Molecular detection and genotypic characterization of Toxoplasma gondii infection in bats in four provinces of China

Background Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts, including humans. Limited information about T. gondii infection in bats is available in China. The objective of the present study was to determine prevalence and genetic characterization of T. gondii infection in bats in Jilin, Liaoning, Jiangxi and Guangdong provinces, China. Methods During May 2005 to August 2013, bats were sampled from Jilin, Liaoning, Jiangxi, and Guangdong provinces, China, and liver tissues were collected for the detection of T. gondii by a nested PCR targeting the B1 gene. The positive samples were genotyped at 11 genetic markers (SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and Apico) using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results A total of 626 bats representing 10 species were examined for T. gondii infection, 38 (6.1%) were tested positive with by PCR, 8 positive DNA samples were completely genotyped, of which 3 samples (2 from Cynopterus sphinx, and 1 from Murina leucogaster) represented ToxoDB#10, and 5 samples (2 from Murina leucogaster, 2 from Myotis chinensis, and 1 from Rhinolophus ferrumequinum) belonged to ToxoDB#9 (http://toxodb.org/toxo/). Conclusions The present study revealed an overall T. gondii prevalence of 6.1% in bats from Jilin, Liaoning, Jiangxi and Guangdong provinces in China, and reported two T. gondii genotypes (ToxoDB#9 and #10) having a wide geographical distribution in China. These results provide new genetic information about T. gondii infection in bats, and have implications for better understanding of the genetic diversity of T. gondii in China and elsewhere.


Background
Toxoplasma gondii is an obligate intracellular protozoan parasite with a worldwide distribution, which can infect a wide variety of warm blooded animals and humans. Approximately one third of the world population and 7.9% of population in China are seropositive for T.
gondii antibodies [1][2][3]. T. gondii infection can cause serious diseases in the developing fetus and immunocompromised individuals [4]. It is transmitted to humans and animals via ingesting T. gondii tissue cysts from undercooked meat, by consuming water or food contaminated with T. gondii oocysts, or through transplacental transmission [5,6].
Bats are considered as important natural reservoir of many zoonotic viruses, such as rabies virus, Hantavirus, Marburg virus, Nipah virus, Ebola virus and severe acute respiratory syndrome coronaviruses [7,8]. Besides viruses, bacteria, fungi, and protozoa have been detected in bats, and can potentially be transmitted to humans [9][10][11]. Bats are important to public health, as they live in different habitats and have high mobility and the possible interactions with humans.
Although bats play significant roles in the transmission of some important zoonotic diseases, there were only limited reports on T. gondii infection in bats. The prevalence of T. gondii infection was 10.4% in British bats [12], and 29.3% in bats in Myanmar [13]. Additionally, there were two cases of toxoplasmosis described in captive bats in Australia [14], and several T. gondii strains have been isolated in Kazakhstan and in Brazil [15,16]. In China, T. gondii seroprevalence in five bat species in four provinces was detected using modified agglutination test (MAT) [17]. However, there was only one previous report on genotypes of T. gondii in bats in Yunnan and Guangxi, southern China [18]. In the present study, we determined the prevalence and characterized T. gondii isolates in bats in other four provinces of China.

Ethics statement
This study was approved by the Ethics Committee of Military Veterinary Institute, Academy of Military Medical Sciences. Bats were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.

Study sites and bat collection
The study was conducted in four provinces of China, including Jilin (41°-46°N, 122°-131°E), Liaoning (38°-43°N , 118°-125°E), Jiangxi (24°-30°N, 113°-118°E), and Guangdong (20°-25°N, 109°-117°E) from May 2005 to August 2013. Jilin and Liaoning provinces are located in northeastern of China, where the average temperature is lower than other two provinces. Jiangxi province, located in southeast China, has an average annual temperature of 11.6-19.6°C. Guangdong province lies on southern edge of mainland, with an average annual temperature of 22.8°C. Bats were captured at roosts with hand nets, and identified to species in the field.

DNA extraction and PCR detection
Bats were euthanized, and liver tissues were collected for genomic DNA extraction using the TIANamp Genomic DNA kit (TianGen, Beijing, China). T. gondii infection was examined by a semi-nested PCR targeting the B1 gene as described elsewhere [19][20][21]. The positive samples were used further for genetic characterization.

Genetic characterization of T. gondii
Multilocus PCR-RFLP was conducted to genetically characterize T. gondii in bats, using 11 genetic markers (i.e., SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) as described previously [12,18]. Briefly, the target sequences were first amplified by multiplex PCR using external primers for all 11 markers. Nine reference strains, namely GT1, PTG, CTG, MAS, TgCgCa1, TgCatBr5, TgCatBr64, TgRsCr1, and TgWtdSc40, were used as the positive controls ( Table 1). The PCR amplification was performed using a thermal cycler (PTC 200, Bio-RAD). The T. gondii B1-positive DNA sample was incubated at 95°C for 5 min to activate the DNA polymerase, then 30 cycles of PCR at 95°C for 30 s, 55°C for 60 s and 72°C for 90 s, and then at 72°C for 7 min. Then 1 μl of the products were used as template DNA for nested PCR with internal primers for each marker, respectively. The nested PCR products were digested with restriction enzymes for 2 h, and the restriction fragments were resolved in 2.5% agarose gel to distinguish single nucleotide polymorphisms (SNPs) using a gel document system (UVP GelDoc-It™ Imaging System, Cambridge, UK).

Results and discussion
A total of 626 bats, belonging to ten species of seven genera, were collected in the present study ( Table 2) (Table 2). Only three species, including Plecotus auritus in Jilin, M. chinensis in Liaoning, and Hipposideros armiger in Guangdong, were detected negative, probably due to the small number of samples, or the low prevalence.
The results of the present study demonstrated that T. gondii infection in bats was widely distributed in Jilin, Liaoning, Jiangxi and Guangdong provinces, with prevalence ranging from 3.7% to 7.3%. Other surveys have shown an overall prevalence of 6.7% in bats in Yunnan, and 20.3% in Guangxi by a nested PCR [18], and a seroprevalence of 18.4% in five bat species in Anhui, Hubei, Guangdong, and Guangxi by MAT [17]. In other countries, the prevalence of T. gondii infection was found in 10.4% British bats using a SAG1-PCR [12], and in 29.3% bats in Myanmar by a nested B1-PCR [13]. The difference may be related to bats species, study regions, and the detection methods.
The present study revealed that T. gondii prevalence was higher in bats in southern China (Guangdong, and Jiangxi) than in northern China (Jilin, and Liaoning). The possible reason is that warm and humid environment in  II/III  II  II  II  II  II  II  II  II  II  II  Reference, Type II, ToxoDB #1   CTG  Cat  United States  II/III  III  III  III  III  III  III  III  III  III I  III  III  III  III  III  I  I  I  u-1 * I  Reference, ToxoDB #19   TgWtdSc40  WTD  USA  u-1  II  II  II  II  II  II  II  I  II  I  Type 12, ToxoDB #5   TgCatBr64  Cat  Brazil  I  I  u-1  III  III  III  u-1  I  III  III  I  Reference, ToxoDB #111   TgRsCr1  Toucan  Costa Rica  u-1  I  II  III  I  III  u-2  I  I  III  I  Reference, ToxoDB #52   TgBatJL1-TgBatJL2  MC  Liver  Jilin, China  u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   TgBatJL3  ML  Liver  Jilin, China  I  I  I  I  I  I  I  I  I  I  I  ToxoDB #10   TgBatJL4  ML  Liver  Jilin, China  I  I  I  I  nd  I  I  I  I  nd nd  =nd   TgBatJX5-TgBatJX7  RF  Liver  Jiangxi, China  u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   TgBatGD8  CS  Liver  Guangdong, China I  I  I  I  I  I  I  nd  I  I  nd  nd   TgBatGD9, TgBatGD10 CS  Liver  Guangdong, China I  I  I  I  I  I  I  I  I  I  I  ToxoDB #10   TgBatGD11  CS  Liver  Guangdong, China nd  I  nd  I  I  I  I  I  I  nd  southern China is more suitable for survival of T. gondii oocysts [1]. The 38 T. gondii-positive bat samples were directly genotyped, and only 8 positive DNA samples were completely genotyped, possibly due to low DNA concentration. Of which, 3 samples (2 from C. sphinx, and 1 from M. leucogaster) represented ToxoDB Genotype #10, and 5 samples (2 from M. leucogaster, 2 from M. chinensis, and 1 from R. ferrumequinum) belonged to ToxoDB Genotype #9 (Table 1).
Two genotypes, namely ToxoDB#10 and ToxoDB#9, were found in bats in this study, which was consistent with a previous study [18], showing a limited diversity of T. gondii genotypes in bats in China. Unfortunately, T. gondii genotype in bats was not successfully identified in Liaoning, probably due to the small size of sampled bats and low intensity of infection.
Recent studies have demonstrated that bats may share the same T. gondii genotypes as in wild and domestic animals, and humans. Although several genotypes of T. gondii have been described in China, there are two main genotypes, including types I (ToxoDB#10) and an atypical genotype ToxoDB#9. In particular, genotype #9 has been reported in Microtus fortis in Jilin province [22], pigs in Jiangxi and Yunnan provinces [20,23], cats in Beijing, Yunnan and Guangdong provinces [24][25][26], bats in Yunnan and Guangxi provinces [18], and humans [27,28], suggesting that the genotype ToxoDB#9 is the most common lineage in mainland China. It is not only present in China, but also in Vietnam and Sri Lanka, which indicated a widespread distribution in Eastern Asia [29]. ToxoDB#10 is also common in China, found in Plateau pikas and Qinghai voles in Qinghai [19], M. fortis in Jilin [22], tree sparrows in Fujian [30], pigs in Henan, Hubei, Hunan, and Jiangsu [27,31], sheep in Qinghai [27], and human in Shanghai [27]. These results have shown a wide distribution of the two genotypes identified from bats in China.

Conclusions
The present study revealed an overall T. gondii prevalence of 6.1% in bats from Jilin, Liaoning, Jiangxi and Guangdong provinces, China, and reported two T. gondii genotypes (ToxoDB#9 and #10). The wide geographical distribution of the two genotypes implied an important role of bats in transmission of T. gondii. These results provide new genetic information about T. gondii infection in bats, and have implications for better understanding of the genetic diversity of T. gondii in China.