Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system.

Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii.

[Establishment of LAMP Detection for Toxoplasma gondii Based on the 529 bp Repeat Sequence].

Objective: To establish the loop mediated isothermal amplification（LAMP） technique for determining Toxoplama gondii. Methods: The primers for LAMP of the conserved 529 bp sequence was designed by the Primer Explorer 4.0 software. The LAMP reaction was made on the constructed pMD-19T-529 bp recombinant plasmid as a template, and was optimized in loop primer, concentrations of betaine and MgSO4, and reaction temperature. The optimized LAMP and PCR were performed in ultra-pure water, on pig genome, Cryptosporidium parvum genome, Isospora suis genome, and pMD-19T-529 bp, respectively, to test the sensitivity of LAMP. The pMD-19T-529 bp was serially diluted to 109-100 copies/ml to test the specificity of LAMP. Results: LAMP using the designed primers amplified the 529 bp fragment of T. gondii. The optimized LAMP system had a total reaction volume of 25 µl, with the optimal concentrations of betaine and MgSO4 being 0.4 mol/L and 6 mmol/L, respectively. The amplification efficiency of 30 min reaction in the presence of loop primer was comparable to that of 60 min reaction without loop primer, which indicated that addition of loop primer shortened the reaction time by 30 min. The optimal reaction condition was 63 ℃ 30 min, 80 ℃ 3 min. The established LAMP method specifically amplifed the 529 bp fragment of T. gondii, and its efficiency was 10 times of PCR（detection threshold 103 copies/ml vs. 104 copies/ml）. Conclusion: The established LAMP for detecting Toxoplama gondii 529 bp repeat sequence shows high specificity and sensitivity.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene.

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

Rhoptry protein 47 gene sequence: A potential novel genetic marker for population genetic studies of Toxoplasma gondii.

Toxoplasma gondii, an obligate intracellular parasite, is able to infect many animal species and humans, and can cause toxoplasmosis of the host. In this study, we examined sequence variation in rhoptry protein 47 (ROP47) gene among T. gondii isolates originating from different hosts and geographical regions. The entire genome region of the ROP47 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ), based on the ROP47 gene sequences. The results of sequence alignments showed that all ROP47 gene sequences were 396 bp in length. There were 19 variable nucleotide positions in the coding region, resulted in 16 amino acid substitutions (12.21%) among all examined T. gondii strains and the existence of polymorphic restriction sites for endonucleases SacI and AflIII, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analysis of ROP47 gene sequences showed that three major clonal lineage types I, II and III were clustered differently, consistent with PCR-RFLP results. These results suggest that ROP47 gene sequence may represent a potential novel genetic marker for population genetic studies of T. gondii isolates.

First report of Babesia bigemina infection in white yaks in China.

White yaks, a unique yak breed and the pearl of the plateau, only live in Tianzhu Tibetan Autonomous County (TTAC), Gansu Province, northwest China, contributing significantly to local economy. However, there was no information on the prevalence of Babesia bigemina in white yaks. In this study, a total of 974 serum samples collected from white yaks in TTAC were examined for specific antibodies against B. bigemina using a commercially available ELISA kit. The overall seroprevalence of B. bigemina in white yaks was 17.76% (173/974). A multivariate logistic regression analysis was performed to determine the risk factors associated with B. bigemina seroprevalence, and the results indicated that age, gender and the numbers of pregnancies of white yaks were not the significant risk factors. However, the white yaks in spring (OR=3.523, 95% CI=1.899-6.538, P<0.001) and summer (OR=3.439, 95% CI=1.909-6.193, P<0.001) encountered higher risk of being exposed to B. bigemina than that in winter. Thus, season was considered as a risk factor associated with B. bigemina infection. This is the first survey of B. bigemina seroprevalence in white yaks in China, which extends the host range for B. bigemina and provides useful information for controlling B. bigemina infection in white yaks.