Prevalence and molecular characterization of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Ningxia, northwestern China.

Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are common intestinal pathogens that infect humans and animals. To date, research regarding these three protozoa in the Ningxia Hui Autonomous Region (Ningxia) has mostly been limited to a single pathogen, and comprehensive data on mixed infections are unavailable. This study aimed to evaluate the zoonotic potential of these three protozoa. In this study, small subunit ribosomal RNA (SSU rRNA) and 60 kDa glycoprotein (gp60) genes of Cryptosporidium; internal transcribed spacer (ITS) gene of E. bieneusi; and SSU rRNA, glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis were examined. DNA extraction, polymerase chain reaction, and sequence analysis were performed on fecal samples collected from 320 dairy cattle at three intensive dairy farms in Ningxia in 2021 to determine the prevalence and genetic characteristics of these three protozoa. The findings revealed that 61.56% (197/320) of the samples were infected with at least one protozoan. The overall prevalence of Cryptosporidium was 19.38% (62/320), E. bieneusi was 41.56% (133/320), and G. duodenalis was 29.38% (94/320). This study identified four Cryptosporidium species (C. bovis, C. andersoni, C. ryanae, and C. parvum) and the presence of mixed infections with two or three Cryptosporidium species. C. bovis was the dominant species in this study, while the dominant C. parvum subtypes were IIdA15G1 and IIdA20G1. The genotypes of E. bieneusis were J, BEB4, and I alongside the novel genotypes NX1-NX8, all belonging to group 2, with genotype J being dominant. G. duodenalis assemblages were identified as assemblages E, A, and B, and a mixed infection involving assemblages A + E was identified, with assemblage E being the dominant one. Concurrently, 11 isolates formed 10 different assemblage E multilocus genotypes (MLGs) and 1 assemblage A MLG and assemblage E MLGs formed 5 subgroups. To the best of our knowledge, this is the first report on mixed infection with two or three Cryptosporidium species in cattle in Ningxia and on the presence of the C. parvum subtype IIdA20G1 in this part of China. This study also discovered nine genotypes of E. bieneusis and novel features of G. duodenalis assemblages in Ningxia. This study indicates that dairy cattle in this region may play a significant role in the zoonotic transmission of Cryptosporidium spp., E. bieneusi, and G. duodenalis.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in Central Inner Mongolia, Northern China.

BACKGROUND: Cryptosporidium is a gastrointestinal protozoan that widely exists in nature, it is an established zoonotic pathogen. Infected cattle are considered to be associated with cryptosporidiosis outbreaks in humans. In the present study, we aimed to assess the prevalence and species distribution of Cryptosporidium in dairy cattle in Central Inner Mongolia. METHODS: We focused on the small subunit ribosomal RNA gene (SSU rRNA) of Cryptosporidium and 60-kDa glycoprotein gene (gp60) of Cryptosporidium parvum. We collected 505 dairy cattle manure samples from 6 sampling sites in Inner Mongolia in 2021; the samples were divided into 4 groups based on age. DNA extraction, polymerase chain reaction (PCR), sequence analysis, and restriction fragment length polymorphism (RFLP) using SspI and MboII restriction endonucleases were performed. RFLP analysis was performed to determine the prevalence and species distribution of Cryptosporidium. RESULTS: SSU rRNA PCR revealed that the overall prevalence of Cryptosporidium infection was 29.90% (151/505), with a prevalence of 37.67% (55/146) and 26.74% (96/359) in diarrheal and nondiarrheal samples, respectively; these differences were significant. The overall prevalence of Cryptosporidium infection at the 6 sampling sites ranged from 0 to 47.06% and that among the 4 age groups ranged from 18.50 to 43.81%. SSU rRNA sequence analysis and RFLP analysis revealed the presence of 4 Cryptosporidium species, namely, C. bovis (44.37%), C. andersoni (35.10%), C. ryanae (21.85%), and C. parvum (11.92%), along with a mixed infection involving two or three Cryptosporidium species. Cryptosporidium bovis or C. andersoni was the most common cause of infection in the four age groups. The subtype of C. parvum was successfully identified as IIdA via gp60 analysis; all isolates were identified as the subtype IIdA19G1. CONCLUSIONS: To the best of our knowledge, this is the first report of dairy cattle infected with four Cryptosporidium species in Inner Mongolia, China, along with a mixed infection involving two or three Cryptosporidium species, with C. bovis and C. andersoni as the dominant species. Moreover, this is the first study to identify C. parvum subtype IIdA19G1 in cattle in Inner Mongolia. Our study findings provide detailed information on molecular epidemiological investigation of bovine cryptosporidiosis in Inner Mongolia, suggesting that dairy cattle in this region are at risk of transmitting cryptosporidiosis to humans.

Prevalence and molecular characterization of Giardia duodenalis in dairy cattle in Central Inner Mongolia, Northern China.

Giardia duodenalis is a gastrointestinal protozoan ubiquitous in nature. It is a confirmed zoonotic pathogen, and cattle are considered a source of giardiasis outbreaks in humans. This study aimed to evaluate the prevalence and multilocus genotype (MLG) of G. duodenalis in dairy cattle in Central Inner Mongolia. This study was based on the small subunit ribosomal RNA (SSU rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed on 505 dairy cattle fecal samples collected in 2021 from six sampling sites and four age groups in Central Inner Mongolia to determine the prevalence and MLG distribution of G. duodenalis. The PCR results of SSU rRNA revealed that the overall prevalence of G. duodenalis was 29.5% (149/505) and that the overall prevalence of the diarrhea and nondiarrhea samples was 31.5% (46/146) and 28.5% (103/359), respectively; the difference was not significant (p > 0.05). SSU rRNA sequence analysis revealed that G. duodenalis assemblage E (91.1%, 133/146) was primarily detected and that assemblage A (8.9%, 13/146) was detected in 13 samples. The G. duodenalis-positive samples were PCR amplified and sequenced for gdh, tpi, and bg, from which 38, 47, and 70 amplified sequences were obtained, respectively. A combination of G. duodenalis assemblages A and E were detected in seven samples. Multilocus genotyping yielded 25 different assemblage E MLGs, which formed six subgroups. To the best of our knowledge, this is the first report regarding G. duodenalis infection in dairy cattle in Inner Mongolia, China. This study revealed that Inner Mongolian cattle pose a risk of giardiasis transmission to humans and that the distribution of local cattle G. duodenalis assemblage E MLGs is diverse. The findings of this study can bridge the knowledge gap in the molecular epidemiological investigation of giardiasis in Central Inner Mongolia.

Effects of Percutaneous Coronary Intervention and Coronary Artery Bypass Grafting on Clinical Outcomes in Patients with Reduced Ejection Fraction Heart Failure and Coronary Heart Disease: A Meta-Analysis.

OBJECTIVE: To clarify the effects of percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) on the clinical outcomes of patients with coronary heart disease (CHD) complicated with reduced ejection fraction heart failure (HFrEF) through meta-analysis. METHODS: Three major literature databases - PubMed, Web of Science, and Cochrane - were searched by search terms and the literature retrieval time was publications dating from January 2007 to December 2021. To search for observational studies and randomized controlled trials (RCT) comparing the efficacy of PCI and CABG in patients with CHD and HFrEF, the abstract or full text of the literature was read and the final included literature was determined, according to inclusion and exclusion criteria. The quality of the included literature was evaluated using the Ottawa scale and data extraction was further completed. Data analysis was made using RevMan5.4 and R4.1 software; relevant forest plots and funnel plots were made, according to the extracted data. Egger's test was used to evaluate whether the data had publication bias. Outcomes were the major adverse cardiovascular events (MACE). RESULTS: A total of 10 studies were included and 11,032 subjects were included, made up of 5,521 cases of PCI and 5,511 cases of CABG. The results showed no significant difference between the two groups in cardiac mortality (CM) (RR=1.13, 95% CI 0.98-1.30, P = 0.10) and in overall all-cause mortality (ACM) (RR=1.12, 95% CI 0.92-1.37, P = 0.25). In the subgroup analysis of ACM, in the subgroups with left ventricular ejection fraction (LVEF) less than 35% and exceeding 35% and less than 50% (RR=1.12, 95% CI 0.92-1.37, P = 0.25) between the two groups, there was no statistical difference. However, among other MACE, compared with the PCI group, the CABG group had a lower risk of MACE (RR=1.58, 95%CI 1.49-1.70, P < 0.00001), myocardial infarction (MI) (RR=1.99, 95% CI 1.02-3.88, P = 0.04), heart failure (HF) (RR=1.29, 95% CI 1.17-1.43, P < 0.00001) and revascularization (RR=2.74, 95% CI 1.93-3.90, P < 0.00001). Finally in the CABG group, the risk of stroke or transient ischemic attack (TIA) was higher (RR=0.71, 95% CI 0.58-0.86, P = 0.0006) than the PCI group. CONCLUSIONS: The mortality rates of PCI and CABG were similar in patients with CHD complicated with HFrEF. Compared with PCI, CABG had a lower incidence of MACE, MI, HF, and revascularization, and a higher incidence of stroke or TIA.

Association of Ethnicity, Sex, and Age With Cancer Diagnoses and Health Care Utilization Among Children in Inner Mongolia, China.

Importance: China is experiencing a sustained increase in childhood cancer. However, whether differences exist in disease burden by ethnicity remains unclear. Objective: To compare differences in cancer diagnoses and health care utilization in Inner Mongolia among children subgrouped by ethnicity (Han vs Mongolian), sex, and age. Design, Setting, and Participants: This retrospective cohort study in Inner Mongolia, China, used data on children aged 0 to 14 years with cancer from the Inner Mongolia Regional Health Information Platform, which comprises the National Basic Medical Insurance database and the Inner Mongolia cause-of-death reporting system, from January 1, 2013, to December 31, 2019. Ethnicities analyzed included Han and Mongolian; patients of other ethnicities were not included in the analysis because of the small sample size. Cancer was broadly defined as a primary malignant tumor or hematologic cancer; benign central nervous system tumors were also included. A 2-year washout period was used to exclude prevalent cases. After diagnosis, the patients were followed up until the date of death or the end of the insured status, whichever came first. Exposures: Ethnicity (Han vs Mongolian), sex (male vs female), and age (0-4, 5-9, and 10-14 years). Main Outcomes and Measures: Crude incidence, 5-year prevalence, and survival rates at 1 year and 3 years after diagnosis; health care utilization, represented by medical costs during the first year and first 3 years after diagnosis; and hospital attendance with level (tertiary vs secondary and lower-level hospitals) and location of each unique visit. Results: From 2013 to 2019, 1 106 684 (2013), 1 330 242 (2014), 1 763 746 (2015), 2 400 343 (2016), 2 245 963 (2017), 2 901 088 (2018), and 2 996 580 (2019) children aged 0 to 14 years were registered in the NBMI database. Among the 2 996 580 children enrolled in 2019, the mean (SD) age was 6.8 (4.3) years, of whom 1 572 096 (52.5%) were male, 2 572 091 (85.8%) were Han, and 369 400 (12.3%) were Mongolian. A total of 1910 patients with cancer were identified (1048 were male [54.9%]; 1559 were Han [81.6%], and 300 were Mongolian [15.7%]). There were 764 hematologic cancers (40.0%) and 1146 solid tumors (60.0%). The overall crude incidence of cancer from 2015 to 2019 was 129.85 per million children (95% CI, 123.63-136.06), with a higher incidence among Mongolian than among Han children (155.12 [95% CI, 136.81-173.43] vs 134.39 [95% CI, 127.46-141.32]). The 5-year prevalence was 428.97 per million (95% CI, 405.52-452.42) in 2020, with a higher prevalence among Mongolian than among Han children (568.49 [95% CI, 91.62-645.36] vs 404.34 [95% CI, 379.77-428.91]). The combined 1-year (2015-2019) and 3-year (2015-2017) survival rates were 72.5% (95% CI, 67.5%-77.5%) and 66.8% (95% CI, 61.6%-71.9%), respectively. The 1-year (median [IQR], $1991 [$912-$10 181] vs $3991 [$1171-$15 425]) and 3-year (median [IQR], $2704 [$954-$13 909] vs $5375 [$1283-$22 466]) postdiagnosis costs were lower among Mongolian than among Han children. A higher proportion of Mongolian patients attended low-level hospitals (45.9% vs 17.4%). Conclusions and Relevance: In this cohort study, Mongolian children had a higher incidence and prevalence of cancer but a lower demand for medical care, suggesting that further investigations are needed to identify mechanisms underlying ethnic disparities and ensure that care is equitable.

[Influence of Uranium in Pteris vittata L. Inoculated by Arbuscular Mycorrhizal Fungus].

A pot experiment was carried out to explore the annual changes by bioremediation inoculated with 30 g Glomus versiorme in Pteris vittata L. The results showed that mycorrhizal colonization was the lowest in September 2013 (57.14%), and was the highest in March 2014 (75.20%), following the tendency firstly increasing and then decreasing. The dry biomass was markedly high in Gv than that in CK, especially in roots. The total U was significantly higher in Gv than that in CK, and was fixed predominantly into roots. The media in Gv showed less U than that in CK. It was absorbed the most to iron and manganese oxidable U and sulfide U, and each U species declined accompanying the time prolongation. In addition, bioconcentration factors were higher in Gv compared to those of CK, and both treatments were above 1. Positive relationship was found between mycorrhizal colonization and bioconcentration factors. Therefore, U uptake was enhanced inoculated by Gv, and the symbiont in arbuscular mycorrhizal fungus and Pteris vittata L. had a potential to remediate U polluted soil.

Different roles of PD-L1 and FasL in immunomodulation mediated by human placenta-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from either bone marrow (BMSCs) or placenta (PMSCs) have the capacity to suppress immune responses to mitogenic and allogeneic stimulations. Both cell contact and soluble factor dependent mechanisms have been proposed to explain this immunosuppression. This study explored the roles of some of cell surface molecules expressed on human PMSCs (hPMSCs) in hPMSC mediated immunomodulation. hPMSCs strongly suppressed mitogen and allogeneic peripheral mononuclear cells (PBMCs) induced T cell activation and proliferation. hPMSCs constituently expressed programmed death-ligand 1 (PD-L1) and Fas ligand (FasL) molecules. Neutralising antibodies to-PD-L1 and FasL significantly reduced the suppressive effect of hPMSCs on T cell proliferation. However, only anti-PD-L1 antibody partially restored early T cell activation suppressed by hPMSCs. Anti-FasL antibody but not anti-PD-L1 antibody reduced apoptosis of activated T cell indicating that FasL molecule plays a role in inducing apoptosis of activated T cells, although overall hPMSCs diminished T cell apoptosis. Different effects of PD-L1 and FasL molecules on T cell activation and activated T cell apoptosis suggest that these two molecules influence T cell response at different stages. hPMSCs significantly prevented activated T cells from going into S phase. Both antibodies to PD-L1 and FasL had significant effect on reversing the effect of hPMSCs on cell cycles. hPMSCs reduced INF-γ but increased IL-10 production by mitogen activated T cells. Both antibodies partially abolished the effect of hPMSCs on INF-γ and IL-10 production. These data demonstrated that PD-L1 and FasL molecules play significant roles in immunomodulation mediated by hPMSCs. This study provides a rational basis for modulation of negative costimulators on hPMSCs to increase their immunosuppressive properties in their therapeutic applications.

[Effects of exogenous nitric oxide on physiological characteristics of longan (Dimocarpus longana) seedlings under acid rain stress].

This paper studied the effects of exogenous nitric oxide donor sodium nitroprusside (SNP) on the chlorophyll content, antioxidant enzyme activities, and osmotic regulation substances of longan (Dimocarpus longana 'Fuyan') seedlings under acid rain (pH 3.0) stress. Under the acid rain stress, the seedling leaf superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities and chlorophyll, soluble protein and soluble sugar contents decreased obviously, while the leaf malondialdedyde content had a remarkable increase, suggesting the toxic effect of the acid rain on the seedlings. Exogenous nitric oxide had dual nature on the physiological characteristics of longan seedlings under acid rain stress. Applying 0.1-0.5 mmol x L(-1) of SNP improved the SOD, POD and CAT activities and the chlorophyll, soluble protein and soluble sugar contents significantly, and decreased the malondialdedyde content. Low concentrations SNP reduced the oxidative damage caused by the acid rain stress, and 0.5 mmol x L(-1) of SNP had the best effect. Under the application of 0.5 mmol x L(-1) of SNP, the total chlorophyll, soluble protein, and soluble sugar contents and the SOD, POD and CAT activities increased by 76.0%, 107.0%, 216.1%, 150. 0%, 350.9% and 97.1%, respectively, and the malondialdedyde content decreased by 46.4%. It was suggested that low concentration (0.1-0.5 mmol x L(-1)) SNP could alleviate the toxic effect of acid rain stress on longan seedlings via activating the leaf antioxidant enzyme activities and reducing oxidative stress, while high concentration SNP (1.0 mmol x L(-1)) lowered the mitigation effect.

A novel transfection method for eukaryotic cells using polyethylenimine coated albumin microbubbles.

Albumin microbubbles have been intensively studied for their application in gene delivery. However, with negative surface potential, albumin microbubbles hardly bind plasmid DNA, which might contribute to their low transgene efficiency. In this study, we developed polyethylenimine (PEI) coated albumin microbubbles (PAMB) which were prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol and glucose. CHO cells, COS cells and 293T cells were transfected with PEI, PEI+albumin, PAMB and Lipofectamine 2000, respectively. Our results showed that the surface potential was elevated and PAMB could bind plasmid DNA. The transgene efficiency of PAMB was higher than PEI and PEI+albumin (P<0.05), and PAMB performed the same transgene effect as Lipofectamine 2000 did but with lower cytotoxicity than Lipofectamine 2000. Albumin microbubbles modified by PEI has high transgene efficiency and low cytotoxicity even without ultrasound medication, making it a useful non-virus gene delivery method in vitro.

[Effects of inoculation with arbuscular mycorrhizal fungi on AIPO4 uptake by Poncirus trifoliata].

With sand culture in greenhouse, this paper studied the effects of inoculation with arbuscular mycorrhizal (AM) fungi Glomus mosseae and G. versiforme on the utilization of insoluble phosphate AlPO4 (Al-P) by trifoliate orange (Poncirus trifoliata) seedlings. The results indicated that inoculation with AM fungi notably increased the dry matter, P content, and P absorption rate of P. trifoliata. The contribution of mycorrhiza, P uptake by plant and AM fungi, and the contribution of AM fungi in P uptake by P. trifoliata increased greatly with increasing Al-P level in the medium. Inoculation with AM fungi greatly increased the excretion of phosphatase, especially acid- and neutral phosphatase, from plant root and hyphae, but the excretion amount decreased with increasing Al-P level. The contribution of the fungi in P uptake by P. trifoliata was significantly positively correlated with the amounts of acid-, neutral-, alkaline-, and total phosphatase.

[Effects of MSC on cell cycle, phenotype and cytokine secretion of T cells activated by PHA].

AIM: To study the effect of human bone marrow derived mesenchymal stem cell (MSC) on T cell cycle and activation, and to investigate the inhibitory effect of MSC on T cell proliferation and the underlying mechanism. METHODS: Human bone marrow derived MSC were isolated by gradient centrifugation. then in vitro MSC were cultured, expanded,and were used in test after third passage. FCM analysis and ELISA were used to investigate the effects of MSC on the early activation marker expression of T cells, cell cycle and cytokine secretion. RESULTS: T cells stimulated by PHA in the presence of MSC were arrested at G0/G1 phase. The expression of the early activation marker CD25 and CD69 of T cells was inhibited in the presence of MSC both in CD4(+) and CD8(+) T cell subpopulation. MSC caused a sharp decrease of cytokine secretion in IL-2 and IFN-gamma. CONCLUSION: Human bone marrow derived MSC can suppress the activation and proliferation of T cells by altering T cell cycle.

[Human bone marrow derived multipotent adult progenitor cells differentiating into hepatocyte-like cells after being induced by co-culturing with human hepatocyte line L02].

OBJECTIVE: To investigate the possibility of marrow derived multipotent adult progenitor cells (MAPCs) differentiating into hepatocytes by co-culturing with human hepatocyte line L02, and to evaluate the potential use of MAPCs in tissue-engineering either experimentally or clinically. METHODS: (1) Co-culturing without cell-to-cell contact: MAPCs and L02 hepatocytes were spread on coverslips separately (both with a cell density of 1x10(5)/ml), and then they were put in a culture dish (10 cm). The expressions of Alb, AFP, CK18, and CK19 in MAPCs were detected by immunocytochemistry at different time points. A separate culture of L02 hepatocytes served as a positive control and a separate culture of MAPCs served as a negative control. (2) Co-culturing with cell-to-cell contact: MAPCs labeled with CFSE were mixed with L02 hepatocytes (both with a cell density of 1x10(4)/ml), and then the mixed cells were seeded on specific dishes for detection by laser scanning confocal microscope (LSCM). Five days later, the cells were double-stained with SABC-Cy3. The expressions of Alb, AFP, CK18 in MAPCs were observed under LSCM. Similarly, separately cultured L02 hepatocytes served as a positive control and separately cultured MAPCs served as a negative control. RESULTS: (1) Results of co-culturing without cell-to-cell contact: On the first day, the MAPCs expressed a high level of AFP. Then AFP expression tapered daily and there was hardly any expression of AFP on day 7. The expression of Alb was very weak on day 1, but increased significantly by day 3, reached its peak on day 5, and still maintained a high level on day 7. The initial expression of CK18 appeared on day 5 and reached a higher level on day 7. The expression of CK19 was always negative. The positive control cells had a high expression of Alb and CK18, while there was a weak expression of AFP and a negative expression of CK19. The negative control cells had no expressions for the four markers. (2) Results of co-culturing with cell-to-cell contact: On day 5, there were three colors of fluorescence under LSCM: yellow cells were MAPCs differentiating into hepatocytes; green cells were undifferentiated MAPCs; red cells were L02 hepatocytes. The result showed that Alb and CK18 were expressed in many cells and AFP appeared in only a few cells. CONCLUSION: Human MAPCs can be induced to differentiate into mature hepatocyte-like cells by co-culturing with L02 hepatocytes, either with or without cell-to-cell contact, but the former way may be more effective.

First Thelazia callipaeda infestation report in a dog in Taiwan.

An ocular Thelazia callipaeda infestation was found in a male domestic dog in Taiwan during regular examination. This is the first report of the parasite from pet animal in Taiwan. The infested dog showed normal bodily condition but slight conjunctival congestion. This parasite has been reported in humans in southeast Asia, China and Korea. In 1998, first human case was reported in the central area of Taiwan. The dogs infected with T. callipaeda may have important implication in the infestation of this parasite to humans as reservoir hosts.