miR-181d targets BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to Cryptosporidium parvum infection via the intrinsic apoptosis pathway.

Cryptosporidium parvum is an important zoonotic pathogen that is studied worldwide. MicroRNAs (miRNAs) act as post-transcriptional regulators and may play a key role in modulating host epithelial responses following Cryptosporidium infection. Our previous study has shown that C. parvum downregulates the expression of miR-181d through the p50-dependent TLRs/NF-κB pathway. However, the mechanism by which miR-181d regulates host cells in response to C. parvum infection remains unclear. The present study found that miR-181d downregulation inhibited cell apoptosis and increased parasite burden in HCT-8 cells after C. parvum infection. Bioinformatics analysis and luciferase reporter assays have shown that BCL2 was a target gene for miR-181d. Moreover, BCL2 overexpression and miR-181d downregulation had similar results. To further investigate the mechanism by which miR-181d regulated HCT-8 cell apoptosis during C. parvum infection, the expression of molecules involved in the intrinsic apoptosis pathway was detected. Bax, caspase-9, and caspase-3 expression was decreased at 4, 8, 12, and 24 hpi and upregulated at 36 and 48 hpi. Interfering with the expression of miR-181d or BCL2 significantly affected the expression of molecules in the intrinsic apoptosis pathway. These data indicated that miR-181d targeted BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to C. parvum infection via the intrinsic apoptosis pathway. These results allowed us to further understand the regulatory mechanisms of host miRNAs during Cryptosporidium infection, and provided a theoretical foundation for the design and development of anti-cryptosporidiosis drugs.

Host specific Eimeria genus diagnosis and qPCR development in Ovis aries and Capra hircus.

The objective of the present study was to develop a real-time PCR (qPCR) technique for the diagnosis of Eimeria spp. in Ovis aries and Capra hircus. The qPCR technique was developed using SYBR Green, resulting in a PCR with high sensitivity, specificity, and reproducibility.

Micro-RNA expression profile of BALB/c mouse glandular stomach in the early phase of Cryptosporidium muris infection.

Cryptosporidiosis is a zoonotic disease in humans and animals that is caused by infection with the oocysts of Cryptosporidium. MicroRNAs (miRNAs) are important players in regulating the innate immune response against parasitic infection. Public miRNAs data for studying pathogenic mechanisms of cryptosporidiosis, particularly in natural hosts, are scarce. Here, we compared miRNA profiles of the glandular stomach of C. muris-infected and uninfected BALB/c mice using microarray sequencing. A total of 10 miRNAs (including 3 upregulated and 7 downregulated miRNAs) with significant differential expression (|FC| ≥ 2 and P value < 0.05) were identified in the glandular stomach of BALB/c mice 8 h after infection with C. muris. MiRWalk and miRDB online bioinformatics tools were used to predict the target genes of differentially expressed miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to annotate the target genes. GO analysis indicate that gene transcription-related and ion transport-related GO terms were significantly enriched. In addition, the KEGG analyses showed that the target genes were strongly related to diverse types of tumor disease progression and anti-pathogen immunity pathways. In the current study, we firstly report changes in miRNA expression profiles in the glandular stomach of BALB/c mice at the early phase of C. muris invasion. This dysregulation in miRNA expression may contribute to our understanding of cryptosporidiosis pathology. This study provides a new perspective on the miRNA regulatory mechanisms of cryptosporidiosis, which may help in the development of effective control strategies against this pathogen.

Acaricidal and repellent activities of ethanol extracts of nine chinese medicinal herbs against Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus is a major threat to the cattle industry worldwide. The intensive use of acaricides and repellents has resulted in drug resistance. Hence, effective and eco-friendly pest control alternatives are urgently needed, especially from natural plant resources. In this study, the acaricidal and repellent activities of nine herbs against the larvae and eggs of R. microplus were evaluated. The results showed that ethanol extracts of star anise (Illicium verum), chaulmoogra (Hydnocarpus anthelmintica), motherwart (Leonurus artemisia), mandarin orange peel (citri reticulatae pericarpium, i.e., peel of Citrus reticulata fruit), and stemona (Stemona sessilifolia) had good contact acaricidal activities of 100, 98, 94, 88 and 86%, respectively, whereas star anise and clove (Syzygium aromaticum) had good fumigant acaricidal activities of 98 and 96%, respectively. The hatching inhibition rate of star anise against R. microplus eggs was 100%. All nine herbs had good real-time repellent rates, but only castor bean and star anise had repellent effects after 48 h (81.3 and 79.6%, respectively). This is the first report of the acaricidal and repellent activities of these medicinal herbs against R. microplus. Ethanol extracts of these herbs might be considered as potential alternatives to chemical acaricides for control of R. microplus.

MiR-3976 regulates HCT-8 cell apoptosis and parasite burden by targeting BCL2A1 in response to Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium is second only to rotavirus as a cause of moderate-to-severe diarrhea in young children. There are currently no fully effective drug treatments or vaccines for cryptosporidiosis. MicroRNAs (miRNAs) are involved in regulating the innate immune response to Cryptosporidium parvum infection. In this study, we investigated the role and mechanism of miR-3976 in regulating HCT-8 cell apoptosis induced by C. parvum infection. METHODS: Expression levels of miR-3976 and C. parvum burden were estimated using real-time quantitative polymerase chain reaction (RT-qPCR) and cell apoptosis was detected by flow cytometry. The interaction between miR-3976 and B-cell lymphoma 2-related protein A1 (BCL2A1) was studied by luciferase reporter assay, RT-qPCR, and western blotting. RESULTS: Expression levels of miR-3976 were decreased at 8 and 12 h post-infection (hpi) but increased at 24 and 48 hpi. Upregulation of miR-3976 promoted cell apoptosis and inhibited the parasite burden in HCT-8 cells after C. parvum infection. Luciferase reporter assay indicated that BCL2A1 was a target gene of miR-3976. Co-transfection with miR-3976 and a BCL2A1 overexpression vector revealed that miR-3976 targeted BCL2A1 and suppressed cell apoptosis and promoted the parasite burden in HCT-8 cells. CONCLUSIONS: The present data indicated that miR-3976 regulated cell apoptosis and parasite burden in HCT-8 cells by targeting BCL2A1 following C. parvum infection. Future study should determine the role of miR-3976 in hosts' anti-C. parvum immunity in vivo.

Crack mechanism and experimental verification on straightening of AZ31B magnesium alloy plate.

When plates with edge cracks in the rolling process is straightened by cyclic tensile and compressive stress, the tip of edge crack always accompanied by stress concentration, which leads to crack propagation. In this paper, damage parameters are imported into the plate straightening model based on determining the GTN damage parameters of magnesium alloy materials by inverse finite element calibration method, the influence of different straightening process schemes and prefabricated V-shaped crack geometry on crack growth is analyzed through the way of the combination of simulation and straightening experiment. The results show that the peak values of equivalent stress and equivalent strain under each straightening roll appear at the crack tip. The value of longitudinal stress and equivalent stain decrease with the distance to crack tip becomes larger. The peak value of longitudinal stress appears when the crack circumferential angle is about 100°, and the crack tip is easy to form crack propagation; when the plate passes roll 2 and roll 4, the equivalent stress and strain concentration at the crack tip are most obvious; when the reduction reaches a certain degree, the void volume fraction (VVF) reaches the VVF of the material breaking; with the increase of the entrance reduction, the number of VVF at the crack tip which reaches the material fracture increases, and the length of crack propagation increases; the stress concentration at the tip of V-shaped crack with large length-width ratio is obvious, and the VVF is more likely to reach the VVF at the time of material fracture, crack initiates and propagates easily.

Whole transcriptome analysis of HCT-8 cells infected by Cryptosporidium parvum.

BACKGROUND: Cryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed. METHODS: The expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs. RESULTS: After strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA. CONCLUSIONS: The whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection.

Research Note: Prevalence and zoonotic concern of Blastocystis in farmed chickens in southern China.

Blastocystis is a gastrointestinal protozoan parasite commonly reported in humans and animals globally, including poultry, and it can cause zoonotic transmission of blastocystosis. However, comprehensive information is not available on the prevalence, subtype distribution and zoonotic potential of Blastocystis in chickens in China. In this study, a total of 1,000 individual fecal samples of free-range broiler chickens of 4 breeds were collected from 43 farms in 5 cities of Guangdong Province and investigated for the occurrence of Blastocystis infection. Blastocystis was determined by nested PCR analysis of the small subunit ribosomal RNA (SSU rRNA) gene. The overall prevalence was 20.1% (201/1,000) in chicken samples and 69.8% (30/43) in screened farms, and considerable variation in prevalence between farms was evident, with a range of 0 to 76.9%. Population differences of Blastocystis in broilers among sites, breeds, and ages were assessed. The highest infection rates were observed in Yangjiang city (35.8%, 38/106), Sanhuang chickens (29.7%, 104/350), and the >80-day-old chicken group (30.5%, 40/131). DNA sequencing and phylogeny analyses identified 2 zoonotic subtypes, ST6 and ST7. A large predominance was observed for ST7, and genetic polymorphisms were confirmed at the intra-ST7 level with the identification of 5 divergent ST7 types. The incidence of both STs varied largely based on the breed, site, farm, and age. This is the first large-scale study to explore the prevalence and genetic characteristics of Blastocystis in chickens in China. The widespread distribution and avian adaptation of both zoonotic subtypes were demonstrated. The findings of this study highlight a potential threat to humans and will provide a better understanding of the epidemiology and public health impact of poultry Blastocystis.

Lockdown measures during the COVID-19 pandemic strongly impacted the circulation of respiratory pathogens in Southern China.

A range of public health measures have been implemented to suppress local transmission of coronavirus disease 2019 (COVID-19) in Shenzhen. We examined the effect of these measures on the prevalence of respiratory pathogens in children. Clinical and respiratory pathogen data were collected for routine care from hospitalized children with acute respiratory infections in Shenzhen Children's Hospital from July 2018 to January 2022. Nasopharyngeal swabs were collected and respiratory pathogens were detected using standardized clinical diagnostics as part of routine care. Data were analyzed to describe the effects of COVID-19 prevention procedures on other common pathogens. A total of 56,325 children under 14 years of age were hospitalized with an acute respiratory infection during the study period, 33,909 were tested from July 2018 to January 2020 (pre-lockdown), 1168 from February 2020 to May 2020 (lockdown) and 21,248 from July 2020 to January 2022 (post-lockdown). We observed a 37.3% decline of routine care in respiratory infection associated hospital admission in the 19 months' post-lockdown vs. the 19 months' pre-lockdown. There were 99.4%, 16.0% and 1.26% reductions measured for Mycoplasma pneumoniae, influenza virus A and adenovirus, respectively. However, a 118.7% and 75.8% rise was found for respiratory syncytial virus (RSV) and human para-influenza virus (HPIV) during the 19 months' post-lockdown in comparison to the pre-pandemic period. The detection of RSV especially increased in toddlers after the lockdown. Lockdown measures during the COVID-19 pandemic led to a significant reduction of Mycoplasma pneumoniae, influenza virus A and adenovirus infection. In contrast, RSV and HPIV infection increased.

Genotyping of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi from sheep and goats in China.

BACKGROUND: Few studies have molecularly characterized the potential zoonotic protozoa, Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in sheep and goats in China, therefore total 472 fecal samples were collected from eight provinces and infection rates of three protozoa were determined by PCR analysis of corresponding loci. All PCR positive samples were sequenced to identify the genotype. RESULTS: The overall infection rates for Cryptosporidium, G. duodenalis, and E. bieneusi were 1.9% (9/472), 20.6% (97/472), and 44.5% (210/472), respectively. C. xiaoi (n = 5), C. ubiquitum (n = 3), and C. anderson (n = 1) were identified in goats. 97 G. duodenalis strains were successfully detected, and assembly E (n = 96) and assembly A (n = 1) were identified. Two novel G. duodenalis multilocus genotype (MLGs) were identified, with one belonging to subgroup AI and the other to subgroup E5. Nine known genotype (BEB6, CD6, CHC8, CHG3, CHG5, Peru6, CHG1, CHG2, and COS-I) and four new genotype (CHG26, CHG27, CHG28, and CHS18) were identified in E. bieneusi, with CHG3 dominant in this group. CONCLUSIONS: The present results highlight the role of sheep and goats as reservoir hosts for this three gastrointestinal pathogens. In summary, we provided a platform for more detailed research on genotyping or subtyping intestinal pathogens to better understand their risks and modes of transmission.

MiR-942-5p targeting the IFI27 gene regulates HCT-8 cell apoptosis via a TRAIL-dependent pathway during the early phase of Cryptosporidium parvum infection.

BACKGROUND: MicroRNAs (miRNAs) are involved in the regulation of both the innate and adaptive immune response to Cryptosporidium parvum infection. We previously reported that C. parvum upregulated miR­942­5p expression in HCT­8 cells via TLR2/TLR4­NF­κB signaling. In the present study, the role of miRNA-942-5p in the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated HCT-8 cell apoptosis induced by C. parvum was investigated. METHODS: Quantitative real-time polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence were used for analysis. RESULTS: Forced expression of miRNA-942-5p resulted in decreased apoptosis and an increased C. parvum burden in HCT-8 cells. The opposite results were observed using the suppressed expression of miRNA-942-5p. The miRNA-942-5p led to the translational suppression of IFI27 gene through targeting the 3'-untranslated region of the IFI27 gene. Moreover, overexpression of the IFI27 gene produced a high apoptotic ratio and low C. parvum burden. In contrast, a low apoptotic ratio and a high C. parvum burden were observed following downregulation of the IFI27 gene. Both miR-942-5p and the IFI27 gene influenced TRAIL and caspase-8 expression induced by C. parvum in HCT-8 cells. Moreover, TRAIL promoted HCT-8 cell apoptosis in a concentration-dependent manner. CONCLUSIONS: These data suggested that C. parvum induced the downregulation of IFI27 via relief of miR-942-5p-mediated translational suppression. IFI27 downregulation was affected the burden of C. parvum by regulating HCT-8 cell apoptosis through TRAIL-dependent pathways. Future studies should determine the mechanisms by which C. parvum infection increases miR-942-5p expression and the role of miR-942-5p in hosts' anti-C. parvum immunity in vivo.

Molecular detection and phylogeny of Anaplasma spp. closely related to Anaplasma phagocytophilum in small ruminants from China.

The genus Anaplasma comprises eight bacterial species that are obligate intracellular pathogens that affect human and animal health. The zoonotic species A. phagocytophilum is the causative agent of tick-borne fever in ruminants, and of granulocytic anaplasmosis in horses, dogs, and humans. Recently, novel strains related to A. phagocytophilum (A. phagocytophilum-like 1/Japanese variant and A. phagocytophilum-like 2/Chinese variant) have been identified. The aim of this study was to reveal the prevalence and phylogeny of A. phagocytophilum and related stains in small ruminants and ticks in China based on sequences of the 16S rRNA combined restriction fragment length polymorphism (RFLP) and groEL genes. PCR-RFLP and phylogenetic analyses based on the 16S rRNA gene showed the presence of A. phagocytophilum-like 1 and 2 variants in sampled animals from China, with prevalence rates of 22.6% (303/1338) and 0.7% (10/1338), respectively. Only A. phagocytophilum-like 1 DNA was found in Haemaphysalis longicornis. The phylogeny based on the groEL gene showed inclusion of A. phagocytophilum-like 1 and some A. phagocytophilum-like 2 strains in two unique clades distinct from, but related to, Japanese and Chinese strains of related A. phagocytophilum, respectively. One noteworthy result was that the SSAP2f/SSAP2r primers detected Ehrlichia spp. strains. Moreover, the A. phagocytophilum-like 1 and 2 strains should be considered in the differential diagnosis of caprine and ovine anaplasmosis. Further investigations should be conducted to provide additional epidemiological information about A. phagocytophilum and A. phagocytophilum-like variants in animals and ticks.

Cryptosporidium parvum downregulates miR-181d in HCT-8 cells via the p50-dependent TLRs/NF-κB pathway.

Cryptosporidium spp. can cause diarrhea and even death in humans and animals. Host microRNAs (miRNAs) play an important role in the post-transcriptional regulation of the innate immune response to Cryptosporidium infection. To study host miRNA activity in the innate immune response to C. parvum infection, we examined the expression of miR-181d in HCT-8 cells infected with C. parvum and found that it was significantly downregulated, while TLR2, TLR4, NF-κB, and myD88 involved in the TLR/NF-κB signaling pathway were significantly upregulated at the early stages of C. parvum infection. We transfected cells with short-interfering RNAs (siRNA) as TLR2, TLR4, and NF-κB inhibitors. Analysis by quantitative real-time polymerase chain reaction (qPCR) and western blot confirmed that C. parvum downregulates miR-181d expression via the p50 subunit-dependent TLR2/TLR4-NF-κB signaling pathway in HCT-8 cells. This study provides a new theoretical foundation to elucidate the regulatory mechanism of host miRNAs against Cryptosporidium infection.

Occurrence and subtyping of Blastocystis in coypus (Myocastor coypus) in China.

BACKGROUND: Blastocystis is an anaerobic unicellular protist frequently detected in the gastrointestinal tracts of humans and animals worldwide. However, the prevalence and subtype distribution of Blastocystis in the coypu (Myocastor coypus) population have not been reported so far. The aim of this study was to determine the prevalence, genetic characteristics, and zoonotic potential of Blastocystis isolates detected in coypus in China. RESULTS: A total of 308 fecal samples were collected from coypus in seven regions across China and subsequently examined. Blastocystis was detected in 44 (14.3%) specimens by nested PCR amplification of the small subunit ribosomal rRNA (SSU rRNA) gene. Further DNA sequencing and phylogenetic analyses resulted in the identification of two zoonotic known subtypes, ST4 and ST5, and an unknown subtype. ST4 was the most predominant subtype observed in the samples. ST5 infections were only observed in three coypus. Factors that were associated with prevalence of Blastocystis included age, geographical region and subtype. Interestingly, this is the first report about a potentially novel subtype infecting coypus. CONCLUSIONS: This is the first comprehensive report of Blastocystis in M. coypus across a wide geographic range of China. A moderate degree of genetic divergence was observed. The presence of zoonotic subtypes in farmed M. coypus suggests that these animals have the potential to transmit blastocystosis to both humans and domestic animals. These findings provide a better understanding of the genetic diversity of Blastocystis in rodents and contribute towards the establishment of efficient blastocystosis control strategies in the investigated areas.

Globally elevated chemical weathering rates beneath glaciers.

Physical erosion and chemical weathering rates beneath glaciers are expected to increase in a warming climate with enhanced melting but are poorly constrained. We present a global dataset of cations in meltwaters of 77 glaciers, including new data from 19 Asian glaciers. Our study shows that contemporary cation denudation rates (CDRs) beneath glaciers (2174 ± 977 Σ*meq+ m-2 year-1) are ~3 times higher than two decades ago, up to 10 times higher than ice sheet catchments (~150-2000 Σ*meq+ m-2 year-1), up to 50 times higher than whole ice sheet means (~30-45 Σ*meq+ m-2 year-1) and ~4 times higher than major non-glacial riverine means (~500 Σ*meq+ m-2 year-1). Glacial CDRs are positively correlated with air temperature, suggesting glacial chemical weathering yields are likely to increase in future. Our findings highlight that chemical weathering beneath glaciers is more intense than many other terrestrial systems and may become increasingly important for regional biogeochemical cycles.

Hyaluronan-induced alterations of the gut microbiome protects mice against Citrobacter rodentium infection and intestinal inflammation.

Hyaluronan is a glycosaminoglycan polymer that has been shown to play an important role in homeostasis of the gastrointestinal tract. However, its mechanistic significance in gastrointestinal epithelial barrier elements remain unexplored. Here, our results revealed that hyaluronan treatment resulted in significant changes in the gut microbiota in mice. To demonstrate the functional consequences of hyaluronan-treatment and hyaluronan-induced microbiota alterations, Citrobacter rodentium- and DSS-induced colitis models and microbiota transplantation approaches were utilized. We showed that hyaluronan alleviated intestinal inflammation in both pathogen and chemically induced intestinal mucosal damage. The protection in bacterial colitis was associated with enhanced C. rodentium clearance and alleviation of pathogen-induced gut dysbiosis. Microbiota transplantation experiments showed that the hyaluronan-altered microbiota is sufficient to confer protection against C. rodentium infection. Colonization with Akkermansia muciniphila, a commensal bacterium that is greatly enriched by hyaluronan treatment, alleviated C. rodentium-induced bacterial colitis in mice. Additionally, Akkermansia-induced protection was found to be associated with the induction of goblet cells and the production of mucins and epithelial antimicrobial peptides. Collectively, these results provide novel insights into the regulatory role of hyaluronan in modulating the gut microbiota and immunity in enteric infection and inflammation, with therapeutic potential for gut microbiome-targeted immunotherapy.

PREVALENCE AND MOLECULAR CHARACTERISTICS OF BLASTOCYSTIS SP. FROM PEAFOWL (PAVO CRISTATUS) IN CHINA.

This study is the first description of Blastocystis infection in peafowls in China. In total, 143 fecal specimens collected from a peafowl breeding farm in Henan Province were tested for Blastocystis infection by PCR assay targeting the small subunit ribosomal RNA (SSU rRNA) gene, and a total of 50 specimens (35.0%) were positive. Based on sequences and phylogenetic analysis, 2 genetically distinct subtypes (STs) were determined: ST9 and ST7. ST9 was the predominant subtype, accounting for 82% (41/50). The rare zoonotic subtype ST7 was also identified in peafowls, with the infection rate of 18% (9/50). Altogether, the present study is the first report of the prevalence and molecular characteristics of Blastocystis in peafowls in central China. The presence of zoonotic subtypes in peafowls suggests the potential risk of zoonotic transmission of Blastocystis to workers at peafowl farms.

Comparison of 11 respiratory pathogens among hospitalized children before and during the COVID-19 epidemic in Shenzhen, China.

BACKGROUND: The effect of SARS-CoV-2 on existing respiratory pathogens in circulation remains uncertain. This study aimed to assess the impact of SARS-CoV-2 on the prevalence of respiratory pathogens among hospitalized children. METHODS: This study enrolled hospitalized children with acute respiratory infections in Shenzhen Children's Hospital from September to December 2019 (before the COVID-19 epidemic) and those from September to December 2020 (during the COVID-19 epidemic). Nasopharyngeal swabs were collected, and respiratory pathogens were detected using multiplex PCR. The absolute case number and detection rates of 11 pathogens were collected and analyzed. RESULTS: A total of 5696 children with respiratory tract infection received multiplex PCR examination for respiratory pathogens: 2298 from September to December 2019 and 3398 from September to December 2020. At least one pathogen was detected in 1850 (80.5%) patients in 2019, and in 2380 (70.0%) patients in 2020; the detection rate in 2020 was significantly lower than that in 2019.The Influenza A (InfA) detection rate was 5.6% in 2019, but 0% in 2020. The detection rates of Mycoplasma pneumoniae, Human adenovirus, and Human rhinovirus also decreased from 20% (460), 8.9% (206), and 41.8% (961) in 2019 to 1.0% (37), 2.1% (77), and 25.6% (873) in 2020, respectively. In contrast, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased from 6.6% (153), 9.9% (229), and 0.5% (12) in 2019 to 25.6% (873), 15.5% (530), and 7.2% (247) in 2020, respectively (p < 0.0001). CONCLUSIONS: Successful containment of seasonal influenza as a result of COVID-19 control measures will ensure we are better equipped to deal with future outbreaks of both influenza and COVID-19.Caused by virus competition, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased in Shenzhen,that reminds us we need to take further monitoring and preventive measures in the next epidemic season.

Development of a duplex PCR assay for detecting Theileria luwenshuni and Anaplasma phagocytophilum in sheep and goats.

Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962 bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641 bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13 fg/µL for T. luwenshuni and 1.53 fg/µL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.