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Background: Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out., Results: We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16 M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/μl. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive)., Conclusions: The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30 min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objective: Heyi disease, Xila disease, and Badagan disease are three common diseases in Mongolian medicine. The changes in intestinal microbiota may be associated with the occurrence, development, and treatment of these diseases. This study aimed to investigate the effects of herbal treatment on intestinal microbiota and serum metabolites in rats with these three diseases., Methods: Firstly, Heyi, Xila, and Badagan disease model rats were established by environmental, diet, and drug intervention. Then, 16S rRNA gene sequencing and metabolomics analysis were used to analyze the changes in intestinal microbiota and serum metabolites after treatment. PICRUSt analysis was applied to predict the potential functions of intestinal microbiota, and OPLS-DA multivariate model was applied to screen differential serum metabolites., Results: 16S rRNA gene sequencing showed that herbal treatment significantly increased the species diversity and changed the composition of intestinal microbiota in Heyi disease and Xila disease rats. After treatment, there were 10, 9, and 3 bacterial biomarkers that were increased in Heyi, Xila, and Badagan disease rats, respectively. In the Heyi disease model, treatment resulted in 45 differential serum metabolites, involving 4 pathways. In the Badagan disease model, treatment resulted in 62 differential serum metabolites, involving 4 pathways. However, there was no significant difference in serum metabolites between TreatB and ConB in the Xila disease model., Conclusions: Herbal treatment significantly changed the intestinal microbiota and serum metabolites of rats with three Mongolian medicine diseases., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this article., (Copyright © 2022 Guniang Jiu et al.)

Utilizing recycled crushed clay brick (RCB) from C&D waste in road engineering construction as the substitute for natural aggregates has attracted a lot of attention, which would be a promising step forward towards sustainable development and green construction. The objective of this study is to assess the feasibility of cement-stabilized macadam (CSM), incorporating various RCB fine aggregate substitution ratios. For this purpose, the physical and chemical properties of RCB fine aggregate was tested, and RCB exhibited a porous surface micro-morphology, high water absorption and pozzolanic activity. Subsequently, a comprehensive experimental investigation of modified CSM with RCB has been carried out based on laboratory tests concerning the mechanical and shrinkage properties. Results showed that higher RCB fine aggregate substitution ratio resulted in lower unconfined compressive strength, and the negative influence of RCB on unconfined compressive strength would decrease gradually, varying curing time; however, the higher the RCB substitution ratio was, the larger the indirect tensile strength at 90 d curing time of the late curing period was. CSM containing RCB had an overall increasing accumulative water loss rate, accumulative strain of dry shrinkage and average coefficient of dry shrinkage, except that 20% RCB resulted in an excellent dry shrinkage property. Moreover, RCB with pozzolanic activity reacted very slowly mainly at later ages, enhancing the interfacial transition zone.

Background: Mongolian medicine is a systematic theoretical system, which is based on the balance among Heyi, Xila, and Badagan. However, the underlying mechanisms remain unclear. This study aimed to explore the characteristics of intestinal microbiota and metabolites in different rat models of Mongolian medicine., Methods: After establishing rat models of Heyi, Xila, and Badagan, we integrated 16S rRNA gene sequencing and metabolomics., Results: Heyi, Xila, and Badagan rats had significantly altered intestinal microbial composition compared with rats in the MCK group. They showed 11, 18, and 8 significantly differential bacterial biomarkers and 22, 11, and 15 differential metabolites, respectively. The glucosinolate biosynthesis pathway was enriched only in Heyi rats; the biosynthesis of phenylpropanoids pathway and phenylpropanoid biosynthesis pathway were enriched only in Xila rats; the isoflavonoid biosynthesis pathway, the glycine, serine, and threonine metabolism pathway, and the arginine and proline metabolism pathway were enriched only in Badagan rats., Conclusions: The intestinal microbiota, metabolites, and metabolic pathways significantly differed among Heyi, Xila, and Badagan rats compared with control group rats., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this article., (Copyright © 2021 Riao Dao et al.)

We surveyed Rickettsiales bacteria, including Rickettsia, Ehrlichia, Anaplasma, and Neoehrlichia, in wild sika deer (Cervus nippon nippon) from Shizuoka prefecture, Japan. In spleen samples from 187 deer, Anaplasma phagocytophilum (deer type), A. bovis, and A. centrale were successfully detected by PCR assay targeting to 16S rDNA or p44/msp2, and their positive rates were 96.3% (180/187), 53.5% (100/187), and 78.1% (146/187), respectively. Additionally, 2 or 3 Anaplasma species could be detected from a single deer in 165 spleen samples (88.2%), indicating dual or triple infection. In contrast, A. phagocytophilum (human type) 16S rDNA, Rickettsia gltA, Ehrlichia p28/omp-1, and Neoehrlichia 16S rDNA could not be amplified. The serological test of 105 deer serum samples by immunofluorescence assay showed that the detection of antibodies against antigens of A. phagocytophilum HZ (US-human isolate) and Rickettsia japonica YH were 29.5% (31/105) and 75.2% (79/105), respectively. These findings suggest that A. phagocytophilum (deer type), A. centrale, and A. bovis are highly dominant and prevalent in wild sika deer from Shizuoka, a central region of Japan, and that the antibodies against some Rickettsiales bacteria have also been retained in deer blood.

We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected.

Anaplasma phagocytophilum is an obligate intracellular bacterium and causes a febrile illness in humans and livestock. In nature, this bacterium is sustained in a tick-mammal cycle. Several p44/msp2-related genes are expressed from a single expression locus by gene conversion. In this study, we obtained 119 cDNA sequences of p44/msp2 transcripts from A. phagocytophilum in 6 Haemaphysalis ticks and 3 wild sika deer (Cervus nippon) in Japan. These 119 sequences were classified into 36 different variant sequences based on their similarities. The 36 cDNA sequences were phylogenetically grouped into 2 major clusters--tick- and deer-associated. The tick-associated sequences were further classified into 4 distinct subclusters, suggesting that A. phagocytophilum in ticks seems to selectively express specific p44/msp2 transcripts, such as the transcripts in the 4 subclusters that were closely related to previously identified p44/msp2 genes. The deer-associated sequences were also grouped into 4 subclusters, but these transcripts were probably more diverse than the transcripts derived from ticks. This might be due to the relatively nonselective expression of p44/msp2 in deer or the strain differences in A. phagocytophilum from ticks and deer in separate geographic regions or both. Thus, this study may contribute to the understanding of A. phagocytophilum p44/msp2 expression in nature in Japan.