Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces, Eastern China.

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

[Pregnancy Outcomes and Prognosis of Fetal Ventriculomegaly: A Retrospective Cohort Study].

Objective: To evaluate the pregnancy outcomes and neurodevelopment prognosis of subjects prenatally diagnosed with fetal ventriculomegaly (VM). Methods: All the subjects with VM diagnosed by ultrasound and were admitted and treated at West China Second Hospital, Sichuan University between March 2011 and September 2020 were retrospectively enrolled for a chohort study, while non-VM subjects of the same period were selected with a random number table to form the control group. Pregnancy outcomes of the two groups were compared, and the fetuses of both groups were followed up after birth for further assessment and comparison of their neurodevelopmental prognosis. Results: The live birth rate of the VM group was lower than that of the control group (77.63% [229/295] vs. 94.31% [265/281], P<0.001). Furthermore, the proportion of subjects that were transferred to NICU for monitoring and observation after birth was higher in the VM group than that of the control group (20.96% [48/229] vs. 4.53% [12/265], P<0.001). During the follow-up, it was found that the rate of neurodevelopmental abnormalities of the VM group was significantly higher than that of the control group (11.79% [27/229] vs. 1.90% [5/265], P<0.001). Moreover, neurodevelopmental abnormalities of VM fetuses were correlated to the following factors, the degree of VM ( P=0.010), intrauterine progression of VM ( P=0.024), and whether the postnatal cranial ultrasound result was suggestive of VM ( P=0.001). In addition, postnatal cranial ultrasound suggestive of VM was found to be an independent risk factor for neurodevelopmental abnormalities ( OR=9.434, 95% CI: 1.791-49.688, P=0.008). Conclusion: VM reduces the fetal live birth rate and may increase the risks of neurodevelopmental abnormalities after birth. All VM fetuses should be closely followed up for neurodevelopment status after birth, especially those with severe VM, intrauterine progression, and postnatal cranial ultrasound indicative of VM.

Characterization of Starch in Cucurbita moschata Germplasms throughout Fruit Development.

Pumpkins (Cucurbita moschata; Cucurbitaceae) are the rich source of nutrients and valued for their biologically active substances to be used for the treatment of several diseases. The contents, composition, and conformation of starch are the significant quality traits of C. moschata. Two germplasms were targeted for analysis regarding the taste difference. Results indicated that the total starch contents and amylose/amylopectin ratio were high in CMO-X as compared to CMO-E during each fruit development stage. Scanning electron microscopy and transmission electron microscopy observations revealed that smooth surface starch granules fused together to enhance the starch accumulation. For a comparison of fruit development in CMO-E and CMO-X, the putative pathway for starch metabolism was developed and homologs were identified for each key gene involved in the pathway. GBSS and SBE were correlated with the difference in the amylose/amylopectin ratio of CMO-E and CMO-X. Conclusively, the developmental regulation of genes associated with starch accumulation can be considered as an important factor for the determination of fruit quality.

Metabolic and transcriptomic analysis of two Cucurbita moschata germplasms throughout fruit development.

BACKGROUND: Pumpkins (Cucurbita moschata; Cucurbitaceae) are valued for their fruits and seeds and are rich in nutrients. Carotenoids and sugar contents, as main feature of pumpkin pulp, are used to determine the fruit quality. RESULTS: Two pumpkin germplasms, CMO-X and CMO-E, were analyzed regarding the essential quality traits such as dry weight, soluble solids, organic acids, carotenoids and sugar contents. For the comparison of fruit development in these two germplasms, fruit transcriptome was analyzed at 5 different developmental stages from 0 d to 40 d in a time course manner. Putative pathways for carotenoids biosynthesis and sucrose metabolism were developed in C. moschata fruit and homologs were identified for each key gene involved in the pathways. Gene expression data was found consistent with the accumulation of metabolites across developmental stages and also between two germplasms. PSY, PDS, ZEP, CRTISO and SUS, SPS, HK, FK were found highly correlated with the accumulation of carotenoids and sucrose metabolites, respectively, at different growth stages of C. moschata as shown by whole transcriptomic analysis. The results of qRT-PCR analysis further confirmed the association of these genes. CONCLUSION: Developmental regulation of the genes associated with the metabolite accumulation can be considered as an important factor for the determination of C. moschata fruit quality. This research will facilitate the investigation of metabolic profiles in other cultivars.

A high-density linkage map and QTL mapping of fruit-related traits in pumpkin (Cucurbita moschata Duch.).

Pumpkin (Cucurbita moschata) is an economically worldwide crop. Few quantitative trait loci (QTLs) were reported previously due to the lack of genomic and genetic resources. In this study, a high-density linkage map of C. moschata was structured by double-digest restriction site-associated DNA sequencing, using 200 F2 individuals of CMO-1 × CMO-97. By filtering 74,899 SNPs, a total of 3,470 high quality SNP markers were assigned to the map spanning a total genetic distance of 3087.03 cM on 20 linkage groups (LGs) with an average genetic distance of 0.89 cM. Based on this map, both pericarp color and strip were fined mapped to a novel single locus on LG8 in the same region of 0.31 cM with phenotypic variance explained (PVE) of 93.6% and 90.2%, respectively. QTL analysis was also performed on carotenoids, sugars, tuberculate fruit, fruit diameter, thickness and chamber width with a total of 12 traits. 29 QTLs distributed in 9 LGs were detected with PVE from 9.6% to 28.6%. It was the first high-density linkage SNP map for C. moschata which was proved to be a valuable tool for gene or QTL mapping. This information will serve as significant basis for map-based gene cloning, draft genome assembling and molecular breeding.

Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.

The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.

Molecular cloning, sequencing, and expression of the outer membrane protein P2 gene of Haemophilus parasuis.

Haemophilus parasuis is the etiological agent of Glässer's disease characterized by fibrinous polyserositis, polyarthritis, and meningitis in young pigs. But it is difficult to develop universal serological diagnostic tools and effective vaccines against this disease because of the serovar diversity of the isolates. In this study, enterobacterial repetitive intergenic consensus-polymerase chain reaction, were performed to investigate the gene profile of 111 isolates of H. parasuis from China. And a specific common gene of H. parasuis was cloned and identified as the outer-membrane protein (OMP) P2 gene. Sequencing results of OMP P2 genes of 22 isolates showed that they had high homology and could be divided into 2 genetic types. Moreover, the OMPP2 protein was expressed in Escherichia coli expressing system. And the purified recombinant protein provided partial protection against H. parasuis infection in mice. It suggested the OMP P2 was an immunogenic protein and had great potential to serve as a vaccine and diagnostic antigen.

Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens.

From December 2003 to July 2006, a total of 131 (28.4%) Haemophilus parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. These strains were isolated from clinically diseased pigs, and 50 of them along with 15 reference strains of all known serovars were subjected to PCR-FRLP (restriction fragment length polymorphism) analysis by tbpA gene. The analysis of the 1.9-kb tbpA amplicon using TaqI, AvaI and RsaI endonucleases produced 9 RFLP patterns for the 15 reference strains and 13 patterns for the 50 field isolates. And the first three prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). Meanwhile, co-infection of H. parasuis, PRRSV and PCV2 was examined in the 462 pig herds. It is indicated that 11.5% cases (53), 27.9% cases (129) and 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens. It is confirmed that H. parasuis existed widely in southeast China with numerous genotypes.