Protection against Mycoplasma hyorhinis infection in commercial pigs via immunization with inactivated vaccines prepared with homologous or heterologous strains.

Mycoplasma hyorhinis is a highly prevalent pathogen in pig farms worldwide, causing polyserositis and polyarthritis, resulting in great economic losses. Previous genotyping and pathogenic studies have revealed significant genetic and antigenic diversity among M. hyorhinis strains. While there are reports on M. hyorhinis vaccine development, the cross-protection between different M. hyorhinis strains has not been clarified. In this study, two M. hyorhinis strains (HEF-16 and JS-54), belonging to different sequence types, were inactivated to produce vaccines. Pigs were vaccinated respectively and subsequently infected with strain HEF-16. The protection against challenge with homologous or heterologous strains was determined and compared. Both vaccinated groups of pigs exhibited a high antibody titer two weeks after the first vaccination, and significant decreases in pathogen load in joints, along with an increase in average daily weight gain compared to the challenged group after M. hyorhinis challenge. Pigs immunized with the HEF-16-derived vaccine showed a significant reduction in joint swelling and lameness, similar to pigs immunized with the JS-54-derived vaccine. At necropsy, animals in the challenged group exhibited moderate-to-severe polyserositis and arthritis, whereas pathological changes were greatly reduced in animals from the vaccinated groups. No significant differences were observed in clinical symptoms nor pathological damages between the two vaccinated groups. Overall, our study demonstrates the effective protection of the inactivated M. hyorhinis vaccines against challenges with homologous or heterologous strains in commercial pigs. This indicates a promising clinical application prospect for inactivated bacterin vaccines in preventing M. hyorhinis-related diseases in pig farms.

ASFV subunit vaccines: Strategies and prospects for future development.

African Swine Fever (ASF) is an acute, highly contagious, and lethal disease caused by the African Swine Fever Virus (ASFV), posing a severe threat to the global pig farming industry. Although live vaccines are currently available, preventing and controlling ASF remains a considerable challenge. Several factors have impeded vaccine development, including the complexity of ASFV particles and the suppressive effects of its gene-encoded proteins on the host's immune system. This article delves into the immunological responses elicited by ASFV, encompassing both innate and adaptive immunity, and examines how ASFV evades host immune defenses. Special attention is given to the current progress in the development of ASFV subunit vaccines, including protein-based vaccines, DNA vaccines, and viral vector vaccines. The advantages, challenges, and future directions of different vaccine types are discussed, offering new perspectives and insights for the future of ASFV vaccine development.

Mycoplasma synoviae surface-located elongation factor G functions as a cytoadhesin to promote adhesion to synovial sheath cells through binding to vimentin.

Mycoplasma synoviae infection has caused serious economic losses to the poultry industry worldwide. The molecular mechanism by which M. synoviae colonizes the synovium and induces synovitis is unclear. In this study, desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry analyses were used to screen M. synoviae membrane proteins that bind the membrane proteins of synovial sheath cells (SSCs). Among the 128 screened proteins, elongation factor G (EF-G) of M. synoviae was identified as a surface-located protein using colony blotting and dual fluorescence analyses. The immunogenicity of EF-G was confirmed by the preparation of a rabbit polyclonal antibody. EF-G was identified as a cytoadhesin that directly binds to SSCs using indirect immunofluorescence assay and ELISA plate binding assay. In addition, antibody adhesion inhibition and protein adhesion inhibition demonstrated that EF-G could significantly promote the adhesion of M. synoviae to SSCs. Co-IP, GST pull-down, bacterial two-hybridization, and ELISA plate binding assays were performed to demonstrate the binding of EF-G and vimentin in vivo and in vitro. Antibody adhesion inhibition, protein adhesion inhibition, and siRNA interference adhesion inhibition assays demonstrated that vimentin significantly affected M. synoviae adhesion to SSCs. These studies indicate that two interacting proteins, EF-G, a novel cytoadhesin, and vimentin, an important cell surface receptor, play important roles in the adhesion of M. synoviae to SSCs, laying a foundation for subsequent studies on the mechanism of M. synoviae-induced synovitis and providing meaningful targets for screening target drugs against M. synoviae infection.

[Development of an ELISA method for detection of antibodies against Mycoplasma hyopneumoniae based on Mhp336 protein].

This study aims to establish an ELISA method with high specificity for the detection of antibodies against Mycoplasma hyopneumoniae. Firstly, we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen. Then, we optimized the ELISA parameters, including antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, colorimetric reaction time, and cut-off value. Afterwards, reproducibility experiments were conducted, and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined. Finally, 226 porcine serum samples were detected using the method established in this study, a commercial ELISA kit for M. hyopneumoniae antibody detection, and a convalescent serum ELISA kit for M. hyopneumoniae antibody detection. The detection results of the three methods were compared to evaluate the sensitivity and specificity of the ELISA method established in this study. For this method, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, and colorimetric reaction time were 0.05 µg/mL, PBS containing 2.5% skim milk, 1 h, 1:500, 0.5 h, 1:10 000, 1 h, and 5 min, respectively. Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332. The coefficients of variation of both intra-batch and inter-batch kits were below 7%. The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M. hyopneumoniae antibody detection in terms of sensitivity and specificity. We successfully established an ELISA method for detecting the antibodies against M. hyopneumoniae based on Mhp336 protein. This method demonstrated high sensitivity and specificity, serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.

Enhancing Point-of-Care Diagnosis of African Swine Fever Virus (ASFV) DNA with the CRISPR-Cas12a-Assisted Triplex Amplified Assay.

Accurate, ultrasensitive, and point-of-care (POC) diagnosis of the African swine fever virus (ASFV) remains imperative to prevent its spread and limit the losses incurred. Herein, we propose a CRISPR-Cas12a-assisted triplex amplified colorimetric assay for ASFV DNA detection with ultrahigh sensitivity and specificity. The specific recognition of recombinase aided amplification (RAA)-amplified ASFV DNA could activate the Cas12a/crRNA/ASFV DNA complex, leading to the digestion of the linker DNA (bio-L1) on magnetic beads (MBs), thereby preventing its binding of gold nanoparticles (AuNPs) network. After magnetic separation, the release of AuNPs network comprising a substantial quantity of AuNPs could lead to a discernible alteration in color and significantly amplify the plasmonic signal, which could be read by spectrophotometers or smartphones. By combining the RAA, CRISPR/Cas12a-assisted cleavage, and AuNPs network-mediated colorimetric amplification together, the assay could detect as low as 0.1 copies/µL ASFV DNA within 1 h. The assay showed an accuracy of 100% for the detection of ASFV DNA in 16 swine tissue fluid samples, demonstrating its potential for on-site diagnosis of ASFV.

Public expenditure on health care and the incidence of non-communicable diseases (NCDs) in China from 2008 to 2018.

OBJECTIVES: This study investigates whether differences in individual-level and provincial-level health funding could explain or mitigate health inequalities among older people in terms of non-communicable diseases within a population served by fragmented health insurance schemes. STUDY DESIGN: A national repeated cross-sectional analysis was done of the 2008, 2011, 2014, and 2018 Chinese Longitudinal Healthy Longevity Surveys. These provided a total of 44,623 persons aged 60 and over. MAIN OUTCOME MEASURES: Respondents were asked whether they had been diagnosed with any types of non-communicable diseases by doctors. A dichotomous outcome variable was constructed to indicate whether older people had any diagnosed non-communicable diseases. RESULTS: Compared with uninsured older persons, those who were enrolled in social health insurance schemes designed for civil servants as cadres, urban employees and urban residents were more likely to report a higher incidence of non-communicable diseases. There were no significant differences in the prevalence of non-communicable diseases between uninsured older people and those in the New Rural Cooperative Medical Scheme. Although the incidence of non-communicable diseases among older persons increased over the study period, greater health expenditure was significantly associated with a lower risk of non-communicable diseases. The interaction results between individual social health insurance schemes and public health expenditure indicate that disparities in the incidence of non-communicable diseases among different health insurance schemes diminish as public health expenditure increases. Older individuals with Public Free Medical Services benefited the most in provinces with higher public health expenditure compared with other health insurance schemes. CONCLUSIONS: Given the evidence of the beneficial effects of universal health coverage on non-communicable diseases among older persons, these results should encourage policy makers to increase public health funding and to raise the overall benefit packages for social health insurance schemes.

Necroptosis in Pneumonia: Therapeutic Strategies and Future Perspectives.

Pneumonia remains a major global health challenge, necessitating the development of effective therapeutic approaches. Recently, necroptosis, a regulated form of cell death, has garnered attention in the fields of pharmacology and immunology for its role in the pathogenesis of pneumonia. Characterized by cell death and inflammatory responses, necroptosis is a key mechanism contributing to tissue damage and immune dysregulation in various diseases, including pneumonia. This review comprehensively analyzes the role of necroptosis in pneumonia and explores potential pharmacological interventions targeting this cell death pathway. Moreover, we highlight the intricate interplay between necroptosis and immune responses in pneumonia, revealing a bidirectional relationship between necrotic cell death and inflammatory signaling. Importantly, we assess current therapeutic strategies modulating necroptosis, encompassing synthetic inhibitors, natural products, and other drugs targeting key components of the programmed necrosis pathway. The article also discusses challenges and future directions in targeting programmed necrosis for pneumonia treatment, proposing novel therapeutic strategies that combine antibiotics with necroptosis inhibitors. This review underscores the importance of understanding necroptosis in pneumonia and highlights the potential of pharmacological interventions to mitigate tissue damage and restore immune homeostasis in this devastating respiratory infection.

Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain.

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.

Conserved Domains in Variable Surface Lipoproteins A-G of Mycoplasma hyorhinis May Serve as Probable Multi-Epitope Candidate Vaccine: Computational Reverse Vaccinology Approach.

Mycoplasma hyorhinis (M. hyorhinis) is responsible for infections in the swine population. Such infections are usually cured by using antimicrobials and lead to develop resistance. Until now, there has been no effective vaccine to eradicate the disease. This study used conserved domains found in seven members of the variable lipoprotein (VlpA-G) family in order to design a multi-epitope candidate vaccine (MEV) against M. hyorhinis. The immunoinformatics approach was followed to predict epitopes, and a vaccine construct consisting of an adjuvant, two B cell epitopes, two HTL epitopes, and one CTL epitope was designed. The suitability of the vaccine construct was identified by its non-allergen, non-toxic, and antigenic nature. A molecular dynamic simulation was executed to assess the stability of the TLR2 docked structure. An immune simulation showed a high immune response toward the antigen. The protein sequence was reverse-translated, and codons were optimized to gain a high expression level in E. coli. The proposed vaccine construct may be a candidate for a multi-epitope vaccine. Experimental validation is required in future to test the safety and efficacy of the hypothetical candidate vaccine.

A Whole-Genome Analysis of the African Swine Fever Virus That Circulated during the First Outbreak in Vietnam in 2019 and Subsequently in 2022.

Since its initial report in Vietnam in early 2019, the African swine fever (ASF), a highly lethal and severe viral swine disease worldwide, continues to cause outbreaks in other Southeast Asian countries. This study analyzed and compared the genomic sequences of ASF viruses (ASFVs) during the first outbreak in Hung Yen (VN/HY/2019-ASFV1) and Quynh Phu provinces (VN/QP/2019-ASFV1) in Vietnam in 2019, and the subsequent outbreak in Hung Yen (VN/HY/2022-ASFV2) in 2022, to those of other ASFV strains. VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 genomes were 189,113, 189,081, and 189,607 bp in length, encoding 196, 196, and 203 open reading frames (ORFs), respectively. VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 shared a 99.91-99.99% average nucleotide identity with genotype II strains. Variations were identified in 28 ORFs in VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 compared to 20 ASFV strains, and 16 ORFs in VN/HY/2022-ASFV2 compared to VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1. Vietnamese ASFV genomes were classified as IGR II variants between the I73R and I329L genes, with two copy tandem repeats between the A179L and A137R genes. A phylogenetic analysis based on the whole genomes of 27 ASFV strains indicated that the Vietnamese ASFV strains are genetically related to Estonia 2014, ASFV-SY18, and Russia/Odintsovo_02/14. These results reveal the complete genome sequences of ASFV circulating during the first outbreak in 2019, providing important insights into understanding the evolution, transmission, and genetic variation of ASFV in Vietnam.

Mucosal and cellular immune responses elicited by nasal and intramuscular inoculation with ASFV candidate immunogens.

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) that is highly contagious and has an extremely high mortality rate (infected by virulent strains) among domestic and wild pigs, causing huge economic losses to the pig industry globally. In this study, SDS-PAGE gel bands hybridized with ASFV whole virus protein combined with ASFV-convalescent and ASFV-positive pig serum were identified by mass spectrometry. Six antigens were detected by positive serum reaction bands, and eight antigens were detected in ASFV-convalescent serum. In combination with previous literature reports and proteins corresponding to MHC-II presenting peptides screened from ASFV-positive pig urine conducted in our lab, seven candidate antigens, including KP177R (p22), K78R (p10), CP204L (p30), E183L (p54), B602L (B602L), EP402R-N (CD2V-N) and F317L (F317L), were selected. Subunit-Group 1 was prepared by mixing above-mentioned seven ASFV recombinant proteins with MONTANIDETM1313 VG N mucosal adjuvant and immunizing pigs intranasally and intramuscularly. Subunit-Group 2 was prepared by mixing four ASFV recombinant proteins (p22, p54, CD2V-N1, B602L) with Montanide ISA 51 VG adjuvant and immunizing pigs by intramuscular injection. Anticoagulated whole blood, serum, and oral fluid were collected during immunization for flow cytometry, serum IgG as well as secretory sIgA antibody secretion, and cytokine expression testing to conduct a comprehensive immunogenicity assessment. Both immunogen groups can effectively stimulate the host to produce ideal humoral, mucosal, and cellular immune responses, providing a theoretical basis for subsequent functional studies, such as immunogens challenge protection and elucidation of the pathogenic mechanism of ASFV.

The prevalence of hypertension and hypertension control among married Namibian couples.

BACKGROUND: Previous studies suggest that having a marital partner with hypertension is associated with an individual's increased risk of hypertension, however this has not been investigated in sub-Saharan Africa despite hypertension being a common condition; the age-standardised prevalence of hypertension was 46.0% in 2013 in Namibia. OBJECTIVE: To explore whether there is spousal concordance for hypertension and hypertension control in Namibia. METHODS: Couples data from the 2013 Namibia Demographic and Health Survey were analysed. Bivariable and multivariable logistic regression models were used to explore the odds of individual's hypertension based on their partner's hypertension status, 492 couples. and the odds of hypertension control in individuals based on their partner's hypertension control (121 couples), where both members had hypertension. Separate models were built for female and male outcomes for both research questions to allow independent consideration of risk factors to be analysed for female and males. RESULTS: The unadjusted odds ratio of 1.57 (CI 1.10-2.24) for hypertension among individuals (both sexes) whose partner had hypertension compared to those whose partner did not have hypertension, was attenuated to aOR 1.35 (CI 0.91-2.00) for females (after adjustment for age, BMI, diabetes, residence, individual and partner education) and aOR 1.42 (CI 0.98-2.07) for males (after adjustment for age and BMI). Females and males were significantly more likely to be in control of their hypertension if their partner also had controlled hypertension, aOR 3.69 (CI 1.23-11.12) and aOR 3.00 (CI 1.07-8.36) respectively. CONCLUSIONS: Having a partner with hypertension was positively associated with having hypertension among married Namibian adults, although not statistically significant after adjustment. Partner's hypertension control was significantly associated with individual hypertension control. Couples-focused interventions, such as routine partner screening of hypertensive individuals, could be developed in Namibia.

Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.

Identification of the multiple roles of enolase as an plasminogen receptor and adhesin in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent underlying porcine enzootic pneumonia, a chronic respiratory disease worldwide. The recruitment of plasminogen to the surface and subsequently promotion of plasmin conversion by the surface-located receptor, have been reported to assist the adhesion and invasion of Mycoplasmas. The surface localization and plasminogen-binding ability of M. hyopneumoniae enolase were previously confirmed; however, the biological functions were not be determined, especially the role as a plasminogen receptor. Here, using ELISA and SPR analyses, we confirmed the stable binding of M. hyopneumoniae enolase to plasminogen in a dose-dependent manner. The facilitation of the activation of plasminogen in the presence of tPA and direct activation of plasminogen at low efficiency without tPA addition by M. hyopneumoniae enolase were also determined using a plasmin-specific chromogenic substrate. Notably, the C-terminal and N-terminal regions located in M. hyopneumoniae enolase play an important role in plasminogen binding and activation. Additionally, we demonstrate that M. hyopneumoniae enolase can competitively inhibit the adherence of M. hyopneumoniae to PK15 cells. These results provide insight into the role of enolase in M. hyopneumoniae infection, a mechanism that manipulates the proteolytic system of the host.

Apigenin suppresses mycoplasma-induced alveolar macrophages necroptosis via enhancing the methylation of TNF-α promoter by PPARγ-Uhrf1 axis.

BACKGROUND: Mycoplasma-associated pneumonia is characterized by severe lung inflammation and immunological dysfunction. However, current anti-mycoplasma agents used in clinical practice do not prevent dysfunction of alveolar macrophages caused by the high level of the cytokine tumor necrosis factor-α (TNF-α) after mycoplasma infection. Apigenin inhibits the production of TNF-α in variet inflammation associated disease. PURPOSE: This study aimed to investigate apigenin's effect on mycoplasma-induced alveolar immune cell injury and the mechanism by which it inhibits TNF-α transcription. METHODS: In this study, we performed a mouse model of Mycoplasma hyopneumoniae infection to evaluate the effect of apigenin on reducing mycoplasma-induced alveolar immune cell injury. Furthermore, we carried out transcriptome analysis, RNA interference assay, methylated DNA bisulfite sequencing assay, and chromatin immunoprecipitation assay to explore the mechanism of action for apigenin in reducing TNF-α. RESULTS: We discovered that M. hyopneumoniae infection-induced necroptosis in alveolar macrophages MH-S cells and primary mouse alveolar macrophages, which was activated by TNF-α autocrine. Apigenin inhibited M. hyopneumoniae-induced elevation of TNF-α and necroptosis in alveolar macrophages. Apigenin inhibited TNF-a mRNA production via increasing ubiquitin-like with PHD and RING finger domains 1 (Uhrf1)-dependent DNA methylation of the TNF-a promotor. Finally, we demonstrated that apigenin regulated Uhrf1 transcription via peroxisome proliferator activated receptor gamma (PPARγ) activation, which acts as a transcription factor binding to the Uhrf1 promoter and protected infected mice's lungs, and promoted alveolar macrophage survival. CONCLUTSION: This study identified a novel mechanism of action for apigenin in reducing alveolar macrophage necroptosis via the PPARγ/ Uhrf1/TNF-α pathway, which may have implications for the treatment of Mycoplasma pneumonia.

Genotyping and biofilm formation of Mycoplasma hyopneumoniae and their association with virulence.

Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson's index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.

Th1 and Th17 mucosal immune responses elicited by nasally inoculation in mice with virulence factors of Mycoplasma hyopneumoniae.

Nicotinamide Adenine Dinucleotide-Dependent (NADH) flavin oxidoreductase and NADH oxidase (NOX) are important virulence factors of Mycoplasma hyopneumoniae (Mhp), which are devoted to the function of adhesion, oxidative stress damage and apoptosis to host cells in our previous studies. Here, immune responses of NADH flavin oxidoreductase (NFOR) and NOX in mice and immune efficacy inoculated with intramuscular (IM), intranasal (IN), intramuscular unite intranasal (IM + IN) approaches were evaluated and compared. Cellular immunity levels, systemic immune and local mucosal immune responses were investigated by indirect enzyme-linked immunosorbent assay (iELISA) and quantitative reverse transcription PCR (qRT-PCR). Mice inoculated with NFOR and NOX by IM and IN or IM + IN could induce obvious secretion of specific immunoglobulin G (IgG) and secretory immunoglobulin A antibodies (sIgA) compared to those in negative control group. IM + IN inoculation resulted in systemic and local mucosal immune responses that were strongly produced. Moreover, Mhp NFOR and NOX could activate local mucosal immune responses mediated by Th1 and Th17 cells by IN. Our finding supported the notion that IM + IN was an effective immunization route for Mhp, which lays a foundation for more effective prevention of Mhp, and provides theoretical basis for the development of new subunit vaccines of Mhp.

Phenotypic characteristics and protective efficacy of an attenuated Mycoplasma hyopneumoniae vaccine by aerosol administration.

With the improvement of large-scale breeding in pig farms, conventional head-by-head immunization has disadvantages with low efficiency and high cost. Considering that most pathogens leading to pulmonary diseases circulate from the respiratory mucosa, immunization through the respiratory tract route has been a highly attractive vaccine delivery strategy. In this study, to develop an effective Mycoplasma hyopneumoniae (Mhp) aerosol vaccine, a customized ultrasonic atomizer was developed. The aerodynamic diameter, activity, and content of the Mhp aerosol vaccine were measured. In addition, piglets were immunized with the Mhp aerosol vaccine, and the immunity of the animal challenge protection test was evaluated. At the end of nebulization, the mass median aerodynamic diameters (MMAD) and geometric standard deviation (GSD) of the aerosol were 2.98 ± 0.02 µm and 1.51 ± 0.02, respectively. Moreover, 10 min after nebulization, the MMAD and GSD of the aerosol were 2.76 ± 0.02 µm and 1.51 ± 0.01, respectively, which were hardly changed. Compared with theoretical value, the actual titer of aerosol vaccines presented in 50% color changing unit (CCU50) after nebulization decreased 0.6. The shape, size, and uniformity of collected aerosols are relatively stable. The proportion of Mhp in aerosol produced by vaccine stock solution and 10 times diluted vaccine solution was 76.52% and 58.82%, respectively, and the average number of Mhp in a single aerosol was 3.06 and 1.51, respectively. In addition, the aerosol vaccine antigen particles could be transported to the lower respiratory tract, a local mucosal immune response was induced in piglets. The vaccine colonized the respiratory tract and significantly decline the lung lesion index after aerosol vaccination. In conclusion, an effective aerosol vaccine against Mhp infection was developed. And this is the first effective assessment for Mhp live vaccine with aerosolization against infection in piglets.

Protocol: evaluation of an optimised couples-focused intervention to increase testing for HIV in rural KwaZulu-Natal, South Africa, the Igugu Lethu ('Our treasure') cohort study.

BACKGROUND: Between 2012 and 2015, the Uthando Lwethu (UL) study demonstrated that a theory-based behavioural couples-focused intervention significantly increased participation in couples HIV testing and counselling (CHTC) among South African couples who had never previously tested for HIV together or mutually disclosed their HIV status, 42% compared to 12% of the control group at 9 months follow-up. Although effective, we were nonetheless concerned that in this high prevalence setting the majority (58%) of intervention couples chose not to test together. In response we optimised the UL intervention and in a new study, 'Igugu Lethu', we are evaluating the success of the optimised intervention in promoting CHTC. METHODS: One hundred eighty heterosexual couples, who have been in a relationship together for at least 6 months, are being recruited and offered the optimised couples-focused intervention. In the Igugu Lethu study, we have expanded the health screening visit offered to couples to include other health conditions in addition to CHTC. Enrolled couples who choose to schedule CHTC will also have the opportunity to undertake a random blood glucose test, blood pressure and BMI measurements, and self-sample for STI testing as part of their health screening. Individual surveys are administered at baseline, 4 weeks and 4 months follow-up. The proportion of couples who decide to test together for HIV will be compared to the results of the intervention arm in the UL study (historical controls). To facilitate this comparison, we will use the same recruitment and follow-up strategies in the same community as the previous UL study. DISCUSSION: By strengthening communication and functioning within the relationship, the Igugu Lethu study, like the previous UL study, aims to transform the motivation of individual partners from a focus on their own health to shared health as a couple. The Igugu Lethu study findings will answer whether the optimised couples-focused behavioural intervention and offering CHTC as part of a broader health screening for couples can increase uptake of CHTC by 40%, an outcome that would be highly desirable in populations with high HIV prevalence. TRIAL REGISTRATION: Retrospectively registered. ISRCTN Registry ISRCTN 46162564 Registered on 26th May 2022.

Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.