An alternative PCR assay with high sensitivity and speci city for the detection of swine toxoplasmosis based on the GRA14 gene

Xi He South China Agricultural University College of Veterinary Medicine Derong Zhou South China Agricultural University College of Veterinary Medicine Yanwu Sun South China Agricultural University College of Veterinary Medicine Yuan Zhang South China Agricultural University College of Veterinary Medicine Xiaogang Zhang Foshan Standard Bio-Tech Co., Ltd., Foshan, Guangdong Province Yabiao Weng South China Agricultural University College of Veterinary Medicine Ruiqing Lin (  rqlin@scau.edu.cn ) South China Agricultural University College of Veterinary Medicine


Background
Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and speci c PCR is crucial for the diagnosis of swine toxoplasmosis.

Methods
To established a high speci city and sensitivity PCR detection method for swine toxoplasmosis, we used T. gondii GRA14 gene as target to design speci c primers and established a PCR detection method for swine toxoplasmosis. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016-2017 were assessed by the newly established GRA14 gene PCR method.

Conclusions
These results indicate that the new PCR method based on the T. gondii GRA14 gene would be useful for the diagnosis of swine toxoplasmosis and that it would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

Page 3/19
Background Toxoplasmosis is a global endemic parasitic disease caused by Toxoplasma gondii, which infects humans and various animals via ingestion of raw or undercooked meat from chronically infected animals or by ingestion of sporulated oocysts found in food, soil and water contaminated with cat feces [1]. In humans, the majority of infections are asymptomatic; however, on rare occasions, mild u-like symptoms are observed [2,3,4], but when mothers are contaminated during pregnancy, congenital toxoplasmosis ranges from asymptomatic cases to fetal death [5]. Additionally, in immunocompromised individuals, it also ranges from pauci-symptomatic cases to fatal visceral involvement [6]. Although toxoplasmosis is often associated with cats and their shedding of oocysts [7], meat is the most common source of infection in developed countries. Among livestock, pigs are susceptible to infection with T. gondii, and infected pigs are considered one of the most important sources of T. gondii infection in humans [3]. Research has reported that 41% of the cases of foodborne human toxoplasmosis in the USA are caused by the consumption of pork, which can thus be considered a main source of infection for humans [8,9].
Pig production is spreading worldwide, and there are two main production systems: specialized intensive systems and free-range organic production. In most experimental studies on the epidemiology of swine toxoplasmosis, endemic outbreaks of swine toxoplasmosis still occur frequently, especially in intensive breeding pig farms, and the morbidity and mortality of pigs are high [10,11]. With the development of the pig industry, the smooth circulation of breeding pigs and hogs has accelerated the global spread and prevalence of toxoplasmosis in pigs, causing severe economic losses to the pig industry and threatening human health. Therefore, the accurate diagnosis of toxoplasmosis in pigs is a prerequisite for the effective prevention and control of human toxoplasmosis.
Pigs are one of the intermediate hosts of T. gondii. Pigs can become infected by food or water contaminated with oocysts. Then, T. gondii sporozoites released from sporulated oocysts invade enterocytes and convert to tachyzoites, and tachyzoites rapidly divide by endodyogeny and disseminate through the blood to other organs and tissues during the acute phase of infection [12]. The immune response of the host causes tachyzoites to switch back to bradyzoites that encyst in skeletal muscle and brain tissues to establish a chronic infection [13]. Moreover, the current diagnosis of T. gondii infection relies mainly on serological detection of speci c IgG and IgM [14,15]. However, serological detection has been limited. Among them, IgM is the rst antibody detected during infection, indicating that the host has recently been infected, and these antibodies can persist for months or even years after acute infection. High levels of IgG antibodies are present in sera, and serological detection does not distinguish a recent infection or previous infection [16]. In addition, serological detection may fail during the active phase of T. gondii infection because the antibody titers are low. PCR methods are useful for the identi cation and diagnosis of the T. gondii acute infection phase [17,18]. Some PCR methods have been used to detect toxoplasmosis, such as those targeting the B1 gene [19], internal transcript spacer (ITS) sequence [20] and 529 bp repetitive element [21], but these methods are mainly used in laboratory research, and the applicability of these methods is ambiguous because the data are limited regarding their use in largescale clinical detection.
Dense granule protein (GRA) is a type of secreted metabolic antigen released from dense granules into nasal cysts after T. gondii invades host cells. It plays an important role in parasite survival and intracellular replication [22] and is considered a potential target for the diagnosis and prevention of T. gondii infection [23,24]. The GRA14 gene has been found in multiple stages of the life cycle of T. gondii, such as tachyzoites, bradyzoites, schizonts, etc. [25], and it can be used as a new diagnostic target for toxoplasmosis. In this study, we used T. gondii GRA14 genes as targets to design speci c primers for T. gondii and established a high-speci city and high-sensitivity PCR detection method for swine toxoplasmosis. By the new PCR methods, we examined the prevalence of T. gondii in intensive breeding pig farms in Guangdong, Fujian, Guizhou, Shaanxi, and Anhui. The data generated improve our understanding of the transmission of T. gondii between pigs and humans.

Specimens
T. gondii, Staphylococcus aureus, Clostridium welchii, Salmonella, and Escherichia coli kept in the Laboratory of Veterinary Parasitology of South China Agricultural University; Isospora suis and Eimeria suis were donated by Foshan Standard Bio-Tech Co., Ltd., Foshan, Guangdong Province.
From January 2016 to December 2017, a total of 5602 blood, semen, wastewater, waste feed, feces, urine, and tissue specimens were collected from intensive breeding pig farms in 7 provinces, Guangdong, Fujian, Guizhou, Anhui, Shaanxi, Anhui and Hunan, China (Fig. 1). Among them, 4645 specimens were collected from 54 farms in Guangdong, 391 specimens were collected from 3 farms in Fujian, 280 specimens were collected from 3 farms in Guizhou, 139 specimens were collected from 3 farms in Shaanxi, 95 specimens were collected from 2 farms in Anhui, 32 specimens were collected from 1 farm in Hunan and 20 specimens were collected from 1 farm in Jiangxi. These farms were selected based on willingness to participate in the study. The main specimens were blood, including 5462 blood specimens.
Blood specimens were obtained by venous blood collection and saved in blood protection solution (Foshan Standard Bio-Tech Co., Ltd., China), and the freshly collected semen, wastewater, waste feed, feces, urine, and tissue specimens were placed into clean plastic bags and transported immediately to the laboratory. The blood, semen, wastewater, waste feed, feces, and urine specimens were stored at 4 ~ 8°C, and the tissues were stored at -20°C.

Genomic DNA extraction
The extraction of T. gondii DNA used different protocols for different samples. For blood specimens, 300 µL of blood was collected and mixed with three times the volume of Red Blood Cell Lysis Buffer (Qcbio Science & Technologies Co., Ltd., China), gently shaken, stored at room temperature for 5 min, and centrifuged at 10,000 rpm for 1 min; then, the supernatant was removed, leaving leukocyte sediment. For semen specimens, frozen semen was removed from liquid nitrogen and immediately placed in a 37°C water bath, shaken gently for 10 s, placed for 20 ~ 30 s and thawed. The DNA of blood and semen specimens was extracted by using a TIANamp Blood DNA Kit (Tiangen Biotech (Beijing) Co., Ltd., China).
For fecal, feed, urine and wastewater specimens, before the DNA was extracted, these specimens were allowed to rest undisturbed at room temperature overnight. The next day, the supernatant uid was removed by vacuum aspiration, and the precipitation liquid was centrifuged at 2,000 ×g for 10 min. Two hundred milligrams of precipitation liquid was used for DNA extraction by using a QIAamp DNA Mini Kit (QIAGEN, Korea). For tissue specimens, the tissue was shredded with sterilized scissors, and 200 mg of tissue specimen was used for DNA extraction with the Wizard SV Genomic DNA Puri cation System (Promega (Beijing) Biotech Co., Ltd., China). The DNA was stored at -80°C before PCR analyses.

Primer design
A conserved region of the dense granule protein 14 (GRA14) gene (GenBank accession number: MH213492.1) was selected to design speci c primers for detecting toxoplasmosis. Primers were synthesized and puri ed by Sangong Company (Shanghai, China). After experimental veri cation, one primer set designated GRA14-3 was screened for further development of the PCR assay. PCR ampli cation was performed using the primers GRA14F-3: (5'-ATGCAGGCGATAGCGCG-3') and GRA14R-3: adjusted to 3 mM, 1.25 µL of each primer at a concentration of 10 pmol/µL, 2 µL of DNA and 9.5 µL of sterile distilled water. Reaction conditions were initial denaturation at 95°C for 5 min; followed by 35 cycles of 95°C for 30 s, 50°C to 66°C, which was chosen to optimize the PCR annealing temperature, and 72°C for 1.5 min; followed by a nal extension step at 72°C for 7 min.
Speci city and sensitivity of the GRA14 gene PCR assay PCR speci city was evaluated using extracted DNA from the blood and tissue of T. gondii-free pigs and other pathogens, such as I. suis, E. suis, S. aureus, C. welchii, Salmonella and E. coli. To determine the sensitivity of the PCR assay, tenfold serial dilutions of T. gondii DNA (RH strain) from tachyzoites at concentrations ranging from 2.35×10 4 to 0.023 tachyzoites were prepared, DNA was extracted from each dilution, and PCR of these dilutions was carried out for each gene target.
Comparison of the GRA14 PCR assay with the RE sequence and B1 gene PCR assays A comparison of the traditional detection primers (RE sequence, B1 gene) and the primers screened in this study (GRA14) showed that 15 pig blood specimens from different farms were detected at the same time.
The targets were the B1 gene (GenBank accession number AF179871) and the 529-bp repeat element sequence (RE) (GenBank accession number AF146527) [21]. The primers were 5'-CGCTGCAGGGAGGAAGACGAAAGTTG-3' (TOX4) and 5'-CGCTGCAGACACAGTGCATCTGGATT-3' (TOX5) for the RE sequence and 5'-GAAAGCCATGAGGCACTC CA-3' and 5'-TTCACCCGGACCGTTTAGC-3' for the B1 gene [26]. The RE sequence reaction conditions were initial denaturation at 95°C for 5 min; followed by 35 cycles of 94°C for 30 s, annealing at 58°C for 30 s, and 72°C for 30 s; and a nal extension step at 72°C for 5 min. The B1 gene reaction conditions were initial denaturation at 94°C for 5 min; followed by 35 cycles of 94°C for 1 min, annealing at 58°C for 30 s, and 72°C for 30 s; and a nal extension step at Clinical specimen detection by the GRA14 PCR assay A total of 5602 specimens collected from 67 intensive breeding pig farms in 7 provinces (specimen types included blood, semen, wastewater, waste feed, feces, urine, and tissues) were tested to date by PCR assay, and the primers were the GRA14 primers screened in this study. These specimens were collected from 7 provinces, Guangdong, Fujian, Guizhou, Anhui, Shaanxi, Anhui and Hunan, China.

PCR product sequencing and statistical analysis
Some positive PCR products of the expected size were sequenced on an ABI3730 autosequencer by Sangon Biotech (Shanghai, China) in both directions using the PCR primers. DNA sequence analysis was undertaken using the National Center for Biotechnology Information (NCBI) BLAST programs and database.
The χ 2 test implemented in SPSS Statistics 21.0 (IBM Corp., New York, NY, USA) was used to compare differences in infection rates among sampling date, season, and sample types. Differences were considered signi cant at P < 0.05.

Optimization of GRA14-PCR assays
Conserved regions inside the GRA14 gene were used to design candidate primer pairs; among them, one of the primer pairs named GRA14-3 had a high positive rate and no nonspeci c bands (Fig. 2a). The optimal reaction annealing temperature was 65°C for the established PCR assay (Fig. 2b).
The PCR assay was found to be highly speci c for the detection of T. gondii DNA, and there was no ampli cation from any of the heterologous control samples, including pig blood DNA, pig tissue DNA, I. suis, E. suis, S. aureus, C. welchii, Salmonella and E. coli. (Fig. 3a).
The sensitivity of the GRA14 PCR assay was determined by 10-fold serial dilution of T. gondii tachyzoite amount (2.35×10 4 ~0.0235). The results showed that the method had a limit of detection of 2.35 T. gondii tachyzoites (Fig. 3b).
Speci city and sensitivity comparison of the B1, RE and GRA14 PCR assays The speci city of the GRA14 PCR assay was compared with that of the protocols used to amplify B1 and RE. We performed PCR using the same 15 blood specimens from the different farms and showed that the GRA14 PCR assay had better speci city than the B1 and RE PCR assays. Ampli cation was observed in 15 DNA blood specimens by the RE sequence PCR assay, but there were nonspeci c bands, and it was not easy to determine positivity (Fig. 4a). Only two specimens were positive, and 11 T. gondii-positive samples detected by the GRA14 gene were negative by the B1 gene PCR assay ( Fig. 4b and 4c). Furthermore, the positive PCR products were con rmed by sequencing. gondii than other cities (χ 2 = 84.2, P < 0.0001). Specimens collected in 2017 (19.1% or 886/4639, P < 0.05) had a higher infection rate than those collected in 2016 (16.1% or 155/963, P < 0.05). There were signi cant differences in detection rates for samples collected during the seasons (Table 1), and the infection rates of T. gondii in pigs were 22.8% (670/2940), 3.8% (57/1479), 39.4% (187/474) and 18.2% (129/709) in spring, summer, autumn and winter, respectively. Of the 5602 specimens analyzed, the kind of specimens, including blood, wastewater, tissue, waste feed, semen and urine, varied, and the main specimens were blood; the infection rates of T. gondii in pigs were 18.9% (1033/5462), 3.8% (2/53), 3.0% (1/33), 33.3% (5/15), and 0% (0/95), and 12.5% (1/8) in blood, semen, wastewater, tissues, and urine ( Table 2). Of the 963 blood specimens collected from pigs at different growth stages in 2016, including sows, boars, fattening pigs and piglets, 132 (16.3%) were positive for T. gondii. The detection rates among the different growth stages ranged from 0% (0/29) to 18.6% (122/655) (Fig. 5).

Discussion
The pig industry is important to the economy of many countries because pork is a main source of food for humans. Infected pigs are a source of T. gondii infection for humans and animals in many countries [27,28]. Therefore, it is of great importance for public health to establish a rapid, highly sensitive and speci c detection method for swine toxoplasmosis, especially during acute infection. In this study, based on the GRA14 gene of T. gondii, a sensitive and speci c PCR-based method for the rapid and direct detection of swine toxoplasmosis was developed.
A comparison of the detection of GRA14 and previously reported genes (B1 and the 529-bp RE) showed that PCR using GRA14-speci c primers ampli ed Toxoplasma-speci c DNA more e ciently. In the past few decades, different primer sets have been developed to detect T. gondii, including those targeting the B1 gene and the 529-bp RE [21,29]. B1 ampli cation has been used more often for T. gondii detection, although it has shown lower speci city in some cases than other primer sets [30]. Although the 529-bp RE showed greater sensitivity, and some studies showed that it is 10 times more sensitive than B1 [31,32], some strains have partially or entirely lost repetitive units, which results in lack of detection of T. gondii infection. In this study, we developed a new PCR method to detect T. gondii in pigs based on the GRA14 gene, which was more sensitive than PCR ampli cation of the B1 gene and more speci c than PCR ampli cation of the 529-bp RE. To ensure the validity of our investigation, all samples were randomly selected from clinical samples, including some that were positive samples. Evaluation of the detection sensitivities and speci city of PCRs using primers targeting the genes examined here demonstrated that GRA14-PCR is the most effective in amplifying Toxoplasma DNA.
GRA14-PCR could detect a minimum of 2.35 tachyzoites of T. gondii, and it can be used for detection during early T. gondii infection; the detection limit of diluted tachyzoites was four tachyzoites for 529-bp RE PCR and 40 for B1 PCR [21]. These results indicate that GRA14-PCR is an excellent method for the diagnosis of toxoplasmosis.
Furthermore, we found that GRA14-PCR was effective in amplifying T. gondii DNA in blood, tissue, semen, urine and waste feed. In this study, we used the GRA14-PCR method to detect mainly blood specimens, including 5462 specimens because, during the acute phase of T. gondii infection, the sporozoites released from sporulated oocysts invade enterocytes and convert to tachyzoites, and tachyzoites rapidly divide by endodyogeny and disseminate through lymphatic and blood circulation to other organs and tissues. Therefore, the detection of blood specimens can better identify the acute infection period of toxoplasmosis and result in timely treatment for pigs.
A total of 5462 blood specimens from 5 provinces in China were further applied in epidemiological studies with the GRA14-PCR method.  [39] and Brazil (12.5% or 38/296) [40] but lower than that reported in Mexico (50.8% or 218/429) [41] and Italy (57.1% or 12/21) [42]. The signi cant difference in the prevalence of T. gondii infection may come from the geographical distribution, animal species, or different detection methods.
In this study, statistical analysis showed that there was a signi cant difference in pigs among various sampling regions (P < 0.0001). According to our statistical analysis, the prevalence of T. gondii infection in pigs varied from 0 to 31.7%. Shaanxi had highest infection rates of T. gondii in this study, and Jiangxi and Anhui provinces had the lowest infection rates, where the specimen numbers were much different. We need to obtain additional specimens from Anhui, Jiangxi and Hunan in a later study.
Our data showed that there were signi cant differences in detection rates for specimens collected during the different seasons (P < 0.0001). Autumn (39.5%, 187/474) had higher infection rates of T. gondii than the other seasons, followed by spring and winter infection rates of 22.8% (670/2940) and 18.2% (129/709), respectively. Lower rates were observed in the summer (3.9% or 57/1479). This result is different from that in a previous report in which higher infection rates of T. gondii were observed in autumn and summer, and lower rates were observed in winter [37]. We need to obtain additional specimens from different climates in future studies.
This study showed that samples collected in 2017 (19.1% or 886/4639, P < 0.05) had a higher rate of exhibiting T. gondii infection than those collected in 2016 (16.1% or 155/963, P < 0.05), possibly due to the rapid development of the pig industry in China and the weak public awareness of sanitation husbandry practices in pig production; the agricultural sector's propaganda and guidance need to be strengthened.

Conclusions
In this study, we demonstrated that the GRA14 gene would be an excellent target for primer design for swine T. gondii infection detection, particularly in the acute infection period. The GRA14-PCR assay targeting conserved regions in the GRA14 gene showed more sensitivity and speci city than the 529-bp RE and B1 PCR assays. Based on this assay, the higher the T. gondii infection rates in the studied intensive breeding pigs are, the higher the risk of eating undercooked or raw pork meat in some southern and northern regions of China, raising a major public health concern. It is essential to establish e cient strategies to prevent and control T. gondii infection in intensive breeding pigs and humans in these investigated regions. These ndings contribute to improving the diagnosis of toxoplasmosis, especially in the incubation period when the correct and precise diagnosis is fundamental to early treatment in areas.