Low prevalence of Toxoplasma gondii in pork from slaughter houses in the central of China

Background: Toxoplasma gondii is widely distributed and can infect many species of warm-blooded animals , including swine. This study aimed to evaluate the prevalence of T. gondii infection in pork from the cent er of China. A total of 2 798 samples, including 305 hearts, 2086 diaphragm s , and 4 0 7 sera were collected from Henan Province, China . The modified agglutination test was used to detect antibodies against T. gondii in ser a from jugular vein blood and heart blood (cut-off: 1:25) , diaphragm exudate (cut-off: 1:10) . T. gondii DNA was screened from the digestive juice of all diaphragm tissue samples , and attempt to isolate viable T. gondii strain by bioassay in mice. Results: A total of 9.94% (278/2798) swine showed sera conversion of T. gondii antibodies. Region , but not gender , was associated with T. gondi infection in swine. T. gondii nucleic acid w as not found in the meat digestive juice s ( 2090 swine ) . Three groups of mice showed T. gondii antibodies after having been bioassayed with diaphragm samples (3/81). Unfortunately, no viable T. gondii strain was isolated from pork. Conclusions : This is the first large-scale survey T. gondi infection in pork from central China. Overall, the prevalence of viable T. gondii in pork was extremely low. Nevertheless, T. gondi i infection is present in swine from center China. Consumers could acquire T. gondii infection from ingestion of raw or undercooked pork.


Background
Toxoplasma gondii is an obligate intracellular parasitic protozoa that is widely distributed worldwide and can infect warm-blooded animals, including swine [1]. Swines are susceptible to T. gondii infection and may become infected with T. gondii by consumption of oocysts, tachyzoite transplacental transmission, and consumption of meat containing tissue cysts [1]. China is the largest country of producer and consumer of pork, and its annual production was estimated at 55 million metric tons. Pork is known as one of the most common sources of human T. gondii infection in China [2].
The overall estimated seroprevalence for T. gondii in swine is 32.9% from published papers of 2000-2017 in China, and T. gondii strains have been isolated from swine and genotyped as ToxoDB#9, ToxoDB#3, and ToxoDB#1 [3][4]. Overall, the infection of T. gondii in swine samples from hospitals is higher than that of farms and slaughterhouses [4]. This phenomenon suggests that T. gondii infection may be beneficial for subsequent infection by other pathogens. Few clinical toxoplasmosis in pregnant sows and in fattening swine was reported [4]. Young swine may die from toxoplasmosis without entering the human food chain, however, most T. gondii infection in swine was subclinical, and thereby the pork could serve as a source of human infection [1]. However, no pork inspection strategy for T. gondii contamination has been established, and no performance standards for processing T. gondii-positive meat have been developed. Diaphragm, tongue and heart are the most successful isolation T. gondii tissues for the swine [1]. The objective of this investigation was to estimate the seroprevalence of T. gondii infection in pork from slaughterhouses, and try to isolate viable T. gondii. To our knowledge, the present study is the first large-scale investigation of T. gondii infections in pork from center China.

Results
In the present study, 9.94% (278/2798) (95% CI, 8.88%-11.10%) of the examined swine was seropositive for T. gondii infection by MAT (Table 1). A total of 95 sera, four heart, and 179 diaphragm samples were determined to be positive for the T. gondii antibody. The seroprevalence of T. gondii infection in swine from 13 cities ranged from 0-33%. The seroprevalence rates of T. gondii varied with regions. A high prevalence was observed in samples from Xuchang and Xinyang, compared to the other regions (P < 0.05), and no seropositive serum from swine was observed in Zhoukou and Puyang.
Risk factors in relation to geographic location and gender were analyzed. The seroprevalence of T. gondii by region is shown in Table 2. The seroprevalence of T. gondii in swine from the south of Henan (18.23%, 64/391) was higher than that in the center of Henan (7.50%, 103/1,353) and the north of Henan (7.49%, 34/454), with a statistical significance of P < 0.05. Only gender information from 235 swine samples was available.

Discussion
T. gondii detection methods include serological methods and etiology assay. Serological methods are more appropriate in detecting T. gondii for monitoring meat safety, which could detect specific anti-T. gondii immunoglobulin in serum and meat juice within the food chain [5]. However, because the concentration of blood in the meat juice was low compared to that in the serum, it was low sensitivity, and adjusting the dilution factor of meat juice could improve sensitivity [6,7]. For swine, from serological assays, it has been shown that meat juice from hearts should be diluted to approximately a fifth of the serum dilution; diaphragm samples had T. gondii titers less than one tenth of the serum titer [7].
Previous reports have screened for T. gondii antibodies in swine sera at a 1:25 dilution or higher by MAT [1,3], prompting us to use this as a cut-off in the present study. The MAT has been extensively used to detect IgG antibodies to T. gondii in sera or body fluids of animals, and its validity was supported by the isolation of T. gondii from pigs [1,3,8,9]. We adopted a cut-off value of 1:25 for serum and serum from heart blood, and a cut-off value of 1:10 for the diaphragm exudate. They gave the best agreement with the serum results according our previous work [8]. However, serological testing has its limitations; for instance, it may fail to detect the active phase of T. gondii infection, or the test may not detect T. gondii infections in immunocompromised animals. Therefore, testing meat exudate may lead to underestimation of the number of positive samples.
The seroprevalence of T. gondii based on screening swine was 11.6% worldwide [1], and 32.9% for China [4]. In this study, 9.94% of the examined swine samples were found to be seropositive for T. gondii infection, which is lower than that of the rest of the world as well as China's average infection rate. It is also lower than the seroprevalence of T. gondii in free-range chickens, ostriches, sheep, swine, domestic cats, and large cats in Henan Province [10][11][12][13][14][15]. The reasons for the differences were as follows: the samples in this study were collected from healthy swine in slaughterhouses, improved management and intensive swine farming was developed in China recently. The maximum titer against T. gondii antibodies in pork was 1:12800 in this study (one diaphragm exudate), this was higher than that other reports (1:64-1:1024) [1,16,17]. After bioassayed analyze in this study, mice from diaphragm with low MAT titer (< 1:10) showed T. gondii positive antibody in mice (Table 3). This result indicated that seronegative sample does not guarantee that the pork is free of T. gondii.
The seroprevalence of 9.94% in this survey indicates that swine from the central of China are widely exposed to T. gondii. The route of T. gondii infection in swine is probably by ingestion of T. gondii oocysts or cysts. This finding suggests that swine from Henan province had contacted with T. gondii oocysts from cats or from soil, water, feed, infected rodents, or cysts from kitchen garbage. The seroprevalence was highest in south of Henan province, suggesting that higher temperature and humid environment favors the survival of oocysts in this region. The results of the present study agree with those of previous reports [18][19]. In this study, female swine were found more susceptible to T. gondii than male swine, which may be related to the fact that male swine as breeding pigs are often kept alone, and have less contact with cats and rodents reducing the opportunity of contact with T. gondii oocysts. This agreed with prior report in swine [20].
PCR and histopathology were insensitive for the detection of T. gondii cysts in pork. PCR could detect trace amounts of T. gondii DNA in blood and tissues. However, the prevalence of T. gondii chronic infection or natural infection cases is often underestimated due to limited information on the distribution and quantity of parasites. Furthermore, the cysts are not randomly and evenly distributed in tissues [1]. In this study, isolating T. gondii DNA from the homogenate of a large meat samples (50 gram or 5 gram meat for one swine) rather than using a small sample would increase PCR sensitivity. However, T. gondii DNA was not detected in the pork digestion fluids during this survey (81 tissue homogenate, which came from 2090 swine), which is less than our previously survey (2.06%, 34/1647) from tissue samples in Henan province by T. gondii B1 gene [21]. Our result indicated that the relative low density of T. gondii parasites in pork.
Bioassays using mice or cats are the gold standard for detecting viable T. gondii in meat, which can detect even just a few parasites [1,[22][23]. Bioassays using cats are more sensitive than mice. However, using cats is more expensive and opposed by animal protection organizations. Therefore, the mouse bioassay was used for the present survey.
However, bioassays involving mice or cats require at least six weeks of monitoring, which is a relatively long time and thus is not suitable for application to slaughterhouses.
The result of T. gondii isolation reports showed that diaphragm, coppa muscle, tongue, and heart are the more viable tissues in swine [16,24]. The diaphragm and heart were selected in this study, and the sera of three group mice showed T. gondii positive antibody. However, no T. gondii parasite was observed from mice and no viable T. gondii was isolated from pork. However, we could not be sure the safety of pork that only relatively small amounts (50 g or 5 g) of pork have been tested for viable parasites, compared the whole swine (100 Kg). The result may be explained by the relative low density or avirulence of T. gondii in pork.

Conclusion
The results of present study showed 9.94% prevalence of T. gondii antibody, low T. gondii DNA detection rate, and low viable T. gondii strainisolated rate was obtained from pork in center China. Considering that pork is the meat with the highest consumption, and its rate of consumption is still steadily growing in China, there is a risk of humans being infected by T. gondii through pork. Consumers, human and other carnivores, should take precautions to avoid becoming infected T. gondii by eating raw or undercooked pork and swine by-products. These swine were males or females, and their ages ranged from 5 to 8 months. The names of the slaughterhouses and sample collection dates were recorded. The samples were allowed us to survey for T. gondii infection. Fluide samples (sera, sera from heart blood, and muscle exudate from diaphragm) and tissue samples were stored at 4°C and tested for T. gondii antibodies or bioassay in mice within one week.

Assessment of T. gondii antibodies in swine samples
The serum, serum from heart blood, and diaphragm exudate samples were tested for antibodies against T. gondii by modified agglutination test (MAT) [25]. Serum or serum from heart blood with MAT titers of 1:25 or higher were considered positive for T. gondii
After the inoculation, the clinical symptoms of the mice were observed daily and recorded.
Smears of the lungs, mesenteric lymph node, and brain of dead mice were examined for T. gondii tachyzoite or cysts within 60 days post inoculation (DPI). After 60 DPI, the mice were bled, and sera were tested for T. gondii antibodies using MAT with the dilution of    "-" It was not tested.   This map has been provided by the authors.