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Feline calicivirus (FCV) is a contagious cat pathogen that causes oral ulceration and/or upper respiratory disease. In this study, we collected 61 samples from a pet hospital in Beijing and used PCR or RT-PCR to detect FCV and feline herpesvirus 1 (FHV-1). Approximately 44.3% (27/61) of the samples were FCV positive, and 23.0% (14/61) were coinfected with FCV and FHV-1. FCV was isolated from 15 samples. One isolate was from a cat with virulent systemic disease (VSD) signs, and 14 isolates were from cats with stomatitis or upper respiratory diseases. The range of genome sequence identity among these isolates was 76.1–100.0%. Four of the isolates were considered to be of the same strain, with sequence identity ranging from 99.5 to 99.7%, and two isolates, BJ-280 and BJ-288, had completely identical sequences. The genomic sequence identity ranged from 76.0 to 88.5% between the 15 isolates and several reference strains, including the F4 and F9 vaccine strains. These results demonstrate that many FCV strains are co-circulating in Beijing. Due to the diversity of FCV in Beijing, it is necessary to monitor the current prevalence of the virus. This study provides more information for the development of effective measures to control FCV.

A multiplex PCR (m-PCR) with primers targeting the 16S rRNA, Rv3873 and a 12.7-kb fragment in the genomes of a Mycobacterium tuberculosis complex was designed for the differential diagnosis of M. tuberculosis, M. bovis, M. bovis BCG and non-tuberculosis Mycobacterium (NTM). The specificity of this assay was 100%, and the detection limit was 15 pg of genomic DNA. Of the 206 blinded clinical samples, the detection rate of M. bovis infection by m-PCR was lower than that of the interferon gamma (IFN-γ) release assay; however, the false-positive rate by the tuberculin skin test and false-negative samples in the IFN-γ release assay were reduced. Our findings indicated that our m-PCR method is a useful tool for complementation to differentiate M. bovis from M. tuberculosis and NTM species.

Gingivitis is evidenced by inflammation of the free gingiva, and still reversible. If left untreated, it may then progress to periodontitis. In the present study, the therapeutical effect of ketotifen fumarate on gingivitis was explored. Domestic cats with varying degrees of gingivitis naturally were enrolled in this study. Subgroups of animals were treated twice daily for one week with or without ketotifen fumarate (5 mg/kg). Effects of ketotifen fumarate were measured on gingival index, cells accumulation, mediators release, receptor-ligand interaction, oxidative stress, MAPK and NF-κB pathways, epithelial barrier and apoptosis. Ketotifen fumarate attenuated the initiation and progression of gingivitis, inhibited the infiltrations of mast cells, B lymphocytes, T lymphocytes, macrophages, neutrophils and eosinophils as well as the release of IgE, β-hexosaminidase, tryptase, chymase, TNF-α, IL-4, and IL-13, influenced endothelial cells, fibroblasts and epithelial cells proliferation and apoptosis, and induced Th2 cells polarization, where ketotifen fumarate also might affect their interactions. Ketotifen fumarate reduced the oxidative stress, and inhibited NF-κB and p38 MAPK related with mast cells and macrophages accumulation. Ketotifen fumarate improved the aberrant expression of ZO-1 and inhibits the following apoptosis. On the other hand, these cells and mediators augmented functional attributes of them involving SCF/c-Kit, α4β7/VCAM-1 and IL-8/IL-8RB interactions, thus creating a positive feedback loop to perpetuate gingivitis, where an inflammation microenvironment was modeled. Our results showed a previously unexplored therapeutic potential of ketotifen fumarate for gingivitis and further suggest that, in addition to biofilms, targeting inflammation microenvironment could be new strategy for the treatment of gingivitis/periodontitis.

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.

Fifty-five samples (15.62%) collected from dogs and cats were identified as canine parvovirus (CPV) infection in Beijing during 2010-2013. The nucleotide identities and aa similarities were 98.2-100% and 97.7-100%, respectively, when compared with the reference isolates. Also, several synonymous and non-synonymous mutations were also recorded for the first time. New CPV-2a was dominant, accounting for 90.90% of the samples. Two of the 16 samples collected from cats were identified as new CPV-2a (12.5%), showing nucleotide identities of 100% with those from dogs. Twelve samples (15.78%) collected from completely immunized dogs were found to be new CPV-2a, which means CPV-2 vaccines may not provide sufficient protection for the epidemic strains.

The TB10.4 antigen of Mycobacterium bovis/Mycobacterium tuberculosis induces a strong Th1 CD4+ T-cell response. Thus, it is currently under intensive study as a possible vaccine candidate. However, how TB10.4 activates innate immune cells is unclear. How TB10.4 interacts with toll-like receptors (TLRs) and signaling pathways responsible for active inflammation have also not been fully elucidated. Here, as stimulated RAW264.7 cells with recombinant TB10.4 (rTB10.4), derived from M. bovis, increased TNF-α, IL-6 and IL-12 p40 secretin in a dose-dependent manner. Blocking assays showed that TLR2-, but not TLR4-neutralizing antibody reduced expression of TNF-α, IL-6 and IL-12 p40 in RAW264.7 cells. rTB10.4 stimulation activated p38 kinase (p38) and extracellular-regulated kinase (ERK) was TLR2-dependent, whereas inhibition of p38 and ERK activity significantly reduced TNF-α, IL-6 and IL-12 p40 production. Furthermore, rTB10.4 stimulation of RAW264.7 cells resulted in TLR2-mediated activation of NF-κB and induced translocation of NF-κB p65 from the cytoplasm to the nucleus via IκBα degradation. rTB10.4-induced TNF-α, IL-6 and IL-12 p40 release was attenuated by the specific IκB phosphorylation inhibitor, BAY 11-7082. These findings indicate that the M. bovis-derived rTB10.4 induced production of TNF-α, IL-6 and IL-12 p40 involves p38, ERK and NF-κB via the TLR2 pathway.

Graphical abstract, Tylvalosin, a new broad-spectrum, third-generation macrolides, may exert a variety of pharmacological activities. Here, we report on its anti-oxidative and anti-inflammatory activity in RAW 264.7 macrophages and mouse treated with lipopolysaccharide (LPS) as well as piglet challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin treatment markedly decreased IL-8, IL-6, IL-1β, PGE2, TNF-α and NO levels in vitro and in vivo. LPS and PRRSV-induced reactive oxygen species (ROS) production, and the lipid peroxidation in mice lung tissues reduced after tylvalosin treatments. In mouse acute lung injury model induced by LPS, tylvalosin administration significantly attenuated tissues injury, and reduced the inflammatory cells recruitment and activation. The evaluated phospholipase A2 (PLA2) activity and the increased expressions of cPLA2-IVA, p-cPLA2-IVA and sPLA2-IVE were lowered by tylvalosin. Consistent with the mouse results, tylvalosin pretreatment attenuated piglet lung scores with improved growth performance and normal rectal temperature in piglet model induced by PRRSV. Furthermore, tylvalosin attenuated the IκBα phosphorylation and degradation, and blocked the NF-κB p65 translocation. These results indicate that in addition to its direct antimicrobial effect, tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury through suppression of NF-κB activation.

Cargo transportation equipment is commonly used for ground loading in aerospace. Taking flexible cargo bags as a transport carriers and taking a cargo ship hatch as the target container, a kind of cargo bag transportation system is designed according to the transportation process and function requirements. On the basis of the analysis of structure and working procedure, the working principle of the mechanical subsystem as well as the structure design of key components is proposed in detail. To improve the performance of support plate’s stiffness, a topological optimization is implemented and the optimized result is hence achieved. Finite element simulation shows that the performance of the stiffness is improved significantly. The approach presented in this research benefits the design of cargo transportation equipment.

A CpG-enriched recombinant plasmid (pUC18-CpG) as an adjuvant of FMD killed vaccine was tested for immunization and vaccination challenge in a porcine model. Our preliminary results had indicated that the recombinant plasmid could enhance the humoral immune response triggered by the traditional oil-adjuvant vaccine after the initial inoculation. A subsequent vaccination-challenge test showed an increased PD50 value. Thus, coadministration of the recombinant plasmid with the oil-adjuvant vaccine helped illicit an immune response earlier than that elicited by giving the vaccine alone. Our results showed that pUC18-CpG can be a potent immunoadjuvant for the traditional FMD killed vaccine and can greatly enhance the traditional vaccine's efficacy when given in combination with it.

A swine-specific CpG motif enriched plasmid (pUC18-CpG) was constructed in this study. Its immunostimulant property was tested in vitro via lymphocyte transformation assay using swine peripheral blood mononuclear cells (PBMCs). The recombinant plasmid showed higher Stimulation Index (SI) compared to the positive control (LPS). In a following animal experiment, pUC18-CpG was co-administered with a commercial swine FMD killed vaccine. Animals in the pUC18-CpG adjuvanted groups showed much higher antibody titers during the vaccination period.

Bovine tuberculosis (bTB) is a worldwide zoonosis caused mainly by Mycobacterium bovis. The traditional diagnostic method used often is the tuberculin skin test, which uses bovine purified protein derivatives (PPD-B). However, it is difficult to maintain uniformity of PPD-B from batch to batch, and it shares common antigens with nonpathogenic environmental mycobacteria. To overcome these problems, M. bovis -specific antigens that showed good T cell stimulation, such as CFP-10, ESAT-6, Rv3615c, etc., have been used in the skin test, but there have been no large-scale clinical studies on these antigens. In this study, two combinations (CFP-10/ESAT-6/TB10.4 protein cocktail and CFP-10/ESAT-6/Rv3872/MPT63 protein cocktail) were developed and used as stimuli in the skin test. Cattle were double-blind tested to assess the efficiency of the protein cocktail-based skin tests. The results showed that the CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test can differentiate TB-infected cattle from Mycobacterium avium -infected ones and that it shows a high degree of agreement with the traditional tuberculin skin test (κ = 0.8536) and gamma interferon (IFN-γ) release assay (κ = 0.8154). Compared to the tuberculin skin test, the relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 87% and 97%, respectively., The relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 93% and 92%, respectively, on comparison with the IFN-γ release assay. The correlation between the increases in skin thickness observed after the inoculation of stimuli was high (PPD-B versus CFP-10/ESAT-6/TB10.4, Spearman r of 0.8435). The correlation between the optical density at 450 nm (OD 450 ) obtained after blood stimulation with PPD-B and the increase in skin thickness observed after inoculation of the CFP-10/ESAT-6/TB10.4 protein cocktail was high (Spearman r = 0.7335). Therefore, the CFP-10/ESAT-6/TB10.4-based skin test responses correlate to traditional measures of bovine TB evaluation, including skin test and gamma interferon release assay.