Staphylococcus chromogenes is one of the most prevalent non-aureus staphylococci associated with persistent intramammary infections (IMIs) in dairy cattle. However, the local cellular immune response to naturally occurring S. chromogenes IMI remains poorly characterized. Thus, this study aimed to assess milk T cell subsets, macrophages, neutrophils, and key intracellular cytokine production by milk T cells in mammary gland quarters naturally infected with S. chromogenes. Milk samples were used from non-infected and S. chromogenes-infected quarters from clinically healthy Holstein-Friesian dairy cows. The intensity of intracellular IFN-γ production by CD4+ (P = 0.048) and CD8+ (P = 0.035) T lymphocytes was higher in S. chromogenes-infected quarters than in non-infected controls, with a similar trend observed for γδ T cells (P = 0.06). Furthermore, the proportion of milk γδ T cells was higher in S. chromogenes-infected quarters (P = 0.05). Differences were observed neither in intracellular IL-17A production by T cell subsets nor in the proportions of milk CD4+ and CD8+ T lymphocytes, macrophages and neutrophils. Our findings identify T cell responses as a key component of the mammary immune response to S. chromogenes IMI and open new avenues for future studies investigating its potential in vivo protective response against major mastitis pathogens.
The recent emergence of foot and mouth disease virus (FMDV) serotype SAT1 in Egypt necessitated the development of a specific vaccine directed against the newly introduced serotype. The present study describes the preparation and evaluation of a monovalent inactivated SAT1 vaccine with potential for inclusion in national FMD control programs. Three SAT1 field isolates were adapted in BHK-21 cells through serial passage, after which an optimal master seed virus was selected based on reproducible and stable infectivity characteristics. The vaccine was formulated as a double oil emulsion using Montanide ISA 206. The vaccine potency was assessed in guinea pigs through determination of the protective dose (GPPD50) following challenge with virulent guinea pig adapted virus that revealed satisfactory protective potency. The humoral immune response induced by such vaccine was subsequently evaluated in vaccinated cattle (2 mL dose), buffaloes (2 mL dose), and sheep (1 mL dose) through monitoring of their serum neutralizing antibody titers. Protective antibody levels were detected at 14 days in cattle and buffaloes and at 21 days in sheep post vaccination. The exhibited immunity had a duration extended to 32 weeks in cattle and buffaloes and to 28 weeks in sheep. These findings indicate that the prepared SAT1 vaccine was capable of inducing protective and sustained humoral immunity in the principal livestock species targeted within Egyptian vaccination campaigns and support its potential application in national FMD control programs.
The gut microbiota plays a crucial role in immune regulation and metabolic homeostasis in ruminants; however, the systemic effects of rumen fluid transfer (RFT) on the gut-liver axis remain unclear. In this study, Xizang sheep were used as the animal model to investigate the effects of RFT using multi-omics approaches, including transcriptomics, metabolomics, and 16S rRNA sequencing. The results showed that RFT was associated with changes in immune- and metabolism-related genes (e.g., TLR4, NOD2, PPARA, and ABCG2) and alterations in several key metabolites, including tryptophan-related compounds, amino acids, and bile acid-associated metabolites. RFT was also associated with shifts in gut microbial composition, as reflected by changes in the relative abundance of genera such as Prevotella, Ruminococcus, and Lactobacillus. Integrated multi-omics analysis indicated coordinated associations among gut microbiota, metabolites, and host gene expression along the gut-liver axis. These findings indicate that RFT may be linked to immune and metabolic regulation in Xizang sheep, providing a systems-level framework for understanding microbiota-host interactions.
Streptococcus equi subsp. zooepidemicus (SEZ) primarily functions as a zoonotic pathogen, with the capacity to infect a wide variety of animal species, including humans. The present study has captured the importance role of GSDMD in mice against SEZ, here, with the help of published works, we chose miR-325-3p as a regulator to GSDMD and investigated the role of miR-325-3p during SEZ infection in vitro and vivo. In this study, SEZ significantly increased the expression of GSDMD and release of cytokines in macrophages. Both prediction assay using Target Scan databases and luciferase reporter assay showed that miR-325-3p directly targeted to GSDMD. Moreover, overexpression of miR-325-3p significantly decreased the expression of GSDMD and the release of IL-1β and IL-18 in macrophages infected with SEZ. And inhibition of miR-325-3p expression exhibited the opposite effect. In vivo experiments, we found that injection of miR-325-3p mimics into mice alleviated lung injury induced with SEZ, and miR-325-3p mimics also reduced the IL-1β and IL-18 contents in bronchoalveolar lavage fluid (BALF) and bacterial loads in lung tissue. Results in this study revealed that miR-325-3p suppressed inflammatory response triggered by SEZ both in vitro and vivo. Taken together, our findings provided evidence to identify miR-325-3p as a potential therapeutic agent to SEZ infection.
Myeloperoxidase (MPO), primarily produced by neutrophils, is highly conserved between species and exists as a glycosylated monomeric pro-MPO prior to storage as dimeric MPO in azurophilic granules. MPO is increasingly recognized as a biomarker of inflammatory diseases and is commonly measured using antibody-based assays. We compared two commercial enzyme linked immunosorbent assays (ELISAs): a canine assay detecting MPO based on antibodies produced against pro-MPO, and a human assay detecting MPO based on antibodies produced against the MPO monomer (MPO-intermediate), in the detection of MPO in canine serum and urine samples. Neither assay demonstrated cross-reactivity with the MPO-standard of the other assay. In urine samples (n = 87), median MPO concentrations were 78.0 pg/mL (IQR:78.0218.0) using the canine assay and 99.6 pg/mL (IQR:31.25107.4) using the human assay, with no correlation observed between the two assays. When segregating urine samples based on the presence of bacteriuria, only the human MPO-assay showed differences between groups, with higher and more variable concentrations in bacteriuric samples and consistently low concentrations in non-bacteriuric samples. Only the concentrations obtained with the human MPO-assays demonstrated an association with urinary IL-8 concentrations and urinary leukocyte degree. In serum samples (n = 56), the canine MPO-assay measured the highest median concentrations, 86.85 ng/mL (IQR:75.17 97.11), compared to the human MPO-assay, 1.2 ng/mL (IQR: 0.761.54), with no correlation between the two assays. These findings highlight the complexity of MPO measurements and underscore the importance of assay selection and biochemical understanding when investigating MPO as a biomarker in different biological matrices.
INTRODUCTION:Canine transmissible venereal tumor (CTVT) is a naturally occurring transmissible cancer and an important model in comparative oncology due to its immune evasion mechanisms and tumor-host interactions. Although CTVT usually responds favorably to chemotherapy, variations in tumor behavior suggest the involvement of inflammatory and molecular pathways. CXCL8 is a key mediator of inflammation and immune modulation, whereas the biological significance of TP53 intronic polymorphic loci in CTVT remains poorly understood. MATERIALS AND METHODS:CTVT samples from dogs in Colombia and Brazil were evaluated for CXCL8 and P53 expression by immunohistochemistry. Polymorphic loci were investigated by Sanger sequencing followed by MUSCLE alignment, genomic remapping, and comparative analysis against canine TP53 reference sequences and previously published canine transmissible tumor genomic datasets. Tumor-associated CD8 + T-cell infiltration was assessed by flow cytometry. Associations among polymorphic loci, inflammatory biomarkers, cytomorphological features, and histopathological parameters were analyzed. RESULTS AND DISCUSSION:High CXCL8 immunoreactivity was observed in tumor cells and was significantly associated with increased cellular cannibalism. Three TP53 polymorphic loci were identified within an intronic/non-coding region relative to the canine reference sequence KJ511265.1. Two loci showed concordance with previously reported high-confidence (PASS) variants. Tumors harboring polymorphic loci showed increased neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, CD8 + T-cell infiltration, and cytomorphological indicators associated with malignancy. CONCLUSIONS:CXCL8 appears to play an important role in the inflammatory microenvironment of CTVT. TP53 intronic polymorphic loci may reflect regulatory variability associated with tumor-host interactions, reinforcing the value of CTVT as a comparative oncology model.
Acute thymic involution frequently occurs in calves and is associated with impaired immune function, yet its detailed pathogenesis remains unclear. To investigate the involvement of inflammatory cells in acute thymic involution in calves, this study examined mast cell dynamics in the calf thymus. Thymus samples were collected at necropsy from Japanese Black calves under 300 days of age and classified histopathologically into five stages. Mast cells were specifically identified by toluidine blue staining and immunohistochemistry for tryptase, whereas immunohistochemistry for chymase showed positive reactions in various cell types, including thymocytes. The densities of toluidine blue-metachromatic and tryptase-positive cells were increased in acute thymic involution. These cell densities were consistently higher in the medulla than in the cortex. Tryptase-positive cell density was higher than toluidine blue-metachromatic cell density. In double immunofluorescence for tryptase and PCNA, PCNA-positive nuclei were observed in thymocytes, but not in tryptase-positive cells at any stages of acute thymic involution. Analysis of the publicly available DNA microarray data showed that a cluster of microarray spots annotated to genes encoding mast cell chemoattractants exhibited relatively higher gene expression levels in thymuses with more advanced acute thymic involution than in thymuses with less advanced acute thymic involution. The expression levels of ADM, CCL16, CCL24, and TGFB2 showed positive correlations with the stage of acute thymic involution. These results suggest that mast cells increase in the calf thymus during acute thymic involution without relying on local cell proliferation in the thymus.
Dogs are the definitive hosts of Echinococcus granulosus sensu stricto (s.s.), the causative agent of cystic echinococcosis (CE) in livestock and humans. As canine infections are usually asymptomatic, early and reliable detection of active adult infections is essential for effective control and surveillance. In this study, two in-house sandwich ELISA assays were developed using purified polyclonal rabbit antibodies raised against E. granulosus s.s. soluble crude antigen (EgSCA) and a recombinant fibronectin antigen (rEgFN162). Experimental infection was established in two dogs (n = 2) by oral administration of viable protoscoleces, while one dog was maintained as a non-infected negative control. Fecal samples were collected throughout the infection period and following anthelmintic treatment. Cross-reactivity was assessed using fecal samples from dogs naturally infected with Toxocara canis (n = 5) and Ancylostoma caninum (n = 2). The diagnostic performance of both assays was evaluated in terms of timing of positivity, sensitivity, specificity, and cross-reactivity. The EgSCA-based sandwich ELISA detected coproantigens as early as 4-5 days post-infection and showed an overall sensitivity of 80% and specificity of 86.7%, with limited cross-reactivity with T. canis (20%) and A. caninum (50%). In contrast, the rEgFN162-based assay showed delayed positivity (18-23 days post-infection), lower sensitivity (46.7%), but higher specificity (93.3%). In both in-house sandwich ELISAs, coproantigen positivity persisted for an extended period after anthelmintic treatment, indicating prolonged antigen detectability despite parasite elimination. Western blot and indirect ELISA confirmed strong antigen-specific humoral responses against both antigens in immunized rabbits. Overall, the findings of this proof-of-concept study suggest that the EgSCA-based in-house sandwich ELISA is more suitable for early coproantigen-based detection of active E. granulosus s.s. infections in dogs, while rEgFN162 may serve as a complementary or confirmatory antigen in combined diagnostic approaches, contributing to improved CE surveillance and control in endemic areas.
The use of monoclonal antibodies to block PD-1 binding to its ligand PD-L1 as a strategy for cancer treatment has already proven successful in the clinical management of human cancers. However, the clinical application of anti-PD-1 monoclonal antibodies in canine cancer immunotherapy still requires systematic and in-depth research. In this study, an anti-canine PD-1 monoclonal antibody (designated 2H4) was developed that can be used for therapeutic purposes. In an Indirect Enzyme-Linked Immunosorbent Assay (ELISA), the minimum binding concentration of 2H4 for PD-1E (a recombinant canine PD-1 extracellular domain protein) was 0.48 ng/mL. In the PD-1/PD-L1 blocking assay, the minimum concentration of 2H4 required to block PD-1/PD-L1 interaction was 3.9 ng/mL. Further studies identified the spatial region containing three PD-1 amino acid residues-Y68, K78, and E136-as the binding site for 2H4. In therapeutic trials using a Beagle tumor model, the 2H4 antibody demonstrated the ability to activate the Beagles' anti-tumor immune response without causing any side effects. However, due to the limitations of xenograft animal models and the potential impact of murine antibodies on therapeutic efficacy, there was no significant difference in the rate of tumor shrinkage between the 2H4 antibody treatment group and the control group. Nevertheless, following subsequent tumor implantation, tumor volume in the antibody treatment group decreased significantly. The results suggest that 2H4 has potential for the treatment of tumors. In future, this antibody will need to undergo canine-specific modification, and dogs with spontaneous cancer will need to be selected to further evaluate its clinical efficacy.
Native cows are known for their extraordinary resilience, thanks to a long process of adaptation to specific environmental and management conditions that has shaped their physiological and metabolic traits. Despite the growing interest in these breeds, many biological mechanisms remain largely unexplored. Among these, the peripartum period represents a delicate phase in which the ability to maintain homeostasis is challenged by endocrine and immune adaptations. This preliminary study aimed to investigate the peripartum period in the native Rubia gallega breed by evaluating serum concentrations of cortisol, haptoglobin, leptin, IL-1β, IL-6, and TNF-α. In this study, 15 multiparous cows were enrolled and monitored from -15 to + 15 days before calving. With the exception of leptin (P > 0.05), all parameters investigated were affected by time to calving (P < 0.05). The cows examined showed the highest cortisol concentrations at calving compared to all other observation time points. Haptoglobin levels were significantly higher at -3 days compared to -15 and -7 days, as well as at calving. IL-1β showed a steady increase throughout the pre-calving period, followed by a decrease at calving and a subsequent increase during the post-calving phase. TNF-α concentrations progressively decreased starting at -7 days, continuing this downward trend until the end of the monitoring period. IL-6 increased from -15 days to calving, then decreased at + 7 days. Overall, the picture presented by these preliminary results indicates a transient and well-regulated inflammatory activation that supports the physiological demands of calving and lactation.
Respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV‑3) are important pathogens causing respiratory infections in humans and animals worldwide. This study aimed to investigate the presence of RSV and PIV‑3 in lambs with pneumonia, including single and mixed infections, and to evaluate the role of different lymphocyte types in immunopathogenesis. The material consisted of lung samples from 100 lambs (aged 4-12 months) with pneumonia, collected between July and November 2024. PCR and immunohistochemistry (IHC) were used to detect viral presence, while hematoxylin-eosin (HE) staining and IHC were applied to assess pathological changes. PCR results showed PIV3 alone in 7%, RSV alone in 8%, and co-infection in 7% of cases. IHC findings revealed PIV3 in 6%, RSV in 6%, and mixed infections in 6% of the samples. In addition, immune responses to PIV and RSV viral infections were evaluated by determining the expression levels of CD3+, CD4+, and CD8+ T lymphocytes and CD19+ B lymphocytes via IHC analysis. It was demonstrated that these viruses specifically elicit responses from helper T cells and B cells. Given the limited availability of pharmaceutical and therapeutic options against RSV and PIV3, and the lack of a licensed vaccine for these infections particularly in small ruminants it is believed that the data obtained from this study will contribute to future vaccine development efforts. It has also been concluded that the cytokine profiles involved in the pathogenesis of these infections should be examined in greater detail from a broader perspective.
Neutrophil chemotaxis and phagocytosis are critical for protection against bacteria, but can be compromised by methicillin-resistant Staphylococcus aureus (MRSA). MRSA can also circumvent macrophage surveillance by affecting polarization and reactive oxygen species (ROS) production. We previously demonstrated that the secretome of equine mesenchymal stromal cells (MSCs), comprised of all secreted bioactive factors and collected as conditioned medium (CM), reduces the growth of MRSA both in vitro and in vivo. This study aimed to determine if the equine MSC secretome has additional anti-MRSA properties by studying its effects on equine neutrophil and macrophage functions in vitro. Transwell assays demonstrated that CM from adipose tissue- and bone marrow-, but not peripheral blood-, derived MSCs significantly enhanced neutrophil migration. In addition, CM from all three MSC sources significantly reduced the phagocytic capacity of neutrophils but did not alter ROS production. MSC CM from all three tissue sources promoted macrophage polarization toward both CD86-positive (M1-like) and CD206-positive (M2-like) phenotypes, but did not change phagocytic capacity or ROS production. Further experiments showed that the complete CM, rather than the soluble and extracellular vesicle (EV) subfractions, was responsible for the increased neutrophil chemotaxis. Additionally, the primary effect of EVs on macrophages was cell death, possibly through autophagy, which can be beneficial if tissue damaging inflammation is diminished with decreased numbers of viable macrophages. Collectively, our findings show that the equine MSC secretome modulates various innate immune responses in vitro and may have therapeutic potential for managing dysregulated inflammation associated with bacterial diseases in vivo.
Streptococcus iniae, an emerging pathogen, significantly impacts the productivity of marine and freshwater aquaculture by causing streptococcosis. Vaccination is a promising strategy to address this challenge. This study investigates an innovative vaccination approach utilizing a biomaterial-based scaffold encapsulated with a lectin adjuvant to combat S. iniae in Nile tilapia (Oreochromis niloticus). The scaffold was prepared via ion gelation using chitosan and alginate, and it was adjuvanted with lectin. The fish were administered with both injection and oral routes, by the formulated vaccine. Both the i.p. and oral vaccinated groups exhibited significantly higher levels of lysozyme, myeloperoxidase, and superoxide dismutase compared to the control group (P < 0.05). Furthermore, increased levels of specific antibody Immunoglobulin M (IgM) were observed, along with notable fold changes in adaptive immune genes in the spleen and head kidney. The treatment group vaccinated with the encapsulated vaccine adjuvanted with lectin demonstrated high survival rates in both routes. These findings highlight the potential of this scaffold-based vaccine delivery and adjuvant for its long-term efficacy both i.p. and oral administration as a novel and effective approach for aquaculture. This strategy could also be applied for the delivery of other vaccines and therapeutics in aquatic species.
DNA vaccines are a promising against avian coccidiosis but they require comprehensive safety evaluation before clinical application. This study assessed the safety and environmental impact of pVAX-ROP27 DNA vaccine against Eimeria tenella in both chickens and mice. Chickens received intramuscular immunizations: single low dose at 1 day (100 μg), repeated doses (100 μg at 7, 14, 21 days), or a high dose (300 μg at 14 days) using three vaccine batches. Mice received a single dose or overdose. Clinical signs, body weight, and histopathology were evaluated. Distribution and persistence of EtROP27 and Kana genes in tissues, faeces, and water were detected by PCR. No adverse reactions or significant weight differences were observed (P > 0.05). Both genes were detected in all tested tissues (heart, liver, spleen, lung, kidney, blood, small intestine, testis, injection site muscle) at 0.5 months but became undetectable by 2 months, suggesting clearance within the detection limit of conventional PCR, with no evidence of genomic integration by this method. No histopathological lesions or environmental gene release (in faeces/water up to 8 weeks) were detected, confirming a favorable safety profile with no detectable tissue damage, integration, or environmental spread within the observation period.
BACKGROUND:In bovine species, the epitheliochorial placenta prevents prenatal transfer of immunoglobulins, rendering calves dependent on colostrum intake for passive immunity. Efficient passive immunity transfer (PIT) is critical for neonatal health, yet conventional assessments primarily focus on immunoglobulin quantification without evaluating structural integrity. OBJECTIVE:This pilot study aimed to comprehensively evaluate PIT in a cohort of Holstein calves using an integrated approach combining field-based diagnostic tests, quantitative protein assays, and molecular validation techniques. METHODS:A total of 13 Holstein cow-calf dyads were initially enrolled in this study. However, two calves were excluded from the longitudinal serum analysis due to mortality. Consequently, the final cohort for the longitudinal assessment comprised 11 calves and their respective dams. Serum samples were collected from the 11 cows during the pre-partum period (3 and 2 weeks before calving, and at parturition) and from the 11 surviving calves at 0, 48, and 72 h postpartum. Immunoglobulin concentrations were assessed using sodium sulfite precipitation and zinc sulfate turbidity (ZST) tests. Protein concentration of the immunoglobulin-enriched fraction was determined using the bicinchoninic acid (BCA) assay. Structural integrity and immunoreactivity were evaluated using SDS-PAGE and Western blotting. Colostrum quality was assessed via density measurement. All subsequent immunological, molecular, and statistical analyses were performed on this final cohort (n = 11). RESULTS:Maternal IgG concentrations declined progressively approaching parturition. In the neonates, serum IgG levels increased significantly following colostrum ingestion, with 100% of the longitudinally tracked surviving calves (n = 11) successfully surpassing the critical passive immunity transfer threshold (>10 mg/mL) by 48-72 h postpartum. Electrophoretic and immunoblot analyses confirmed the presence and structural integrity of the transferred immunoglobulins. Statistical analysis using a repeated-measures ANOVA revealed a highly significant effect of time on serum immunoglobulin levels (P = 0.001). CONCLUSION:The findings suggest adequate passive immunity transfer within the study cohort and highlight the potential value of integrating quantitative and molecular approaches for a more comprehensive assessment of neonatal immune status. Given the exploratory nature of this pilot study and the lack of standardization in colostrum administration, these results provide a foundational model for future, larger-scale investigations into the structural and functional aspects of PIT.
Successful establishment of pregnancy requires maternal immune tolerance to paternal antigens introduced by insemination. Regulatory T cell (Treg)-associated responses are thought to play a central role in this process, whereas pro-inflammatory T helper 1 (Th1) responses may disrupt pregnancy success. In other species, seminal plasma (SP) has been shown to modulate maternal immune adaptation; however, this relationship remains poorly defined in mares, where SP is frequently reduced during routine breeding practices. The objectives of this study were to (1) characterize temporal changes in systemic Treg- and Th1-associated transcripts during early pregnancy, (2) determine the association between these immune mediated transcripts and pregnancy diagnosis, and (3) evaluate the influence of SP exposure on T cell development. Peripheral blood mononuclear cells (PBMCs) were collected from mares prior to insemination and during early pregnancy. Relative expression of Treg-associated (FoxP3, CD25, TGF-β) and Th1-associated (TBX21, IFN-γ) transcripts were quantified by qRT-PCR. Statistics were performed using SAS 9.4®, where the effect of gestational length, pregnancy diagnosis, and breeding method were assessed. FoxP3 expression increased temporally in pregnant mares (p = 0.04), while no changes were observed for other transcripts. Pregnancy outcome was associated with IFN-γ expression (p = 0.05), which was higher in mares that failed to establish pregnancy, particularly pre-breeding and day 7 post-ovulation. SP exposure influenced CD25 (p = 0.04) and trended for TGF-β (p = 0.06), with highest expression in mares bred with fresh semen at day 14. These findings suggest that SP exposure at insemination may promote Treg-associated signaling and contribute to maternal immunotolerance during early pregnancy in the mare.
Suture materials act as foreign bodies capable of modulating host immune responses and influencing wound healing outcomes. In resource-limited veterinary settings, non-medical threads are frequently used for cutaneous closure; however, their immunological effects remain poorly characterized. This study evaluated systemic and local inflammatory cytokine responses to commonly used non-medical suture materials compared with standard silk sutures in a caprine model. Twenty Kano Brown bucks were allocated to four groups (n = 5 per group), they underwent vasectomy followed by skin closure using embroidery thread, cobbler's thread, hair-braiding thread, or silk sutures. Serum and wound-fluid concentrations of interleukin-12 (IL-12) and interferon-gamma (IFN-γ) were quantified using goat-specific ELISA at defined postoperative intervals and analysed using two-way repeated-measures ANOVA. All suture materials induced significant postoperative increases in IL-12 and IFN-γ, consistent with physiological inflammatory responses to tissue injury. Silk sutures exhibited comparatively higher cytokine concentrations and delayed peak responses, whereas non-medical threads demonstrated earlier cytokine peaks and lower or more rapidly resolving inflammatory profiles. Temporal effects were significant for both cytokines (P < 0.01), while differences among non-medical materials were not significant (P > 0.05). Wound-level cytokine dynamics paralleled serum findings, indicating more sustained inflammatory signalling in the silk group. These findings suggest that non-medical suture materials do not elicit exaggerated IL-12 or IFN-γ responses relative to silk and may be immunologically comparable for cutaneous closure in resource-constrained veterinary practice.
