Seroprevalence, Isolation, Genotyping, and Pathogenicity of Toxoplasma gondii Strains from Sheep in China

Toxoplasma gondii is an important cause of reproductive failure in small ruminants that also poses a risk to consumers who consume undercooked meat. However, little is known about sheep toxoplasmosis in China for the world. Therefore, this study was conducted to assess the prevalence of T. gondii infection in sheep from China, to isolate T. gondii via bioassay in mice and to evaluate the virulence of the isolated T. gondii based on vero cell invasion and mice. A total of 840 samples (304 unfrozen hearts and 536 sera) from sheep in China were collected from 2014 to 2016. Heart samples (n = 36) of T. gondii seropositive sheep (MAT, ≥25) were bioassayed in mice individually. DNA derived from cell cultured tachyzoites of the isolated T. gondii was characterized by PCR-RFLP of 10 loci (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The virulence of the T. gondii was evaluated based on the mortality and encystation in mice, as well as their growth characteristics in cell culture. Antibodies to T. gondii were found in 174 of 840 (20.71%, 304 hearts juice and 536 sera) sheep by the modified agglutination test (cut-off 1:25). Viable T. gondii was isolated from the hearts of two of 36 seropositive sheep hearts. Both genotypes of the sheep heart isolates were ToxoDB#9. The virulence of the two ToxoDB#9 isolations varied significantly. To the best of our knowledge, this is the first report of isolation of ToxoDB#9 strain of T. gondii from sheep in China.


INTRODUCTION
The parasite Toxoplasma gondii is a major cause of reproductive failure in small ruminants, including sheep. Veterinary Investigation Diagnosis Analysis data from 2014 showed that about 25% of ovine production problems were caused by T. gondii (www.gov.uk/government/ statistics). Moreover, viable T. gondii has been isolated from goat meat, milk and cheese (Dubey et al., 2014a,b). T. gondii infection is widespread among humans. The prevalence of T. gondii is higher in Latin America than in North America and East Asia (Dubey, 2010); however, the cause of this difference is not known. T. gondii cause lymphadenopathy, Abbreviations: MAT, modified agglutination test; DPI, days post-inoculation; PCR, polymerase chain reaction; ROP, rhoptry protein; GRA, dense granule. retinochoroiditis, encephalitis, abortion, and the death of immunocompromised patients (Hide, 2016). In China, mutton is the main ingredient of hotpot, which often results in meat being undercooked. Therefore, the consumption of undercooked meat containing T. gondii tissue cysts could pose a health risk to consumers.
Isolation of viable T. gondii from feline has been the most successful model in China. Among 122 viable T. gondii isolates from animals and humans in China, 85 strains (69.7%) were isolated from tissue or fecal samples from cats. Moreover, 73 (85.9%) T. gondii isolates from cats were genotyped as ToxoDB#9, while 10 were genotyped as ToxoDB#1(1), ToxoDB#2(1), ToxoDB#10(1), ToxoDB#17(1), ToxoDB#18(2), and ToxoDB#205(4), and the genotypes of the other two strains were not determined (Dubey et al., 2007;Zhou et al., 2009;Chen et al., 2011;Qian et al., 2012;Wang et al., 2013a;Yang et al., 2015;Wang D. et al., 2016;Wang Q. Q. et al., 2016). However, only one isolate of T. gondii has been obtained from sheep in Qinghai (type II) (Zhou et al., 2009), and no studies have reported isolation of viable T. gondii from other small ruminants in China. China has the largest number of sheep in the world, with an estimated 187 million domestic sheep (https://top5ofanything. com/list/d4d1ef5e/Countries-With-the-Most-Sheep) and an unknown number of wild sheep. The clinical and economic importance of sheep toxoplasmosis remains uncertain, and most epidemiological literature was published in Chinese. Therefore, the present study was conducted to summarize these Chinese papers and present the sero-prevalence in sheep from different geographical areas. Further, the prevalence of T. gondii infections in sheep from China was investigated, and an attempt to isolate viable T. gondii was made.

Sheep Sample Collection
A total of 840 domestic sheep samples (304 fresh hearts and 536 sera) were collected from individual farms in Henan, Xinjiang, Zhejiang, and Jiangsu Province (Table 1, Figure 1).  the climatic transition belt between the warm-temperate and subtropical zones. Xinjiang (41 • N, 85 • E) has a semi-arid and desert climate. Domestic sheep in China are part of the farmers's household, and live with whatever other animals the farmers own, including cats. Unfrozen hearts and serum from sheep were collected between 2014 and 2016. In addition, juice was obtained from 304 hearts. Blood was obtained from jugular veins of 536 sheep. The juice or blood samples were allowed to clot, then centrifuged at 2000 × g for 10 min, after which the supernatant were separated and stored at −20 • C until tested. Available background information is summarized in Table 1.

Serological Examination
Serum and heart juice samples from 840 sheep were tested for antibodies to T. gondii using the modified agglutination test (MAT) (Dubey and Desmonts, 1987). Whole formalin fixed RH T. gondii tachyzoites were kindly provided by Dr. J. P. Dubey (ARS, USDA). A titer of 1:25 was considered indicative of exposure to T. gondii. In addition, sera and heart juice were double diluted further with 0.01 M phosphate buffered saline (PBS), then tested for T. gondii parasites. Briefly, 100 mL 0.01 M PBS was amended with 8.5 g NaCl, 0.308 g NaH 2 PO 4 (M.W. 120), and 1.08 g Na 2 HPO 4 (M.W. 142), after which the pH was adjusted to 7.2 (Dubey, 2010).

Isolation of Viable T. gondii from Sheep Hearts by Bioassay in Mice
Specific-pathogen-free Kunming mice were supplied by the Zhengzhou University Laboratory Animal Center. Eight-weekold female Kunming mice were used in this study. Heart samples of T. gondii seropositive sheep (MAT, ≥25) were bioassayed in mice separately. The myocardium (50 g) was then homogenized, digested in pepsin (5.2 g pepsin, 10.0 g NaCl, 14 mL HCl, diluted to 1 l with deionized water, pH 1.1-1.2). The heart homogenate was subsequently incubated at 37 • C in a shaking water bath for 60 min. After which, the sample was filtered by double gauze and centrifuged at 1200 × g for 10 min. The supernatant was then removed and the pellet was suspended in 0.01 M PBS (pH 7.2) and neutralized by mixing with 1.2% sodium bicarbonate. Following mixing, the sample was centrifuged at 1200 × g for 10 min, after which the supernatant was removed and 5-10 mL of saline containing 1000 units penicillin and 100 µg of streptomycin per ml was added. Myocardium digested liquid was then inoculated subcutaneously into four Kunming mice (1 mL per mouse) that had been maintained on drinking water supplemented with dexamethasone phosphate (DXM, 10 µg/ml) for 3 days before inoculation (Dubey, 2010). DXM treated mice were utilized as a control group, while DXM untreated mice were utilized as a blank group. Lung or brain impression smears of dead mice were examined for T. gondii tachyzoites or cysts. Survivors were bled on day 60 post-inoculation (DPI) and 120 DPI. 1:25 and 1:200 dilutions of sera from each mouse were tested for T. gondii antibodies with the MAT. Mice were killed 120 DPI and their brains were examined for tissue cysts after a squash preparation. All brains of survivors were homogenized and sub-passaged into new groups of mice subcutaneously.

In vitro Cultivation and Genotyping
Brain homogenates of T. gondii positive mice were seeded into vero cell culture flasks as previously described (Dubey, 2010). The number of cysts in the brains of mice were counted microscopically using the method reported by Dubey et al. (2012). Briefly, whole mouse brain was homogenized with 1 mL of saline (0.85% NaCl), tissue cysts were counted microscopically in 50 µl of the homogenate, and the count multiplied by 20 was the number of tissue cysts per brain. The time required for tachyzoites grow up in cell culture was recorded. DNA was extracted from cell culture derived tachyzoites using a commercial DNA extraction kit (Tiangen Biotec Company, DP304, China). The multiplex PCR of the T. gondii isolates was performed using 10 PCR-RFLP genetic markers, SAG1, SAG2 (5 ′ -3 ′ SAG2, alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico as previously described by Su et al. (2010). Reference T. gondii DNA was included in all batches.

Evaluation the Virulence of T. gondii Tachyzoites Isolated from Sheep by Mice
Fresh tachyzoites were collected from cell culture, counted in a disposable hemocytometer and diluted 10-fold from 10 −1 to 10 −6 to reach an end-point of <1 tachyzoite. Next, <1, 10 0 , 10 1 , 10 2 , 10 3 , or 10 4 tachyzoites were inoculated intraperitoneally into five Kunming mice for each dilution. Clinical symptoms, illness and death of mice were observed and recorded every day. Lung or mesenteric lymph node impression smears of dead mice were examined for T. gondii tachyzoites. At 30 DPI, sera from mice were analyzed for T. gondii antibody. The virulence was evaluated based on the percentage of dead mice among T. gondii positive mice.

Ethics Approval and Consent to Participate
This study was carried out in accordance with the recommendations of the institutional animal use committee of the Henan Agricultural University (China). The protocol was approved by the Beijing Association for Science and Technology (approval SYXK [Beijing] 2007-0023).

Statistical Analysis
Statistical analysis was performed using the Graph Pad Prism 4.0 software (Graph Pad Software Inc., San Diego, CA, USA). Data were analyzed by the chi-squared test or Fisher's exact test. A P < 0.05 was considered statistically significant.

Seroepidemiology of T. gondii in Sheep
Antibodies to T. gondii were found in 174 of 840 (20.71%) sheep with titers of 1:25 in 48, 1:50 in 2, 1:100 in 24, and 1:200 or above in 100. Seropositivity rates varied with respect to source of sheep. The difference in seroprevalence of T. gondii in sheep from Henan Province (29.33%), Zhejiang Province (21.15%) and Jiangsu Province (18.22%) was not significant. However, the prevalence was higher in all of these regions than in Xinjiang (1.96%) (P < 0.05) ( Table 1).

Isolation and Virulence of T. gondii from Sheep
A total of 36 T. gondii seropositive sheep heart homogenates were bioassayed in four Kunming mice individually. T. gondii antibodies were only detected in 10 groups of mice at 60 DPI. Specifically, one mouse was positive in eight groups, three mice were positive in one group, and four mice were positive in one group. However, T. gondii antibodies were only detected in mice from the last two groups at 120 DPI, while the other eight groups were negative. Viable T. gondii were isolated from the two positive groups by Kunming mice ( Table 1). All mice remained asymptomatic. T. gondii tissue cysts from the brain were detected in these mice when killed at 120 DPI. The average number of brain cysts in T. gondii infected mice was 1900 ± 141 from sheep heart sample 20151110#24, while 100 ± 53 cysts were observed in heart sample 20151110#28. The brain homogenates of T. gondii positive mice were sub-inoculated into mice, and seeded onto cell cultures for propagation of tachyzoites. Two isolates (TgSpHn1, TgSpHn2) were successfully propagated in cell culture and mice. Genetic typing of the isolates from sheep hearts revealed that they were all ToxoDB genotype #9 (Chinese 1) ( Table 2).

DISCUSSION
In this study, the prevalence of antibodies and titers to T. gondii in sheep from Henan, Jiangsu, and Zhejiang province was higher than in sheep from Xinjiang ( Table 1). The climate of Henan, Zhejiang, and Jiangsu is subtropical, whereas that of Xinjiang is semi-arid and desert. The arid climate of Xinjiang may contribute to the low prevalence of toxoplasmosis in the region. These results are consistent with those of other reports (Table 4, Figure 1). We have summarized available reports on sheep toxoplasmosis in Table 4. The prevalence of antibodies and titers to T. gondii was 2-39% in sheep from different parts of China (Table 4), indicating widespread environmental contamination with T. gondii oocysts. These findings are in accordance with  Figure 1.
Frontiers in Microbiology | www.frontiersin.org those of our previous investigation of T. gondii in free-range chickens (Feng et al., 2016). Oral ingestion of T. gondii oocysts is the main source of infection for sheep, and poses a risk for exogenous transplacental transmission in pregnant sheep (Innes et al., 2009). Moreover, a previous study confirmed that reactivation of T. gondii cysts in chronically infected sheep serves as another important risk for endogenous transplacental transmission in sheep during pregnancy (Williams et al., 2005;Hide, 2016). Additionally, mutton containing T. gondii tissue cysts poses a threat to human health. Mutton is the main ingredient of hot pots, instant-boiled mutton, dumpling and kebabs in China, however, these cooking methods are often not sufficient to eradicate T. gondii. Moreover, the high prevalence of T. gondii in aborting sheep observed in the present study indicate that T. gondii is a risk factor for abortion. Increasing rates of prevalence of T. gondii antibodies in older sheep indicated post-natal exposure of T. gondii infection, which agreed with the results of previous studies (Dubey, 2010).
T. gondii antibodies were detected in mice of 10 groups at 60 DPI, but were no longer present in eight groups at 120 DPI. Moreover, mice in eight groups were all negative for T. gondii after sub-passage in mice. After checking the T. gondii DNA of the eight injected sheep heart pepsin digested liquid by PCR, none of them were positive. We do not know the reason for this phenomenon, therefore, more studies investigating this interesting observation should be conducted.
The MAT we used has been extensively employed for the detection of T. gondii antibodies in many species, including humans and sheep (Dubey, 2010). Viable T. gondii was isolated from 100% (3/3) of sheep with MAT antibodies above 1:800 (Dubey et al., 2014a). The isolation of viable T. gondii is the gold standard for detecting live T. gondii parasites. However, the success of isolation depends on the tissues tested and the methods used. The density of T. gondii cysts in the heart has been shown to be higher than that in the brain or muscle, and the heart is the ideal choice for isolation of T. gondii (Dubey et al., 2015). Felid bioassay is the most sensitive method for identification of T. gondii (Dubey, 2010). For murine bioassay, the use of immunosuppressed mice facilitates early detection of T. gondii. Immunosuppression of mice by dexamethasone has been shown to be a useful method of isolating T. gondii (Qian et al., 2012). Moreover, viable ToxoDB#9 strains of T. gondii were isolated from cats, pigs, voles and humans from China in a previous study (Dubey et al., 2007;Chen et al., 2011;Wang et al., 2013a,b). Additionally, DNA from Hipposideros larvatus, sika deer, goat, Cebus apella and masked palm civets was genotyped and identified as ToxoDB#9 (Jiang et al., 2014;Miao et al., 2015;Cong et al., 2016;Hou et al., 2016). When combined with our results, these findings indicate that ToxoDB#9 is predominant and widespread in animals from China, including sheep. These results indicate the limited genetic diversity of T. gondii from China.
The virulence of T. gondii was assessed based on their growth rates in cell culture and outbred mice after intraperitoneal injection of dilutions of tachyzoites. The pathogenicity, encystation and growth rate in cell culture of TgSheepHn1 were all stronger than those of TgSheepHn2. ToxoDB#9 isolates were previously reported to have different virulence and pathogenicity in mice (Cheng et al., 2015), which is in accordance with the virulence of the two isolates from sheep observed in the present study. Continued passages of a strain in mice or cell culture can alter the virulence (Dubey, 2010). In the present study, these factors were considered before making conclusions concerning the virulence of strains. Virulence and genome structure analyses of different ToxoDB#9 stains of T. gondii showed remarkable variation in ROP 16 and GRA15 (Li et al., 2014). The diversity of ToxoDB #9 stains of T. gondii may be connected to the invasion and immune response of this successful parasite.
The results of the present study showed that there is widespread exposure of sheep to T. gondii in China. Two viable ToxoDB#9 stains of T. gondii were isolated from sheep hearts and found to have different virulence. To the best of our knowledge, this is the first report of isolation of ToxoDB#9 from sheep in China. Because this organism remains present in the tissues of sheep and can therefore infected people via consumption of undercooked meat, sheep pose a risk of T. gondii infection and have the potential to impact public health.