Impact of apitherapy on canine, equine, and chicken lymphocytes, in vitro.

Apitherapy is a form of alternative medicine that utilizes products from the western honeybee (Apis mellifera), including honey, propolis, and honeybee venom, to improve the health status of human patients by altering host immunity. An added benefit of these products is that they are nutraceuticals and relatively inexpensive to aquire. Currently, little is known about the use of honeybee products in veterinary species, as well as their impact on host immunity. In the present in vitro study, honey, propolis, and honeybee venom were co-cultured with enriched canine, equine, and chicken peripheral blood lymphocytes (PBLs) with cell proliferation, cell viability/apoptosis, and cellular morphology evaluated. Concanavalin A (Con A) and dexamethasone were used as stimulatory and suppressive controls, respectively. Honeybee products' effects on the three veterinary species varied by product and the species. Honey stimulated the PBLs proliferation in all three species but also displayed some increased cytotoxicity. Propolis stimulated proliferation in canine and equine PBLs, however, it suppressed proliferation in the chicken PBLs. Honeybee venom was the strongest PBL stimulant for all three species and in the equine, surpassed the stimulant response of Con A and yet, enhanced PBL cell viability post culture. In summary, the results of this preliminary in vitro study show that these three honeybee products do impact lymphocyte proliferation and viability in dogs, horses, and chickens, and that more research both in vitro and in vivo will be necessary to draw conclusions regarding their future use as immune stimulants or inhibitors.

Validation of a commercially available photometric analytical system for assessment of plasma oxidative status in healthy horses.

Reactive oxygen species (ROS) are the end-products of physiologic functions in health. Oxidative stress occurs when endogenous antioxidants are insufficient to neutralize ROS in the system. As a result, ROS can damage DNA, RNA, proteins, lipids, and cell organelles. To obtain accurate measurements of plasma oxidative stress, levels of both oxidants and antioxidants must be measured. This study validates a commercially available, semi-quantitative, photometric analytical system that measures systemic determinants of reactive oxygen metabolites (dROM) and plasma antioxidant capacity (PAC) in stored equine plasma. The objectives of this work were: 1) to validate a photometric analytical system to quantify dROM and PAC in equine plasma; and 2) to determine expected results for these tests in healthy adult horses. We hypothesized that this system would reliably and reproducibly assess dROM and PAC in equine plasma. We observed expected, dose-dependent increases in dROM generated by adding increasing concentrations of H2O2 or ascorbic acid to equine plasma to provide samples containing a known quantity of oxidants or antioxidants respectively. Mean dROM value in healthy horses was 103.3 ±20.7 U. Carr and mean PAC was 2881.0 ± 313.9 U. Cor. This system reliably and reproducibly quantified dROM and PAC in equine plasma samples.

Hematologic profiles and development of innate immune function in healthy Holstein calves during the pre-weaning period.

BACKGROUND: The development and state of innate immune cell function during the first 90 days of life in dairy calves have not been fully described. OBJECTIVE: This transversal study attempted to examine the changes that occur in circulating blood cells and the innate immune response in healthy calves from birth to 89 days of age. METHODS: Healthy Holstein calves represent three windows of development, G1 from 1 to 7 days old (n = 26), G2 from 30 to 40 days old (n = 28), and G3 from 60 to 89 days old (n = 36) were sampled once each from a single herd. A few biomarkers of the general health and innate and inflammatory immune responses were measured. RESULTS: The youngest calves had the lowest red blood cell (RBC) counts, cell hemoglobin concentration means (CHCMs), red cell distribution widths (RDWs), and cell hemoglobin contents of mature red blood cells (CHm) compared with the other groups. They also had the lowest iron concentrations and highest intracellular myeloperoxidase indices. However, white blood cell (WBC) and lymphocyte concentrations gradually increased from G1 to G3. G2 calves had the lowest serum protein concentrations and highest number of innate immune cells compared with the other groups. Calves were able to mount phagocytic and ROS responses from birth. CONCLUSIONS: The physiologic responses of circulating blood cells and innate immune responses in dairy calves are shown according to age. Neonates had limitations in several RBC and WBC indices and immunologic responses that would likely impact overall vigor and health. Fortunately, these limitations resolve by 90 days of age.

Evaluation of equine xenogeneic mixed lymphocyte reactions using 5-ethynyl-2'-deoxyuridine (EdU).

Allogeneic solid organ transplantation is currently the only treatment option for end stage organ disease. The shortage of available donor organs has driven efforts to utilize xenogeneic organs for transplantation. In vitro methods for evaluating immune-compatibility are a quick and low cost means of screening novel tissue products prior to more involved, expensive, and invasive live animal studies. Recently, a new analog of the DNA base thymidine, 5-ethynyl-2'-deoxyuridine (EdU), was developed. It may be used in a fast, efficient and specific means of evaluating cell proliferation via flow cytometry. This study was designed to test and optimize this platform for assessing equine xenogeneic one-way mixed lymphocyte reaction (MLR) to porcine stimulator cells. Furthermore, it was hypothesized that an enriched T-lymphocyte (T-cell) population would generate a stronger proliferative response to stimulation, and higher levels of cytokine production when compared to unfractionated peripheral blood mononuclear cells (PBMCs). PBMCs and T-cells were isolated from 3 horses and 4 pigs. Equine xenogeneic MLRs were set up using porcine allogeneic MLRs as a reference for clinically acceptable levels of cell proliferation. Equine T-cells showed significantly greater EdU incorporation in one-way xenogeneic MLRs than equine PBMCs. However, there was no significant difference in cell proliferation between porcine T-cell and PBMC as responders in allogenic one-way MLRs. Given the results of this study, we consider that enriched equine T-cells should be used in preference to unfractionated PBMCs when attempting to evaluate the equine xenogeneic response using the EdU assay as an indicator of suitability for transplant in vivo.

Influence of exposure to microbial ligands, immunosuppressive drugs and chronic kidney disease on endogenous immunomodulatory gene expression in feline adipose-derived mesenchymal stem cells.

OBJECTIVES: Feline autologous mesenchymal stem cells (MSCs) show promise for immunomodulatory activity, but the functional impact of chronic kidney disease (CKD), concurrent immunosuppressive drug administration or infection is unknown. The study objectives compare endogenous cytokine gene expression (interleukin [IL]-6, IL-10, IL-12p40, IL-18 and transforming growth factor beta [TGF-ß]) in adipose-derived MSCs (aMSCs) from cats with and without CKD, following in vitro exposure to microbial ligands and treatment with common immunosuppressive drugs. METHODS: Previously obtained aMSCs, phenotype CD44+, CD90+, CD105+ and MHCII-, from cats with (n = 6) and without (n = 6) CKD were compared via real-time PCR (RT-PCR) for immunomodulatory gene expression. aMSCs were exposed in vitro to lipopolysaccharide (LPS), peptidoglycan or polyinosinic:polycytidylic acid (Poly I:C), simulating bacterial or viral exposure, respectively. aMSCs were also exposed to ciclosporin, dexamethasone or methotrexate. Gene expression was measured using RT-PCR, and Cq was utilized after each run to calculate the delta cycle threshold. RESULTS: aMSCs isolated from healthy and CKD cats showed no significant differences in gene expression in the five measured cytokines. No significant changes in measured gene expression after drug treatment or microbial ligand stimulation were observed between normal or CKD affected cats. Proinflammatory genes (IL-6, IL-12p40 and IL-18) showed altered expression in aMSCs from both groups when compared with the same cells in standard culture after exposure to methotrexate. Poly I:C altered IL-6 and TGF-ß gene expression in aMSCs from both healthy and CKD cats when compared with the same cells in standard culture. CONCLUSIONS AND RELEVANCE: The five genes tested showed no statistical differences between aMSCs from healthy or CKD cats. There was altered cytokine gene expression between the control and treatment groups of both healthy and CKD cats suggesting feline aMSCs have altered function with immunosuppressive treatment or microbial ligand exposure. Although the current clinical relevance of this pilot study comparing brief exposure to select agents in vitro in aMSCs from a small number of cats is unknown, the study highlights a need for continued investigation into the effects of disease and concurrent therapies on use of cell-based therapies in feline patients.

Assessing the infiltration of immune cells in the upper trachea mucosa after infectious laryngotracheitis virus (ILTV) vaccination and challenge.

The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8ß+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8ß+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

Activation of Cytotoxic Lymphocytes and Presence of Regulatory T Cells in the Trachea of Non-Vaccinated and Vaccinated Chickens as a Recall to an Infectious Laryngotracheitis Virus (ILTV) Challenge.

While the protective efficacy of the infectious laryngotracheitis virus (ILTV) vaccines is well established, little is known about which components of the immune response are associated with effective resistance and vaccine protection. Early studies have pointed to the importance of the T cell-mediated immune responses. This study aimed to evaluate the activation of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and to quantify the presence of regulatory T cells (Tregs) in the larynx-trachea of chickens vaccinated with chicken embryo origin (CEO), tissue culture origin (TCO) and recombinant Herpesvirus of Turkey-laryngotracheitis (rHVT-LT) vaccines after challenge. Our results indicated that CEO vaccine protection was characterized by early CTLs and activated CTLs enhanced responses. TCO and rHVT-LT protection were associated with a moderate increase in resting and activated CTLs followed by an enhanced NK cell response. Tregs increase was only detected in the non-vaccinated challenged group, probably to support healing of the severe trachea epithelial damage. Taken together, our results revealed main differences in the cellular immune responses elicited by CEO, TCO, and rHVT-LT vaccination in the upper respiratory tract after challenge, and that activated CTLs rather than NK cells play a main role in vaccine protection.

Investigation of Long COVID Prevalence and Its Relationship to Epstein-Barr Virus Reactivation.

Coronavirus disease 2019 (COVID-19) patients sometimes experience long-term symptoms following resolution of acute disease, including fatigue, brain fog, and rashes. Collectively these have become known as long COVID. Our aim was to first determine long COVID prevalence in 185 randomly surveyed COVID-19 patients and, subsequently, to determine if there was an association between occurrence of long COVID symptoms and reactivation of Epstein-Barr virus (EBV) in 68 COVID-19 patients recruited from those surveyed. We found the prevalence of long COVID symptoms to be 30.3% (56/185), which included 4 initially asymptomatic COVID-19 patients who later developed long COVID symptoms. Next, we found that 66.7% (20/30) of long COVID subjects versus 10% (2/20) of control subjects in our primary study group were positive for EBV reactivation based on positive titers for EBV early antigen-diffuse (EA-D) IgG or EBV viral capsid antigen (VCA) IgM. The difference was significant (p < 0.001, Fisher's exact test). A similar ratio was observed in a secondary group of 18 subjects 21-90 days after testing positive for COVID-19, indicating reactivation may occur soon after or concurrently with COVID-19 infection. These findings suggest that many long COVID symptoms may not be a direct result of the SARS-CoV-2 virus but may be the result of COVID-19 inflammation-induced EBV reactivation.

Comparison of the immune response following subcutaneous versus intranasal modified-live virus booster vaccination against bovine respiratory disease in pre-weaning beef calves that had received primary vaccination by the intranasal route.

This study was performed to elucidate whether the route of booster vaccination affects the immune response against respiratory vaccine viruses in pre-weaning beef calves that receive primary intranasal (IN) vaccination during the first month of life. The objective was to compare the serum neutralizing antibody (SNA) titers to BHV1, BRSV, and BPI3V, cytokine mRNA expression and mucosal BHV1- and BRSV-specific IgA in nasal secretions following administration of IN or subcutaneous (SC) modified-live virus (MLV) booster vaccines 60 days after primary IN vaccination in young beef calves. Twenty-one beef calves were administered 2 mL of an IN MLV vaccine containing BHV1, BRSV, and BPI3V (Inforce3®) between one and five weeks of age. Sixty days after primary vaccination, calves were randomly assigned to one of two groups: IN-MLV (n = 11): Calves received 2 mL of the same IN MLV vaccine used for primary vaccination and 2 mL of a SC MLV vaccine containing BVDV1 & 2 (Bovi- Shield GOLD® BVD). SC-MLV (n = 10): Calves were administered 2 mL of a MLV vaccine containing, BHV1, BRSV, BPI3V, and BVDV1 & 2 (Bovi-Shield GOLD® 5). Blood and nasal secretion samples were collected on days -61 (primary vaccination), -28, -14, 0 (booster vaccination), 14, 21, 28, 42 and 60 for determination of SNA titers, cytokine gene expression analysis and nasal virus-specific IgA concentrations. Statistical analysis was performed using a repeated measures analysis through PROC GLIMMIX of SAS®. Booster vaccination by neither IN nor SC routes induced a significant increase in SNA titers against BHV1, BRSV, and BPI3V. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA titers (on day 42) and IgA concentration in nasal secretions (on days 21 and 42) compared to calves receiving IN booster vaccination. Both IN and SC booster vaccination were able to stimulate the production of BHV1-specific IgA in nasal secretions. In summary, booster vaccination of young beef calves using either SC or IN route two months after IN MLV primary vaccination resulted in comparable SNA titers, cytokine gene expression profile and virus-specific IgA concentration in nasal secretions. Only a few differences in the systemic and mucosal immune response against BHV1 and BRSV were observed. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA and secretory IgA titers compared to IN booster vaccination.

Relationships among Indicators of Metabolism, Mammary Health and the Microbiomes of Periparturient Holstein Cows.

During the period called "transition", from the ceasing of milk production to the reestablishment of full milk production, it is postulated that the microbiota of cows undergo changes in composition driven by the fluxes in systemic energetics and that these changes appear to impact the health of cows. The primary objective of this study was to document the make-up of the microbiota in the mammary gland compared with those in the vagina and in feces in an attempt to determine any correlations between the composition of the microbiota, the impact of blood indicators of energetic metabolites and the health of the mammary gland at the time of calving. Samples were collected from 20 Holstein dairy cows immediately following calving to assess their general health and measure the microbiomes associated with each cow using 16S rRNA sequencing. The results indicated that the microbiomes found within each maternal niche were different. A set of significant negative associations between the blood energetic biomarkers (NEFAs, BHB, triglycerides and cholesterol) and the taxa Pseudomonas, Christensenellaceae and Methanobrevibacter were observed in this study. In contrast, Escherichia and Romboutsia were positively correlated with the same energetic metabolites. Therefore, it was concluded that there appears to be a set of relationships between the microorganisms that colonize several niches of cows and the sufficiency of systemic energy metabolism. Furthermore, both the microbiome and energy dynamics impact the health of the mammary gland of the host.

Analysis of Measles-Mumps-Rubella (MMR) Titers of Recovered COVID-19 Patients.

The measles-mumps-rubella (MMR) vaccine has been theorized to provide protection against coronavirus disease 2019 (COVID-19). Our aim was to determine whether any MMR IgG titers are inversely correlated with severity in recovered COVID-19 patients previously vaccinated with MMR II. We divided 80 subjects into two groups, comparing MMR titers to recent COVID-19 severity levels. The MMR II group consisted of 50 subjects who would primarily have MMR antibodies from the MMR II vaccine, and a comparison group of 30 subjects consisted of those who would primarily have MMR antibodies from sources other than MMR II, including prior measles, mumps, and/or rubella illnesses. There was a significant inverse correlation (rs = -0.71, P < 0.001) between mumps virus titers (mumps titers) and COVID-19 severity within the MMR II group. There were no significant correlations between mumps titers and severity in the comparison group, between mumps titers and age in the MMR II group, or between severity and measles or rubella titers in either group. Within the MMR II group, mumps titers of 134 to 300 arbitrary units (AU)/ml (n = 8) were found only in those who were functionally immune or asymptomatic; all with mild symptoms had mumps titers below 134 AU/ml (n = 17); all with moderate symptoms had mumps titers below 75 AU/ml (n = 11); all who had been hospitalized and had required oxygen had mumps titers below 32 AU/ml (n = 5). Our results demonstrate that there is a significant inverse correlation between mumps titers from MMR II and COVID-19 severity.IMPORTANCE COVID-19 has presented various paradoxes that, if understood better, may provide clues to controlling the pandemic, even before a COVID-19 vaccine is widely available. First, young children are largely spared from severe disease. Second, numerous countries have COVID-19 death rates that are as low as 1% of the death rates of other countries. Third, many people, despite prolonged close contact with someone who is COVID-19 positive, never test positive themselves. Fourth, nearly half of people who test positive for COVID-19 are asymptomatic. Some researchers have theorized that the measles-mumps-rubella (MMR) vaccine may be responsible for these disparities. The significance of our study is that it showed that mumps titers related to the MMR II vaccine are significantly and inversely correlated with the severity of COVID-19-related symptoms, supporting the theorized association between the MMR vaccine and COVID-19 severity.

The effect of foal or adult horse plasma on equine monocyte-derived dendritic cell phenotype and function.

Immunological and endocrine immaturity in foals increases foal morbidity and mortality from bacterial sepsis. Dendritic cells (DC) are critical in activating the adaptive immune response, but foal DC are phenotypically and functionally different than those of adult horses. Age-related variations in availability of some soluble plasma factors, such as hormones, might govern some age-related differences in DC function. Effects of exposure to plasma factors on equine DC phenotype and function have not been described. We hypothesized that exposure to plasma from foals or adult horses would differentially impact monocyte-derived DC (MoDC) phenotype and function. Eight healthy adult horses and 8 healthy foals were divided into pairs of one adult horse and one foal. Blood was collected from each pair for MoDC generation when foals were 1 and 30 days of age. MoDC from horses and foals were then exposed to killed whole-cell bacteria in the presence of their own age-matched plasma, plasma from the opposite-aged animal in the pair, and serum-free medium alone (control). Expression of DC-relevant surface markers (MHC class-II, CD86, and CD14) and endocytosis capability were measured by flow cytometry. Supernatant cytokine concentrations (IL-4, IL-17, IFN-γ, and IL-10) were quantified with a validated bead-based immunoassay. Data were analyzed using linear mixed-effects and Tobit regression models (P < 0.05). The percentage of MoDC expressing surface markers MHC class-II and CD86 was reduced in MoDC derived from 1-day-old foals in comparison to adult horse MoDC when cultured in medium alone or with either source of plasma (P = 0.0001). Foal and adult horse MoDC cultured in either source of plasma expressed more CD86 and less CD14 than cells cultured in serum-free medium alone (P ≤ 0.02). Adult horse and foal MoDC exposed to bacterial antigen in the presence of 1-day-old foal plasma secreted less IL-10 (P ≤ 0.0008) compared to those cultured in adult horse plasma. Endogenous production of IL-17 by MoDC from foals at day 1 of age cultured in adult plasma was increased compared to foal MoDC cultured in serum-free medium (P = 0.004). Phagocytosis of killed, labeled Staphylococcus aureus was reduced when MoDC generated from foals or adult horses were exposed to plasma from foals at day 1 or 30 of age (P ≤ 0.03). Age-related variation in soluble plasma factors appear to regulate equine MoDC function, but specific plasma factors capable of regulating MoDC phenotype or function were not defined in this study.

Immune response and onset of protection from Bovine viral diarrhea virus 2 infection induced by modified-live virus vaccination concurrent with injectable trace minerals administration in newly received beef calves.

Strategies to improve the onset of protective immunity induced by vaccination against respiratory pathogens may have a significant impact on health of newly received beef calves. The objective was to determine if the use of injectable trace minerals (ITM; Se, Zn, Cu, and Mn) concurrent with a modified-live virus (MLV) vaccine enhances the immune response and onset of protection in beef calves challenged with BVDV2 five days after vaccination. Forty-five calves were randomly assigned to one of three groups (15/group): VAC + ITM, received MLV-vaccine and ITM (Multimin®90) subcutaneously (SC); VAC + SAL, received the same vaccine and saline SC; or UNVAC, unvaccinated. Five days after vaccination (d.0), calves were challenged with BVDV2 strain 890. Health status was evaluated and blood samples were collected for leukocyte counts, BVDV1 and 2 serum neutralizing antibodies (SNA), BVDV-PCR, and percentage of CD4+, CD8+, WC1+ and CD25+ T-cells. VAC + ITM had lower health scores than UNVAC (d.8 and 9). VAC + ITM had higher BVDV1 & 2 SNA titers than VAC + SAL and UNVAC on d.21 and 28. Lymphocyte counts decreased in UNVAC but not in VAC + ITM or VAC + SAL (d.3 to 11). CD4+ T-cells significantly decreased in UNVAC and VAC + SAL (d.3). VAC + ITM had higher percentage of CD4+ T-cells than UNVAC (d.3 and 7). VAC + ITM had lower percentage of activated CD4+ and CD8+ T-cells than UNVAC (d.7). In summary, vaccination induced a rapid protection against BVDV2 infection. Administration of ITM was associated with increased SNA response to BVDV1 & 2, enhanced health status, mitigation of CD4+ T-cells decrease, and reduction of T-cell activation in calves challenged with BVDV2 five days after immunization. These results support the strategic use of ITM concurrent with vaccination, especially when a rapid protection is needed in newly received beef calves.

Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.

Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8+ and CD21+ were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.

Phenotypic characterization of equine monocyte-derived dendritic cells generated ex vivo utilizing commercially available serum-free medium.

The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric analysis of surface markers (MHC class-II, CD86 and CD14). Data were analyzed using paired t-tests and repeated measures ANOVA. Two MoDC populations that differed in size and phenotype were identified: larger MoDC (LgMoDC) and smaller MoDC (SmMoDC). Medium type, plate chemistry, or length of monocyte adhesion time did not impact MoDC viability or yield. LgMoDC generated in serum-free medium expressed more MHC class-II and CD86 (P ≤ 0.03). A prolonged duration in culture reduced MoDC yield (P ≤ 0.04). MoDC can be consistently and reliably generated using AIM-V serum-free medium in standard tissue culture plates with a recommended culture duration of 3-4 days.

The comparative efficacy of disinfectant wipes on common-use computer keyboards in a veterinary teaching hospital.

The efficacies of 3 disinfectant wipes at reducing bacterial contamination on keyboards in a veterinary teaching hospital were studied. Thirty common-use keyboards were randomized into "dirty" and "clean" halves. Cultures were obtained from the "dirty" halves. The "clean" halves were disinfected with a randomly assigned wipe [peroxygen (AHP)-, alcohol-, quaternary ammonium (QAC)-based] or untreated (NT) and cultured. Colony-forming units (CFU) were enumerated after 48 hours. Mean reduction in CFU was 91.5%, 65.3%, 94.9%, and 78.8% for the AHP, alcohol, QAC, and NT groups, respectively. There was a significant reduction in CFUs between the dirty and clean keyboard halves within each group but no statistically significant differences were noted between groups. The reduction in CFUs in the NT group was attributed to the mechanical action of wiping the keyboard surface for culture. The use of disinfectant wipes reduced CFUs on keyboards and may be a useful component of veterinary infection control programs.

Efficacité comparative de lingettes désinfectantes sur des claviers d'ordinateurs en usage commun dans un hôpital d'enseignement vétérinaire. L'efficacité de trois lingettes désinfectantes à réduire la contamination bactérienne sur des claviers dans un hôpital d'enseignement vétérinaire fut étudiée. Trente claviers en usage commun furent séparés de manière aléatoire en moitié « sale ¼ et « propre ¼. Des cultures furent obtenues de la moitié « sale ¼. La moitié « propre ¼ fut désinfectée avec une lingette assignée de manière aléatoire [à base de peroxygène (AHP), alcool, ou ammonium quaternaire (QAC)] ou non traitée (NT) et échantillonnée pour culture. Le nombre d'unités formatrices de colonies (CFU) fut énuméré après 48 heures. La réduction moyenne de CFU était de 91,5 %, 65,3 %, 94,9 %, et 78,8 % pour les groupes AHP, alcool, QAC, et NT, respectivement. Il y avait une réduction significative dans les CFUs entre les claviers des moitiés sale et propre dans chaque groupe mais aucune différence statistiquement significative ne fut notée entre les groupes. La réduction en CFU dans le groupe NT fut attribuée à l'action mécanique de frottage de la surface des claviers. L'utilisation de lingettes désinfectantes a réduit le nombre d'UFC sur les claviers et pourrait être une composante utile des programmes de surveillance des infections vétérinaires.(Traduit par Dr Serge Messier).

Immune Responses in the Eye-Associated Lymphoid Tissues of Chickens after Ocular Inoculation with Vaccine and Virulent Strains of the Respiratory Infectious Laryngotracheitis Virus (ILTV).

Infectious laryngotracheitis (ILT) is an acute respiratory disease of poultry caused by infectious laryngotracheitis virus (ILTV). Control of the disease with live attenuated vaccines administered via eye drop build upon immune responses generated by the eye-associated lymphoid tissues. The aim of this study was to assess cytokine and lymphocyte changes in the conjunctiva-associated lymphoid tissues (CALT) and Harderian gland (HG) stimulated by the ocular inoculation of the ILTV chicken embryo origin (CEO) vaccine strain and virulent strain 63140. This study offers strong evidence to support the roles that the CALT and HG play in the development of protective ILTV immune responses. It supports the premise that ILTV-mediated immunomodulation favors the B cell response over those of T cells. Further, it provides evidence that expansions of CD8α+ cells, with the concomitant expression of the Granzyme A gene, are key to reducing viral genomes in the CALT and halting ILTV cytolytic replication in the conjunctiva. Ultimately, this study revealed that the early upregulation of interleukin (IL)-12p40 and Interferon (IFN)-γ cytokine genes, which shape the antigen-specific cell-mediated immune responses, retarded the decline of virus replication, and enhanced the development of lesions in the conjunctiva epithelium.

Characterization and comparison of cell-mediated immune responses following ex vivo stimulation with viral and bacterial respiratory pathogens in stressed and unstressed beef calves1.

The goal of this study was to compare the cell-mediated immune responses of highly commingled, sale-barn origin calves (STR; n = 10) to those of single source calves that had been weaned for 60 d (UNS; n = 10). Peripheral blood mononuclear cells and neutrophils (PMNs) were isolated from jugular venous blood of each calf. Peripheral blood mononuclear cells were stimulated with Concanavalin A (ConA), BVDV-1, BVDV-2, BHV-1, Mannheimia haemolytica, and Pasteurella multocida and evaluated for clonal proliferation and secretion of IL-8 into cell culture supernatants. The native functional capacities of PMNs were evaluated in response to stimulation with heat-killed Escherichia coli and Staphylococcus aureus. Complete blood counts and serum biochemical profiles were performed for each animal at the time of sample collection. Compared with STR calves, UNS calves had greater lymphocyte proliferative responses following stimulation BVDV1 (P = 0.041), BVDV2 (P = 0.002), BHV-1 (P = 0.001), M. haemolytica (P = 0.016), and P. multocida (P = 0.049). In addition, PMNs isolated from UNS calves had a greater ability to phagocytose E. coli (P = 0.001) and S. aureus (P = 0.003) when compared with STR calves. Serum nonesterified fatty acids were higher in STR calves (P < 0.001). Serum ß-hydroxybutyrate was lower in STR calves (P < 0.003). These data suggest that immunologic and physiologic differences exist between STR and UNS calves. Although the underlying mechanisms for these differences are not clear, it is possible that combinations of energy imbalances, stress-induced immunosuppression, and general immune naiveté may predispose STR calves to an increased risk of morbidity and mortality due to bovine respiratory disease.

The effect of age on foal monocyte-derived dendritic cell (MoDC) maturation and function after exposure to killed bacteria.

Neonatal foals are uniquely susceptible to certain infections early in life. Dendritic cells (DC) are vital in the transition between the innate and adaptive immune response to infection, but DC biology in foals is not fully characterized. Monocyte-derived DC represent a suitable in vitro model similar to DC that differentiate from monocytes recruited from circulation. We hypothesized that foal monocyte-derived DC (MoDC) would exhibit age-dependent phenotypic and functional differences compared to adult horse MoDC. MoDC generated from 9 horses (collected once) and from 8 foals (collected at 1, 7, and 30 days-of-age) were exposed to killed whole cell Escherichia coli or Staphylococcus aureus bacteria. MoDC expression of MHC class II (MHC class-II), CD86, and CD14 were measured by flow cytometry, and supernatant cytokine concentrations of IL-4, IL-17, IFN-γ, and IL-10 were quantified with a validated immunoassay. The percentage of MoDC expressing MHC class-II and CD86 was lower and CD14 was higher for cells generated from 1-day-old foals compared to cells generated from adult horses (P < 0.0001). Bacterial exposure increased the percentage of cells expressing CD86 at all ages (P < 0.0001). Bacteria-exposed MoDC from 1-day-old foals produced significantly less IL-4, IL-17, and IFN-γ than adult MoDC produced in response to bacterial exposure (P ≤ 0.04). Following bacterial exposure, foal MoDC phenotype and cytokine secretion were different than those of mature horses. These differences could reduce the ability of foals to generate a protective immune response against bacterial infection.