Impacts of feeding a Saccharomyces cerevisiae fermentation product on productive performance, and metabolic and immunological responses during a feed-restriction challenge of mid-lactation dairy cows.

Saccharomyces cerevisiae fermentation products are commonly used in dairy cattle ration to improve production efficiency and health. However, whether these benefits will persist during feed-restriction-induced negative energy balance is unknown. The objective of this experiment was to examine the effect of a Saccharomyces cerevisiae fermentation product (NT, NutriTek, Diamond V) on performance, metabolic, inflammatory, and immunological responses to a feed-restriction challenge in mid-lactation dairy cows. Sixty Holstein cows were blocked by parity, days in milk, and milk yield and then randomly assigned to 1 of the 2 supplements: NT or placebo (CTL). The supplements were mixed in total mixed ration before feeding at a rate of 19 g/d per cow. The production phase of the experiment lasted 12 wk. Intake and milk yield were recorded daily, and milk composition was measured weekly. After the production trial, a subset of cows (NT: n = 16; CTL: n = 16) were immediately enrolled in a 5-d feed-restriction challenge with 40% ad libitum intake followed by a 5-d realimentation. Milk yield and composition were measured at each milking from d -2 to 10 relative to feed restriction. Blood samples were collected on d -2, -1, 1, 2, 3, 4, 5, 6, 8, and 10 relative to the initiation of feed restriction to measure circulating metabolites, insulin, cortisol, IL-10, tumor necrosis factor-α, lipopolysaccharide binding protein, and haptoglobin. Immune function assessments, including peripheral mononuclear cell proliferation and functional assays of circulating granulocytes, were performed on d -3 and 4 of the feed restriction. No differences were observed in dry matter intake, milk yield, or concentrations or yield of components except for fat yield. An interaction of parity and treatment was observed for milk fat yield that was lower for CTL than NT in primiparous cows, but no differences were observed among treatments in milk fat yield of multiparous cows. Feed restriction successfully induced negative energy balance and its associated metabolic changes, including reduced concentrations of plasma glucose and increased nonesterified fatty acids and ß-hydroxybutyrate. Cows fed NT had a similar decrease in milk yield but had a more pronounced reduction in plasma glucose concentration and greater ß-hydroxybutyrate concentration during feed restriction than those fed CTL. Feed restriction did not induce evidence of systemic inflammation but did reduce granulocyte functional activity. Compared with CTL, feeding NT improved the reactive oxygen species production by granulocytes after stimulation by extracellular antigens. In conclusion, feeding NT increased milk fat production of first-lactation cows but did not affect overall productive performance. However, supplementation with NT improved induced granulocyte oxidative burst. This may explain the greater glucose utilization by cows fed NT rather than CTL during feed restriction.

Ocular exposure to infectious laryngotracheitis virus alters leukocyte subsets in the head-associated lymphoid tissues and trachea of 6-week-old White Leghorn chickens.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease primarily infecting the upper respiratory tract and conjunctiva. Administration of live attenuated ILTV vaccines via eye drop, drinking water, or by coarse spray elicits protective mucosal immunity in the head-associated lymphoid tissues (HALT), of which conjunctiva-associated lymphoid tissue (CALT) and the Harderian gland (HG) are important tissue components. The trachea, a non-lymphoid tissue, also receives significant influx of inflammatory cells that dictate the outcome of ILTV infection. The objective of this study was to evaluate leukocyte cellular and phenotypic changes in the CALT, HG and trachea following ocular infection with a virulent ILTV strain. At 1, 3, 5, 7 and 9 days post-infection, CALT, HG, and trachea of 6-week-old specific pathogen free (SPF) chickens ocularly-exposed to vehicle or virulent ILTV strain 63140 were dissociated, the cells enumerated and then phenotyped using flow cytometry. The CALT had the highest viral genomic load, which peaked on day 3. In ILTV-infected birds, the CALT had a decreased percentage of leukocytes. This was reflected by decreased numbers of MHCI+MHCII-, MHCI+MHCIIlow+, and CD4+ cells, while IgM+ and MHCI+MHCIIHigh+ expressing cell populations increased. In the HG, the most notable change in cells from ILTV-infected birds was a decrease in IgM expressing cells and histologically, an increase in Mott cells. In summary, an acute, ocular exposure to ILTV strain 63140 in young birds shifts subsets of lymphocyte populations in the CALT and HG with minimal impact on the trachea.

Lead alters intracellular protein signaling and suppresses pro-inflammatory activation in TLR4 and IFNR-stimulated murine RAW 264.7 cells, in vitro.

Lead (Pb) is a persistent environmental pollutant that has a structure and charge similar to many ions, such as calcium, that are essential for normal cellular function. Pb may compete with calcium for protein binding sites and inhibit signaling pathways within the cell affecting many organ systems including the immune system. The aim of the current study was to assess whether the calcium/calmodulin pathway is a principal target of environmentally relevant Pb during pro-inflammatory activation in a RAW 264.7 macrophage cell line. RAW 264.7 cells were cultured with 5 µM Pb(NO3)2, LPS, rIFNγ, or LPS+rIFNγ for 12, 24, or 48 hr. Intracellular protein signaling and multiple functional endpoints were investigated to determine Pb-mediated effects on macrophage function. Western blot analysis revealed that Pb initially modulated nuclear localization of NFκB p65 and cytoplasmic phosphorylation of CaMKIV accompanied by increased phosphorylation of STAT1ß at 24 hr. Macrophage proliferation was significantly decreased at 12 hr in the presence of Pb, while nitric oxide (NO) was significantly reduced at 12 and 24 hr. Cells cultured with Pb for 12, 24, or 48 hr exhibited altered cytokine levels after specific stimuli activation. Our findings are in agreement with previous reports suggesting that macrophage pro-inflammatory responses are significantly modulated by Pb. Further, Pb-induced phosphorylation of CaMKIV (pCaMKIV), observed in the present study, may be a contributing factor in metal-induced autophagy noted in our previous study with this same cell line.

Technical note: Development of a challenge model for Streptococcus uberis mastitis in dairy heifers.

A challenge model for experimentally inducing Streptococcus uberis mastitis in bred dairy heifers was developed. Qualifying heifers (n=7) exhibited antibody titers of <1:10,000 against Strep. uberis antigens and were free of intramammary infections (IMI). Two contralateral quarters of each heifer were assigned to receive an infusion of Strep. uberis (1,000 to 2,000 cfu); remaining quarters served as unchallenged controls. For a successful challenge and infection, 3 of 4 consecutive mammary secretion samples had to culture positive for Strep. uberis. Six of the 7 heifers were challenged successfully in both infused quarters with a mean dose of 1,080 cfu; once confirmed, infections were treated with a one-time infusion of nonlactating cow therapy. Before challenge, mammary secretion leukocyte counts averaged 8.4×10(6)/mL in all quarters. At 24h after challenge, leukocyte count increased to 18.4×10(6)/mL in challenged quarters, peaking on d 5 at 24.3×10(6)/mL; unchallenged quarters remained at ≤10.4×10(6)/mL, but increased to 15.2×10(6)/mL on d 7 and then decreased. Before challenge, macrophages predominated (81%) in mammary secretions followed by lymphocytes (15.3%) and neutrophils (3.7%). By 24h after challenge, neutrophils increased in challenged quarters and predominated for the duration of the trial (65.3 to 70%), whereas macrophages predominated in unchallenged control quarters (65.2 to 75.2%). The challenge model was successful in establishing Strep. uberis IMI in 85.7% of animals, and IMI were controlled (100% cure) by administering nonlactating cow therapy. All heifers calved free of IMI and antimicrobial residues, with milk production similar to that of herd mates and with somatic cell counts (SCC) <200,000 cells/mL.

Changes in serum testosterone and anti-Müllerian hormone concentration in bulls undergoing scrotal insulation.

While changes in semen quality after heat stress are well characterized in the bull, changes in endocrine function have not been critically evaluated. It was hypothesized here that scrotal insulation results in alterations in Sertoli cell and Leydig cell function, as measured by changes in serum testosterone and anti-Müllerian hormone (AMH) concentration. Scrotal insulation bags were placed in 10 bulls for 8 d. Blood was collected on days -22 and -2, and weekly from days 5 to 96 (day 0 = first day of scrotal insulation) for measurement of serum concentration of AMH and testosterone using ELISA. The concentration of AMH decreased on day 5, followed by an increase on day 54 (P = 0.014). When AMH concentration was normalized to pre-insulation values, the percent increase in serum concentration of AMH was significant between days 26 and 54, with another peak at 75 d (P = 0.031). The serum concentration of testosterone (P = 0.0001) and the percentage of change in testosterone concentration (P < 0.0001) increased on day 5, followed by a decrease from days 33 to 96. Scrotal insulation was associated with Sertoli and Leydig cell dysfunction, as measured by serum testosterone and AMH concentration. The persistently low concentration of testosterone at the end of the study suggests a long term effect of scrotal insulation on Leydig cell function.

Laser-assisted vitrification of large equine embryos.

The major difficulty in providing the benefits of embryo cryopreservation for equine agriculture is the mismatch between the optimal embryo age for collection from the mare (7-8 days after ovulation was detected) and the optimal age for freezing under current methods (6.5 days after ovulation). To overcome this limitation, we tested a method to enhance penetration of cryopreservative across the capsule and trophoblast of day 7 and 8 embryos combined with rapid freezing by vitrification. Six small embryos (<300 µm in diameter) were collected on day 6-7 after ovulation and twelve larger embryos were recovered on day 7-8. In the treatment group, replacement of blastocoelic fluid with cryopreservative solution was facilitated by a laser system used to create a small opening in the embryonic capsule and trophectoderm. All embryos were vitrified using a CryoLeaf freezing support. After recovery from freezing and embryo transfer, three of four small untreated embryos (<300 µm in diameter, 75%) and four of nine large blastocysts in the treatment group (>300 µm in diameter, 44%) resulted in a vesicle as detected by ultrasonography approximately one week after transfer. However, only one recipient mare was still pregnant on day 23, and she delivered a live foal. Further investigation is required to determine why most of the embryos in this experiment were lost between day 13 and day 23 of gestation.

Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa.

Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival.

Optimization of cryopreservation protocols for cooled-transported stallion semen.

Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P<0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P<0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.

An immunomodulatory feed additive enhances in vitro viral vaccine recall antigen responses in dairy heifers.

Enhancing immunological responses to vaccination is an important goal in many herd health management systems. OmniGen-AF®(OG) is an immunomodulatory feed additive that has been shown to enhance innate immune function in ruminants and its effects on adaptive immunity require additional study. The objective of this study was to evaluate post-vaccine antibody titers and circulating cellular memory development in heifers fed OG and administered a commercially available modified-live bovine respiratory disease (BRD) vaccine. Twenty-four Holstein heifers were assigned to one of two diets for 170 days: Control TMR (CON; n = 11), or TMR plus OG (TRT; 9 g/100 kg BW/day; n = 13). Samples for hematology, serology, and cellular assays were collected on D-110, 0, 21, 42, and 60 of the trial. Heifers were administered two priming doses of a modified-live BRD vaccine, with a third dose given on D0. There were no significant differences in total WBC and absolute number or the percentage of circulating lymphocytes, monocytes, neutrophils, RBC, or platelets on D-110 through D21. On D42 and D60, CON had significantly higher numbers of lymphocytes. On D0, mean serum neutralizing (SN) titer to BHV-1 was significantly higher for CON compared to TRT. SN titers were not significantly different between CON and TRT at any other time point for BHV-1, BVDV type 1, or BVDV type 2. TRT mounted a significantly stronger recall proliferative response to 0.5 multiplicity of infection (MOI) of BHV-1, BVDV type 1 and BVDV type 2 on D42 and D60; 0.25 MOI of BVDV type 1 on D21 and D42; and 0.25 MOI BVDV type 2 on D42 compared to CON. IL-4 production induced by 0.5 and 1.0 MOI BHV-1 (D42 and D60); 0.25 MOI of BVDV type 1 (D21); and 0.25 and 0.5 MOI of BVDV type 2 (D60) were significantly higher for TRT than CON. IL-17 production induced by 0.25 MOI of BVDV type 1 was significantly higher on D60 for TRT compared to CON. IFN-gamma and IL-10 were not significantly different between treatments. These data indicate feeding OG has a beneficial effect on responses to vaccine antigens in Holstein dairy heifers.

Ambient ammonia does not appear to inhibit the immune response to infectious bronchitis virus vaccination and protection from homologous challenge in broiler chickens.

Commercial broilers are commonly exposed to gaseous ammonia (NH3) originating from degradation of nitrogen-containing excreta in the litter during the grow-out period. Ammonia concentrations in the air are higher in poorly ventilated houses and appear to coincide with the elevated incidence of respiratory disease occurring during the winter months. This study examined the effect of NH3 on the immune response to infectious bronchitis virus (IBV) vaccination and protection against homologous serotype challenge in commercial broiler chickens. One-day-old chicks were administered IBV vaccine and exposed to 30-60 ppm of NH3. At 28 DOA, birds were challenged oculonasally with a pathogenic homologous IBV, and protection was measured by viral detection, clinical signs, ciliostasis, and presence of airsacculitis. IBV-specific serum IgG and lacrimal fluid IgA titers, as well as Harderian gland (HG) immune cell phenotypes, were evaluated. Ammonia exposure was associated with an increased incidence of airsacculitis among non-vaccinated, challenged birds. Vaccinated, NH3-exposed birds were completely protected from IBV challenge. Ammonia had subtle effects on cilia morphology and function but did not affect vaccine or challenge virus replication and clearance, clinical signs, ciliostasis, tracheal histopathology scores, or immune responses. In the HG of vaccinated birds, the percent of leukocytes, MHC I+/MHC IIhi expression, IgM+ expression, and CD8+ expression was increased, while mucosal IgA and serum IgG titers were nominal. Non-vaccinated, IBV-challenged birds exhibited an increased percent of leukocytes, MHC I+/MHC IIhi expression, and IgM+ expression in the HG at 5 dpc, followed by increased mucosal IgA and serum IgG titers and CD8+ expression at 10-14 dpc. In contrast, vaccinated, IBV-challenged birds had a minimal increase in MHC I+/MHC IIhi expression, and serum IgG antibody titers in vaccinated birds increased rapidly. The results indicate that commercial broilers exposed to moderate levels of ambient NH3 are equally protected against IBV challenge if appropriately vaccinated, and the absence of robust immune activation in vaccinated, challenged birds suggests that the challenge virus was efficiently neutralized before establishing infection. In contrast, ambient NH3 exposure was associated with a higher incidence of airsacculitis in non-vaccinated, challenged birds, despite the apparent lack of differences in the immune response between birds in the NH3-exposed and NH3 control groups.

Effects of an immunomodulatory feed additive on intramammary infection prevalence and somatic cell counts in a dairy herd experiencing major health issues.

A trial was conducted to determine if feeding OmniGen-AF® (OG) to 22 late lactation cows 60 days prior to and during the early dry period, a time of increased susceptibility to mastitis, could reduce disease incidence in a dairy herd experiencing major health issues. Treated cows (n = 11) consumed a ration containing OG [9 g/100 kg of body weight/day] beginning 60 days before dry-off, during the dry period, and through 30 days in milk (DIM). Control cows received the same ration during the dry period through 30 DIM only. Body weights, body condition scores (BCS), intramammary infection (IMI) prevalence, new IMI rates, somatic cell counts (SCC), milk yield, and adverse health events were measured. No differences were found between treatments for body weight or BCS. Adverse health event data at calving showed no differences between treatments except for percentage of cows with hyperketonemia, which was lower among treated cows (63.6% vs 100%). Prevalence of IMI from calving through 30 DIM for treated cows (6.1%) was lower than controls (11.05%); likewise, new IMI rate during this time for treated cows (0.61%) was lower than controls (5.81%). The SCC from calving through 30 DIM for treated cows (215,000/ml) was lower than controls (493,000/ml). Average production/day at the first DHIA test (~33 DIM) showed that treated cows produced more milk (39.9 kg) than controls (35.34 kg). In conclusion, feeding OG 60 days prior to dry-off reduced hyperketonemia and mastitis, lowered SCC, and numerically increased milk yield in a dairy herd experiencing major health issues.

Transfer of maternal colostral leukocytes promotes development of the neonatal immune system I. Effects on monocyte lineage cells.

Although it has been established that maternal leukocytes traffic from colostrum into the neonatal circulation, the effects of these cells on neonatal immunity are only beginning to be understood. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal antigen presenting cells. At birth, groups of neonatal calves received whole or cell-free colostrum (CFC) from their respective dams. Peripheral blood samples were obtained over the first 4 weeks of life, and expression of surface markers associated with cellular activation and physiological stress were monitored on monocyte lineage cells. Calves receiving cell-free colostrum at birth expressed elevated levels of CD11a, CD11c, and CD14, compared to calves receiving whole colostrum (C). Calves receiving cell-free colostrum had an elevated number of monocytes in the peripheral blood during the first 2 weeks of life, however, these cells expressed lower levels of expression of CD25 and MHC class I compared to calves receiving whole colostrum. The most significant differences in marker expression occurred within the first 7 days of life.

Transfer of maternal colostral leukocytes promotes development of the neonatal immune system Part II. Effects on neonatal lymphocytes.

It has been established that maternal leukocytes, conditioned by the mammary environment, cross the neonatal gut and circulate in the newborn calf. However, the impact of these cells on the development of neonatal immunity remains to be determined. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal adaptive immunity by examining the expression of surface markers on neonatal lymphocytes. At birth, neonatal calves were fed whole colostrum, or colostrum that had the maternal cells removed (cell-free colostrum), from their respective dams. Peripheral blood samples were collected at regular intervals over the first 4 weeks of life and lymphocytes were evaluated for surface expression of cellular markers. The results of these studies demonstrated that calves receiving whole colostrum had fewer CD11a positive lymphocytes in circulation during the first 2 weeks of life and this marker was expressed at a lower density than calves receiving cell-free colostrum. In addition, calves receiving whole colostrum also had a higher percentage of lymphocytes expressing the activation markers CD25 and CD26 by 7 days after birth. During the first week of life, lymphocytes from calves receiving whole colostrum had a higher density of MHC class I expression on their surfaces than cells from calves receiving cell-free colostrum. In general, these results indicate that transfer of maternal cells with colostrum allows for more rapid development of lymphocytes and maternal cells appeared to enhance their activation.

Advancements in large animal embryo transfer and related biotechnologies.

Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non-surgical embryo recovery and transfer are well-established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstration of a new cryopreservation method which involves reduction of the blastocoelic volume and microinjection of cryopreservative. Successful cryopreservation will improve the ability of practitioners to preserve and implant embryos in recipient mares. Recent advances in the use of equine FSH to induce superovulation in mares brings to the forefront the issue of how to best preserve the large number of embryos that are produced. Finally, the use of sexed semen after superovulation will provide the bovine and equine breeding industry the offspring of the desired sex.

Effects of injectable trace minerals administered concurrently with a modified live virus vaccine on long-term protection against bovine viral diarrhea virus acute infection in dairy calves.

The objective was to evaluate the effects of injectable trace minerals (ITM) concurrent with modified-live virus (MLV) vaccination on protection from bovine viral diarrhea virus (BVDV) infection in dairy calves. In a previous study (Palomares et al., 2016), thirty dairy calves received two doses of a MLV vaccine subcutaneously (SC), concurrently with ITM (n = 15) or saline (n = 15), SC. Five months later, 20 of these calves received ITM (G1, n = 10) or saline (G2, n = 10) according to their previous groups and were challenged intranasally with BVDV2. Five unvaccinated calves were also challenged with BVDV2 (G3). Blood samples were collected on days 0 (BVDV challenge), 3, 5, 6, 7, 8, 9, 11, 14, 18, 21, 32 and 61 for leukocyte count, virus isolation and BVDV serum neutralizing antibodies (SNA). Mild-moderate clinical signs were observed in G3 after BVDV challenge. Group 1 showed lower sum health score and nasal score on d5 and fecal score on d8 compared to G2. Rectal temperature and leukocyte counts were not different between G1 and G2. In contrast, G3 calves had significant leukopenia and lymphopenia from d3 to d7 (P < .05) and higher rectal temperatures on d6 to d8, compared to values on d0 (P < .05). All unvaccinated calves became viremic, while viremia was not detected in G1 or G2. Average daily gain was not different between vaccinated groups, however, only G1 calves had significantly greater (P = .04) ADG compared to non-vaccinated calves during the first 14 days post challenge. Vaccinated calves treated or not with ITM were protected from BVDV2 infection five months post-vaccination.

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses.

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.

Serum, uterine, and vaginal mucosal IgG antibody responses against Tritrichomonas foetus after administration of a commercial killed whole T foetus vaccine in beef cows.

The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus-purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus-specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of beef cows with a commercial vaccine containing T foetus induced significant increase in the levels of IgG to the T foetus TF1.17 surface antigen in serum, vaginal secretions, and uterine fluid, which remained elevated through Days 43, 75, and 182 in uterine fluids, vaginal secretions, and serum, respectively. Since purified TF1.17 antigen has been shown to protect against experimental T foetus infection in heifers, the vaccine-induced TF1.17-specific IgG response is likely to be important in the prevention of trichomoniasis in beef cattle.

Evaluation of multiple immune parameters after vaccination with modified live or killed bovine viral diarrhea virus vaccines.

The development of immunity to vaccine antigen was examined using three prime/boost strategies and the progression of immune activities was evaluated over the course of 8 weeks. Calves were vaccinated and multiple immune parameters were evaluated using several methods to assess humoral or cellular immunity from the same samples in parallel. The three vaccination protocols used were a killed vaccine followed by a killed boost (killed/killed), MLV vaccine and boost (MLV/MLV), or a MLV vaccine and killed boost (MLV/killed). All the vaccines used included modified live IBR/PI3 viruses to make the bystander context as similar as possible. The Singer strain of BVDV was used as the source antigen in the killed vaccine, and the NADL strain of BVDV was used in the MLV vaccine. Controls received a vaccine containing only MLV IBR/PI3. The assessment panel measured SN titers, as well as lymphocyte proliferation, cytokine mRNA expression, intracellular cytokine production, and released IFN-gamma after in vitro stimulation with three strains of BVDV virus. MLV/MLV and MLV/killed groups developed significant SN titers to the type 1 BVDV virus strains, Singer and NADL, and low crossover titers were also seen to the type 2 strain, 890 over the evaluation period. These two groups showed significant proliferation in response to the NADL virus as compared to controls. Multiple immune assessments were conducted simultaneously to attempt to provide a broader, more in depth evaluation of immune response to these BVDV vaccination protocols. We observed that the correlation among most of the assays conducted were weak; the correlation between SN titers and cellular proliferation assays demonstrated a moderate correlation.

Colostrum induced phenotypic and trafficking changes in maternal mononuclear cells in a peripheral blood leukocyte model for study of leukocyte transfer to the neonatal calf.

The relationship between the colostral environment and the function of leukocytes in colostrum is not clearly defined. This study examined the effects of defatted, acellular colostrum (AC) on the phenotype of peripheral blood mononuclear cells (PBMC) and their capacity to enter the circulation of neonatal calves after ingestion as a model of this relationship. Maternal PBMC were exposed to medium alone or medium supplemented with 25% AC. Expression of CD11a, CD11b, CD11c, CD43, CD49d, CD49e, and CD62L was assessed on freshly isolated and treated PBMC. Exposure to AC increased the percentage of cells expressing CD11a, CD11c and CD43, but decreased the percentage of cells expressing CD62L relative to freshly isolated PBMC. The density of expression of CD11b and CD11c was reduced, but increased for CD43 after exposure to AC relative to freshly isolated PBMC. Density of CD62L expression and percentage of cells expressing CD11a and CD43 were significantly different for cells treated with AC relative to medium alone. Further, these changes could not be attributed to occult bacterial contamination of the AC, as treatment of PBMC with LPS in the same medium yielded none of the observed changes. Maternal PBMC (treated as described) were labeled with the fluorescent tracer, PKH26-GL, and fed to neonatal calves within 6 h of birth. The circulation of these cells in the neonate was monitored by flow cytometry. We observed that: (1) cells exposed to AC, but not medium alone, entered the circulation; (2) peak trafficking occurred 12-24 h after ingestion; (3) a large fraction of labeled cells appeared in the neonatal circulation; and (4) labeled cells disappeared from circulation by 36 h after ingestion. This study indicates that exposure to the colostral environment induced phenotypic changes facilitating trafficking of colostral cells into the neonate.

Differentiation of peanut lectin positive suppressor T-cells from peanut lectin negative precursors in bovine cells by 12-O-tetradecanoylphorbol-13-acetate.

Although 12-O-tetradecanoylphorbol-13-acetate (TPA) can synergize with lectins to enhance lymphocyte proliferation, pretreatment of lymphocytes with TPA decreases their response. Pretreatment also inhibits the response to allogeneic cells in the mixed lymphocyte culture. In this study we determined that at least part of this inhibition was due to the generation of T-lymphocytes with the ability to suppress proliferation in vitro. By using populations enriched in lymphocytes or macrophages we determined that the interaction of TPA with lymphocytes, but not macrophages, was required to mediate the suppression. The number of macrophages present in culture (range, 0.5-10%) was irrelevant to the generation of inhibitory activity. Moreover, the TPA-induced suppressor activity copurified with T-cells. Furthermore, when peanut lectin (agglutinin) (PNA) was used to separate T-cells after treatment with TPA, essentially all of the activity copurified with the PNA positive cells. When PNA separations were carried out before treatment with TPA, the suppressor activity arose from the PNA negative fraction. Therefore, TPA appeared to cause phenotypically PNA negative T-cells to gain the PNA positive marker, as well as to function as suppressor cells in vitro. Suppressor activity was also found in the culture medium. Thus the suppression observed may be mediated through a soluble factor released by the TPA-treated cells. Although the suppressor cell activity induced by TPA can only partially account for its in vitro inhibition of lymphocyte proliferation, the development of suppressor cells merits further study with respect to lymphocyte phenotypic and functional differentiation. The results also suggest the possibility that similar processes could occur in vivo, possibly during the course of tumor promotion.