii IgG-antibody in the raw milk and sera of domestic cows in China. In total, 894 cows were randomly selected from rural areas in northeastern China. The positive rate of T. gondii in the milk and serum samples were 6.38% (57/894) and 7.16% (64/894), respectively. Moreover, a history of abortion (OR = 2.03, 95% CI: 1.11–3.72, p = 0.022) was identified as the only risk factor for T. gondii infection in the studied cows. This study investigated the seroprevalence of T. gondii in the raw milk and sera of cows in China; it provided timely and useful data for public health and food safety, especially in rural areas.


Introduction
Toxoplasma gondii is an important food-related parasitic causative agent; it is believed to infect almost the whole species of warm-blooded animals, including humans, terrestrial and aquatic mammals [1][2][3]. Feline and other warm-blooded animals are the definitive and intermediate hosts for this parasite, respectively [1]. The most relevant impact of this parasitic infection is mainly on immune-compromised individuals or pregnant women [1]. This parasitic infection gives rise to huge public health safety problems and economic losses in the veterinary field as well [1].
Until now, T. gondii DNA and anti-T. gondii antibodies have already been detected in the milk of some kinds of animals, such as goat [4,5], sheep [6], donkey [7], cow [8], buffalo [9], camel [10], cat [11], and even human [4]. Thus, it is believed that the transmission of T. gondii can occur via the milk of all animals if consumed raw [12]. Indeed, humans have been reported to present with symptoms of acute T. gondii infection after drinking raw goat milk [13]. However, it is believed that it is extremely unlikely to be infected by T. gondii via consuming cow milk because cow has already been considered as a poor host for this parasite. Surprisingly, seroepidemiology surveys suggested that the consumption of raw cow milk was significantly related to T. gondii infection [14,15].
In China, the demand for milk is ever-increasing and forecast analysis suggests that the demand will be an increase of over 3× compared with 2010 [16]. Thus, it is very important that public health understands the contamination situation of this parasite in raw cow milk. Unfortunately, at present, relevant information is still unavailable. Therefore, we firstly conducted this study to investigate the prevalence of an anti-T. gondii IgG-antibody in raw cow milk collected from rural areas in China; this is because rural areas are considered to be important risk areas. The present data will provide new evidences about cow milk contaminated by T. gondii in China; in addition, the date highlight that more monitoring and prevention measures should be taken to safeguard the safety of dairy products.

Results
In this study, 6.38% (57/894) of milk samples and 7.16% (64/894) of serum samples were identified to be positive for T. gondii, respectively. Of these, seven negative milk samples tested positive in their correspondent serum samples. A high correlation (Spearman's coefficient = 0.804, p-value < 0.001; and Kendall's tau = 0.621, p-value < 0.001) between the S/P% values acquired in the serum and milk samples was found. Moreover, significant agreements between serum and milk were found among all the studied groups (Table 1). In addition, a similar trend in antibody levels in milk and serum was found in the present study: in the first half month, the ELISA S/P% values of the milk and serum samples were both low; subsequently, in the second half month (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), the S/P% values increased to a peak both in the milk and serum samples. The decline time of seric IgG was the thirtieth day of lactation. From 31 to 75 days after the beginning of lactation, the level of seric IgG fluctuated slightly in both the sera and milk samples ( Figure 1).

Discussion
Due to the abundant production and low production cost, cow milk is the most consumed milk all over the world; accordingly, it can provide essential substances for organism development, including minerals, proteins, and vitamins [9,16]. It is widely believed that it is unlikely to become infected with T. gondii through consuming cow's milk because In this study, the examined cows were divided into five age groups. The highest T. gondii prevalence in the studied milk and serum samples were both found in the age group (>60 Mon) without a significant difference (Table 2). During the univariate analysis, a history of abortion was found to be related to T. gondii seroprevalence in both the milk (p = 0.030) and serum (p = 0.045) samples (Table 2). After the multivariate regression analysis, a history of abortion was also the only variable that was inferred to be related to T. gondii prevalence in the investigated cows (OR = 2.03, 95% CI: 1.11-3.72, p = 0.022) ( Table 3).

Discussion
Due to the abundant production and low production cost, cow milk is the most consumed milk all over the world; accordingly, it can provide essential substances for organism development, including minerals, proteins, and vitamins [9,16]. It is widely believed that it is unlikely to become infected with T. gondii through consuming cow's milk because cows are recognized as a poor host for T. gondii. In recent years, a study conducted in Brazil found that the ingestion of untreated cow's or/and goat's milk is popular in seropositive pregnant women [17]. Constantly, the ingestion of raw cow's milk is found to be highly related with T. gondii infection [15,18,19]. Thus, the investigation of the prevalence of T. gondii in cow's milk is very important; especially for domestic cows, which is of great significance to food safety. Consequently, we conducted the present study for the first time to investigate the prevalence of T. gondii in cow's milk in China; this will give us valuable data for formulating relevant prevention and control measures for toxoplasmosis in cows and humans.
It is well known that severe reproductive problems in small ruminants could be induced by T. gondii infection; which can cause huge economic losses in animal husbandry [24,25]. More worrisome, the contamination of T. gondii was found on animal by-products, including meat, milk, and other processed products; this is a major hidden issue for public health [26]. Moreover, the contamination will greatly influence global livestock production [27]. Several studies have showed the relation between T. gondii infection and the rate of abortion in cows [28][29][30]. Dubey et al. indicated that abortion in cows would only be associated with a primary infection [28]. One cause of abortion and congenital toxoplasmosis in cattle was considered to be maternal toxoplasmosis [29]. Similarity, a history of abortion was confirmed to be highly related with the seroprevalence of T. gondii in both the serum and milk samples in the present study. It is noteworthy that another very important protozoan, Neospora caninum, cannot be disregarded, it has been considered to play a crucial role in the reproductive failure of cattle and widely distributed in China [31]. Thus, it is necessary to prepare and implement routine surveillance of T. gondii and N. caninum infection at all levels, including individual, herd, and farm levels. Moreover, farms with history of miscarriages should be more concerned.
Children living in rural areas are at a high risk of T. gondii infection because the ingestion of unpasteurized milk is frequent; this is recognized as a possible pathway for T. gondii transmission [32]. Clinical evidence shows that a breast-fed child became infected from a T. gondii-positive mother, suggesting the important role of milk in the transmission of T. gondii [33]. Tachyzoites in the milk and suckling trauma were recognized to make for the transmission of T. gondii [33]. Moreover, T. gondii cysts could be harbored in relatively stable mammary cells during prelactation. The mammary gland cells could also secrete these silent cysts via exocytosis [34], resulting in T. gondii infection via the ingestion of milk. Thus, rural areas may be more affected by T. gondii because the pasteurization of milk is unlikely to be implemented due to objective conditions [35].
We are not aware of any simpler studies in China and the present study reports a seroprevalence of 6.38% (57/894) of T. gondii in cow milk in China. A history of abortion was confirmed to be significantly related to the T. gondii infection in the studied cows. However, one of the most important limitations should not be neglected. We did not calculate the number of necessary samples through a statistical point of view. Thus, the present results could not be used as representative of all the dairy cows in the study area. Moreover, we only detected the IgG antibodies of the samples and it is still not certain that live strains exist in the samples. However, the data of this study is valuable for public health and food safety. Thus, future studies should be conducted to explain the unresolved issues; in addition, it is necessary to prepare and implement relevant prevention and control measures, especially in rural areas.

Ethical Certificate
The study was approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural Science and Technology University. The owners of domestic cows gave us the permission to collect the samples. In this case, the owners of the domestic cows were invited to collect the milk samples; and the serum samples were collected by local veterinarians.

Sampling
The present study was conducted in three cities (Changchun, Jilin, and Baicheng) of the Jilin province (40 • 50 -46 • 19 N; 121 • 38 -131 • 19 E); and three cities (Daqing, Shuangyashan, and Suihua) of the Heilongjiang province (43 • 26 -53 • 33 N; 121 • 11 -135 • 05 E), in northeastern China between January 2019 and September 2020 (Figure 2). At last, 894 domestic cows were randomly selected as target animals for this study to collect the serum and milk. In this study, all the included cows were kept to produce milk for sale or self-supply. The first step was to collect the milk samples due to its convenience. , 11, x FOR PEER REVIEW 6 of 8 and milk. In this study, all the included cows were kept to produce milk for sale or selfsupply. The first step was to collect the milk samples due to its convenience. Approximately 10 mL of milk was collected from each cow after disinfecting the udder.
Subsequently, blood samples were collected; a blood sample of approximately 5 mL Approximately 10 mL of milk was collected from each cow after disinfecting the udder. Subsequently, blood samples were collected; a blood sample of approximately 5 mL was obtained from the jugular vein of each cow and stored in a blood collecting tube. After sampling, all the collected samples were kept in medical incubator; and immediately delivered to a laboratory. We firstly isolated the serum from the blood samples according to our previous study [5] and stored them at −20 • C. Then, we processed the collected milk samples, and removed the fatty components and the somatic cells based on the reported procedure [36]; we kept the remaining ingredients at −20 • C until they were used. The individual data of each cow was obtained from the owner by oral inquiry. We referred to the method of the previous study to calculate the day of lactation [37].

Serologic Examination
In this study, we used a commercial enzyme-linked immunosorbent assay (ELISA) kit (ID Screen ® Toxoplasmosis Indirect MultiSpecies, IDVET, Montpellier, France) to test the specific anti-T. gondii IgG-antibodies in the milk and serum samples according to the reported procedure [32]. For the positive criteria, an S/P% of the serum samples of ≥50% (sample to positive ≥50% = positive) and an S/P% of the milk samples of ≥21.8% (sample to positive ≥21.8% = positive) were considered as positive [38].

Statistical Analysis
In this study, a SPSS 25.0 software package was employed to perform the statistical analysis. The cut-off value of statistically significant was a p-value < 0.05. Moreover, Spearman and Kendall rank correlation coefficients were used to evaluate the correspondence between all the serum and milk results according to the day of lactation. A chi-squared test was used to evaluate the univariate associations between T. gondii seroprevalence in the domestic cows and the risk factors. Finally, we identified the possible risk factors for T. gondii infection in the included cows by multivariate logistic regression analysis.