Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

Epidemiological and clinical profile of pediatric hepatitis B virus infections in Wuhan: a retrospective cohort study.

BACKGROUND: Hepatitis B virus (HBV) remains a substantial public health safety concern drawing considerable attention in China and globally. The detection of HBV serological markers can enable the assessment of HBV infection and replication status in vivo and evaluate the body's protection against HBV. Therefore, this study aims to identify the epidemiological and clinical characteristics of HBV infection in children to prevent and control HBV infection in Wuhan areas. METHODS: We conducted an extensive retrospective cohort analysis of 115,029 individuals aged 0-18 years who underwent HBV serological markers detection for HBV infection in hospital between 2018 and 2021 using Electrochemiluminescence immunoassay. We generated descriptive statistics and analysed HBV infection's epidemiological and clinical characteristics between different sex and age groups. RESULTS: The overall positive detection rates of HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb in all participants were 0.13%, 79.09%, 0.17%, 2.81%, and 5.82%, respectively. The positive rate of HBeAb and HBcAb in males was significantly lower than that in females (2.64% vs. 3.13%, 5.56% vs. 6.29%) (P < 0.05). Twenty-two distinct HBV serological expression patterns were revealed. Among them, 8 common expression patterns accounted for 99.63%, while the remaining 14 uncommon expression patterns were primarily observed in neonatal patients with HBV infection. There are no significant differences in serological patterns based on sex (P < 0.05). The overall HBV infection detection rate was 5.82% [range 5.68-5.95] and showed a declining yearly trend. The rate in females was higher than that in males 6.29% [6.05, 6.35] vs. 5.56% [5.39, 5.59]. The overall HBV diagnostic rate over 4 years was 0.20% [0.17, 0.22], and the rate declined yearly. The prevalence of acute infection was higher than that of other infection types before 2019, but the incidence of unclassified infection showed a significant upward trend after 2019. CONCLUSIONS: While the overall HBV infection detection rate in children has decreased year by year, the infection rate remains high in children under one year and between 4 and 18 years. This continued prevalence warrants heightened attention and vigilance.

An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission.

SARS-CoV-2 is primarily transmitted through droplets and airborne aerosols, and in order to prevent infection and reduce viral spread vaccines should elicit protective immunity in the airways. The neonatal Fc receptor (FcRn) transfers IgG across epithelial barriers and can enhance mucosal delivery of antigens. Here we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized spike; the resulting S-Fc bound to S-specific antibodies and FcRn. Intranasal immunization of mice with S-Fc and CpG significantly induced antibody responses compared to the vaccination with S alone or PBS. Furthermore, we intranasally immunized mice or hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2 and its variants. Intranasal immunization also significantly reduced viral airborne transmission in hamsters. Nasal IgA, neutralizing antibodies, lung-resident memory T cells, and bone-marrow S-specific plasma cells mediated protection. Hence, FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission.

Serum level of S100A8/A9 as a biomarker for establishing the diagnosis and severity of community-acquired pneumonia in children.

Background: S100A8/A9, which is a member of S100 proteins, may be involved in the pathophysiology of Community-acquired pneumonia (CAP) that seriously threatens children's health. However, circulating markers to assess the severity of pneumonia in children are yet to be explored. Therefore, we aimed to investigate the diagnostic performance of serum S100A8/A9 level in determining the severity of CAP in children. Methods: In this prospective and observational study, we recruited 195 in-hospital children diagnosed with CAP. In comparison, 63 healthy children (HC) and 58 children with non-infectious pneumonia (pneumonitis) were included as control groups. Demographic and clinical data were collected. Serum S100A8/A9 levels, serum pro-calcitonin concentrations, and blood leucocyte counts were quantified. Results: The serum S100A8/A9 levels in patients with CAP was 1.59 ± 1.32 ng/mL, which was approximately five and two times higher than those in healthy controls and those in children with pneumonitis, respectively. Serum S100A8/A9 was elevated parallelly with the clinical pulmonary infection score. The sensitivity, specificity, and Youden's index of S100A8/A9 ≥1.25 ng/mL for predicting the severity of CAP in children was optimal. The area under the receiver operating characteristic curve of S100A8/A9 was the highest among the indices used to evaluate severity. Conclusions: S100A8/A9 may serve as a biomarker for predicting the severity of the condition in children with CAP and establishing treatment grading.

Application value of antibody titres and RNA detection in the early prediction of Mycoplasma pneumoniae pneumonia in children: a retrospective study.

BACKGROUND: Children with Mycoplasma pneumoniae pneumonia (MPP) are prone to a missed diagnosis at the early stages of the disease, which greatly affects the prognosis of children. In this study, the application value of Mycoplasma pneumoniae (MP) antibody titres and RNA detection for diagnosing MP infection in children with community-acquired pneumonia (CAP) was evaluated. The present study aimed to seek appropriate detection methods and strategies for early rapid diagnosis in children with MPP. METHODS: A retrospective study was conducted on 563 paediatric patients aged 1 month to 15 years with CAP who were admitted to Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2021 and February 2022. In all patients, throat swabs were collected for MP-RNA detection (simultaneous amplification and testing, SAT), and paired serum samples were collected for MP total antibody detection (particle agglutination, PA). RESULTS: The classification as MPP or non-MPP was based on clinical diagnosis, serum MP antibody titre, and clinical or laboratory evidence of infection by other pathogen(s). Among the 563 patients with pneumonia, 187 patients were in the MPP group, and 376 patients were in the non-MPP group. The Kappa values between the particle agglutination test at different titres (1:80, 1:160) and MP-RNA detection were 0.612 and 0.660 (P<0.01), and the consistency of the three methods was acceptable. When the single screening method was used, MP-RNA had the highest sensitivity (93.05%), while PA (1:160) had the highest specificity (100%). PA (1:80), with an area under the curve (AUC) of 0.822, was better than PA (1:160), with an AUC of 0.783, and there was a significant difference. When the combined screening methods were used, the AUC of MP-RNA parallel PA (1:160) was significantly higher than that of titres (1:80) (z=-4.906, P < 0.01). Except for MP-80, the efficacy of the other three test methods in females was slightly better than that in males. Among the differences in age distribution, PA (1:80) was slightly less effective in the 13-72 months age group than at other ages, and MP-RNA parallel PA (1:160) was slightly better than the younger age group (≤ 36 m). In the older age group (> 36 m), PA (1:160) was just the opposite, while MP-RNA was slightly better than other age groups in the 13-72 months age group. CONCLUSIONS: For the diagnosis of MPP in children at the early of the disease, the antibody titre (1:160) parallel MP-RNA should be given preference, and then the disease should be further classified according to the antibody titre level and the age of the child. The combined application of the two detection methods could complement each other and strengthen the advantages, providing reliable laboratory evidence for the clinical diagnosis and timely treatment of MPP. When using the PA method alone to provide a reference standard to clarify MP infection, the differential diagnosis ability of 1:80 for MPP is better than 1:160, especially for children younger than 36 months.

An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission.

SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and airborne aerosols. To prevent viral infection and reduce viral spread, vaccine strategies must elicit protective immunity in the airways. FcRn transfers IgG across epithelial barriers; we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized S protein; the resulting S-Fc bound to S-specific antibodies (Ab) and FcRn. A significant increase in Ab responses was observed following the intranasal immunization of mice with S-Fc formulated in CpG as compared to the immunization with S alone or PBS. Furthermore, we intranasally immunize adult or aged mice and hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2, including Delta and Omicron variants. Intranasal immunization also significantly reduced viral transmission between immunized and naive hamsters. Protection was mediated by nasal IgA, serum-neutralizing Abs, tissue-resident memory T cells, and bone marrow S-specific plasma cells. Hence FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission. Based on these findings, FcRn-targeted non-invasive respiratory immunizations are superior strategies for preventing highly contagious respiratory viruses from spreading.

Single-cell transcriptomic and chromatin accessibility analyses of dairy cattle peripheral blood mononuclear cells and their responses to lipopolysaccharide.

BACKGROUND: Gram-negative bacteria are important pathogens in cattle, causing severe infectious diseases, including mastitis. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and crucial mediators of chronic inflammation in cattle. LPS modulations of bovine immune responses have been studied before. However, the single-cell transcriptomic and chromatin accessibility analyses of bovine peripheral blood mononuclear cells (PBMCs) and their responses to LPS stimulation were never reported. RESULTS: We performed single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) in bovine PBMCs before and after LPS treatment and demonstrated that seven major cell types, which included CD4 T cells, CD8 T cells, and B cells, monocytes, natural killer cells, innate lymphoid cells, and dendritic cells. Bioinformatic analyses indicated that LPS could increase PBMC cell cycle progression, cellular differentiation, and chromatin accessibility. Gene analyses further showed significant changes in differential expression, transcription factor binding site, gene ontology, and regulatory interactions during the PBMC responses to LPS. Consistent with the findings of previous studies, LPS induced activation of monocytes and dendritic cells, likely through their upregulated TLR4 receptor. NF-κB was observed to be activated by LPS and an increased transcription of an array of pro-inflammatory cytokines, in agreement that NF-κB is an LPS-responsive regulator of innate immune responses. In addition, by integrating LPS-induced differentially expressed genes (DEGs) with large-scale GWAS of 45 complex traits in Holstein, we detected trait-relevant cell types. We found that selected DEGs were significantly associated with immune-relevant health, milk production, and body conformation traits. CONCLUSION: This study provided the first scRNAseq and scATAC-seq data for cattle PBMCs and their responses to the LPS stimulation to the best of our knowledge. These results should also serve as valuable resources for the future study of the bovine immune system and open the door for discoveries about immune cell roles in complex traits like mastitis at single-cell resolution.

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.

Immune reactivity and host modulatory roles of two novel Haemonchus contortus cathepsin B-like proteases.

BACKGROUND: Haemonchus contortus is a blood-feeding, gastrointestinal nematode (GIN) that causes significant economic losses to the small ruminant industry worldwide. Despite extensive efforts, our understanding of the molecular mechanisms used by GIN to evade host immune responses is limited. Cathepsin B-like proteins (CBPs) are members of the cysteine protease family and are involved in parasite invasion and thus provide viable vaccine candidates. METHODS: In silico comparative analysis was used to identify conserved proteins among a subset of clade V parasitic nematodes with emphasis on blood-feeding worms, among which CBPs appeared prominently. We identified and characterized two novel CBPs designated Hc-CBP-1 and Hc-CBP-2. Rabbit anti-recombinant (r) Hc-CBP-1 and rHc-CBP-2 were used to detect the presence of native proteins in the excretory secretory products (ESP) and in worm tissues of adult H. contortus. Peptide arrays of rHc-CBP-1 and rHc-CBP-2 were screened with the homologous and heterologous anti-sera and with sera from dexamethasone-treated (Dex+) and non-treated (Dex-) H. contortus-infected animals to identify key immunogenic peptides. Gene transcription of Hc-cbp-1 and Hc-cbp-2 was also performed on H. contortus-infected animals treated with Dex+. Finally, the mature recombinant proteins were used to assess their abilities to modulate cell functions. RESULTS: Immunohistochemistry showed that both Hc-CBP-1 and Hc-CBP-2 are present on the brush borders of the intestine; Hc-CBP-2 was also present in the hypodermis of the body wall. Peptide displays screened with rabbit anti-rHc-CBP-1 and anti-rHc-CBP-2 revealed regions within the proteins where dominant and overlapping epitopes prevailed. ELISA results were consistent with only Hc-CBP-1 being present in H. contortus adult ESPs. H. contortus from Dex+ animals exhibited a threefold increase in Hc-cbp-2 transcript while Hc-cbp-1 expression did not change. In contrast, comparisons of immunoreactivities of rHc-CBP-1 and rHc-CBP-2 peptide arrays to sera from Dex+ and Dex- animals primarily showed changes in Hc-CBP-1 binding. Lastly, rHc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers, including TNFα, IL-1, IL-6 and CD86. CONCLUSIONS: These results suggest that as secreted and cryptic proteins, respectively, Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities that have interesting dynamics during infection and may provide viable immune-related targets for attenuating H. contortus infectivity.

FcRn-Targeted Mucosal Vaccination against Influenza Virus Infection.

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal challenge with influenza A/PR/8/34 virus. Further, mice immunized with a mutant HA-Fc lacking FcRn binding sites or HA alone succumbed to lethal infection. Protection was attributed to high levels of neutralizing Abs, robust and long-lasting B and T cell responses, the presence of lung-resident memory T cells and bone marrow plasma cells, and a remarkable reduction of virus-induced lung inflammation. Our results demonstrate for the first time, to our knowledge, that FcRn can effectively deliver a trimeric viral vaccine Ag in the respiratory tract and elicit potent protection against respiratory infection. This study further supports a view that FcRn-mediated mucosal immunization is a platform for vaccine delivery against common respiratory pathogens.

Repeated, drug-truncated infections with Ostertagia ostertagi elicit strong humoral and cell-mediated immune responses and confer partial protection in cattle.

Bovine ostertagiasis causes significant production losses to the cattle industry. Protective immunity induced by natural infection is slow to develop and anthelmintic resistance is rapidly developing. There is a need to advance alternatives for control of gastrointestinal nematode parasites. The present study investigated the effects of repeated, drug-truncated infections (rDTI) on development of protective immunity and attenuation of a challenge infection by O. ostertagi. Helminth-free calves were randomly assigned to either a rDTI or a control group (n = 5). The rDTI group received daily oral infections of 5000 Ostertagia L3 for 5 consecutive days, then were drug-treated on 14 and 15 days post infection (dpi), to attenuate O. ostertagi at the late fourth larval (L4) through young adult stages. DTI was repeated 3 weeks after the drug treatment. A total of 5 DTIs were administered to the DTI-treated animals. Non-DTI-treated, control animals received tap water as infection control. All animals were drug-treated at the same time. Animals were challenge-infected 4 weeks following the final round of rDTI. The results show that eggs per gram of feces (EPG) in the rDTI group were significantly reduced (P < 0.05) from 21 to 39 dpi, with an overall reduction in cumulative EPG. The control group exhibited reduced (P = 0.0564) average weight gains when compared to those of the rDTI group during weeks 4-5 post infection, a period coinciding with peak EPG output of control animals. Antigen-specific IgG, IgE and IgA responses were detected after the 2nd DTI, and stronger antibody recall responses were elicited by challenge infection. High levels of antigen-specific peripheral blood mononuclear cell (PBMC)/T cell proliferation to whole worm and excretory-secretory (ES) antigens were detected in rDTI-treated animals. These data indicate that partial protective immunity against ostertagiasis, involving cell-mediated and humoral responses, can be attained by rDTI which allowed for maximal antigen exposure from staggered parasitic developmental stages. The data suggest that rDTI can be used as a model to study host-parasite interactions and identify parasite antigens responsible for eliciting host protective immune responses.

Vaccination with Neospora caninum-cyclophilin and -profilin confers partial protection against experimental neosporosis-induced abortion in sheep.

The purpose of this study was to evaluate the protective efficacy of a vaccine consisting of recombinant Neospora caninum-cyclophilin (NcCyP) and -profilin (NcPro) in sheep. At 42 d and 21 d prior to mating, adult Dorset ewes were immunized with the rNcCyP-rNcPro vaccine (Group 1) or co-purifying non-recombinant (NR) control vaccine (Group 2). At 90 days post-mating, all immunized ewes and were challenged by intravenous injection with 106Nesopora caninum Illinois tachyzoites (NcTZ). Significant protection (P < 0.05) was observed in Group 1 with 9 out of 13 ewes giving birth to live-born lambs (69.2%), whereas all Group 2 ewes aborted (6/6). Neospora caninum was detected by PCR in both fetal and placental tissues from all Group 2 aborting ewes and in the placental tissues of Group 1 aborting ewes. In contrast, tissues and placentas of Group 1 live-born lambs were Neospora DNA-negative. Immunoreactive Neospora antigens were demonstrated in placentas associated with abortions, but not in tissues of aborted fetuses or those of the live-born lambs and their associated placentas. Anti-NcCyP and anti-NcPro titers were high in sera from Group 1 ewes and were further boosted by challenge infection, resulting in long-lasting (≥14.5 mos.) elevated titers. Lambs born to Group 1 ewes also had high NcCyP and NcPro titers in pre-colostrum sera. Immunofluorescence staining (IFA) of NcTZ with Group 1 post-immunization sera revealed both surface and internal TZ staining, a pattern consistent with that observed with rabbit sera to rNcCyP or rNcPro. Infection of NR-vaccinated ewes produced high but transient anti-NcCyP and anti-NcPro Ab titers. The results indicate that the NcCyP-NcPro vaccine elicited strong anti-N. caninum responses and conferred significant protection against abortion and transplacental transmission of N. caninum TZ in sheep.

Ostertagia ostertagi Mediates Early Host Immune Responses via Macrophage and Toll-Like Receptor Pathways.

Ostertagia ostertagi is an abomasal parasite with significant economic impact on the cattle industry. Early host immune responses are poorly understood. Here, we examined time course expression of Toll-like receptors (TLRs) in peripheral blood mononuclear cells (PBMC) during infection where PBMC macrophages (Mϕ) generated both pro- and anti-inflammatory responses when incubated with excretory/secretory products (ESP) from fourth-stage larvae (OoESP-L4) or adult worms (OoESP-Ad). First, changes in cell morphology clearly showed that both OoESP-L4 and OoESP-Ad activated PBMC-Mϕ in vitro, resulting in suppressed CD40 and increased CD80 expression. Expression of mRNAs for TLR1, -4, -5, and -7 peaked 7 days postinfection (dpi) (early L4), decreased by 19 dpi (postemergent L4 and adults) and then increased at 27 dpi (late adults). The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) (transcript and protein) increased in the presence of OoESP-Ad, and the anti-inflammatory cytokine interleukin 10 (IL-10) (protein) decreased in the presence of OoESP-L4 or OoESP-Ad; however, IL-10 mRNA was upregulated, and IL-6 (protein) was downregulated by OoESP-L4. When PBMC-Mϕ were treated with ligands for TLR4 or TLR5 in combination with OoESP-Ad, the transcripts for TNF-α, IL-1, IL-6, and IL-10 were significantly downregulated relative to treatment with TLR4 and TLR5 ligands only. However, the effects of TLR2 ligand and OoESP-Ad were additive, but only at the lower concentration. We propose that O. ostertagi L4 and adult worms utilize competing strategies via TLRs and Mϕ to confuse the immune system, which allows the worm to evade the host innate responses.

Immunoproteomic and mass spectrometric analysis of Eimeria acervulina antigens recognized by antisera from chickens infected with E. acervulina, E. tenella or E. necatrix.

BACKGROUND: Coccidiosis is caused by Eimeria spp. and can result in severe economic losses to the global poultry industry. Due to anticoccidial drug resistance rapidly developing in the parasites and drug residues in poultry products, efficacious and safe alternative coccidia control measures are needed. The objective of the present study was to identify common protective antigens which may be used as vaccine candidates in the development of subunit, multivalent, cross-protective vaccines against most of the economically important Eimeria species. METHODS: Whole sporozoite proteins of Eimeria acervulina were prepared and analyzed by 2-dimensional gel electrophoresis (2-DE) followed by western blotting using immune sera specific to E. tenella, E. acervulina, or E. necatrix. The protein spots detected by all three immune sera were then excised from the preparative gel and protein ID was performed by MALDI-TOF-MS/MS. RESULTS: Approximately 620 E. acervulina sporozoite protein spots were demonstrated by 2-DE with silver staining, among which 23 protein spots were recognized by immune sera specific to all three Eimeria species. The results showed that 21 putative E. acervulina proteins were identified, which include proteins with known enzymatic properties, and those which are involved in protein translation, transport and trafficking, and ribosomal biogenesis and functions. There is one protein which may be involved in transcription and one heat-shock protein. Two proteins contain predicted domains, but with no apparent functions known. There were 2 protein spots which had no detectable proteins. None of the proteins has a predicted signal peptide or a transmembrane domain; however, 6 of the 21 putative proteins were predicted to be potentially secretory through the non-classical pathway. CONCLUSIONS: Our study identified a diverse group of antigens immunologically common to all three Eimeria species, none of which was previously characterized and tested as a vaccine candidate. Further research on immunogenicity and cross-protective potential of these individual proteins as vaccine candidates will aid the development of vaccines against the most common and pathogenic Eimeria spp.

TRPV4 is dispensable for the development of airway allergic asthma.

Allergic asthma is one of the most common immune-mediated disorders affecting the lungs. It is characterized clinically by airway hyperresponsiveness, eosinophilia, enhanced IL-4 and IL-13, peribronchial inflammation with mononuclear cell infiltration, and goblet cell hyperplasia associated with increased mucus production. However, chronic asthma with repeated exposures to inhaled allergens can result in subepithelial pulmonary fibrosis. The transient receptor potential cation channel subfamily V member 4 (TRPV4) protein can promote the generation of myofibroblasts and pulmonary fibrosis. Here, we investigated the possibility that TPRV4 facilitates the development of allergic asthma and subsequent pulmonary fibrosis in the lung. To test this, wild-type (WT) and TPRV4 gene knockout (KO) mice were repeatedly sensitized with chicken ovalbumin (OVA) and repeatedly subjected to aerosol challenge with 1% OVA. We found that there were no significant differences in the development of allergic asthma between the WT and TPRV4 KO mice. Both groups of mice exhibited similar levels of airway hyperresponsiveness, IL-13, IL-5, OVA-specific IgE, eosinophilia, mucus-secreting goblet cell hyperplasia, and deposition of collagen fiber, which is a hallmark of the pulmonary fibrosis. Thus, these data suggest that TPRV4 protein is dispensable in the initiation and development of airway asthma and subsequent fibrosis.

Neospora caninum cytoplasmic dynein LC8 light chain 2 (NcDYNLL2) is differentially produced by pathogenically distinct isolates and regulates the host immune response.

Neospora caninum is the causative agent of bovine neosporosis. A N. caninum cytoplasmic dynein LC8 light chain (NcDYNLL) protein was characterized in this study. Cytoplasmic dyneins, including DYNLLs, belong to the microtubule minus-end-directed motor proteins and are involved in many cellular processes. Previous microarray studies revealed that NcDYNLL was downregulated in the non-pathogenic clone, Ncts-8, when compared with the wild-type NC1 isolate. The present study showed that DYNLLs from different species are highly conserved (>85% identity), and the NcDYNLL belongs to the DYNLL2 family. NcDYNLL2 and Toxoplasma gondii DYNLL2 have identical amino acid sequences, although they are slightly divergent at the genetic level (89% identity). NcDYNLL2 was cloned and expressed in Escherichia coli and purified. NcDYNLL2 was identified in soluble and insoluble fractions of tachyzoite lysate. As expected, soluble NcDYNLL2 was lower in the Ncts-8 lysate when compared with that of NC1 isolate. NcDYNLL2 release by the tachyzoites was low; however, it was increased when tachyzoites were treated with either calcium ionophore or ethanol. The data indicate that NcDYNLL2 may be actively secreted at low levels, but the secretion was upregulated by agents that also augment microneme protein secretions. Immunostaining of NcDYNLL2 in isolated and intracellular Neospora tachyzoites showed a diffuse distribution pattern. Furthermore, rNcDYNLL2 was internalized by the host immune cells and stimulated tumour necrosis factor-α) and interleukin-12 (IL-12) production by murine dendritic cells. Taken together, these results suggest that NcDYNLL2 is a secretory protein that cross-regulates host immunity.

Characterization of IL-10-producing neutrophils in cattle infected with Ostertagia ostertagi.

IL-10 is a master regulator of immune responses, but its cellular source and function in cattle during the initial phase of immune priming have not been well established. Despite a massive B cell response in the abomasal draining lymph nodes in Ostertagia ostertagi (OO)-infected cattle, protective immunity is slow to develop, and partial protection requires years of repeated exposure. In addressing this problem, our initial hypothesis was that B cells produce IL-10 that downregulates the host protective immune response. However, our results showed that neutrophils made up the majority of IL-10-producing cells in circulation and in secondary lymphoid tissues, particularly the spleen (80%). Conversely, IL-10-producing B cells were rare. In addition, approximately 10% to 20% of the neutrophils in the blood and spleen expressed MHC II and were IL-10 negative, suggesting that neutrophils could also participate in antigen presentation. In vitro investigation of bovine neutrophils revealed that exposure thereof to OO extract increased IL-10 and MHC II expression in these cells in a dose-dependent manner, consistent with IL-10+/MHC II+ neutrophils detected in cattle shortly after experimental OO infection. Co-culture of untreated neutrophils with anti-CD3 antibody (Ab)-stimulated CD4+ T cells led to enhanced T cell activation; also, IL-10 depletion with neutralizing Ab enhanced the stimulatory function of neutrophils. OO extract depressed neutrophil stimulation of CD4+ T cells in the presence of IL-10-neutralizing Ab, suggesting that OO utilizes both IL-10-dependent and independent mechanisms to manipulate the bovine immune response. Finally, contact and viability were required for T cell-stimulatory neutrophil function. This report, to the best of our knowledge, is the first to demonstrate that neutrophil-derived IL-10 is directly involved in T cell regulation in cattle. Our data suggest that neutrophils and neutrophil-derived IL-10 are co-opted by nematode parasites and other pathogens to attenuate host immune responses and facilitate pathogen survival.

Bovine neutrophils form extracellular traps in response to the gastrointestinal parasite Ostertagia ostertagi.

Ostertagia ostertagi (OO) is a widespread parasite that causes chronic infection in cattle and leads to annual losses of billions of dollars in the cattle industry. It remains unclear why cattle are unable to mount an effective immune response despite a large influx of immune cells to the infected abomasal mucosa and draining lymph nodes. Neutrophils, the immune system's first responders, have the capacity to release neutrophil extracellular traps (NETs) to contain various pathogens, including some parasites. In the present study, the mechanisms by which O. ostertagi influences bovine NET formation were investigated. O. ostertagi larval soluble extract (OO extract) was able to induce typical NETs by purified neutrophils in vitro, confirmed by co-localization of extracellular DNA with typical NET-associated proteins histone and neutrophil elastase (NE). Consistent with existing literature, inhibition assays demonstrated that these OO extract-induced NETs were dependent upon the enzymes NADPH oxidase and myeloperoxidase (MPO). Live OO stage 4 larvae (L4) stimulated neutrophils to form NETs similar to those induced by OO extract. Bovine neutrophils also released NETs in response to Caenorhabditis elegans, a free-living soil nematode, suggesting that bovine NET production may be a conserved mechanism against a broad range of nematodes. This is the first report demonstrating O. ostertagi-induced NET formation by bovine neutrophils, a potentially underappreciated mechanism in the early immune response against nematode infections.

Characterization of Ostertagia ostertagi annexin-like proteins at different developmental stages.

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host's gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P < 0.05) was higher in adult worms than in L3 or L4. The results indicate that the production of Oos-ANXL-2s is developmentally regulated and most abundant in the adult worm. This rather large family of proteins could be a potential vaccine target against O. ostertagi infection and warrants further investigation.