Modified ear canal ablation and lateral bulla osteotomy for management of otitis media in an alpaca.

OBJECTIVE: To report surgical treatment of severe otitis media in an alpaca by a modification of a subtotal ear canal ablation and lateral bulla osteotomy technique used in dogs. STUDY DESIGN: Case report. ANIMALS: An 11-week-old female alpaca cria. METHODS: The cria had a 2-week history of right otitis media, nonresponsive to medical treatment, as well as right facial nerve paralysis, and a melting corneal ulcer of the right eye. Otitis media was confirmed by computed tomography. Right subtotal ear canal ablation and lateral bulla osteotomy were performed using a modification of a technique reported in dogs. RESULTS: There were no surgical complications and the alpaca was discharged from the hospital 5 days later. At 10 months, moderate motor function had been restored to the pinna with the ear standing partially erect. The otitis had resolved, and the alpaca was reportedly well integrated into the herd. CONCLUSION: Subtotal ear canal ablation and lateral bulla osteotomy, a technique modified from that performed in dogs, were successful in providing complete clinical resolution of otitis media in an alpaca.

The effect of foot-and-mouth disease vaccination on early pregnancy loss in beef heifers in Argentina.

The threat of Foot and Mouth Disease (FMD) in South America has global economic implications and retaining a FMD Free status under the World Organization for Animal Health (OIE) remains a top priority. In Argentina the Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), the national service of agri-food health and quality, requires cattle located in the Pampean region of the Salado River basin to receive two foot-and-mouth disease (FMD) vaccinations per year, which results in one vaccination coinciding with beef cattle breeding season. While the vaccination program remains necessary, there is a growing concern amongst food animal veterinarians, that the overlap of FMD vaccination with the first 35 days of the breeding season is associated with early pregnancy loss (EPL). To address this concern, a preliminary randomized controlled trial t study was conducted to investigate the risk ratio (RR) of EPL in vaccinated, pregnant Aberdeen Angus heifers. Initially (Day 0), 858 heifers underwent fixed time-AI (FTAI). Subsequently, on day 33, following pregnancy diagnosis by transrectal ultrasonography pregnant heifers (n = 311) were randomly allocated to two treatment groups. Group 1 (162 animals) received an inactivated oil emulsion FMD vaccine, and Group 2 (149 animals) received a saline injection (control). On day 51 (18 days post vaccination), pregnancy status was re-evaluated by ultrasonography. The initial pregnancy rate (PR) on Day 33 was 58% (498/858 animals). On Day 51 (18 days post vaccination), PR in Group 1 was 96.3% (156/162 animals), and in Group 2 (control) was 98.6% (147/149 animals). The EPL in Group 1 was 3.7% (6/162 animals) and in Group 2 was 1.3% (2/149 animals). The RR of EPL in Group 1, compared to Group 2, was 2.8 (95% confidence interval: 0.6-13, p-value: 0.20). With such a wide range in confidence intervals and a p value of 0.20 a larger prospective study would be necessary to establish an unequivocally statistically significant link between heifer vaccination 33 days post FTAI and an increased risk of EPL.

Characterization of equine P-selectin glycoprotein ligand-1 by using a specific monoclonal antibody.

P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recognized a protein with molecular weight 165 and 280kDa under reducing and non-reducing condition, respectively, likely corresponding to ePSGL-1. Seventy percent of equine monocytes bound by both ePL1 and HECA-452, an antibody defining sLex-like carbohydrate epitope. Both ePL1 and HECA-452 recognized ePSGL-1 protein precipitated by equine P-selectin-IgG chimera. Neuraminidase treatment increased ePL1 binding and the molecular weight of ePSGL-1, O-sialoglycoprotein endopeptidase digestion and tyrosine mutation abolished ePL1 staining and recognition. The ePL1 specific binding epitope appears to be the polypeptide backbone of ePSGL-1 in the presence of tyrosine but the process is independent of sialylation modification. In conclusion, we provide evidence that this antibody can be used for cell surface staining and immune-blot analyses.

Coagulation values in normal ferrets (Mustela putorius furo) using selected methods and reagents.

BACKGROUND: Accurate determination of commonly measured coagulation values would be useful in the diagnosis and management of coagulopathies in domestic ferrets (Mustela putorius furo). We are unaware of reports of coagulation times in this species. OBJECTIVES: The purpose of this study was to determine reference values for prothrombin time (PT), activated partial thromboplastin time (PTT), fibrinogen concentration, and antithrombin (AT) activity in ferrets using selected methods and reagents. METHODS: Blood samples obtained from 18 clinically healthy ferrets were anticoagulated with 0.129 M sodium citrate in a ratio of 9 parts blood to 1 part anticoagulant. Plasma was collected and stored at -70 degrees C until analysis. PT and PTT were measured with a fibrometer and with an ACL 3000 automated system. PTT was measured with and without the addition of ellagic acid. Fibrinogen was assayed by a turbidimetric method. AT activity was determined using a chromogenic assay and pooled ferret plasma (100% activity). Differences in methods and reagents were evaluated using paired t tests. RESULTS: PT was significantly longer using the fibrometer (12.3+/-0.3, 11.6-12.7 seconds) compared with the ACL (10.9+/-0.3, 10.6-11.6 seconds) (P<.01). PTT was not significantly different with the fibrometer (18.7+/-0.9, 17.5-21.1 seconds) vs the ACL (18.1+/-1.1, 16.5-20.5 seconds), but was significantly longer on both analyzers when ellagic acid was added (fibrometer 20.4+/-0.8, 18.9-22.3 seconds; ACL 20.0+/-1.0, 18.6-22.1 seconds) (P<.01). Fibrinogen concentration was 107.4+/-19.8 mg/dL (90.0-163.5 mg/dL), and AT activity was 96%+/-12.7% (69.3-115.3%). CONCLUSION: These coagulation results for healthy ferrets will be useful in the evaluation of ferrets with coagulopathies, provided similar reagents and methods are used.

Cloning and functional characterization of recombinant equine P-selectin.

The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the P-selectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Pre-incubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse.

Molecular cloning and characterization of bovine P-selectin glycoprotein ligand-1.

Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH(2)-terminal of PSGL-1, the putative P-selectin binding domain. In the NH(2)-terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular P-selectin binding domain.

Prognostic value of clinicopathologic variables obtained at admission and effect of antiendotoxin plasma on survival in septic and critically ill foals.

This prospective study compared survival rates of critically ill and septic foals receiving 1 of 2 different types of commercial equine plasma and analyzed admission variables as possible predictors of survival. Standardized clinical, hematologic, biochemical, and hemostatic admission data were collected and foals received either conventional commercially available hyperimmune equine plasma or equine plasma specifically rich in antiendotoxin antibodies in a double-blinded, coded fashion. Sepsis was defined as true bacteremia or sepsis score >11. Overall survival rate to discharge was 72% (49/68). Foals that were nonbacteremic and demonstrated a sepsis score of < or = 11 at admission had a 95% (18/19) survival rate. The survival rate to discharge for septic foals was 28/49 (57%), with truly bacteremic foals having a survival rate of 58% (14/24), whereas that for nonbacteremic, septic foals was 56% (14/25). Sensitivity and specificity for sepsis score >11 as a predictor of bacteremia were 74 and 52%, respectively. For the entire study population, a higher survival rate to discharge was documented for those foals receiving hyperimmune plasma rich in antiendotoxin antibodies (P = .012, odds ratio [OR] 6.763, 95% confidence interval [CI]: 1.311, 34.903). Administration of plasma rich in antiendotoxin antibodies also was associated with greater survival in septic foals (P = .019, OR 6.267, 95% CI: 1.186, 33.109). Statistical analyses demonstrated that, among 53 clinical and clinicopathologic admission variables, high sepsis score (P < .001), low measured IgG concentration (P = .01), high fibrinogen concentration (P = .018), low segmented neutrophil count (P = .028), and low total red blood cell numbers (P = .048) were the most significant predictors of overall mortality.

The Impact of Lactobacillus casei on the Composition of the Cecal Microbiota and Innate Immune System Is Strain Specific.

The probiotic function to impact human health is thought to be related to their ability to alter the composition of the gut microbiota and modulate the human innate immune system. The ability to function as a probiotic is believed to be strain specific. Strains of Lactobacillus casei are commonly utilized as probiotics that when consumed alter the composition of the gut microbiota and modulate the host immune response. L. casei strains are known to differ significantly in gene content. The objective of this study was to investigate seven different L. casei strains for their ability to alter the murine gut microbiota and modulate the murine immune system. C57BL/6 mice were fed L. casei strains at a dose of 108 CFU/day/mouse for seven days and sacrificed 3.5h after the last administration. The cecal content and the ileum tissue were collected for microbiota analysis and immune profiling, respectively. While 5 of the L. casei strains altered the gut microbiota in a strain specific manner, two of the strains did not alter the overall cecal microbiota composition. The observed changes cluster into three groups containing between 1 and 2 strains. Two strains that did not affect the gut microbiota composition cluster together with the control in their impact on pattern recognition receptors (PRRs) expression, suggesting that the ability to alter the cecal microbiota correlates with the ability to alter PRR expression. They also cluster together in their impact on the expression of intestinal antimicrobial peptides (AMPs). This result suggests that a relationship exists between the capability of a L. casei strains to alter the composition of the gut microbiota, PRR regulation, and AMP regulation.

Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X(7).

Previous reports about the nucleotide receptor P2X(7), which exhibits ion channel and pore-forming activity and is known to promote IL-1beta processing, have centered largely on its role in macrophage function, whereas its participation in monocyte activity has been unclear. However, because extracellular ATP has been shown to affect monocytes with respect to IL-1beta release, we hypothesized that the P2X(7) receptor is also present and functional in a subpopulation of blood monocytes. Flow cytometric analysis revealed that about 70% of monocytes isolated from normal human donors expressed the P2X(7) receptor. Activation of P2X(7) receptor-associated pore formation by the agonist BzATP resulted in a 9- to 15-fold increase in the uptake of the membrane-impermeant fluorescent dye YO-PRO, and this dye uptake is markedly inhibited by the P2X(7) receptor antagonists KN-62 and oATP. Evidence supporting the presence of the functional P2X(7) receptor in monocytes also includes the observation that BzATP exposure results in a dose-dependent increase in the activation of mitogen-activated 2protein kinases and the nuclear translocation of the transcription factor NF-kappaB in human monocytes and in THP-1 human monocytic cells. Furthermore, treatment of monocytes with BzATP induced the expression of cyclooxygenase-2 (COX-2) and tissue factor, which are two important endpoints that have not been previously shown to be regulated by nucleotide receptor action in monocytes. Together, these data indicate that a subpopulation of human monocytes express P2X(7) receptors that are functional with respect to pore formation, signal transduction, and mediator production, further supporting a key role for this nucleotide receptor in host immune responses.

The Effect of Lactobacillus casei 32G on the Mouse Cecum Microbiota and Innate Immune Response Is Dose and Time Dependent.

Lactobacilli have been associated with a variety of immunomodulatory effects and some of these effects have been related to changes in gastrointestinal microbiota. However, the relationship between probiotic dose, time since probiotic consumption, changes in the microbiota, and immune system requires further investigation. The objective of this study was to determine if the effect of Lactobacillus casei 32G on the murine gastrointestinal microbiota and immune function are dose and time dependent. Mice were fed L. casei 32G at doses of 106, 107, or 108 CFU/day/mouse for seven days and were sacrificed 0.5h, 3.5h, 12h, or 24h after the last administration. The ileum tissue and the cecal content were collected for immune profiling by qPCR and microbiota analysis, respectively. The time required for L. casei 32G to reach the cecum was monitored by qPCR and the 32G bolus reaches the cecum 3.5h after the last administration. L. casei 32G altered the cecal microbiota with the predominance of Lachnospiraceae IS, and Oscillospira decreasing significantly (p < 0.05) in the mice receiving 108 CFU/mouse 32G relative to the control mice, while a significant (p < 0.05) increase was observed in the prevalence of lactobacilli. The lactobacilli that increased were determined to be a commensal lactobacilli. Interestingly, no significant difference in the overall microbiota composition, regardless of 32G doses, was observed at the 12h time point. A likely explanation for this observation is the level of feed derived-nutrients resulting from the 12h light/dark cycle. 32G results in consistent increases in Clec2h expression and reductions in TLR-2, alpha-defensins, and lysozyme. Changes in expression of these components of the innate immune system are one possible explanation for the observed changes in the cecal microbiota. Additionally, 32G administration was observed to alter the expression of cytokines (IL-10rb and TNF-α) in a manner consistent with an anti-inflammatory response.

Equine platelet CD62P (P-selectin) expression: a phenotypic and morphologic study.

Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet-leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl-L-prolyl-L-arginyl-L-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study alpha-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20-25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet-leukocyte mediated vascular injury in the horse appears warranted.

Effect of inhaled endotoxin on cardiopulmonary function and E-selectin expression in pigs.

OBJECTIVE: To evaluate the effect of controlled exposure to inhaled lipopolysaccharides (LPS) on the pulmonary inflammatory response of anesthetized pigs. ANIMALS: Forty-seven 8- to 12-week-old domestic pigs. PROCEDURE: Pigs were anesthetized with pentobarbital, instrumented for measurement of cardiopulmonary function, and randomly assigned to receive saline (0.9% NaCI) solution or 0.25, 0.5, or 1.0 microg of LPS/kg/h for 2 or 6 hours via nebulization through the endotracheal tube. Cardiopulmonary variables were measured, ex vivo neutrophil superoxide production determined, and postmortem assessment for pulmonary neutrophil influx and modulation of adhesion molecule (E-selectin) expression was done. RESULTS: Mild changes in cardiopulmonary function were observed in response to inhaled LPS in the 2-and 6-hour groups. In pigs inhaling LPS (0.5 or 1.0 microg/kg/h) for 6 hours, there was significant pulmonary neutrophil influx observed postmortem. An increase in expression of E-selectin on pulmonary endothelial cells after 6 hours of LPS inhalation (0.5 microg/kg/h) was also observed. In contrast, there was no significant influx of neutrophils or expression of E-selectin in lungs from pigs inhaling LPS for 2 hours. CONCLUSIONS AND CLINICAL RELEVANCE: inhalation of LPS resulted in localized pulmonary inflammation characterized by neutrophil influx and increased expression of the endothelial cell adhesion molecule, E-selectin. It may be possible to relate our experimental findings to the clinical consequences of airborne LPS exposure in swine confinement facilities.

Plasma endotoxin concentration in horses: a methods study.

BACKGROUND: Accurate determination of plasma endotoxin concentration is critical for ex vivo and in vitro cellular and molecular studies of endotoxemia in horses. However, reports are conflicting with respect to anticoagulant, handling, and sample preparation. OBJECTIVE: The purpose of this study was to determine the effect of blood sample fraction and handling time on measurement of endotoxin concentration in horses. METHODS: Whole blood, anticoagulated with 3.8% (0.12 M) sodium citrate (9:1), was collected from 5 healthy horses. Whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma (PPP) were spiked with endotoxin (2 EU/mL). Endotoxin-spiked WB samples were centrifuged immediately to generate PRP for measurement. Endotoxin concentration was subsequently measured by Limulus amebocyte assay at 0, 15, 30, 45, and 60 minutes. Assays were performed in triplicate and results were analyzed using Student's t-test, with significance set at P <.05. RESULTS: Mean endotoxin concentrations in 2 EU/mL-spiked WB were significantly different from those in PPP at all time points tested. Recovery of endotoxin in PRP generated from WB was significantly diminished after just 15 minutes. CONCLUSION: PRP generated from WB is significantly more reliable than PPP in determining endotoxin concentration ex vivo. Measurement of endotoxin in PRP generated from WB was significantly diminished after 15 min, identifying a time frame within which to process blood samples for endotoxin analysis.

Treatment with aspirin or clopidogrel does not affect equine platelet expression of P selectin or platelet-neutrophil aggregates.

Inflammation-induced P-selectin (CD62P) expression on platelets and endothelial cells facilitates interactions among platelets and polymorphonuclear leukocytes (PMN), and can also promote coagulation. The effects of clopidogrel and aspirin (ASA) on equine platelet CD62P expression were investigated. Six horses were treated in a cross-over design with clopidogrel (2mg/kg PO q 24) or ASA (5mg/kg PO q 24h) for 5 days. Platelets collected at 24, 72, 96, 120, and 168 h after the initiation of therapy were stimulated using 0.1 µg/mL thrombin, followed by flow cytometric analysis using anti-CD41/61 and anti-equine CD62P antibodies. Platelet-PMN aggregates were also enumerated. Baseline CD62P positive platelet numbers were not different between groups (mean ± SD): 4254 ± 1785 (clopidogrel) and 3600 ± 1780 (ASA, P=0. 435). Although expression tended to decrease, there were no significant changes in CD62P+platelets after treatment with either drug (clopidogrel P=0.139, ASA P=0.161). There was also no difference in platelet-PMN aggregates during or after treatment with ASA (P=0.513) or clopidogrel (P=0.543). Due to small numbers of horses, this study may have been underpowered to detect a true decrease in expression, and differences between therapies may have been more pronounced if this study had evaluated horses with systemic inflammation.

Secondary renal tubular acidosis in a Hereford calf.

A 3-month-old Hereford heifer calf was presented for lethargy. Blood gas analysis and plasma biochemical testing revealed severe metabolic acidosis, azotemia, hyponatremia, hyperchloremia, and normal anion gap. Results of a urinalysis were consistent with acute tubular necrosis with inadequate acidification of urine based on the degree of acidemia. Salmonella enterica serovar agona was cultured from both urine and feces. The calf was treated with intravenous polyionic fluids, bicarbonate, and antimicrobials. Acidosis and azotemia resolved, and 4 months following initial presentation the heifer was clinically normal.

Equine PSGL-1 modifications required for P-selectin binding.

Equine PSGL-1 (ePSGL-1) is widely expressed on equine PBMC as a homodimer with sialylation (sLeX) modifications that contribute to P-selectin binding affinity. To investigate the role of other potential post-translational modifications required for high-affinity P-selectin binding, ePSGL-1 was transfected into CHO cells expressing equine FucT-VII and/or C2GnT. P-selectin-IgG chimera binding by ePSGL-1 transfected into CHO cells only occurred when both FucT-VII and C2GnT were expressed, establishing that fucosylation and core-2 branching are required as post-translational modifications for high-affinity P-selectin binding. However, enzymatic removal of N-glycans or site and/or point-mutation preventing N-glycan addition did not inhibit P-selectin binding, indicating that N-glycosylation is not required. Taken together, we hypothesized that sialylation, fucosylation, or core-2 branching must occur on O-glycans. The presence of numerous serine/threonine residues in the ePSGL-1 extracellular domain suggests several potential O-glycans attachment sites. P-selectin binding was also susceptible to OSGP cleavage, providing evidence for the existence of clustered, sialyated O-glycans on ePSGL-1. Because OSGP eliminated ePSGL-1 precipitation the P-selectin binding domain of ePSGL-1 must contain clustered, sialyated, fucosylated, and core-2 branched O-glycans. Using point-mutation deletion techniques, the binding domain was determined to reside between residues 48 and 100 of ePSGL-1. Sulfation, a critical modification for human PSGL-1 binding to P-selectin, was not necessary for equine P-selectin binding, while dimerization of ePSGL-1 was critical. These species-specific features of equine PSGL-1 provide new information that advances our understanding of high-affinity P-selectin binding mediated mononuclear cell trafficking.

Identification of equine P-selectin glycoprotein ligand-1 (CD162).

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19-43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14-16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.

Bovine platelets activated by Haemophilus somnus and its LOS induce apoptosis in bovine endothelial cells.

Haemophilus somnus is a bacterial pathogen that causes respiratory disease and vasculitis in cattle. Thrombotic meningoencephalitis (TME) and other severe forms of H. somnus-mediated vascular disease are characterized histopathologically by vasculitis, thrombosis, and infiltration of polymorphonuclear cells. It has been reported previously that activated human platelets express CD40L, FasL and P-selectin (CD62P). We hypothesized that if these surface markers are up-regulated on bovine platelets after in vitro exposure to H. somnus and its lipooligosaccharide (LOS), they might contribute to endothelial cell damage. Using flow cytometry, we demonstrated low baseline expression of these molecules by bovine platelets and increased expression following in vitro stimulation with ADP, H. somnus or H. somnus LOS. H. somnus stimulated platelets were capable of causing apoptosis in endothelial cells as measured by Hoechst-33342 staining and caspase-3 activity. If these events occur in vivo, they might promote vascular damage and endothelial cell apoptosis, leading to the development of vasculitis and thrombosis that characterize bovine H. somnus infection.