Semiparametric estimation of restricted mean survival time as a function of restriction time.

The restricted mean survival time (RMST) is an appealing measurement in clinical or epidemiological studies with censored survival outcome and receives a lot of attention in the past decades. It provides a useful alternative to the Cox model for evaluating the covariate effect on survival time. The covariate effect on RMST usually varies with the restriction time. However, existing methods cannot address this problem properly. In this article, we propose a semiparametric framework that directly models RMST as a function of the restriction time. Our proposed model adopts a widely-used proportional form, enabling the estimation of RMST predictions across an interval using a unified model. Furthermore, the covariate effect for multiple restriction time points can be derived simultaneously. We develop estimators based on estimating equations theories and establish the asymptotic properties of the proposed estimators. The finite sample properties of the estimators are evaluated through extensive simulation studies. We further illustrate the application of our proposed method through the analysis of two real data examples. Supplementary Material are available online.

Perforin-2 Breaches the Envelope of Phagocytosed Bacteria Allowing Antimicrobial Effectors Access to Intracellular Targets.

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study, we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2's bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of Ags contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface Ag was found to be independent of Perforin-2. Consistent with our in vitro results, a protease-sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate, our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria, facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.

Bias-adjusted Kaplan-Meier survival curves for marginal treatment effect in observational studies.

For time-to-event outcomes, the Kaplan-Meier estimator is commonly used to estimate survival functions of treatment groups and to compute marginal treatment effects, such as the difference in survival rates between treatments at a landmark time. The derived estimates of the marginal treatment effect are uniformly consistent under general conditions when data are from randomized clinical trials. For data from observational studies, however, these statistical quantities are often biased due to treatment-selection bias. Propensity score-based methods estimate the survival function by adjusting for the disparity of propensity scores between treatment groups. Unfortunately, misspecification of the regression model can lead to biased estimates. Using an empirical likelihood (EL) method in which the moments of the covariate distribution of treatment groups are constrained to equality, we obtain consistent estimates of the survival functions and the marginal treatment effect. Equating moments of the covariate distribution between treatment groups simulate the covariate distribution that would have been obtained if the patients had been randomized to these treatment groups. We establish the consistency and the asymptotic limiting distribution of the proposed EL estimators. We demonstrate that the proposed estimator is robust to model misspecification. Simulation is used to study the finite sample properties of the proposed estimator. The proposed estimator is applied to a lung cancer observational study to compare two surgical procedures in treating early-stage lung cancer patients.

Targeting Neutrophils in Severe Asthma via Siglec-9.

Severe asthma comprises only 5% of patients with asthma, but the burden it brings to the social health system accounts for more than half of all asthmatics. Clinical evidence shows that severe asthma is often linked to the recruitment and activation of neutrophils in the airways. However, the underlying molecular and immunological mechanisms of neutrophilia in severe asthma are not clear and currently available drugs exert only limited effects on neutrophilic inflammation. Great efforts are underway to address the mystery of neutrophilic inflammation in chronic respiratory disorders. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are members of the immunoglobulin gene family. Of note, Siglec-9 is uniquely expressed by human neutrophils and monocytes, as well as a minor population of natural killer cells. Engaging this structure with antibodies or glycan ligands results in programmed cell death in human neutrophils. Intriguingly, the administration of Siglec-E antibody abolished the recruitment of neutrophils in mouse models of neutrophilic pulmonary inflammation in vivo. Given that neutrophils are probably a major culprit in the generation and perpetuation of inflammation, targeting Siglec-9 could be beneficial for the treatment of severe asthma, chronic obstructive pulmonary disease, and related pulmonary disorders characteristic of neutrophilia.

Effect of diosmetin on airway remodeling in a murine model of chronic asthma.

Bronchial asthma, one of the most common allergic diseases, is characterized by airway hyperresponsiveness (AHR), inflammation, and remodeling. The anti-oxidant flavone aglycone diosmetin ameliorates the inflammation in pancreatitis, but little is known about its impact on asthma. In this study, the effects of diosmetin on chronic asthma were investigated with an emphasis on the modulation of airway remodeling in BALB/c mice challenged with ovalbumin (OVA). It was found that diosmetin significantly relieved inflammatory cell infiltration, goblet cell hyperplasia, and collagen deposition in the lungs of asthmatic mice and notably reduced AHR in these animals. The OVA-induced increases in total cell and eosinophil counts in bronchoalveolar lavage fluid were reversed, and the level of OVA-specific immunoglobulin E in serum was attenuated by diosmetin administration, implying an anti-Th2 activity of diosmetin. Furthermore, diosmetin remarkably suppressed the expression of smooth muscle actin alpha chain, indicating a potent anti-proliferative effect of diosmetin on airway smooth muscle cells (ASMCs). Matrix metallopeptidase-9, transforming growth factor-ß1, and vascular endothelial growth factor levels were also alleviated by diosmetin, suggesting that the remission of airway remodeling might be attributed to the decline of these proteins. Taken together, our findings provided a novel profile of diosmetin with anti-remodeling therapeutic benefits, highlighting a new potential of diosmetin in remitting the ASMC proliferation in chronic asthma.

Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma hyorhinis.

BACKGROUND: To establish a method for sensitive and rapid diagnosis of Mycoplasma hyorhinis in clinical specimens, a simple, sensitive loop-mediated isothermal amplification (LAMP) assay was designed and evaluated. METHODS: Three sets of four special primers, recognizing distinct sequences of the target, were designed for sensitive, specific amplification of nucleic acid under isothermal conditions. The LAMP assay was carried out using 35 clinical specimens of bronchoalveolar lavage fluid (BALF) from pigs. For comparison, these specimens were also tested using conventional PCR, real-time PCR, and nested PCR assays. RESULTS: After optimization of the reaction condition and reaction system, the LAMP reaction successfully detected Mycoplasma hyorhinis within 40 minutes at 61 degrees C. The LAMP assay achieved a sensitivity of 10(1) copies per microL at 61 degrees C in 40 minutes, compared to real-time PCR and nested PCR, and was over 10(3) times more sensitive than conventional PCR. In the test for the specificity of the LAMP assay, only Mycoplasma hyorhinis genomic DNA was positive and no other microorganisms were positive with the primers, indicating that the LAMP assay is specific to Mycoplasma hyorhinis. Mycoplasma hyorhinis was detected in 32 samples using the LAMP and real-time PCR assays and in 27 and 11 samples using the nested PCR assay and conventional PCR assay, respectively. All the positive samples detected by real-time PCR, nested PCR and conventional PCR assays were positive in the LAMP assay. CONCLUSIONS: The LAMP assay is inexpensive, easy to perform, shows a rapid reaction and does not require complex instruments like PCR. Therefore, LAMP is a simple, accurate, fast, and economical assay suitable as an alternative in veterinary practices.

Different responses of abundant and rare bacterial composition to groundwater depth and reduced nitrogen application in summer maize field.

Introduction: It is well known that reduced nitrogen application and groundwater depth can change soil microbial communities, but the associated difference in the response of abundant and rare bacterial composition to these local environmental changes remains unclear. Methods: In this study a lysimeter experiment was carried out to examine the impact of reduced nitrogen and groundwater depth on the composition of abundant and rare bacteria. Results and discussion: Our results demonstrated that the summer maize field soil species composition of rare bacterial sub-communities was significantly regulated by reduced nitrogen application, groundwater depth change and their interactions. However, only reduced nitrogen application had a significant influence on the species composition of abundant bacterial sub-communities. The structural equation model (SEM) indicated that reduced nitrogen application and groundwater depth change also could indirectly regulate the species composition of abundant and rare bacteria by altering soil attributes. The changes in soil pH and TSN had the most significant effects on the community composition of abundant and rare bacteria, respectively. More importantly, rare bacterial sub-communities were more sensitive to the changes in nitrogen input, groundwater depth and soil factors. Collectively, our study first demonstrated that abundant and rare microbial sub-communities responded differently to reduced nitrogen application and groundwater depth change. This study highlights that summer maize farmland production management should take nitrogen input and groundwater depth into consideration to maintain the compositional stability of soil rare microbial sub-communities.

Effect of Fat to Lean Meat Ratios on the Formation of Volatile Compounds in Mutton Shashliks.

This study aimed to investigate the release of volatile compounds in mutton shashliks (named as FxLy, x-fat cubes: 0-4; y-lean cubes: 4-0) with different fat-lean ratios before and during consumption, respectively. In total, 67 volatile compounds were identified in shashliks using gas chromatography/mass spectrometry. Aldehyde, alcohol, and ketone were the major volatile substances, accounting for more than 75% of the total volatile compounds. There were significant differences in the volatile compounds of mutton shashliks with different fat-lean ratios. With the increase of the fat content, the types and content of volatile substances released also increase. However, when the percentage of fat exceeded 50%, the number of furans and pyrazine, which were characteristic of the volatile compounds of roasted meat, was decreased. The release of volatiles during the consumption of mutton shashliks was measured using the exhaled breath test and the results showed that adding an appropriate amount of fat (<50%) helps to enrich the volatile compound components in the mouth. However, shashliks with higher fat-lean ratios (>2:2) shorten the mastication duration and weaken the breakdown of bolus particles in the consumption process, which is not conducive to the release potential of volatile substances. Therefore, setting the fat to lean ratio to 2:2 is the best choice for making mutton shashliks, as it (F2L2) can provide rich flavor substances for mutton shashliks before and during consumption.

Study on the Relationship between AAD and Clinical Features in Emergency Ward Patients and the Application Effect of Probiotics.

In order to investigate the influencing factors of AAD in the emergency ward and the clinical effect of probiotics, 100 AAD patients admitted to the emergency ward of our hospital from January 2021 to January 2022 and 100 healthy physical examination subjects are selected for conducting clinical trials. The subjects are, respectively, included in the AAD group and the healthy group. By using the random number table method, 100 patients in the AAD group are randomly divided into the probiotic group and traditional group, with 50 patients in each group. The traditional group receives conventional treatment, and the probiotic group is treated with Clostridium caseinate bifidobacterial probiotics additionally. Multivariate logistic analysis is performed on the risk factors for AAD, and the comparison of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and the intestinal microflora of the different groups before and after treatment is conducted. The experimental result reveals that probiotics treatment for AAD patients can reduce inflammatory response and promote intestinal colonization of beneficial bacteria such as bifidobacteria.

The application of pre-hospital first aid mode in patients with acute stroke: meta-analysis.

To systematically evaluate the application effect of pre-hospital and in-hospital emergency mode in patients with acute stroke. The study was conducted by systematic search of Chinese (CNKI, Wanfang and VIP) and English (PubMed, EMBASE and Cochrane Library) databases. The case-control studies comparing the role of pre-hospital and in-hospital emergency mode for patients with acute stroke were included in this study. Outcome indicators included the time from admission to thrombolytic therapy (DNT), the time from calling for help to receiving professional treatment, the first aid effect (effective rate, disability rate and mortality), complications and prognosis. Meta-analysis was performed using RevMan 5.3. Seventeen studies were included in the final analysis. Compared with traditional emergency measures, pre-hospital and in-hospital emergency measures can significantly reduce DNT (mean difference [MD] = -22.63, p < 0.00001), time from call to professional treatment (MD: -13.22, p < 0.00001), disability rate (RR = 0.88, p = 0.004), fatality rate (RR = 0.58, p < 0.00001), central cerebral fever (RR = 0.44, p = 0.0009), and gastrointestinal bleeding (RR = 0.44, p = 0.002). In addition, daily living ability (MD = 16.56, p < 0.00001) and emergency response rate (RR = 1.50, p < 0.00001) were significantly improved. The pre-hospital and in-hospital emergency mode has a significant emergency effect in patients with acute stroke, which is a protective factor. This emergency mode can be widely used in clinical practice.

Wogonin attenuates neutrophilic inflammation and airway smooth muscle proliferation through inducing caspase-dependent apoptosis and inhibiting MAPK/Akt signaling in allergic airways.

BACKGROUND: Severe neutrophilic asthma is often characterized by persistent airway inflammation and irreversible airway remodeling, which are overstimulated by the high-mobility group box protein 1 (HMGB1). Although wogonin, an O-methylated flavone, has been widely used to treat inflammatory and allergic diseases, its therapeutic effects and potential mechanisms on severe neutrophilic asthma remain elusive. OBJECTIVE: To evaluate whether wogonin alleviates airway neutrophilia through inducing neutrophil apoptosis and attenuates airway smooth muscle cells (ASMCs) proliferation and migration. METHODS: The effect of wogonin on reducing neutrophilic airway inflammation, including neutrophil infiltration and inflammatory mediators, was examined in a mouse model of severe neutrophilic asthma sensitized with ovalbumin and lipopolysaccharide. Also, the effect of wogonin on inducing human neutrophil apoptosis was manifested using cellular morphology, flow cytometry, and caspase inhibition assays. Furthermore, the effect of wogonin on inhibiting HMGB1-mediated ASMCs proliferation and migration was determined. RESULTS: Wogonin reduced the frequency of neutrophils and inhibited the production of multiple inflammatory mediators, including ovalbumin-specific IgE, tumor necrosis factor-α, interleukin-6, and HMGB1, in bronchoalveolar lavage fluid and lung tissues of the neutrophilic asthmatic mouse model. These data strongly support a significantly suppressed neutrophilic airway inflammation, functionally consistent to the relieved airway hyperresponsiveness by wogonin in vivo. Wogonin induced human neutrophil apoptosis in a dose-dependent manner by activating caspase-8 and caspase-3 in vitro. Wogonin pretreatment abolished HMGB1-induced ASMCs proliferation and migration, which can be explained by the inhibition of phosphorylation in the mitogen-activated protein kinase (MAPK) /Akt singling pathways. CONCLUSION: Our findings demonstrate that wogonin augments caspase-dependent apoptosis in neutrophils to alleviate neutrophilic inflammatory responses and regulates intracellular signaling to inhibit HMGB1-mediated ASMCs activation, providing a promising therapeutic agent for severe neutrophilic asthma.

Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells.

Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH active-site mutant has lost all hemolytic and phospholipase activities as well as pathogenicity. In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated. Erythrocyte membranes formed thin tubular protrusions immediately after exposure to VHH, and membrane corrugations were evident after extended incubation. In contrast, the mutant VHH did not induce any gross morphological changes. With VHH-treated FG-9307 cells, a cell line derived from flounder gill, destruction of organelles and formation of features resembling apoptotic bodies were observed. Immunogold staining showed that a large amount of VHH was deposited on the membranes and membrane debris of erythrocytes and FG-9307 cells after treatment with VHH. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in VHH-treated FG-9307 cells using DAPI staining. Moreover, cell cycle analysis showed that VHH increased the proportion of cells in G1 phase. In addition, VHH significantly increased the percentage of apoptosis, the number of TUNEL positive apoptotic cells, and caspase-3 activity in FG-9307 cells when compared with the untreated controls. These data suggested that VHH killed the cells through apoptosis via the caspase activation pathway.

Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity.

Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.

[Correlation of propagated sensation along meridian and TCM constitution].

OBJECTIVE: To explore the correlation between propagated sensation along meridian (PSM) and TCM constitution at different age stages. METHODS: According to age, 840 participants were divided into a youth group (326 cases), a middle aged group (243 cases) and an elderly group (271 cases). The TCM constitution of all the participants was evaluated, and the PSM test was performed. The distribution of TCM constitution, the occurring rate and transmission of PSM in each group were observed and compared; the correlation between PSM and the TCM constitution was preliminary investigated by Logistic regression analysis. RESULTS: The distribution of nine types of TCM constitution in three groups:the proportion of normal constitution and partial constitution were significantly different (all P<0.05); the occurring rate and transmission of PSM in three groups were not significantly different (all P>0.05); the proportion of occurring rate for nine types of TCM constitutions in the whole population, from high to low, presented special intrinsic quality, neutral quality, yin-deficiency quality, qi-deficiency quality, yang-deficiency quality, damp-heat quality, phlegm-dampness quality, qi-stagnation quality and blood-stasis quality; besides, the proportion of occurring rate for different TCM constitutions in the youth group, middle aged group and elderly group was similar to that of whole population. The Logistic regression analysis results indicated the neutral quality (P=0.025) and special intrinsic quality (P=0.018) were positively while blood-stasis quality (P=0.043) was negatively related with PSM in all subjects; the qi-deficiency quality (P=0.025), phlegm-dampness quality (P=0.019), blood-stasis quality (P=0.012) and qi-stagnation quality (P=0.035) were negatively related with PSM in youth group; the neutral quality (P=0.001) was positively related with PSM inthe middle aged group; the neutral quality (P=0.006) and yin deficiency quality (P=0.004) were positively related with PSM in the elderly group. CONCLUSIONS: The occurrence of PSM in different age stages is related with TCM constitution, which could be increased in clinical treatment to improve acupuncture efficacy.

Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65.

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection.

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1ß, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation.

Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs.

An attenuated Mycoplasma hyopneumoniae vaccine that requires intrathoracic administration is commercially available for use against mycoplasmal pneumonia in China. Given the limitations of such a route of administration, this study was undertaken to assess the capacity of an ISCOM-matrix adjuvant to enhance immunogenicity following intramuscular use. Immune responses in pigs following vaccination and subsequent intra-tracheal bacterial inoculation were examined using lymphocyte proliferation, serology and mucosal IgA in both nasal and saliva swabs. Vaccination induced clear lymphocyte proliferation, but only slight serum antibody responses although these were significantly increased following experimental infection. Mucosal IgA was not detected in either nasal or salivary secretions. Following bacterial challenge, animals vaccinated with the adjuvant-containing live vaccine exhibited less severe pulmonary lesions (median score 3.67) than unvaccinated pigs (median score 13.58). The degree of ciliary loss on the respiratory tract surface was reduced in vaccinated pigs compared with experimentally infected controls. The findings indicated that the adjuvant vaccine administered IM provided protection against experimentally induced mycoplasmal pneumonia and could have commercial potential.

Effect of different adjuvant formulations on the immunogenicity and protective effect of a live Mycoplasma hyopneumoniae vaccine after intramuscular inoculation.

Mycoplasma hyopneumoniae (M. hyopneumoniae) vaccine strain 168 is an intrapulmonically injected attenuated live vaccine that is available in the Chinese market. The aim of this study was to develop suitable adjuvants for this live vaccine to provide effective protection after intramuscular inoculation. Several adjuvant components were screened to assess their toxicity for the live vaccine, and various adjuvant formulations were then designed and prepared. Vaccines supplemented with these adjuvants were used to immunize mice intramuscularly to assess the capacity of the adjuvants to induce a specific immune response. The screened formulations were then evaluated in pigs. Seven of the eight adjuvant components did not affect the viability of the live vaccine, and seven different adjuvant formulations were then designed. In mice, the ISCOM-matrix adjuvant and the levamisole-chitosan mixture adjuvant significantly enhanced serum IgG responses against M. hyopneumoniae, while lymphocyte proliferation was enhanced by the ISCOM-matrix adjuvant, the carbomer-astragalus polysaccharide mixture adjuvant and an oil-in-water emulsion adjuvant. These four adjuvants were evaluated in pigs. Enhancement of specific lymphocyte proliferation responses was observed in the groups vaccinated with the ISCOM-matrix adjuvant and the carbomer-astragalus polysaccharide mixture adjuvant. Significant enhancement of serum IgG antibody production was observed before challenge in pigs vaccinated with the carbomer-astragalus polysaccharide mixture adjuvant and the levamisole-chitosan mixture adjuvant, while after challenge, all of the animals that received vaccines containing adjuvants had higher antibody concentrations against M. hyopneumoniae than unvaccinated animals. Animals inoculated with a vaccine containing the ISCOM-matrix adjuvant (median score 3.57) or the carbomer-astragalus polysaccharide mixture adjuvant (median score 5.28) had reduced lesion scores compared to unvaccinated animals (median score 14.81). These studies will help in the development of appropriate adjuvants for intramuscular administration of this live M. hyopneumoniae vaccine.

Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection.

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection. Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%). It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection.