Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

Abstract Background Enolase is an essential multifunctional glycolytic enzyme that is involved in many biological processes of apicomplexan protozoa, such as adhesion and invasion. However, the characteristics of enolase in Cryptosporidium parvum, including the location on the oocyst and the enzyme activity, remain unclear. Methods The C. parvum enolase gene (cpeno) was amplified by RT-PCR and sequenced. The deduced amino acid sequence was analysed by bioinformatics software. The gene was expressed in Escherichia coli BL21 (DE3) and purified recombinant protein was used for enzyme activity analysis, binding experiments and antibody preparation. The localisation of enolase on oocysts was examined via immunofluorescence techniques. Results A 1,350 bp DNA sequence was amplified from cDNA taken from C. parvum oocysts. The deduced amino acids sequence of C. parvum enolase (CpEno) had 82.1% homology with Cryptosporidium muris enolase, and 54.7–68.0% homology with others selected species. Western blot analysis indicated that recombinant C. parvum enolase (rCpEno) could be recognised by C. parvum-infected cattle sera. Immunolocalization testing showed that CpEno was found to locate mainly on the surface of oocysts. The enzyme activity was 33.5 U/mg, and the Michaelis constant (K m ) was 0.571 mM/l. Kinetic measurements revealed that the most suitable pH value was 7.0–7.5, and there were only minor effects on the activity of rCpEno with a change in the reaction temperature. The enzyme activity decreased when the Ca2+, K+, Mg2+ and Na+ concentrations of the reaction solution increased. The binding assays demonstrated that rCpEno could bind to human plasminogen. Conclusion This study is the first report of immunolocation, binding activity and enzyme characteristics of CpEno. The results of this study suggest that the surface-associated CpEno not only functions as a glycolytic enzyme but may also participate in attachment and invasion process of the parasite

Prevalence and genetic characterization of Cryptosporidium in yaks in Qinghai Province of China.

The objective of this study was to determine the prevalence, species and subtypes of Cryptosporidium infecting yaks in the Qinghai Province of Northwestern China. The prevalence of Cryptosporidium spp. was detected by microscopy and nested-PCR. A total of 586 fecal samples were collected from yaks in 6 counties, of which 142 (24.2%) samples tested positive for Cryptosporidium. The small subunit (SSU) rRNA gene of fifty-five samples were amplified and sequenced successfully and demonstrated that Cryptosporidium bovis (31/55, 56.4%) was the most common species, followed by C. parvum (16/55, 29.1%) and C. ryanae (5/55, 9.0%). Mixed infections of C. parvum and C. bovis (n = 2), C. ryanae and C. bovis (n = 1) were also detected. All three species were found in yaks ranging in age from 2 years. Cryptosporidium was most commonly detected in spring (28.4%), followed by summer (20.9%), then winter (17.5%). Cryptosporidium parvum positive samples were subtyped using the 60 kDa glycoprotein (gp60) gene. Subtypes IIaA15G2R1 (n = 8), IIaA16G2R1 (n = 2), IIaA14G1R1 (n = 1), IIaA14G2R1 (n = 1) and IIaA16G3R1 (n = 1) were detected. All of these subtypes are zoonotic, and may pose a potential threat to human health

Prevalence and molecular characterization of Cryptosporidium in goats across four provincial level areas in China.

This study assessed the prevalence, species and subtypes of Cryptosporidium in goats from Guangdong Province, Hubei Province, Shandong Province, and Shanghai City of China. Six hundred and four fecal samples were collected from twelve goat farms, and the overall infection rate was 11.4% (69/604). Goats infected with Cryptosporidium were found in eleven farms across four provincial areas, and the infection rate ranged from 2.9% (1/35) to 25.0% (9/36). Three Cryptosporidium species were identified. Cryptosporidium xiaoi (45/69, 65.2%) was the dominant species, followed by C. parvum (14/69, 20.3%) and C. ubiquitum (10/69, 14.5%). The infection rate of Cryptosporidium spp. was varied with host age and goat kids were more susceptible to be infected than adult goats. Subtyping C. parvum and C. ubiquitum positive samples revealed C. parvum subtype IIdA19G1 and C. ubiquitum subtype XIIa were the most common subtypes. Other C. parvum subtypes were detected as well, such as IIaA14G2R1, IIaA15G1R1, IIaA15G2R1 and IIaA17G2R1. All of these subtypes have also been detected in humans, suggesting goats may be a potential source of zoonotic cryptosporidiosis. This was the first report of C. parvum subtypes IIaA14G2R1, IIaA15G1R1 and IIaA17G2R1 infecting in goats and the first molecular identification of C. parvum and its subtypes in Chinese goats

Survey and Molecular Characterization of <i>Echinococcus granulosus sensu stricto</i> from Livestock and Humans in the Altai Region of Xinjiang, China

Cystic echinococcosis (CE), caused by the metacestode Echinococcus granulosus sensu stricto (s.s.), is an important zoonotic parasite, endemic in the Altai region of China. It is a serious human health risk and causes livestock losses. To evaluate the prevalence, genetic variation, and population structure of CE, 2898 sheep and 703 cattle were examined from October 2019 to mid-February 2020 in the Altai region (Altai, Habahe, Fuhai, and Buerjin). Sheep had an infection rate of 4.52% (131/2898) and cattle had an infection rate of 4.84% (34/703). In total, 180 cyst isolates were obtained, including 131 sheep, 34 cattle, and 15 from CE human patients. The cysts were investigated using mitochondrial cytochrome C oxidase subunit 1 (cox1). Polymerase Chain Reaction (PCR) results showed that, among the two genotypes of E. granulosus s.s., there were 22 different haplotypes (Haps). Phylogenetic analysis and parsimony network indicated that seventeen (77.27%) Haps belonged to the sheep strain (G1 genotype) and five Haps (22.73%) belonged to the buffalo strain (G3 genotype). Hap3 was the most common haplotype (65.00%, 112/180), which belongs to the G1 genotype. Hap18–Hap22 were found in human samples, indicating that sheep and cattle reservoirs of human CE. Molecular diversity indices revealed the high levels of haplotype diversity and relatively low levels of nucleotide diversity. Tajima’s D and Fu’s Fs tests displayed that the Altai population had a significant deviation from neutrality. Based on pairwise fixation index (Fst) values, a low level of genetic differentiation was found between the populations of E. granulosus s.s. isolated from different regions. The present survey findings represent an epidemiological survey of CE in the Altai region where there were two genotypes simultaneously and will provide more information on the genetic structure of E. granulosus s.s. within this region

Additional file 2: Figure S2. of Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

SDS-PAGE of rCpEno. Lane 1: uninduced pET28a-cpeno transfected E. coli BL21; Lane 2: induced pET28a-cpeno transfected E. coli BL21; Lanes 3 and 4: soluble and insoluble fractions of the induced pET28a-cpeno transfected E. coli BL21 extract, respectively; Lanes 5 and 6: purified rCpEno; Lane M: prestained protein ladder (Thermo Fisher Scientific). (TIF 670 kb

Additional file 1: Figure S1. of Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

Amplification of cpeno by RT-PCR. Lane 1: PCR product of cpeno gene amplified from C. parvum cDNA; Lane M: DL2,000 DNA Marker (Takara); Lane N: negative control. (TIF 325 kb

Additional file 3: Figure S3. of Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

Western blot analysis of purified rCpEno. Purified rCpEno and induced pET-28a (+) plasmid transfected E. coli BL21 (DE3) competent cells were incubated with His-Tag mouse mAb (a) and C. parvum positive serum from cattle (b), respectively. Lane M: prestained protein ladder (Thermo Fisher Scientific). (TIF 902 kb

Seroprevalence and associated risk factors of

Toxoplasma gondii is an intracellular parasite that is extensively prevalent globally. Studies have indicated the presence of T. gondii infection in animals in some provinces of China, but little is known about T. gondii infection in yaks (Bos grunniens) on the Qinghai–Tibetan Plateau. In the current study, to determine the seroprevalence and associated risk factors of T. gondii, a total of 2784 serum samples were collected from 18 different sampling sites in eight counties of the Qinghai and Tibet regions of China from 2018 to 2019. Serum antibodies against T. gondii were detected in 261 yaks (9.38%) via enzyme-linked immunosorbent assay (ELISA). We found that seroprevalence differed significantly among different counties (ranging from 5.41% in Gangcha to 19.79% in Datong), by year in the Tibet Autonomous Region (from 2.34% in 2018 to 13.24% in 2019), and by age (from 5.59% in 0 7) (p < 0.05). Climate, geographical conditions, and age are the main factors influencing T. gondii infection in yaks in these regions. Therefore, our study provides a data reference for public health and prevention of yak toxoplasmosis

Table_1_Wildlife Is a Potential Source of Human Infections of Enterocytozoon bieneusi and Giardia duodenalis in Southeastern China.DOCX

Wildlife is known to be a source of high-impact pathogens affecting people. However, the distribution, genetic diversity, and zoonotic potential of Cryptosporidium, Enterocytozoon bieneusi, and Giardia duodenalis in wildlife are poorly understood. Here, we conducted the first molecular epidemiological investigation of these three pathogens in wildlife in Zhejiang and Shanghai, China. Genomic DNAs were derived from 182 individual fecal samples from wildlife and then subjected to a nested polymerase chain reaction–based sequencing approach for detection and characterization. Altogether, 3 (1.6%), 21 (11.5%), and 48 (26.4%) specimens tested positive for Cryptosporidium species, E. bieneusi, and G. duodenalis, respectively. Sequence analyses revealed five known (BEB6, D, MJ13, SC02, and type IV) and two novel (designated SH_ch1 and SH_deer1) genotypes of E. bieneusi. Phylogenetically, novel E. bieneusi genotype SH_deer1 fell into group 6, and the other genotypes were assigned to group 1 with zoonotic potential. Three novel Cryptosporidium genotypes (Cryptosporidium avian genotype V-like and C. galli-like 1 and 2) were identified, C. galli-like 1 and 2 formed a clade that was distinct from Cryptosporidium species. The genetic distinctiveness of these two novel genotypes suggests that they represent a new species of Cryptosporidium. Zoonotic assemblage A (n = 36) and host-adapted assemblages C (n = 1) and E (n = 7) of G. duodenalis were characterized. The overall results suggest that wildlife act as host reservoirs carrying zoonotic E. bieneusi and G. duodenalis, potentially enabling transmission from wildlife to humans and other animals.</p

Table_2_Wildlife Is a Potential Source of Human Infections of Enterocytozoon bieneusi and Giardia duodenalis in Southeastern China.XLSX

Wildlife is known to be a source of high-impact pathogens affecting people. However, the distribution, genetic diversity, and zoonotic potential of Cryptosporidium, Enterocytozoon bieneusi, and Giardia duodenalis in wildlife are poorly understood. Here, we conducted the first molecular epidemiological investigation of these three pathogens in wildlife in Zhejiang and Shanghai, China. Genomic DNAs were derived from 182 individual fecal samples from wildlife and then subjected to a nested polymerase chain reaction–based sequencing approach for detection and characterization. Altogether, 3 (1.6%), 21 (11.5%), and 48 (26.4%) specimens tested positive for Cryptosporidium species, E. bieneusi, and G. duodenalis, respectively. Sequence analyses revealed five known (BEB6, D, MJ13, SC02, and type IV) and two novel (designated SH_ch1 and SH_deer1) genotypes of E. bieneusi. Phylogenetically, novel E. bieneusi genotype SH_deer1 fell into group 6, and the other genotypes were assigned to group 1 with zoonotic potential. Three novel Cryptosporidium genotypes (Cryptosporidium avian genotype V-like and C. galli-like 1 and 2) were identified, C. galli-like 1 and 2 formed a clade that was distinct from Cryptosporidium species. The genetic distinctiveness of these two novel genotypes suggests that they represent a new species of Cryptosporidium. Zoonotic assemblage A (n = 36) and host-adapted assemblages C (n = 1) and E (n = 7) of G. duodenalis were characterized. The overall results suggest that wildlife act as host reservoirs carrying zoonotic E. bieneusi and G. duodenalis, potentially enabling transmission from wildlife to humans and other animals.</p