Colorimetric biosensor for visual determination of Golgi protein 73 based on reduced graphene oxide-carboxymethyl chitosan-Hemin/platinum@palladium nanozyme with peroxidase-like activity.

A Golgi protein 73 (GP73) colorimetric biosensor based on the reduced graphene oxide-carboxymethyl chitosan-hemin/platinum@palladium nanoparticles (RGO-CMCS-Hemin/Pt@Pd NPs) with peroxidase-like activity was constructed. The RGO-CMCS-Hemin/Pt@Pd NPs with high peroxidase-like activity were successfully synthesized under mild conditions. Then, the aminylated GP73 aptamer (Apt) was bound to the RGO-CMCS-Hemin/Pt@Pd NPs to form the recognition probe. Another unmodified GP73 aptamer (AptI) was served as the capture probe. In the presence of target GP73, the capture probe and the recognition probe specifically bind to GP73 and form a RGO-CMCS-Hemin/Pt@Pd NP-Apt/GP73/AptI sandwich-type structure, which can oxidase the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB in the presence of H2O2. GP73 detection was achieved by measuring the peak UV absorption at 652 nm. Under the optimum conditions, the GP73 concentration was linearly related to the absorbance intensity in the range 10.0-110.0 ng/mL, and the limit of detection (LOD) was 4.7 ng/mL. The proposed colorimetric biosensor was successfully applied to detect GP73 in spiked human serum samples with recoveries of 98.2-107.0% and RSDs of 1.90-5.44%, demonstrating the excellent potential for highly sensitive GP73 detection in clinical detection. A colorimetric biosensor for visual determination of GP73 based on RGO-CMCS-Hemin/Pt@Pd NPs nanozyme with peroxidase-like activity was designed. The GP73 biosensor responses linearly from 10.0-110.0 ng/mL with LOD of 4.7 ng/mL, and shows acceptable specificity and good recovery.

Molecular Detection of Toxoplasma gondii and Neospora caninum in Domestic Ducks in Hunan Province, China.

Toxoplasma gondii and Neospora caninum are protozoan parasites that infect warm-blooded animals, and cause major economic losses in livestock industries worldwide. However, little is known about the genotypes of T. gondii and N. caninum in domestic ducks in China. Herein, brain samples from 588 domestic ducks from Hunan province in China were examined for the presence of T. gondii and N. caninum. Polymerase chain reaction (PCR) was used to detect T. gondii B1 gene and N. caninum NC-5 gene. Forty-five DNA samples (7.7%; 95% CI: 5.5-9.9) were positive for B1 gene, and two (0.3%; 95% CI: 0-0.7) were positive for NC-5 gene. The risk factors significantly associated with T. gondii infection were age and sex. The 45 samples positive for T. gondii were genotyped using multi-locus PCR-RFLP analysis and only one sample was fully genotyped as ToxoDB#9 (Chinese I). These results provide new information about the epidemiology of T. gondii and N. caninum in ducks in Hunan province in China. The data also highlight the importance of a "One Health" approach to dealing with toxoplasmosis.

Seroprevalence, Risk Factors, and Genotypes of Toxoplasma gondii in Free-Range Chickens Intended for Human Consumption in China.

Toxoplasma gondii is a protozoan with worldwide distribution that infects birds and mammals, including humans. The consumption of free-range chicken meat is a common practice in many parts of the world. However, little information is available concerning the molecular prevalence and genotypes of T. gondii infection in free-range chickens intended for human consumption in China. In this study, a total of 1360 serum samples were collected from food markets in Hunan province of China for detecting T. gondii antibodies by indirect hemagglutination assay. In addition, 650 brain tissues were also collected to investigate T. gondii DNA by amplification of B1 gene with a seminested polymerase chain reaction (PCR), and the positive DNA samples were typed at 10 genetic markers using multilocus PCR-restriction fragment length polymorphism. Antibodies to T. gondii were detected in 457 of the examined serum samples (33.6%; 95% confidence interval [CI]: 31.1-36.1), and 72 DNA samples (11.1%; 95% CI: 8.6-13.4) were positive for the T. gondii B1 gene. In this study, region and age of free-range chickens were shown to be risk factors for T. gondii infection (p < 0.01). Two genotypes (ToxoDB#9 and ToxoDB#52) were identified from two samples with complete genotyping results. Our study revealed a high prevalence of T. gondii infection in free-range chickens intended for human consumption in Hunan province, suggesting that recommendations to consumers should be made, especially in some regions of China where consumption of undercooked chicken meat is common. This is the first genetic characterization of T. gondii in free-range chickens intended for human consumption in Hunan province, China, and also the first report of genotype ToxoDB#52 in China.

First report of Neospora caninum infection in pigs in China.

Neospora caninum is a protozoan parasite which can infect many mammals and birds with a worldwide distribution. However, no molecular data are available about the occurrence of N. caninum in pigs. In this study, the serological and molecular prevalence of N. caninum infection in farmed pigs were investigated in Hunan province, China, between January 2017 and December 2018. A total of 1,500 serum samples collected from 10 herds in Hunan province were evaluated using a competitive-inhibition enzyme-linked immunoassay assay (cELISA). The overall seroprevalence of N. caninum in the examined pigs was 1.9%. The seroprevalence of N. caninum ranged from 0.3% to 4.6% among different regions in Hunan province of China (p < .05). DNA was extracted from brain samples, and the Nc-5 gene and ITS-1 region were amplified and then sequenced. Three (0.5%) of the examined 600 brain tissues were found to contain N. caninum DNA. Our phylogenetic analyses indicated that N. caninum samples were classified into two distinct groups. Although the prevalence is low within the pig groups investigated, our results revealed the emergence of N. caninum infection in pigs in China. The finding of the present study provides molecular evidence that the pigs are the natural intermediate host of N. caninum and may have major epidemiological importance.

Identification of Cryptosporidium viatorum XVa subtype family in two wild rat species in China.

BACKGROUND: Cryptosporidium viatorum is a minor Cryptosporidium pathogen in humans. Currently, there is limited information regarding the prevalence and genotypes of C. viatorum in animals in China. METHODS: In this study, 228 faecal samples were collected from two wild rat species (Leopoldamys edwardsi and Berylmys bowersi) in Chongqing Municipality and Guangdong Province, China. These specimens were analyzed for C. viatorum and then subtyped it using PCR and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) and 60-kilodalton glycoprotein (gp60) genes, respectively. RESULTS: A total of 25 (11.0%) faecal samples were tested positive for C. viatorum by SSU rRNA assay. Of these samples, 4 (3.6%) came from L. edwardsi and 21 (18.0%) from B. bowersi. Of the 25 C. viatorum-positive samples, 17 were successfully amplified at the gp60 gene locus, which represented four subtypes belonging to two subtype families, including XVa (XVaA6, XVaA3g, XVaA3h) and XVc (XVcA2G1). Phylogenetic analysis based on the gp60 amino acid sequences indicated that all of the C. viatorum isolates grouped together, supporting the conclusion that C. viatorum from the wild rats represent two subtype families. CONCLUSIONS: These results indicate an occurrence of C. viatorum XVa subtype family from rats which is genetically identical to those found in humans. Our findings suggest that wild rats may be a potential source of human cryptosporidiosis.

Molecular detection of Neospora caninum from naturally infected four passeriforme birds in China.

Neospora caninum is an intracellular protozoan infecting many domestic and wild animals. In the present study, the brain tissues of wild birds collected in Hunan province of China were examined by N. caninum specific nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS-1) region of the nuclear ribosomal DNA. The prevalence of N. caninum was detected in 15.5% (37/239) of wild birds, including 20.5% (9/44) of the examined olive-backed pipit Anthus hodgsoni, 18.3% (24/131) of the examined tree sparrows Passer montanus, 7.9% (3/38) of the examined chestnut bunting Emberiza rutila and 3.8% (1/26) of the examined yellow-breasted bunting E. aureola. Phylogenetic analyses showed that N. caninum from different hosts and geographical origins are genetically diverse and can be further classified into two distinct groups. Our findings indicated that wild birds are potential source of N. caninum for other animals. To our knowledge, this is the first report of N. caninum infection in wild birds in China, which provides a foundation for the prevention and control of this parasite in China and elsewhere.

Molecular Detection and Genotyping of Toxoplasma gondii in Pigs for Human Consumption in Hunan Province, China.

Toxoplasma gondii is one of the most important sources of foodborne diseases. In this study, the molecular prevalence and genotypes of T. gondii were investigated in pigs in Hunan province, China. A total of 339 brain tissue samples of pigs were collected from April 2015 to December 2017 in Hunan province and were used to detect the T. gondii B1 gene. Of these, 34 (10%; 95% confidence interval: 8.7-12.6) samples were tested positive for the T. gondii B1 gene. Positive samples were genotyped at 10 genetic markers (SAG1, SAG2 [5' + 3' SAG2, alter. SAG2], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism technology. Moreover, one sample was identified as genotype ToxoDB#10 (Type I), and another sample was suspected to be unusual genotype ToxoDB#61 that has never been reported in China. This study showed that T. gondii is prevalent in pigs in Hunan province, posing a food safety threat to the public health in the investigated areas. Our result has implications for better understanding the genetic diversity of T. gondii infections in animals in China.