Occurrence and genetic characterization of Toxoplasma gondii and Neospora caninum in slaughtered domestic rabbits in central China

Currently, information on the occurrence and genetic characterization of Toxoplasma gondii and Neospora caninum in tissues of rabbits in China is lacking. In this study, brain and heart samples from 470 slaughtered domestic rabbits were collected in Henan Province, Central China. The occurrence rate of T. gondii and N. caninum DNA detected by nested PCR was 2.8% and 2.1%, respectively. There were no significant differences (p > 0.05) in the frequency of the two parasite infections in relation to sex, breed, and region. Three out of 13 T. gondii-positive samples were completely or partially genotyped at 11 genetic markers using PCR-RFLP, and one was identified as ToxoDB genotype #9. For N. caninum, three different sequences at the ITS1 region and two genotypes at the MS5 microsatellite locus were identified. To our knowledge, this is the first genetic characterization of N. caninum isolates from rabbits.


Introduction
Toxoplasma gondii infections are widely prevalent in warm-blooded vertebrates, including humans and rabbits worldwide, and can cause life-threatening toxoplasmosis in immunocompromized individuals. Fatal cases of toxoplasmosis in domestic rabbits have been reported in a few countries [6,8]. It is estimated that up to one-third of the world's human population has been infected with T. gondii [8]. The consumption of undercooked or raw meat containing tissue cysts is the primary risk factor for human T. gondii infections [8,25]. Rabbit meat is one of the most nutritional white meats, and is very popular in China. Rabbits can be infected by ingestion of food or water contaminated with T. gondii oocysts from feline excrement, or by transplacental transmission of T. gondii to offspring [8].
Humans may become infected by eating undercooked rabbit meat, or from hand-to-mouth processes after slaughtering, skinning rabbits or dealing with undercooked or raw rabbit meat [1]. So far, only a few surveys have focused on T. gondii infection in domestic and wild rabbits in China [4,13,15,24,27]. However, information on the prevalence of T. gondii DNA in the tissues of domestic rabbits in China was not available.
Neospora caninum is similar to T. gondii in morphology and life cycle, and is one of the most important causes of abortion in cattle worldwide [7]. Canids are the definitive hosts, whereas many other animal species, including rabbits, are intermediate hosts [9]. There are no natural or experimental data on neosporosis in rabbits. To date, there has been only one report on the seroprevalence and DNA detection of N. caninum in wild rabbits (Lepus tolai) in China; however, the survey failed to detect N. caninum DNA from the tissues [4]. In spite of the worldwide distribution and broad host range of N. caninum, no data on the genetic characterization of rabbit-derived N. caninum isolates are available internationally.
The aim of this study was to determine the occurrence and genetic characterization of T. gondii and N. caninum from slaughtered domestic rabbits in Henan Province, Central China.

Ethics statement
The research protocol was reviewed and approved by the Research Ethics Committee of Henan University of Science and Technology.

Specimens
Between January 2017 and October 2018, brain and heart samples of 470 slaughtered domestic rabbits were collected from seven food markets in Luoyang, Zhengzhou, and Nanyang cities, Henan Province, central China. Each sample was placed into an individual clean self-sealing bag, and the information on the sex, breed, and market of animals was also recorded. All the tissues collected were frozen at À20°C.

Detection of T. gondii and N. caninum DNA
Approximately 1À2 g tissue was taken selectively from different anatomic regions of each tissue, and the specimen was subsequently homogenized in 3 mL of sterile phosphatebuffered saline (PBS). Genomic DNA was extracted from 300 lL of each homogenized tissue suspension using the commercial TIANamp Genomic DNA kit (TianGen, Beijing, China). Extracted DNA was stored at À20°C until analysis. Molecular detections were carried out by nested PCR amplification based on the T. gondii B1 gene and N. caninum NC5 gene, respectively, as described previously [17,18]. DNA of the T. gondii RH strain and N. caninum Nc-LY-Cow1 strain [19] was used in each analysis as the positive control, respectively. Distilled water was used in each analysis as the negative control.

Genotype identification
Toxoplasma gondii genotyping was carried out using the PCR-RFLP method based on genetic markers SAG1, (3 0 + 5 0 ) SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, as described previously [22]. The ITS1 region of the N. caninum NC5 gene positive DNA samples was amplified by nested PCR using the primers NN1 and NN2 as external primers, and NP1 and NP2 as internal primers, as described previously [10]. Multilocus microsatellite genotyping of N. caninum was done based on genetic markers MS4, MS5, MS6A, MS7, MS8, MS10, and MS12, as described previously [20]. DNA of N. caninum Nc-LY-Cow1 and distilled water were used in each analysis as the positive and negative controls, respectively. Two-directional sequencing of positive PCR products was done by Sangon Biotech Co., Ltd., (Shanghai, China). Nucleotide sequences obtained were aligned with each other and with available sequences in GenBank, using ClustalX 2.0, and the default setting, with manual adjustment. A neighbor-joining tree based on the ITS1 sequences was generated using MEGA7 software. The evolutionary distances were computed using the maximum composite likelihood method, and the reliability of branches in the tree was assessed by bootstrap analysis using 1000 replicates.

Statistical analysis
Chi-square analysis was performed to assess the correlation between the occurrence of T. gondii and N. caninum DNA and the sex, breed, and region of rabbits using SPSS, version 11.5 (Statistical Package for the Social Sciences).

Results and discussion
The occurrence rates of T. gondii and N. caninum DNA in slaughtered domestic rabbits in this study are presented in Table 1. DNA of T. gondii and N. caninum was detected in 13 (2.8%) and 10 (2.1%) of 470 rabbits, respectively. Coinfection with T. gondii and N. caninum was found in one rabbit (0.2%). The occurrence rate of T. gondii DNA in domestic rabbits in this study was lower than that (6.4%) in wild rabbits from Shandong Province, eastern China [4]. To our knowledge, this study is the first molecular evidence of N. caninum DNA in rabbits in China. The survey conducted by Cong et al. [4] failed to detect N. caninum DNA from wild rabbits and showed a seroprevalence rate of 0.8% in Shangdong Province, China. The occurrence rate (2.1%) of N. caninum DNA in domestic rabbits in this study was similar to that (2.8%) in cottontail rabbits (Sylvilagus floridanus) in Italy [26], but lower than that (10.5%) in wild rabbits (Oryctolagus cuniculus) in the UK [12].
Similar occurrence rates of T. gondii and N. caninum DNA were found in the different region, breed, or sex groups, with no significant differences (p > 0.05). In different regions, the occurrence rates were 2.1~3.2% for T. gondii and 1.6~2.4% for N. caninum, respectively. The occurrence rates in Chinese rabbits and New Zealand rabbits were 2.8% and 2.7% for T. gondii, 2.5% and 1.4% for N. caninum, and in male and female rabbits 2.4% and 3.2% for T. gondii, and 1.6% and 2.7% for N. caninum, respectively (Table 1).
In the present study, T. gondii DNA was detected mostly in brain tissue (10/13), consistent with a previous report by de Lima et al. [5]. N. caninum DNA was detected mostly in hearts (8/10), like the findings reported by Gondim et al. [11] in sparrows, and indicating that the heart should be included in molecular epidemiology studies of N. caninum.
In this study, only 1 out of 13 T. gondii-positive samples gave complete genotyping results at all 11 gene loci, and was identified to ToxoDB genotype #9, and two samples were genotyped at four and two genetic loci, respectively ( Table 2). In China, ToxoDB genotype #9 is a predominant genotype, and has been found previously in a number of hosts in different regions including Henan Province [16,23]. However, data on the genetic characterization of rabbit-derived T. gondii isolates in China are limited. So far, only two studies have reported one genotype III isolate from domestic rabbits [27] and two ToxoDB genotype #9 isolates from wild rabbits [4] in China. These data suggest that ToxoDB genotype #9 may be a prevalent lineage in rabbits in China. Further studies on a larger number of samples collected from different regions are needed to understand the genetic diversity of T. gondii from rabbits in China.
ITS1 sequences from the 8 N. caninum-positive rabbits in this study showed 95À100% similarity with N. caninum sequences available in GenBank. Three different ITS1 sequences were identified, namely NcRb1 (from five animals, accession number MK510934), NcRb2 (from two animals, accession number MK510935), and NcRb3 (from one animal, accession number MK510936). Among these ITS1 sequences obtained in this study, NcRb1, identified from 5 out of 8 N. caninum ITS1-positive animals and in two out of three cities, was apparently predominant. NcRb1 was identical to the majority of reference sequences available in GenBank. In the phylogenetic tree of the N. caninum ITS1 region, NcRb1 clusters with several representative sequences from domestic and wild animals (e.g., cattle, dogs, deer, and bats) worldwide, including two Chinese strains (JN634857 from cattle and MF802344 from bats) (Fig. 1). Minor sequence differences were observed in the ITS1 sequences NcRb2 and NcRb3, which contain a 1-bp deletion and a 1-bp mutation, respectively (Fig. 2). NcRb2 was located on an intermediate position between the two clusters where two bat-derived sequences from China also consisting of a 1-bp deletion (located as shown in Fig. 1).  I  I  I  I  I  I  I  I  I  I  I  Type I, ToxoDB #10  PTG, reference Sheep  USA  II/III  II  II  II  II  II  II  II  II  II  II Type II, ToxoDB #1  CTG,  reference   Cat  USA  II/III  III  III  III  III  III  III  III  III  III  III Type III, ToxoDB #2   MAS,  reference   Human France u-1 a  I  II  III  III  III  u-1  I  I  III  I  ToxoDB #17   TgPHN1  Pig  Henan  u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9 [19]  TgRbHN1  Rabbit Luoyang u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9  TgRbHN2  Rabbit Luoyang u-1  II  nd  III  nd  II  nd  nd  nd  nd  nd  TgRbHN3 Rabbit Nanyang u-1 nd nd III nd nd nd nd nd nd nd a u-1 represents unique RFLP genotypes. Figure 1. Phylogenetic relationships among NcRb1 À NcRb3 in this study and different representative sequences in GenBank, as inferred by a neighbor-joining analysis of the partial ITS1 region. Bootstrap values greater than 50% from 1000 pseudoreplicates are shown. The sequences in this study are marked by closed circles.
Due to low DNA concentrations, the N. caninum NC5 gene positive DNA samples from six rabbits gave genotyping results only at the MS5 microsatellite locus. Two MS5 genotypes were identified, including the genotype (TA) 12 (accession number MK510937) from four animals, and genotype (TA) 14 (accession number MK510938) from two animals. After analyses on the complete dataset, the predominate genotype (TA) 12 has been reported previously from dogs in Luoyang, China [18], as well as from dogs and cattle in the United States, Germany, and Spain [20]. The MS5 (TA) 14 genotype was found in China for the first time, and had previously been reported from dogs and cattle in South Korea, Iran, Spain, Portugal, Mexico, Argentina, and Brazil [2,3,14,20,21]. In addition, MS5 genotypes (TA) 8 and (TA) 9 have been reported from cattle in the same region [19]. The above-mentioned analysis on ITS1 and MS5 sequences reveals genetic diversity of N. caninum in Henan Province, central China.

Conclusions
In conclusion, this is the first molecular evidence of N. caninum DNA in rabbits in China. Genetic characterization of rabbit-derived N. caninum was determined based on the ITS1 region and MS5 locus, and the results reveal genetic diversity. One T. gondii isolate detected was identified as ToxoDB genotype #9 which is the major lineage in China. These findings provide new genetic information on both parasites especially N. caninum in rabbits, and have important implications for a better understanding of the genetic diversity of the parasites in China.