Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.

Divergent humoral responses between males and females against 24 kDa excretory-secretory protein of Haemonchus contortus and influence of ovine ß-globin polymorphism.

Lambs harboring the Hb-AA ß-globin haplotype present improved cell-mediated responses and increased resistance against Haemonchus contortus infection. The aim of the present study was to compare the effect of sex and ß-globin haplotypes on specific humoral responses and phenotypes of resistance during H. contortus infection in Morada Nova sheep. As expected, females displayed stronger resistance during the first and second experimental challenges. Differential systemic humoral immune responses were observed comparing sex groups, in which higher levels of specific antibodies targeting 24 kDa excretory-secretory (ES24) protein of H. contortus of IgG and IgM antibodies were respectively observed as predominant isotypes in males and females. The IgM levels were significantly correlated with phenotypes of resistance, evaluated by packed cell volume and fecal egg counts. To our knowledge this is the first study reporting divergent humoral responses profiles to H. contortus infection between male and female sheep. The impact of ß-globin haplotypes was less pronounced in females compared to males. Notably, only males showed significant weight differences across haplotypes, with Hb-AA lambs being the heaviest. Additionally, Hb-AA males had significantly higher PCV (indicating better red blood cell health) and lower FEC (indicating lower parasite burden). These findings suggest a more pronounced effect of ß-globin polymorphisms on H. contortus infection in males, potentially due to their generally weaker resistance compared to females. This study highlights the importance of sex and ß-globin haplotypes in shaping immune responses to H. contortus infection. Specifically, IgM antibodies targeting the ES24 protein appear to play a crucial role in host-parasite interactions and may hold promise for therapeutic development.

Comparative analysis of host immune responses to Hydatid cyst in human and ovine hepatic cystic Echinococcosis.

BACKGROUND: Hydatid disease is caused by the larval stages of the canine tapeworm Echinococcus granulosus. It is one of the most critical helminthic diseases, representing worldwide public health and socio-economic concern. AIM: This study aimed to investigate the expression of apoptosis and immune response within hepatic tissues of humans and sheep infected with the Hydatid cyst. METHODS: Paraffin-embedded tissue was prepared from each tissue sample and used for histopathological examination by Haematoxylin- Eosin. Also, toluidine blue staining was used for mast cell detection, while an immunohistochemical study was performed to assess CD3 T lymphocytes, CD4 helper T lymphocytes, CD8 cytotoxic T lymphocytes, CD20 memory B lymphocytes, CD68 macrophage, and caspase-3 antibodies. RESULTS: The histological examination revealed significant changes, including the infiltration of inflammatory cells, predominantly lymphocytes with scattered giant cells, necrotic hepatic tissue, and fibrosis. Toluidine blue stain revealed a higher number of mast cells (5 cells/field) in humans compared to sheep (3.6 cells/field). The immunohistochemical analysis confirmed that the CD3 were the most predominant inflammatory cell in the hepatic tissue of humans (intensive 70%), and sheep (moderate 38.47%). Caspase-3 was observed in all samples in different grades and mostly in human liver tissue. CONCLUSION: This data could aid in recognizing immunological markers for differentiating disease progression, as well as enhance the understanding of local immune responses to cystic Echinococcosis (CE). The findings could provide preliminary data for future studies on immune responses associated with Hydatid cysts.

Functionally distinct T-helper cell phenotypes predict resistance to different types of parasites in a wild mammal.

The adaptive immune system is critical to an effective response to infection in vertebrates, with T-helper (Th) cells pivotal in orchestrating these responses. In natural populations where co-infections are the norm, different Th responses are likely to play an important role in maintaining host health and fitness, a relationship which remains poorly understood in wild animals. In this study, we characterised variation in functionally distinct Th responses in a wild population of Soay sheep by enumerating cells expressing Th-subset specific transcription factors and quantifying Th-associated cytokines. We tested the prediction that raised Th1 and Th2 responses should predict reduced apicomplexan and helminth parasite burdens, respectively. All measures of Th-associated cytokine production increased with age, while Th17- and regulatory Th-associated cytokine production increased more rapidly with age in males than females. Independent of age, sex, and each other, IL-4 and Gata3 negatively predicted gastro-intestinal nematode faecal egg count, while IFN-γ negatively predicted coccidian faecal oocyst count. Our results provide important support from outside the laboratory that Th1 and Th2 responses predict resistance to different kinds of parasites, and illustrate how harnessing specific reagents and tools from laboratory immunology will illuminate our understanding of host-parasite interactions in the wild.

First study of Brucella ovis antibodies in purebred sheep flocks in the State of Parana, Brazil / [Primeiro estudo de anticorpos para Brucella ovis em rebanhos de ovinos puros de Origem, no estado do Paraná, Brasil]

Brucella ovis, a non-zoonotic species, is the etiological agent of ovine brucellosis, an infectious disease of clinical or subclinical occurrence in sheep flocks. Until then, there is no serological study of anti-Brucella ovis antibodies in purebred sheep herds. This study aimed to determine the presence of anti-Brucella ovis antibodies in purebred sheep flocks with breeding purposes from Parana State. Blood samples from 728 animals, of which 563 were females and 165 males, between 8 and 56 months of age from the six major sheep producing mesoregions of Parana, were submitted to detection of anti-Brucella ovis antibodies by the Agar Gel Immunodiffusion technique using an antigen from the bacteria Brucella ovis (Reo 198). The results indicate the presence of this disease in purebred sheep from Parana State in a low occurrence of 0.27% (2/728). The only two positive animals were rams, Santa Inês breed, from the same flock in the East Center region of Parana, without clinical disease. In conclusion, Brucella ovis is present in purebred sheep in Parana State, Brazil, and this low occurrence may have occurred due to rigorous breeding systems that may contribute to reduce the transmission of this disease.(AU)

Brucella ovis, espécie não zoonótica, é o agente etiológico da brucelose ovina, doença infecciosa de ocorrência clínica ou subclínica. Atualmente, não existe estudo sorológico de anticorpos anti-Brucella ovis em rebanhos de ovinos puros de origem. Este estudo teve como objetivo determinar a presença de anticorpos anti-Brucella ovis em rebanhos ovinos de raça pura de origem, com fins reprodutivos do estado do Paraná. Amostras de sangue de 728 animais, sendo 563 fêmeas e 165 machos, entre oito e 56 meses de idade, pertencentes a seis principais mesorregiões produtoras de ovinos no Paraná, foram submetidas à detecção de anticorpos anti-Brucella ovis pela técnica de imunodifusão em ágar gel usando-se um antígeno da bactéria Brucella ovis (Reo 198). Os resultados indicam a presença da doença em ovinos puros de origem do estado do Paraná em baixa ocorrência de 0,27% (2/728). Os dois únicos animais positivos foram reprodutores da raça Santa Inês, do mesmo rebanho da região Centro Leste do Paraná, sem manifestação clínica. Em conclusão, Brucella ovis está presente em ovinos puros de origem no estado do Paraná, e essa baixa ocorrência pode ter ocorrido devido a sistemas rigorosos de criação, que podem contribuir para a redução da transmissão dessa doença.(AU)

S100A8 expression in oviduct mucosal epithelial cells is regulated by estrogen and affects mucosal immune homeostasis.

Chronic inflammation can cause oviduct mucosal damage and immune dysfunction, leading to infertility, early pregnancy loss, ectopic pregnancy, tumors, and a decrease in reproductive capacities in female animals. Estrogen can suppress immune responses in different tissues and oviducts, and regulate the oviduct immune balance; however, the underlying mechanisms remain unclear. The objective of this study was to explore the mechanism of estrogen-regulated oviduct mucosal immunity and discover new estrogen targets for regulating oviduct mucosal immune homeostasis. Sheep oviduct epithelial cells (SOECs) were treated with 17-ß estradiol (E2). Transcriptome sequencing and analysis showed differentially expressed S100 calcium-binding protein A (S100A) genes that may participate in the oviduct mucosa immunoregulation of estrogen. Quantitative polymerase chain reaction and immunocytochemistry analysis showed that S100A8 expression changed dynamically in E2-treated SOECs and peaked after 7 h of treatment. Estrogen nuclear receptors and G protein-coupled membrane receptors promoted E2-dependent S100A8 upregulation. The S100A8 gene was disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 method. Levels of inflammatory factors interleukin (IL)-1ß and IL-4 were significantly upregulated in S100A8-knockdown SOECs, whereas those of the anti-inflammatory factor IL-10 was downregulated. Following S100A8 knockdown in SOECs treated with E2 for 7 h, IL-10 levels increased significantly. Estrogen affected oviduct mucosa immune function and dynamically regulated S100A8 in SOECs. S100A8 knockdown caused an excessive immune response, indicating that S100A8 is beneficial for maintaining immune homeostasis in the oviduct mucosa. Moreover, estrogen can compensate for the effect of S100A8 knockdown by upregulating IL-10.

Immunization With Bovine Herpesvirus-4-Based Vector Delivering PPRV-H Protein Protects Sheep From PPRV Challenge.

The Morbillivirus peste des petits ruminants virus (PPRV) is the causal agent of a highly contagious disease that mostly affects sheep and goats and produces considerable losses in developing countries. Current PPRV control strategies rely on live-attenuated vaccines, which are not ideal, as they cannot differentiate infected from vaccinated animals (DIVA). Recombinant vector-based vaccines expressing viral subunits can provide an alternative to conventional vaccines, as they can be easily paired with DIVA diagnostic tools. In the present work, we used the bovine herpesvirus-4-based vector (BoHV-4-A) to deliver PPRV hemagglutinin H antigen (BoHV-4-A-PPRV-H-ΔTK). Vaccination with BoHV-4-A-PPRV-H-ΔTK protected sheep from virulent PPRV challenge and prevented virus shedding. Protection correlated with anti-PPRV IgGs, neutralizing antibodies and IFN-γ-producing cells induced by the vaccine. Detection of antibodies exclusively against H-PPRV in animal sera and not against other PPRV viral proteins such as F or N could serve as a DIVA diagnostic test when using BoHV-4-A-PPRV-H-ΔTK as vaccine. Our data indicate that BoHV-4-A-PPRV-H-ΔTK could be a promising new approach for PPRV eradication programs.

Prediction of Genetic Resistance for Scrapie in Ungenotyped Sheep Using a Linear Animal Model.

Selection based on scrapie genotypes could improve the genetic resistance for scrapie in sheep. However, in practice, few animals are genotyped. The objectives were to define numerical values of scrapie resistance genotypes and adjust for their non-additive genetic effect; evaluate prediction accuracy of ungenotyped animals using linear animal model; and predict and assess selection response based on estimated breeding values (EBV) of ungenotyped animals. The scrapie resistance (SR) was defined by ranking scrapie genotypes from low (0) to high (4) resistance based on genotype risk groups and was also adjusted for non-additive genetic effect of the haplotypes. Genotypes were simulated for 1,671,890 animals from pedigree. The simulated alleles were assigned to scrapie haplotypes in two scenarios of high (SRh) and low (SRl) resistance populations. A sample of 20,000 genotyped animals were used to predict ungenotyped using animal model. Prediction accuracies for ungenotyped animals for SRh and SRl were 0.60 and 0.54, and for allele content were from 0.41 to 0.71, respectively. Response to selection on SRh and SRl increased SR by 0.52 and 0.28, and on allele content from 0.13 to 0.50, respectively. In addition, the selected animals had large proportion of homozygous for the favorable haplotypes. Thus, pre-selection prior to genotyping could reduce genotyping costs for breeding programs. Using a linear animal model to predict SR makes better use of available information for the breeding programs.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

The Interplay between Bluetongue Virus Infections and Adaptive Immunity.

Viral infections have long provided a platform to understand the workings of immunity. For instance, great strides towards defining basic immunology concepts, such as MHC restriction of antigen presentation or T-cell memory development and maintenance, have been achieved thanks to the study of lymphocytic choriomeningitis virus (LCMV) infections. These studies have also shaped our understanding of antiviral immunity, and in particular T-cell responses. In the present review, we discuss how bluetongue virus (BTV), an economically important arbovirus from the Reoviridae family that affects ruminants, affects adaptive immunity in the natural hosts. During the initial stages of infection, BTV triggers leucopenia in the hosts. The host then mounts an adaptive immune response that controls the disease. In this work, we discuss how BTV triggers CD8+ T-cell expansion and neutralizing antibody responses, yet in some individuals viremia remains detectable after these adaptive immune mechanisms are active. We present some unpublished data showing that BTV infection also affects other T cell populations such as CD4+ T-cells or γδ T-cells, as well as B-cell numbers in the periphery. This review also discusses how BTV evades these adaptive immune mechanisms so that it can be transmitted back to the arthropod host. Understanding the interaction of BTV with immunity could ultimately define the correlates of protection with immune mechanisms that would improve our knowledge of ruminant immunology.

A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response, and Host-Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry's largest welfare problems. The complex etiology of footrot makes in situ or in vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot-affected interdigital skin compared to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome, which could lead to the identification of future therapeutic targets.

Is vaccination a viable method to control Johne's disease caused by Mycobacterium avium subsp. paratuberculosis? Data from 12 million ovine vaccinations and 7.6 million carcass examinations in New South Wales, Australia from 1999-2009.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johne's disease is increasing worldwide. In an attempt to control an epidemic of ovine Johne's disease (OJD) in New South Wales (NSW), a government/industry sponsored voluntary vaccination/on-farm management program commenced in 2000. We report herein an observational study of changes in disease prevalence as vaccination progressed, based on abattoir surveillance data for OJD from 1999 to 2009. We also discuss the epidemiological, policy, regulatory, research, economic and sociological elements that contributed to the development of a mature control program, whose aim was to halt the epidemic spread of OJD in a naïve sheep population. METHODS: NSW was divided into areas of "High" (HPA), "Medium" (MPA) and "Low" (LPA) OJD prevalence. A killed whole cell vaccine (Gudair®) was administered to sheep from 2000 to 2009. Trained examiners evaluated the viscera of adult sheep carcasses at slaughter for gross evidence of OJD. MAP infection was confirmed by histopathology. PRINCIPAL FINDINGS: From 2000-2009, 12 million vaccine doses were administered in NSW (91%; 10.9 million in the HPA). Many of the vaccinated flocks were suffering > 5% annual mortality in adult sheep, with some individual flocks with 10-15% losses attributable to OJD. A total of 7.6 million carcasses were examined (38%; 2.9 million from the HPA). Overall, 16% of slaughter consignments (sheep consigned to the abattoir from a single vendor) were positive for OJD, of which 94% were from the HPA. In the HPA, the percentage of animals with lesions attributable to OJD at slaughter fell progressively from 2.4% (10,406/432,860) at commencement of vaccination in 2000 to 0.8% (1,573/189,564) by 2009. Herd immunity from vaccination in the HPA was estimated at 70% by 2009, the target commonly espoused for an effective control program based on vaccination. This coincided with a progressive decrease in reports of clinical disease and mortalities in vaccinated flocks. SIGNIFICANCE: We show a decrease in the prevalence of lesions attributable to OJD in NSW concomitant with initiation of voluntary vaccination, on-farm management plans, abattoir monitoring and feedback of animal prevalence data to sheep producers. We conclude that a target of ≤ 1% regional prevalence of OJD affected sheep at slaughter is achievable using these interventions.

Evaluation of antibody response of sheep to foot-and-mouth disease vaccine prepared by using different MontanideTM oil adjuvants.

Despite the widespread use of the conventional inactivated foot-and-mouth disease (FMD) vaccine, its immunogenicity is poor and the duration of its protection is short. In this study, humoral response to commercial ready-to-use MontanideTM ISA 201 VG and MontanideTM ISA 61 VG oil adjuvants and a common adjuvant MontanideTM ISA 206 VG developed by Seppic Inc., were evaluated for FMD antigens in sheep and double oil emulsion (w/o/w) formulations of MontanideTM ISA 201 and 206 and single oil emulsion (w/o) of MontanideTM ISA 61 have been prepared by using current FMDV antigens (O/TUR/07, A/ASIA/G-VII, A/TUR/16 and ASIA/ TUR/15). The animals (n=48) were vaccinated subcutaneously with formulations and five sheep were maintained as an unvaccinated control group. Blood samples were taken at day 0, 7, 14, 21, 28, 60, 90, 120 and 150. Virus neutralization and liquid phase blocking ELISA tests were used to compare antibody response to vaccines prepared by using different MontanideTM mineral oils. The results showed that vaccines prepared by using MontanideTM ISA 61 and 201 gave better antibody response to FMD antigens than MontanideTM ISA 206 formulation, although results were not statistically significant for certain days of sampling. Moreover, the overall type O antibody response of MontanideTM ISA 201 was found to be superior to MontanideTM ISA 61.

Positive selection of IgG+ over IgM+ B cells in the germinal center reaction.

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM+ B cells; IgG+ B cells subsequently increased in frequency, dominating GC responses 14-21 days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM+ and IgG+ GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM+ GC B cells. Instead, high-affinity IgG+ GC B cells outcompeted high-affinity IgM+ GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.

Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics.

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

The specific features of the developing T cell compartment of the neonatal lung are a determinant of respiratory syncytial virus immunopathogenesis.

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.

Effect of climatic factors on urinary cortisol and peripheral blood leukocytes in lambs grazing on a semi-natural grassland in the Hokuriku District of Japan.

The effects of climatic factors on stress and immune functions of grazing lambs in summer and autumn in the Hokuriku District of Japan were evaluated by determining urinary cortisol (U-COR) levels and peripheral blood leukocyte populations and comparing those with lambs kept indoors. Two groups of five lambs, consisting of those grazed on a semi-natural grassland (GRL) and those housed indoors in a domestic animal shelter (INL), were maintained at from July to October. The temperature-humidity index at each location was indicative of heat stress during summer; however, the U-COR elevation was not observed in both groups. The elevation was observed in GRL in autumn and was higher than INL in October. Climatic conditions in autumn were characterized by high humidity and a sudden drop in temperature. U-COR was positively correlated with the relative humidity. The GRL was exposed to low-nutrient conditions for a relatively long time. The CD4+ /CD8+ T cell ratio in GRL decreased in October. Subsequently, the total leucocyte, including granulocytes, monocytes, and lymphocytes, sharply increased. The responses indicated an immune deficiency caused by immunosuppression because of a low nutrition caused by grazing and high-stress conditions in autumn.

Identification of Different Extracellular Vesicles in the Hydatid Fluid of Echinococcus granulosus and Immunomodulatory Effects of 110 K EVs on Sheep PBMCs.

Echinococcosis, mainly caused by Echinococcus granulosus, is one of the 17 neglected tropical diseases. Extracellular vesicles (EVs) play an essential role in the host-parasite interplay. However, the EVs in the hydatid fluid (HF) of E. granulosus are not fully characterized. Herein, three different types of HF EVs, designated as 2 K, 10 K, and 110 K EVs based on the centrifugal force used, were morphologically identified. A total of 97, 80, and 581 proteins were identified in 2 K, 10 K, and 110 K EVs, respectively, 39 of which were commonly shared. Moreover, 11, 8, and 25 miRNAs were detected, respectively, and all of the 7 selected miRNAs were validated by qPCR to be significantly lower abundant than that in protoscoleces. It was further deemed that 110 K EVs were internalized by sheep peripheral blood mononuclear cells (PBMCs) in a time-dependent manner and thus induced interleukin (IL)-10, tumor necrosis factor-α (TNF-α), and IRF5 were significantly upregulated and IL-1ß, IL-17, and CD14 were significantly downregulated (p < 0.05). These data demonstrate the physical discrepancy of three HF EVs and an immunomodulatory effect of 110 K EVs on sheep PMBCs, suggesting a role in immune responses during E. granulosus infection.