Development of a quantum dots based immunochromatographic strip for rapid and on-site detection of African swine fever virus.

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.

Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody.

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies. The developed method exhibited a linear detection range of approximately 6-200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%-102%. These results indicate robust reproducibility of the method. To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits. In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Dual-RPA assay for rapid detection and differentiation of E.granulosus and E.multilocularis.

Echinococcus granulosus (Eg) and Echinococcus multilocularis (Em) are the two most widely prevalent types of echinococcosis. Several diagnostic methods have been developed for detecting Eg and Em. However, some limitations, such as being time-consuming, needing expensive instruments, or exhibiting low sensitivity, make these methods unsuitable for on-site detection. In this study, a dual-RPA assay was established to detect and differentiate Eg and Em. The primer concentration ratio, reaction time, and reaction temperature of the dual-RPA were optimized. The result showed that the primer concentration ratio of Eg:Em was 400 nM:400 nM, and the best amplification efficiency was obtained by reacting at 38 °C for 20 min. The sensitivity, specificity, and repeatability of the assay were also tested. The assay's detection limit for both Eg and Em was 10 copies/µL. The assay showed reasonable specificity by testing ten parasitic nucleic acids. The assay's intra- and inter-batch coefficients of variation were below 10%, which indicates robust reproducibility of the assay. Finally, to validate the performance of the dual-RPA assay, it was compared with real-time PCR by using 86 clinical nucleic acid samples. The coincidence rate of Eg between dual-RPA and TaqMan real-time PCR was 96.51%, and the coincidence rate of Em between dual-RPA and TaqMan real-time PCR was 98.84%, indicating its potential for accurate clinical diagnosis. Therefore, this study established a rapid and sensitive dual-RPA assay that can rapidly detect and differentiate Eg and Em in one reaction tube and provided a new assay for the detection of echinococcosis in the field.

Development and characterization of a novel nanobody with SRMV neutralizing activity.

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

New Insights in Molecular Mechanisms in Antimicrobial Resistance and Strategies in Anti-Biofilms.

This topical collection, entitled "Antimicrobial resistance and anti-biofilms", was first launched in the journal Antibiotics in November of 2020 [...].

A Novel Cost-Effective Nanobody against Fumonisin B1 Contaminations: Efficacy Test in Dairy Milk and Chickens.

BACKGROUND: Fumonisin B1 (FB1) is a secondary metabolite produced mainly by Fusarium verticillioides or Fusarium proliferatum. It poses a huge threat to the sustainable animal industry and human health as well via food chains (egg, meat and milk). Although E. coli-expressed nanobodies are documented for diagnostic applications, nanobodies remain elusive as FB1 detoxifiers in feed and food. RESULTS: In the present study, the E. coli-expressed nanobody was assessed to remove FB1 in fresh milk, embryonated eggs and broilers. Firstly, 2 alpacas received intramuscularly FB1-adjuvanted BSA 6 times, and then the variable domain of the heavy-chain antibody (VHH) of fb1 genes were amplified to clone into the pCANTAB 5 E vector in order to generate a VHH-FB1 phage antibody display library, yielding 3.4 × 1010 capacity with 96.7% positivity. Afterwards, 5 anti-FB1 nanobodies were expressed and identified. Furthermore, maximal 43.2% FB1 was removed from milk by 1:2000 concentration of nanobody 5 (Nb5). Furthermore, SPF-embryonated eggs were inoculated into albumens with nanobody-treated FB1. The Nb5 group yielded an 83.3% hatching rate, higher body weight, lower gizzard ulceration and fewer FB1 residuals. In order to warrant the above results, 50 broilers aged 10 days were received orally with 20 ppm of FB1 for 20 days. At the same time, birds were fed orally with 50 µg of Nb5 or bivalent nanobody 11 (BiNb11). Finally, the Nb5 group showed a higher relative body weight gain and lower gastric ulcerations and fewer inflammations in the thymus and bursa. CONCLUSIONS: Based on the above evidence, the Nb5 nanobody may be considered as an additional FB1 detoxifier, contributing to FB1 decontamination.

Evaluation of porcine GM-CSF during PRRSV infection in vitro and in vivo indicating a protective role of GM-CSF related with M1 biased activation in alveolar macrophage during PRRSV infection.

Granulocyte-macrophage colony stimulating factor (GM-CSF), participates in diverse biological processes associated with innate and adaptive immunity, has unknown effects during PRRSV infection. Here, a double-antibody sandwich ELISA for pGM-CSF was developed in-house for evaluation of pGM-CSF level during PRRSV infection both in vitro and in vivo. In in vitro assay, it was notable that PRRSV-infected porcine alveolar macrophages (PAMs) yielded inconsistent pGM-CSF protein- and mRNA-level, suggesting a post-transcriptional inhibition of pGM-CSF mRNA was employed by PRRSV. Meanwhile, concurrent analysis of pGM-CSF levels in serum samples from PRRSV-infected piglets suggested that effect of PRRSV infection demonstrated minimum effect on pGM-CSF levels regardless of PRRSV virulence phenotypes. Moreover, in vitro treatment of PAMs with pGM-CSF prior PRRSV inoculation did not inhibit PRRSV replication in PAMs although genes downstream of pGM-CSF in PAMs could be upregulated by pGM-CSF treatment. Meanwhile, knockdown of pGM-CSF using siRNA did not enhance PRRSV replication as well. Intriguingly, therapeutic antibody treatment of HP-PRRSV-infected piglets led to significantly increased serum pGM-CSF levels, thus aligning with low pneumonia incidence and low intracellular PRRSV-RNA levels in PAMs of therapeutic antibody treated piglets. Furthermore, transcriptome analysis of PAMs from infected piglets revealed increased serum pGM-CSF levels correlated with activation of downstream signal of pGM-CSF in PAMs as evidenced by a M1-like phenotypes of gene expression pattern, implying a potential host-protective role played by pGM-CSF for PRRSV infection in vivo. In conclusion, our results demonstrated developments of a highly sensitive and specific ELISA for pGM-CSF and revealed a potential protective role conferred by pGM-CSF during PRRSV infection.

Construction and characterization of a contagious ecthyma virus double-gene deletion strain and evaluation of its potential as a live-attenuated vaccine in goat.

Contagious ecthyma is a highly contagious viral disease with zoonotic significance caused by orf virus (ORFV) that affects domestic, ruminants and humans. Live attenuated virus and attenuated tissue culture vaccines are widely used in the fight against ORFV, however, the conventional attenuated vaccine strains have many drawbacks. The aim of this project was to construct a promising contagious ecthyma vaccine strain with safety, high protection efficacy and accessibility by genetic manipulation to against the disease. Using a natural ORFV-GS14 strain as the parental virus, recombinant virus, rGS14-ΔCBP-ΔGIF, with double deletions in the genes encoding the chemokine binding protein (CBP) and granulocyte/macrophage colony-stimulating factor inhibitory factor (GIF) was generated and characterized in vitro and in vivo. Results showed that the growth kinetics curve of rGS14-ΔCBP-ΔGIF and parental virus was consistent, both reaching plateau phase at 48 h post infection, which indicated that the double deletion of cbp and gif genes had little impact on the replication properties of the recombinant virus in primary goat testis (PGT) cell cultures compared with the parental virus. The safety of the double gene-deleted virus was evaluated in lambs. The lambs were monitored for 21 days post infection of the recombinant virus and no ORFV associated symptoms were observed in 21 days post-infection except for slight fever and anorexia in 5 days post-infection, and all lambs inoculated with either recombinant virus or PBS exhibited no clinical signs. To assess the protection efficacy of the rGS14-ΔCBP-ΔGIF, groups of four lambs each were inoculated with rGS14-ΔCBP-ΔGIF, rGS14-ΔCBP, rGS14-ΔGIF or PBS and challenged by a wild type virulent ORFV strain that was isolated from proliferative scabby lesions tissues of infected goat at 21-day post-inoculation. During 14 days post-challenging, lambs inoculated with rGS14-ΔCBP-ΔGIF all remained healthy with unimmunized group all infected, while the single gene-deleted viruses only protected 40% to 50% animals. These results indicated that the double gene-deleted recombinant virus could provide complete protection against virulent ORFV challenging. In conclusion, the double gene-deleted recombinant virus strain, rGS14-ΔCBP-ΔGIF, would be a promising candidate vaccine strains with safety, high protection efficacy and availability.

Establishment and Application of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Leukosis Virus Subgroup J.

Avian leukosis caused by avian leukosis virus (ALV), belonging to the genus Alpharetrovirus of the family Retroviridae, is associated with benign and malignant tumors in hemopoietic cells in poultry. Although several methods have been developed for ALV detection, most of them are not suitable for rapid on-site testing due to instrument limitations, professional operators, or the low sensitivity of the method. Herein, we described the real-time recombinase polymerase amplification (RPA) assay for rapid detection of ALV subgroup J (ALV-J). The major viral structural glycoprotein gp85, highly specific for the subgroup, was used as the molecular target for the real-time RPA assay. The results were obtained at 38°C within 20 min, with the detection sensitivity of 10 copies/µl of standard plasmid pMD18-T-gp85 as the template per reaction. Real-time RPA was capable of ALV-J-specific detection without cross-reaction with other non-targeted avian pathogens. Of the 62 clinical samples tested, the ALV-positive rates of real-time RPA, PCR, and real-time PCR were 66.13% (41/62), 59.68% (37/62), and 67.74% (42/62), respectively. The diagnostic agreement between real-time RPA and real-time PCR was 98.39% (61/62), and the kappa value was 0.9636. The developed real-time ALV-J assay seems promising for rapid and sensitive detection of ALV-J in diagnostic laboratories. It is suitable for on-site detection, especially in a poor resource environment, thus facilitating the prevention and control of ALV-J.

Ovarian Oxidative Stress Induced Follicle Depletion After Zona Pellucida 3 Vaccination Is Associated With Subfertility in BALB/c Mice.

Impaired follicular development associated with autoimmune ovarian disease (AOD), is a typical side effect of ZP3 vaccine-induced subfertility and contributes to the fertility decline, but the mechanism is unknown. In this study, a AOD model was established with recombinant mouse zona pellucida 3 (mZP3) protein in the BALB/c mice, and co-administrated with 0.5 mg/kg antioxidant stress drug sodium selenite (SS), whereas intraperitoneal injection was used and the relationships among oxidant stress (OS), follicle loss and fertility were evaluated. Here we demonstrated that ZP3 vaccination elicited high antibody titers and correlated with reductions of ovarian follicle numbers in both fertile and infertile mice, whereby magnitudes of both factors were negatively correlated with litter size. Moreover, increased OS in ovaries of mZP3-immunized mice was related to high levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and is accompanied by a decrease in the total antioxidant capacity (TAC) of ovaries. Meanwhile, activation of caspase-3 and caspase-9 along with increased Bax and decreased Bcl-2 levels were observed, indicating the ongoing apoptosis of ovarian cells. Notably, inhibition of OS with SS reduced ovarian ROS and apoptosis levels, which was consisted with restoration of follicle numbers. More importantly, SS treatment when co-administered concurrently with mZP3 immunization led to significantly improved fertility (P < 0.05) and the average litter size of the mZP3-vaccinated SS-treated group increased by ~29.2% as compared with that of the vaccinated but untreated group. In conclusion, infertility caused by ZP3 vaccination was mechanistically associated with ovarian OS which triggered depletion of ovarian follicles.

Type and abundance of sialic acid receptors on host cell membrane affect infectivity and viral titer of different strains of Newcastle disease virus.

Cell-based vaccine manufacture is a very cost-effective approach for animal vaccine production. Newcastle disease virus (NDV) can infect a wide range of host cells, but the viral titers of cell culture are too low to meet the vaccine manufacture. In this study, we explored the selectivity of NDV vaccine strains LaSota and Mukteswar to type of sialic acid receptors and demonstrated the relationship between receptor expression levels on cell membrane and viral titers of cell culture. The results suggested that NDV strain LaSota preferentially binds to Neu5Ac-2-S-α-2,6 Gal10Me receptor (SAα2,6 Gal) and strain Mukteswar selectively binds to Neu5Ac-α-2,3 Gal-ß-1,4Glc (SAα2,3 Gal) receptor. Subsequently, the expression levels of SAα2,3 Gal and SAα2,6 Gal receptors on BHK-21 cell membrane were adjusted by overexpression and RNAi assays. The results indicated that the viral titers of NDV strains LaSota and Mukteswar in cell culture were positively correlated with the expression levels of SAα2,6 Gal and SAα2,3 Gal receptors on host cell membrane respectively. In conclusion, our studies provide an understanding of the relationship between infectivity of NDV different strains and receptor types of host cell, and provide a method to increase viral titer of NDV for cell-based vaccine production.

Immunoproteomic analysis of the sporozoite antigens of Eimeria necatrix.

Eimeria necatrix, an apicomplexan protozoa of the genus Eimeria, causes intestinal coccidiosis that can reduce growth performance of poultry and result in high mortality in older chickens. In this report, the whole sporozoite proteins of E.necatrix were studied by two-dimensional electrophoresis (2-DE) and Western blotting using hyper-immune chicken serum containing E.necatrix-specific antibodies. Approximately 680 protein spots for E.necatrix sporozoite were detected by 2-DE with silver staining, where 98 spots were cross-reacted with the E. necatrix-specific immune sera. Out of the 56 spots that were selected for MALDI-TOF-MS/MS analysis, 50 unique proteins were identified using the MASCOT software, 8 proteins were identified as known E.necatrix proteins and the rest were all putative proteins. These proteins have a wide range of known or predicted structures, cellular locations and functions, including proteins in category nuclear location & function, multifunctional- or multifunctional motifs-containing proteins, cellular transport and structure-related proteins, proteins of enzymatic activities, motor proteins-related, cell surface and organelle-related proteins. These new findings will enhance our understandings of parasite immunogenicity and immune evasion mechanisms of E. necatrix and facilitate the discovery phase of highly effective vaccine candidates.

IL-10-/- Enhances DCs Immunity Against Chlamydia psittaci Infection via OX40L/NLRP3 and IDO/Treg Pathways.

Chlamydia psittaci (C. psittaci) is a common zoonotic agent that affects both poultry and humans. Interleukin 10 (IL-10) is an anti-inflammatory factor produced during chlamydial infection, while dendritic cells (DCs) are powerful antigen-presenting cells that induce a primary immune response in the host. However, IL-10 and DCs regulatory mechanisms in C. psittaci infection remain elusive. In vivo and in vitro investigations of the regulatory mechanisms were performed. IL-10-/- mice, conditional DCs depletion mice (zinc finger dendritic cell-diphtheria toxin receptor [zDC-DTR]), and double-deficient mice (DD, IL-10-/-/zDCDTR/DTR) were intranasally infected with C. psittaci. The results showed that more than 90% of IL-10-/- mice, 70% of wild-type mice, and 60% of double-deficient mice survived, whereas all zDC-DTR mice died. A higher lymphocyte proliferation index was found in the IL-10 inhibitor mice and IL-10-/- mice. Moreover, severe lesions and high bacterial loads were detected in the zDC-DTR mice compared with double-deficient mice. In vitro studies revealed increased OX40-OX40 ligand (OX40-OX40L) activation and CD4+T cell proliferation. Besides, the expression of indoleamine 2, 3-dioxygenase (IDO), and regulatory T cells were significantly reduced in the co-culture system of CD4+ T cells and IL-10-/- DCs in C. psittaci infection. Additionally, the activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome increased to facilitate the apoptosis of DCs, leading to rapid clearance of C. psittaci. Our study showed that IL-10-/- upregulated the function of deficient DCs by activating OX40-OX40L, T cells, and the NLPR3 inflammasome, and inhibiting IDO, and regulatory T cells. These effects enhanced the survival rate of mice and C. psittaci clearance. Our research highlights the mechanism of IL-10 interaction with DCs, OX40-OX40L, and the NLPR3 inflammasome, as potential targets against C. psittaci infection.

Polymorphic membrane protein 20G: A promising diagnostic biomarker for specific detection of Chlamydia psittaci infection.

Psittacosis is a zoonotic disease caused by Chlamydia psittaci (C. psittaci), leading to high risk for animal industry and human health. Lack of reliable commercial kits and effective vaccines is hampering control of C. psittaci infection. Polymorphic outer membrane protein Gs (PmpGs) are enriched in diverse C. psittaci, and its role are unclear during C. psittaci infection. In the present study, pmp20G gene was cloned into pET-28a vector and then the constructed plasmid was transferred into Escherichia coli Rossetta (DE3). After denaturation and renaturation, the recombinant Pmp20G-N was identified by SDS-PAGE and Western blot. Afterwards Pmp20G-N was used as the coating antigen to develop an indirect ELISA (I-ELISA) assay. Both the specificity and sensitivity of Pmp20G-N ELISA were 100%, while the MOMP-ELISA had 93.65% sensitivity and 98.94% specificity, respectively. The concordance between MOMP-ELISA and Pmp20G-N ELISA assay was 98.1%. Hence, Pmp20G-N ELISA has the potential to be a diagnostic antigen for detection C. psittaci antibody. However, further studies are needed to be done for differentiating C. psittaci from Chlamydia spp. and other C.psittaci-specific serovars using Pmp20G-N ELISA.

Proteomic Analysis of ISGylation in Immortalized Porcine Alveolar Macrophage Cell Lines Induced by Type I Interferon.

Interferon-stimulated gene product 15 (ISG15), a ubiquitin-like molecule, can be conjugated to protein substrates through a reversible process known as ISGylation. ISG15 and ISGylation are both strongly upregulated by type I interferons and play putative key roles in host innate immunity against viral infection. However, the function of ISGylation and identities of ISGylation substrates are largely unknown. Here, a novel monoclonal antibody (Mab) that specifically recognizes porcine ISG15 (pISG15) was employed to capture ISG15-conjugated proteins from IFNs-stimulated porcine cell lysates. Next, Mab-captured conjugates were analyzed using proteomics-based tools to identify potential ISGylation protein targets in order to elucidate the roles of ISG15 and ISGylation in porcine cells. Subsequently, 190 putative ISGylation sites were detected within 98 identified ISGylation candidates; several candidates contained more than one ISGylation-modifiable lysine residue, including pISG15 itself. Motif enrichment analysis of confirmed ISGylation sites demonstrated a moderate bias towards certain sites with specific upstream amino acid residues. Meanwhile, results of Gene Ontology (GO)-based annotation and functional enrichment and protein-protein interaction (PPI) network analyses of porcine ISG15-conjugated substrate proteins indicated that these substrates were mainly associated with the host metabolism, especially nucleotide metabolic pathways that ultimately may participate in cellular antiviral defenses. Notably, several ISGs (MX1, IFIT1, OAS1, ISG15 and putative ISG15 E3 ligase Herc6) were also identified as putative ISGylation substrates within a regulatory loop involving ISGylation of ISGs themselves. Taken together, proteomics analysis of porcine ISGylation substrates revealed putative functional roles of ISG15 and novel host ISGylation targets that may ultimately be involved in cellular antiviral responses.

Streptococcus suis serotype 2 enolase interaction with host brain microvascular endothelial cells and RPSA-induced apoptosis lead to loss of BBB integrity.

Host proteins interacting with pathogens are receiving more attention as potential therapeutic targets in molecular medicine. Streptococcus suis serotype 2 (SS2) is an important cause of meningitis in both humans and pigs worldwide. SS2 Enolase (Eno) has previously been identified as a virulence factor with a role in altering blood brain barrier (BBB) integrity, but the host cell membrane receptor of Eno and The mechanism(s) involved are unclear. This study identified that SS2 Eno binds to 40S ribosomal protein SA (RPSA) on the surface of porcine brain microvascular endothelial cells leading to activation of intracellular p38/ERK-eIF4E signalling, which promotes intracellular expression of HSPD1 (heat-shock protein family D member 1), and initiation of host-cell apoptosis, and increased BBB permeability facilitating bacterial invasion. This study reveals novel functions for the host-interactional molecules RPSA and HSPD1 in BBB integrity, and provides insight for new therapeutic strategies in meningitis.

Development of Immunochromatographic Assay for the Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae Antibodies.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

Gastric Ulceration and Immune Suppression in Weaned Piglets Associated with Feed-Borne Bacillus cereus and Aspergillus fumigatus.

As a multifactorial cause, gastric ulceration-mediated diarrhea is widely prevalent in the weaned piglets, impairing pig health and economic benefits. With full implementation of antibiotic stewardship programs in China, Bacillus cereus (B. cereus) and Aspergillus fumigatus (A. fumigatus) were identified frequently in porcine feedstuffs and feeds of the animal industry. Association between feed-borne B. cereus and frequent diarrhea remains unclear. In the present study, we conducted a survey of B. cereus and A. fumigatus from feeds and feedstuffs in pig farms during hot season. Interestingly, B. cereus, B. subtilis, B. licheniformis and B. thuringinesis were isolated and identified from piglets' starter meals to sow feeds, accounting for 56.1%, 23.7%, 13.7% and 6.5%, respectively. Obviously, both B. cereus and B. subtili were dominant contaminants in the survey. In an in vitro study, Deoxynivalenol (DON) contents were determined in a dose-dependent manner post fermentation with B. cereus (405 and DawuC). Subsequently, 36 weaned piglets were randomly assigned to four groups and the piglets simultaneously received the combination of virulent B. cereus (Dawu C) and A. fumigatus while animals were inoculated with B. cereus (Dawu C), A. fumigatus or PBS as the control group. Clinically, piglets developed yellow diarrhea on day 5 and significant reductions of relative body weight were observed in the B. cereus group, and co-infection group. More importantly, IgG titers against Classical swine fever virus (CSFV) and Porcine epidemic diarrhea (PED) were reduced dramatically during 14-day observation in co-infection group, the B. cereus (Dawu C) group or the A. fumigatus group. However, lower Foot and mouth disease (FMD) -specific antibodies were reduced on day 7 compared to those of the control group. Additionally, lower lymphocyte proliferations were found in the B. cereus group and the co-infection group compared to the control group. Postmortem, higher lesions of gastric ulceration were observed in the B. cereus group and the co-infection group from day 7 to day 14 compared with those of the A. fumigatus group and the control group. Compared to the A. fumigatus group, higher DON contents were detected in the stomach inoculated with B. cereus and the co-infection with A. fumigatus. In conclusion, our data support the hypothesis that B. cereus might be associated with severe diarrhea by inducing gastric ulcerations and A. fumigatus might aggravate immune suppression, threating a sustainable swine industry. It is urgently needed to control feed-borne B. cereus contamination.

Proteomics analysis of important molecules in serum from meningitic piglets caused by Streptococcus suis serotype 2.

INTRODUCTION: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis in China. This study's aim was comparative analysis of serum proteomics from meningitis and non-meningitis piglets. METHODOLOGY: SS2 meningitis and non-meningitis piglet models were established. The serum samples were collected and analyzed by label-free LC-MS/MS proteomics technology. Differentially expressed proteins (DEPs) from serum were screened out by comparing the meningitis group and non-meningitis group to the healthy group (M/C; N/C), respectively. And then, globally and comparative analysis of DEPs in "M/C" and "N/C" in serum were performed using bioinformatics method. Finally, we comparatively analyzed the serum and cerebrospinal fluid proteomics in piglets that lived with meningitis. RESULTS: We obtained 316 and 191 DEPs from "M/C" and "N/C" which classification visualizations were established. 157 DEPs were common in both groups and 159 DEPs were unique to the "M/C". These DEPs and the signaling pathways which they participated in were visualized. Moreover, some DEPs which participated in multiple pathways were discovered and the interaction between 159 DEPs was also mapped. 39 common DEPs were also screened out in serum and cerebrospinal fluid during meningitis, and signaling pathways associated with these DEPs were further visualized. CONCLUSIONS: DEPs in "M/C" and "N/C" were comparatively analyzed and the similarities and differences of these DEPS which were involved in signal pathways were summarized. Moreover, several important molecules were screened out.

Correction: Chlamydia psittaci PmpD-N modulated Chicken Macrophage Function by Triggering Th2 Polarization and the TLR2/MyD88/NF-κB Signaling Pathway.

The authors would like to make the following corrections to their paper, published in the International Journal of Molecular Sciences [...].