This study aimed to determine the prognostic impact of serum Endocan levels due to E. coli K99 driven systemic inflamatuary response syndrome (SIRS), sepsis (SE) and septic shock (SS). The samples were with 24 diarrhoeic calves and 8 health calves aged 0-7 days. The diarrhoeic calves were divided equally into three groups as the SS group, the SE group and the SIRS group. Repeated serum samples were collected at the 0th, 3rd and 6th hours. The collected samples were used to analyse Endocan and certain proinflammatory cytokines (TNF-α, IL-1β ve IL-6) and acute phase proteins (SAA and Hp). A statistically meaningful difference was observed between the serum Endocan levels of the groups (P < 0.001). The E. coli infected diarrhoeic calves in the SS group had Endocan levels higher than others in other groups. Also, as a prognostic value, a statistically meaningful difference was observed between the repeated Endocan concentrations collected from dead and surviving calves (P < 0.01, P = 0.001, P < 0.001, respectively). When the cut-off point for Endocan 6th hour tests was considered > 131.92, the AUC value was 0.825, the sensitivity was %81.82, the specificity was %77.2, +LR: 3.60, -LR: 0.24 (P < 0.05). As a result, in repeated analysis, serum Endocan levels were shown to be a rigorous indicator of the severity of inflammation in diarrhoeic calves and mortality when the positive correlation between other proinflammatory cytokine and acute phase proteins was taken into consideration.

Mycobacterium bovis (M. bovis), which causes bovine tuberculosis, is endemic in Canada's Wood Buffalo National Park and threatens the conservation of free-ranging bison. It also poses spillover risks to humans and livestock. Accurate diagnostic tools are critical for screening this threatened and culturally significant animal. Interferon gamma (IFNγ) release assays (IGRA) are commonly used for tuberculosis diagnosis in humans and other wildlife species. Hence, this study aimed to develop an IGRA-based approach for detecting M. bovis infection in bison. Animals from a tuberculosis-free captive bison farm (n = 8) were experimentally infected with M. bovis. The whole blood was collected prior to and following M. bovis challenge and incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes and pokeweed mitogen tubes. Plasma was harvested after centrifugation. Concentrations of IFNγ in plasma were quantified using the Mabtech bovine IFNγ ELISA Flex kit. IGRA responses were calculated as the difference in IFNγ concentrations between TB antigen and the nil tubes. A diagnostic cut-off value of 59 pg/ml (Se = 81 %, 95 % CI 57-93 %; Sp =100 %, 95 % CI 89-100 %; AUC = 0.92) was determined using known infection status to distinguish infected bison. Additionally, cut-off values for IFNγ concentrations for plasma from QFT-nil and pokeweed mitogen tubes were calculated to increase confidence in IGRA validity interpretation. The combination of the QFT stimulation platform and Mabtech bovine IFNγ ELISA shows promise as a diagnostic test to distinguish between M. bovis-infected and uninfected bison. These findings support the use of this tool for surveillance in free-ranging and captive Canadian bison populations and warrant further field validation.

Rabies is a fatal but vaccine-preventable zoonotic disease. Global elimination of rabies necessitates multisectoral coordination and transboundary action involving mass animal vaccinations in endemic areas. The current assays for analysing seroconversion against rabies vaccines have limitations for large-scale use due to need for higher bio-containments, low-throughput and a high cost. Here, we report the feasibility of an alternative assay for detecting rabies neutralizing antibodies in vaccinated animals. This pseudovirus neutralization test (PVNT) based on vesicular stomatitis virus (VSV) platform employs secreted alkaline phosphatase (SEAP) as a reporter. The assay replaces the use of live rabies virus with single-round infectious pseudovirions as well as fluorescent microscopy in conventional neutralizing assays such as fluorescent antibody virus neutralization assay (FAVN)/ rapid fluorescent focus inhibition test (RFFIT) with an ELISA reader. We subjected a total of 60 serum samples from vaccinated dogs and cats to both RFFIT and PVNT. The results showed excellent concordance, with the PVNT showing a sensitivity of 93.8 % and specificity of 100 %. Receiver Operator Characteristics (ROC) analysis indicated a high accuracy (area under the curve >0.9) and a kappa value of 0.848 indicating comparable performances of PVNT and RFFIT. Our results show that PVNT can serve as a suitable alternative to traditional live-virus-based neutralization assays for efficacy analysis of animal rabies vaccination.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a zoonosis of global concern due to the absence of effective vaccines or treatments. The conventional diagnostic method, the single intradermal comparative cervical tuberculin test (SICTT), is limited by cost and sensitivity, hindering control strategies. To overcome these limitations, an ELISA that detects specific IgG antibodies against M. bovis using the recombinant protein Mb1454 was developed. The optimized ELISA conditions included 0.5 µg of recombinant Mb1454 protein for plate sensitization, PBMT blocking buffer (PBS + 1 % BSA + 3 % milk + 0.05 % Tween-20), serum dilution of 1:200, secondary antibody dilution of 1:1000, 1 h incubation, and 15 min substrate development time. The analysis of the ELISA-Mb1454 in M. bovis-positive (bTB+, n = 113) and negative (bTB-, n = 120) bovine serum samples showed 92.9 % sensitivity and 90.0 % specificity, with an area under the ROC curve of 0.96, indicating high discrimination capacity in M. bovis detection (p < 0.0001). The assay demonstrated high analytical specificity, with no cross-reactivity observed in sera from animals infected with other common pathogens, including BoAHV-1, BoAHV-5, BVDV, Neospora caninum, Brucella abortus, and Leptospira sp. The results showed no correlation between circulating IgG antibody levels against the Mb1454 antigen and the pathological stage of bovine tuberculosis (bTB) lesions. Nonetheless, our findings demonstrated the immunogenicity of the Mb1454 protein and discriminatory performance of the ELISA-Mb1454 assay in differentiating bTB-positive and -negative cattle, even in the absence of a direct association between antibody titers and disease severity.

This study investigated whether distinct bovine-associated staphylococci can trigger expression of PD-1 and its ligand PD-L1 in RAW 264.7 murine macrophage cell line. Four well-studied strains, Staphylococcus aureus [n = 2; one from a bovine nose (SN) and one udder-adapted strain isolated from a persistent intramammary infection (IMI; S. aureus IM)] and S. chromogenes (n = 2; one udder-adapted strain isolated from a persistent IMI (S. chromogenes IM) and one originating from a teat apex; S. chromogenes TA), were used. The expression of PD-1 and PD-L1, was measured through flow cytometry. The macrophage infection was performed over time using a multiplicity of infection (MOI) of 1, 5, and 10, respectively, up to 6 h. All staphylococcal strains induced higher macrophage expression of PD-1 compared to an unstimulated control. The PD-1 expression was MOI-dependent. Most notably, the S. aureus SN triggered a drastic increase in the macrophage PD-1 expression over time. In contrast, PD-1 expression induced by both staphylococci IM strains dropped significantly over time, whereas the S. chromogenes TA exhibited no significant variation over time. A significant increase, though subtle, in PD-L1 expression was triggered by the S. aureus IM and S. chromogenes IM. A higher intensity of PD-L1 expression, significant yet relatively subtle, was observed during the earlier stages of infection. Thus, our findings underscore strain- and niche-specific patterns of macrophage modulation by staphylococci, encouraging further studies to investigate the potential of PD-1 and/or PD-L1 blockade as a non-antibiotic strategy to tackle bovine IMIs. However, as this study used a murine macrophage cell line, further validation in primary bovine macrophages is required.

Swine influenza viruses (SwIAV) pose a zoonotic threat and contribute to interspecies viral reassortment. Effective vaccination strategies are essential, yet accessible correlates of protection remain limited. We simplified a whole blood (WB) assay to assess immune memory ex vivo in pigs vaccinated with Respiporc FLU3® (n = 6) versus placebo controls (n = 6), followed by homologous challenge with SwIAV (H1avN1). Vaccinated pigs exhibited no lung lesions and minimal viral loads. WB samples, collected pre- and post-vaccination and at day 1 and 5 post-challenge (dpch), were stimulated with SwIAV to quantify IL2, IL4, IFNγ, TNFα, and granzyme B (GZMB) mRNA via qPCR. High IL2 and moderate IL4 mRNA elicited in WB post-booster predicted reduced viral load and lung pathology. Concomitantly, high IFNγ in WB from controls at 5dpch correlated with lesion severity, indicating excessive inflammation in non-vaccinates. Post-challenge, IL4/IL2 mRNA ratios increased in virus-stimulated WB of vaccinates, suggesting Th2-associated resolution of infection. In contrast, controls showed declining IL4/IL2 ratios post-challenge, correlating with disease severity. Thus, the simplified WB assay allows identification of virus-specific immune responses associating with protection, without requiring extensive laboratory infrastructure. By recording the ex vivo-triggered SwIAV-specific IL4/IL2 mRNA dynamics, which develop between booster and challenge monitoring immune memory may simplify determination of vaccination success.

Fish are aquatic vertebrates that are free-living from their embryonic stages and are constantly in contact with the highly diverse and pathogen-rich external aquatic environment. The innate immune system of fish is non-specific and adapted to detect potential pathogens and other stress factors. They can act rapidly and effectively via their key component, the antimicrobial peptides (AMPs). The action of AMP is manifested through the regulation of its expression, and profiling the constitutive expression of key AMPs across various developmental stages in fish provides valuable insights into the transcriptomic mechanisms underlying their defense strategies against infections. Real-time quantitative PCR (RT-qPCR) was employed to quantify the expression of AMPs across the growth stages of Genetically Improved Farmed Tilapia (GIFT), Oreochromis niloticus. The analysis revealed distinct expression patterns of AMPs throughout the developmental stages of GIFT, indicating their critical role in immune preparedness. The expression profiles were specific to the peptide and the growth stages, and enhanced expression was particularly evident during the first post-hatch and first-feeding stages; no specific pattern could be deciphered during the other stages. The dynamic transcriptome, revealed through gene expression studies, provides a window into the intricate interplay of developmental and regulatory pathways. Monitoring the AMP expression profile offers an in-depth understanding of their mechanisms, paving the way for downstream applications in aquaculture, such as early disease detection and prevention, environment management, use of immunostimulants and probiotics, and in functional genomic studies.

Canine mammary tumor (CMT) is the most prevailing neoplasms in female dogs. Matrine demonstrates anti-tumor effects in various organs, its mechanism in CMT treatment remains unclear. This study involved in network pharmacology, molecular docking, dynamics simulations, cell viability, inhibations and migration, to clarify matrine's therapeutic action against CMT. Potential gene/protein targets were identified through PubChem (Matrine), and GeneCards (CMT), with point of intersection targets analyzed via STRING PPI network and Cytoscape. Top 10 hub genes were selected for gene ontology/KEGG pathway analyses (p < 0.05). Molecular docking (JAK2, AR, HDAC2, HDAC6, and NR3C1) revealed strong matrine-JAK2 binding (-7.8 kcal/mol), and dynamics simulations were confirmed by molecular dynamics simulations. Pathway analysis implicated JAK-STAT and PI3K-Akt signaling in matrine's as anti-tumor effects. In vitro results showing that the maximum noncytotoxic concentrations of matrine of canine primary mammary epithelial cells (cPMECs), where the cell viability remained above 90 % at concentrations 280 µM and ≤ 560 µM, respectively, indicating this as the maximum safe concentration for cPMECs, and had a proliferation inhibitory effect time-dependently (12, 24, 48 hrs) on CHMm and CHMp cells within a safe concentration range, and suppression of CMT cells via CCK-8 assays. In 12 h, moderate inhibition was detected, which increased at 24 h, and was most prominent at 48 h. The migration ability of cells decreased at 24 h, respectively. Notably, the 560 µM concentration resulted in over 50 % inhibition in both cell lines after 48 h. Future research should investigate in vivo efficacy to progression matrine as a veterinary oncotherapeutic.

Drinking water quality is a critical factor in poultry production, and suboptimal drinking water can negatively influence immune functions leading to reduced vaccine efficacy. In the field study, we assessed drinking water quality on Alberta layer farms and evaluated its impact on vaccine-induced immune responses. Chemical, physical and microbiological analyses were performed on 26 water samples collected from Alberta layer farms and serological response for poultry viral vaccines were evaluated at the same time. The water hardness, pH, bicarbonates, and dissolved sodium exceeded acceptable limits in 34 %, 50 %, 46 %, and 27 % of the farms, respectively. Farm-level data revealed no significant direct correlation between water quality scores and vaccine induced serological response. For the controlled experiment, specific pathogen-free White Leghorn chicks were assigned to 4 groups: tap water control (TW-control), field water control (FW-control), tap water vaccinated (TW-vaccinated), and field water (FW-vaccinated), two of the groups were vaccinated against infectious bronchitis while maintaining unvaccinated controls and all 4 groups were maintained on their respective water sources for ∼8 weeks. Controlled experiments showed that vaccinated birds receiving FW had 600 units lower mean antibody titers compared to those given TW, which is statistically not significant. The vaccinated FW group also showed reduced CD4⁺ and CD8⁺ T-cell populations in the spleen and lungs, along with altered IFN-γ and significantly increased IL-10 transcription. In conclusion, although the field data showed a lack of correlation between water quality and vaccine induced serological response, the control experiment revealed that poor water quality might influence the infectious bronchitis vaccine effectiveness in layers.

The genital system of female dogs with visceral leishmaniasis (VL) commonly shows lesions on the external genitalia (vulva and vagina). This study aimed to evaluate the inflammatory response in segments of the reproductive tract of female dogs naturally infected with Leishmania infantum, and to correlate these findings with hormonal status (high or low progesterone levels), local parasitism, and local inflammatory infiltrate immunophenotype. Sixteen unspayed, non-pregnant adult female dogs naturally infected with L. infantum, without any other disease, were used. Animals were divided into two groups: G1 (n = 5, high-progesterone phase) and G2 (n = 11, low-progesterone phase). Parasite presence and the immunophenotype of leukocytes infiltrating the genital system (CD3, CD4, CD8, and MCA874) were determined by immunohistochemistry. Chronic inflammatory infiltrate was the predominant lesion and was mainly found in the vulva and vagina. Internal genitalia segments (cervix, uterus, ovary) presented fewer lesions. Animals of G1 group exhibited higher number of immunolabeled parasites in the internal segments of the reproductive tract and of cells positive for CD3 (T lymphocytes) and MCA874 (macrophages) than G2 group. There was no difference for CD4 and CD8 between groups. However, multivariate analysis showed positive correlations with T lymphocyte subtypes, macrophages, and parasite presence in different segments of the reproductive tract of G1 animals. Leukocyte population and number of parasitized cells in the reproductive tract increased with escalating progesterone levels (G1 group), suggesting that estrus and diestrus stages are most favorable to infection maintenance.

Bovine tuberculosis (bTB) is a pulmonary infectious disease caused by Mycobacterium bovis, affecting cattle and a wide range of mammals, including humans. Despite its significant impact on global livestock production, no commercial vaccine is currently available, partly due to potential interference with standard diagnostic tests. In this study, we evaluated the protective efficacy of a triple M. bovis mutant lacking the immunodominant antigens ESAT-6 and CFP-10, as well as the virulence factor Ag85A. This mutant is compatible with DIVA (Differentiation of Infected from Vaccinated Animals) diagnostics based on ESAT-6 and CFP-10 detection. The triple mutant was assayed both alone and in a heterologous prime-boost regimen using recombinant Ag85A conjugated to chitosan nanocapsules. Protection was assessed by quantifying M. bovis colony-forming units (CFUs) in the lungs and spleen following challenge. Organ homogenates were cultured on solid media, and CFUs were enumerated at five and ten weeks post-plating. At five weeks, all vaccinated groups demonstrated comparable protection in the lungs. In the spleen, both the triple mutant and BCG groups showed reduced CFU counts compared to the unvaccinated group. By ten weeks, lung protection was most pronounced in the prime-boost and BCG groups, whereas spleen protection was restricted to the prime-boost group. At this stage, persistence of the triple mutant was detected in both lungs and spleen, highlighting the need for further evaluation of its residual virulence. Post-challenge immune responses were assessed by measuring CD4 +KLRG1-CXCL3 + T cells, a subset previously associated with protective immunity against tuberculosis, among other T cell populations evaluated. Vaccinated mice exhibited a significant expansion of this population compared to unvaccinated controls. Notably, higher frequencies of these cells correlated with reduced pulmonary bacterial burden, reinforcing their potential as a biomarker of protective immunity.

Mycoplasma (M.) hyopneumoniae, the agent of porcine enzootic pneumonia (PEP), a primary pathogen of porcine respiratory disease complex (PRDC), reduces productivity and increases economic losses in swine production, with vaccination being the most important method to control the disease in the producing farms. This study evaluates the pro-inflammatory activity and immunogenicity of an inactivated vaccine for M. hyopneumoniae using an SBA-15 silica-based adjuvant in piglets. Forty-eight weaned piglets were divided into four groups (n = 12/group): a commercial vaccine group (CV); an SBA-15 adjuvanted vaccine group (SBA-15-Vac); a control group, receiving a suspension of SBA-15 with PBS, without the antigen (SBA-15-Cont), and a group that received saline solution (NV). SBA-15-Cont group served as a control for adjuvant-related effects, while the NV group served as negative control for lung lesion analysis, only. All animals were challenged, 21 days post vaccination, with M. hyopneumoniae strain 232. The evaluation of clinical signs was performed, and the inflammatory response was evaluated by measuring acute-phase proteins, as well as anti-M. hyopneumoniae IgG and IgA antibody levels. The SBA-15 adjuvant control group demonstrated mild inflammatory responses maintaining stable transferrin and haptoglobin levels after vaccination, suggesting a controlled inflammatory response compared to the commercial vaccine. The SBA-15 group exhibited a delayed but sustained immune response, suggesting a possible depot effect. Acute-phase protein levels, particularly ceruloplasmin and haptoglobin. Our findings support the potential of SBA-15 as a safe and effective adjuvant and highlight that SBA-15 silica nanoparticles are a promising and safe adjuvant in swine vaccines against M. hyopneumoniae.

The Bos taurus and Bos indicus cattle evolved in different climatic conditions, and they acquired various adaptive traits for their survival. This study unravels the impact of chronic in vitro thermal stress with lipopolysaccharide stress on functional responses of Bos indicus (Kangayam cattle) and Bos taurus (Jersey crossbred cattle) monocyte-derived macrophages. To achieve this, monocyte-derived macrophages from both cattle types were stimulated with lipopolysaccharide (1.0 μg/mL) at either 37°C or 40°C for 24 and 48 h, and their cellular and immune responses were evaluated. The results revealed that Bos indicus monocyte-derived macrophages exhibit enhanced cell viability, oxidative and phagocytosis functions, as well as improved autophagy response to lipopolysaccharide stimulation during thermal stress. Additionally, Bos indicus monocyte-derived macrophages demonstrated reduced variability in transcription of genes related to heat shock, cell signalling, and inflammatory cytokines compared to Bos taurus. Factors such as breed, thermal and lipopolysaccharide stress, period of stress exposure, and their interactions have a significant impact on cellular and immune functions of monocyte-derived macrophages. In conclusion, the monocyte-derived macrophages of Bos indicus possess an enhanced immune-thermal resilience property than Bos taurus.

Equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1 % of infected horses. Antemortem diagnosis has proven challenging as serum antibodies against S. neurona are an indicator of infection but not necessarily disease. Factors governing the occurrence of EPM are largely unknown, although horse immunity might contribute to EPM pathogenesis. Immunoglobulin G is the predominant antibody class in equine serum and consists of four subisotypes; IgG1/2 (IgGa) and IgG4/7 (IgGb) are thought to be indicative of a Th1, cell-mediated immune response, and isotypes IgG3/5 (IgG(T)) and IgG6 (IgGc) are thought to be indicative of a Th2, humoral response. Herein, we examined the hypothesis that EPM occurs due to an aberrant immune response, which will be discernable by IgG subisotypes. A modified ELISA was used to quantify S. neurona antigen-specific IgG sub-isotypes 1/2, 3/5, and 4/7. Based on documented serum concentrations of IgG subisotypes, standard curves were generated using sera from 21 healthy horses and S. neurona-specific IgG subisotype levels were determined in serum and cerebrospinal spinal fluid from infected diseased (n = 93) and infected normal (n = 116) horses. The mean IgG3/5 serum concentration and IgG1/2:IgG3/5 ratio against S. neurona were found to be significantly different between diseased and normal horses, suggesting that the immune response to S. neurona in EPM horses is skewed towards a Th1, cell-mediated response. Unfortunately, these differences were not sufficient for developing a serum-based immunoassay for EPM diagnosis.

Passive immune transfer (PIT) is crucial for the survival and health of neonatal lambs, and its assessment is vital in early-life management. This study evaluated the effectiveness of three field-based diagnostic methods-analog Brix refractometry, digital Brix refractometry, and immunocrit-for estimating PIT in lambs, using serum total protein concentration as the reference standard. Thirteen lambs born from eutocic deliveries were assessed. The mean serum protein concentration was 7.50 ± 0.70 g/dL, exceeding the 5.0 g/dL threshold for adequate PIT. All alternative methods showed strong positive correlations with serum protein: analog Brix (r = 0.9795), digital Brix (r = 0.9870), and immunocrit (r = 0.8945), with perfect categorical agreement (Kappa = 1.0) across all methods. Analog and digital Brix readings were nearly identical, suggesting consistency between instruments. Although immunocrit showed slightly greater variability, it remained a valid diagnostic tool. A weak correlation was observed between colostrum Brix values and lamb serum protein (r = 0.16; p = 0.6115), indicating that colostrum quality alone does not guarantee successful PIT. Factors such as intake timing, volume, and neonatal vigor likely influence PIT outcomes. These findings support the use of Brix refractometry and immunocrit as reliable, rapid, and cost-effective tools for PIT assessment in lambs under field conditions. Their implementation may enhance early detection of PIT failure, reduce neonatal mortality, and improve flock health and productivity.

Myeloid-derived suppressor cells (MDSCs)-immature cells with immunosuppressive properties-are promising therapeutic targets for cancer immunotherapy. However, established therapies targeting MDSCs remain lacking, and novel molecules are needed to regulate MDSCs. Sorafenib, a tyrosine kinase (TRK) inhibitor, suppresses MDSCs. As TRKs regulate chemokine production, we hypothesized that sorafenib inhibits MDSCs by regulating chemokine production. This study aimed to investigate the effect of sorafenib on MDSCs and its underlying mechanism in canine transitional cell carcinoma (cTCC). Sorafenib reduced the number of MDSCs and downregulated CX3CL1 expression in the tumor tissues of cTCC-transplanted mice. Vascular endothelial growth factor A (VEGF-A) and platelet derived growth factor-BB (PDGF-BB) recombinant protein, the target axis of sorafenib, increased CX3CL1 expression in cTCC cell line. Furthermore, a positive correlation was observed between urinary levels of CX3CL1 and VEGF-A in cTCC cases. These results indicated that VEGF-A signaling can regulate CX3CL1 expression. Next, we analyzed whether CX3CL1 accelerates MDSC infiltration. MDSCs expressed CX3CR1, the receptor for CX3CL1, and migrated toward recombinant CX3CL1 protein in a dose-dependent manner. In a cTCC-transplanted mouse model, CX3CL1 depletion or treatment with a CX3CR1 inhibitor showed anti-tumor effects and reduced MDSCs in the tumor tissue. Overall, our results indicated that CX3CL1 promotes MDSC infiltration, and the CX3CL1/CX3CR1 axis could be a therapeutic target for regulating MDSCs in dogs with cTCC.

Brucellosis caused by Brucella abortus remains a major zoonotic challenge in India, causing substantial economic losses in livestock and public health risks. Although India has implemented nationwide S19 vaccination in cattle and buffaloes, comprehensive evaluation of its effectiveness across different regions and between species has been lacking. This study sought to assess post-vaccination sero-conversion rates in calves of aged 4-8 months across five Indian states/union territories (UTs), examine species-specific differences in vaccine response between cattle and buffaloes, and generate practical recommendations to optimize the national brucellosis control program. A multi-phase sero-monitoring study analyzed 19,893 serum samples, comprising 16,085 cattle calves and 3609 buffalo calves during 2021-2024. The samples were collected from Andhra Pradesh, Chandigarh, Haryana, Odisha, and Tamil Nadu and tested using laboratory standardized indirect enzyme-linked immunosorbent assay (iELISA).Significant disparities were observed between species with overall sero-conversion rates of 75.87 % in cattle versus 67.22 % in buffaloes (p < 0.001). Tamil Nadu demonstrated exceptional performance (84.61 %; 95 % CI: 84.20-84.95), with districts like Namakkal achieving 100 % sero-conversion. Other regions showed varied efficacy: Chandigarh (80.77 %), Andhra Pradesh (69.52 %), and Haryana (69.43 %) consistently exhibited 10-15 % lower rates in buffaloes (p < 0.001). Odisha displayed notable phase-wise improvement (71.84 %; CI: 70.02-71.12), with Jagatsinghpur district reaching 97.85 % and buffalo calves improving from 17.64 % (Phase I) to 57.5 %. While, the S19 program achieves moderate efficacy but highlights species-specific disparities and replicable success models such as Tamil Nadu. Targeted buffalo vaccination strategies and adoption of best practices are recommended to achieve > 80 % vaccination coverage thereby herd immunity. Overall, this study provides an evidence-based framework for strengthening India's brucellosis control program and contributing to global eradication efforts.

Infectious bursal disease (IBD) and Newcastle disease (ND) are major infectious diseases that endanger poultry. Despite current vaccination efforts, both diseases still occur worldwide. We have developed bivalent vaccines capable of simultaneously preventing ND and IBD, which does not produce mutant IBDV. Using reverse genetics, we constructed a recombinant Newcastle disease virus (NDV) vector based on the common vaccine strain Clone30 to express the host-protective immunogen VP2L of the IBDV strain and chicken granulocyte-macrophage colony-stimulating factor (GM-CSF). The IBDV-encoded VP2L protein and chicken GM-CSF gene were inserted into different positions of the NDV full-length cDNA in various forms to achieve high-level expression. We successfully rescued the ND-AI bivalent vaccines (rClone30-VP2L(P/M)-GM-CSF(P/M), rClone30-VP2L(NP)-GM-CSF(P/M), rClone30-VP2L(P/M)-GM-CSF(NP) and rClone30-VP2L-IRES-GM-CSF(P/M)). The ND-AI bivalent vaccines maintained genetic stability after at least three consecutive passages in chicken embryos and was confirmed to express VP2L and GM-CSF proteins. The replication titers of the ND-AI bivalent vaccines in chicken embryos and cell cultures were comparable to those of the parental NDV strain rClone30. To assess the immunogenicity of the ND-AI bivalent vaccines, it was administered to 14-day-old commercial chicken chicks, and immune responses were continuously monitored for four weeks post-vaccination. By day 10 post-vaccination, the hemagglutination inhibition (HI) antibody titers of the recombinant NDV vaccine had far exceeded the theoretical protective threshold (4 log2) and remained at high levels for 28 days. Additionally, the levels of IBDV-specific antibodies in the ND-AI bivalent vaccines rapidly increased and remained at high titers for 14 days. Concurrently, the proliferation responses of B cells, CD4 + , and CD8 + T cells were enhanced, and the protein expression levels and mRNA transcription levels of inflammatory factors significantly increased. In summary, the ND-AI bivalent vaccines can stimulate the body to produce a stronger immune response, demonstrating its potential as a vaccine for IBD and ND. Additionally, the addition of GM-CSF can further enhance the immune response.

Enterotoxigenic E. coli infection is a major cause of post-weaning diarrhea in pigs and is associated with systemic inflammation and oxidative stress. This study aimed to characterize temporal changes in biomarkers of inflammation and oxidative stress in response to an E. coli lipopolysaccharide (LPS) challenge, providing insights into host immune responses. Ten female pigs (27.9 kg BW; ∼3 months old) were infused with LPS derived from E. coli O111:B4 at LOW (0.75 µg LPS/kg BW) or MODERATE (1.50 µg LPS/kg BW) dosages. Thirteen blood samples were collected via venous catheter at 0 (pre-infusion), and from 0.5 to 72 h post LPS infusion. Rectal temperature, blood cytokines, acute-phase proteins, and oxidative stress markers were measured. A semi-targeted metabolomics approach was applied to investigate oxidative stress markers, including 8-iso-prostaglandin F₂α (8-iso-PGF₂α). Rectal temperature peaked at 3 h and returned to pre-infusion levels by 8 h. Plasma C-reactive protein (CRP) peaked at 12 h, while haptoglobin peaked at 24 h after LPS infusion. Pig major acute-phase protein (Pig-MAP) peaked at 24 h (LOW) and 36 h (MODERATE). Malondialdehyde (MDA) peaked between 0.5 and 1 h and returned to pre-infusion levels within 12 h. The cytokines IL-6, IFN-γ, IL-10 and IL-1β peaked between 1 and 3 h post-infusion. Moreover, cortisol increased rapidly, peaking at 2 h post LPS infusion. These findings indicate distinct temporal responses of inflammatory and oxidative stress markers following LPS challenge, supporting their use as potential biomarkers for evaluating interventions modulating infection-induced oxidative stress in pigs.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment in humans; however, research on ICIs in cats remains limited, and no clinical trials have been conducted for feline neoplastic diseases. Here, we developed a mouse monoclonal antibody (clone 1A1-2) targeting the feline PD-1 molecule and generated a mouse-feline chimeric antibody (1A1-2-fIgG) by replacing the constant region of 1A1-2 with that of feline IgG. However, administering 1A1-2-fIgG to cats may deplete PD-1-expressing effector T-cells via complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis, as feline IgG binds to CD64, CD16, and C1q. We engineered two 1A1-2-fIgG mutants with amino acid substitutions in the constant region to reduce the interactions between the Fc fragment and C1q or FcγRs and mitigate these effector functions. These mutations successfully abolished the binding to CD64, CD32, and CD16 while preserving the affinity for FcRn, which is essential in maintaining the half-life of antibodies in the blood. Furthermore, the mutants exhibited impaired binding to C1q. Despite these modifications, the mutated antibodies effectively restored IFN-γ production, which had been suppressed by PD-1/PD-L1 signaling in stimulated lymphocytes, to levels comparable to those of the original antibody. These findings reveal that the engineered antibodies have potential for future clinical applications in feline oncology.

Infectious bronchitis virus (IBV) causes infectious bronchitis (IB), which is a major concern for the global poultry industry as a result of substantial economic losses. Although epithelial cells were described as the primary target cells of IBV, other susceptible cell types including macrophages and monocytes, have been identified, where productive infection impairs cellular functions. Avian B cells are central to antibody-mediated immunity in chickens against pathogens, including IBV; however, it remains unknown if IBV can infect and replicate in B cells. This study investigated whether Delmarva (DMV)/1639 IBV can infect B cells in the bursa of Fabricius (BF) in vivo and in DT-40 cells in vitro. In vivo, a significantly higher viral genome load was observed in the BF at 3 days post-infection (dpi), with similar result in sorted B cells from the BF. Viral RNA was found to be localized within B cells of the BF using an in situ hybridization combined with immunohistochemistry. In vitro, a comparable trend in viral genome load was observed in infected DT-40 cells and culture supernatants up to 72 h post-infection (hpi). Immunofluorescence assay revealed a significantly higher percentage of DT-40 cells expressing IBV nucleoprotein. Inoculation of DT-40 cells with virus-containing supernatant confirmed infectivity as did inoculation of embryonated eggs, which resulted in IBV-specific lesions including dwarfing and stunting. These findings demonstrate that IBV can infect and replicate productively in avian B cells; however further studies are warranted to elucidate the impact of IBV infection on B cell function and its role in disease pathogenesis.