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Probiotics may represent a promising approach for reducing Clostridioides (Clostridium) difficile infections (CDIs). A clinical trial conducted by our group demonstrated that CDI patients undergoing adjunctive treatment with Lactobacillus and Bifidobacterium probiotics had a reduction in diarrheal duration and compositional changes in their stool microbiomes. Here, we modified a CDI mouse model to represent clinical outcomes observed in patients and employed this model to identify evidence for the prevention of primary CDI and relapse with the same probiotic. Mice (n = 80) were administered 0.25 mg/ml cefoperazone over 5 days and subsequently challenged with 10(2) C. difficile VPI 10463 spores. A subset of mice (n = 40) were administered 10(8) CFU of probiotics daily alongside cefoperazone pretreatment and until experimental endpoints were reached. Clinical scoring was performed daily on mice and used to evaluate CDI onset and severity. Moderate CDI in mice was defined by survival beyond day 3 postinfection, while mice with severe CDI were those who succumbed to infection prior to day 3 postinfection. Sequencing and analysis of 16S rRNA from stool content were performed to determine compositional alterations to the microbiota. Using total clinical scores, we identified an association between probiotic treatment and delayed onset of primary CDI and relapse by approximately 12 to 24 h (P

Morinda citrifolia (Noni) is a popular traditional medicinal plant consumed in various forms in several countries around the world as a complementary and alternative treatment due to its established health benefits. Noni is rich in bioactive substances and has significantly exhibited pro-oxidant and immunomodulatory effects. In this review, we highlight the pharmacological basis related to the phytochemicals and polysaccharides present in Noni and its potential therapeutic effects. We screened electronic databases such as PubMed, Google Scholar, Scopus for scientific literature. Our results indicate that Noni is beneficial for various diseases with its crude extracts showing therapeutic benefit for a wide range of pathological diseases. We believe that further pharmacological and toxicological studies in addition to well-designed controlled clinical trials can validate Noni to be an effective and novel natural product for prophylactic and therapeutic use of several diseases.

The majority of antibiotic-induced diarrhea is caused by Clostridium difficile ( C. difficile). Hospitalizations for C. difficile infection (CDI) have tripled in the last decade, emphasizing the need to better understand how the organism colonizes the intestine and maintain infection. The mucus provides an interface for bacterial-host interactions and changes in intestinal mucus have been linked host health. To assess mucus production and composition in healthy and CDI patients, the main mucins MUC1 and MUC2 and mucus oligosaccharides were examined. Compared with healthy subjects, CDI patients demonstrated decreased MUC2 with no changes in surface MUC1. Although MUC1 did not change at the level of the epithelia, MUC1 was the primary constituent of secreted mucus in CDI patients. CDI mucus also exhibited decreased N-acetylgalactosamine (GalNAc), increased N-acetylglucosamine (GlcNAc), and increased terminal galactose residues. Increased galactose in CDI specimens is of particular interest since terminal galactose sugars are known as C. difficile toxin A receptor in animals. In vitro, C. difficile is capable of metabolizing fucose, mannose, galactose, GlcNAc, and GalNAc for growth under healthy stool conditions (low Na+concentration, pH 6.0). Injection of C. difficile into human intestinal organoids (HIOs) demonstrated that C. difficile alone is sufficient to reduce MUC2 production but is not capable of altering host mucus oligosaccharide composition. We also demonstrate that C. difficile binds preferentially to mucus extracted from CDI patients compared with healthy subjects. Our results provide insight into a mechanism of C. difficile colonization and may provide novel target(s) for the development of alternative therapeutic agents.

Background Periodic lack of availability and high cost of commercially produced isotonic fluids for intravenous (IV) use in horses have increasingly led to use of home-made or commercially compound fluids by veterinarians. Data regarding the quality control and safety of compounded fluids would be of benefit to equine veterinarians. Objectives To compare electrolyte concentrations, sterility, and endotoxin contamination of commercially available fluids to 2 forms of compounded isotonic crystalloid fluids intended for IV use in horses. Methods Prospective study. Two methods of preparing compounded crystalloids formulated to replicate commercial Plasma-Lyte A (Abbott, Chicago, IL) were compared. One formulation was prepared by a hand-mixed method involving chlorinated drinking water commonly employed by equine practitioners, and the other was prepared by means of ingredients obtained from a commercial compounding pharmacy. The variables for comparison were electrolyte concentrations, sterility, and presence of endotoxin contamination. Results Electrolyte concentrations were consistent within each product but different between types of fluids (P

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.

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 (2 mg/kg PO q 24) or ASA (5 mg/kg PO q 24 h) 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.

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.

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 score11. Overall survival rate to discharge was 72% (49/68). Foals that were nonbacteremic and demonstrated a sepsis score ofor = 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 score11 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.

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.

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.

Although myocardial injury can be a significant component of multiple organ dysfunction (MODS) in association with septicemia in critically ill human patients, it is as yet an undefined clinical entity in equine septicemia. With septicemia as the leading cause of death in neonatal foals, a better understanding of the pathophysiology, diagnosis and treatment of MODS will be important in further improving survival rates. We designed a prospective study to establish normal ranges for cardiac troponin I (cTnI), T (cTnT) and CKMB mass in healthy 24–48 hour old foals, as well as septicemic neonatal foals seen over a 2-year period in a teaching hospital. We also performed a comparison of these biomarkers in surviving and non-surviving septicemic foals. Sepsis was judged on the basis of the presence of any of the 3 following criteria: blood culture positive at admission, admission sepsis score ≥11, or 3 or more sites of infection during hospitalization in foals ≤14 days of age. cTnI was measured by the ACCESS® (Beckman Coulter), cTnT was measured using the Elecsys 2010® Immunoassay (Roche), and CKMB mass measurements were performed using the Elecsys 2010®. Each parameter was described using range and 95th and 50th percentile. Comparisons were made for each parameter between normal and septic foals as well as surviving and non-surviving septic foals using the non-parametric Wilcoxon's rank sum test. Significance was set at p

The determination of prothrombin time (PT) has been standardized for domestic chickens; however, validated tests of coagulation are lacking for nondomestic avian species, limiting the investigation of acquired hemostatic disorders in these species. The purpose of this study was to validate the PT assay for chickens in our laboratory using both fresh and frozen plasma and to apply the assay to psittacine bird plasma to establish reference intervals for PT of Hispaniolan parrots (Amazona ventralis) and umbrella cockatoos (Cacatua alba). We used avian tissue thromboplastin, prepared from brain tissue of 2-week-old chickens, for determining all PT values. The reference intervals for PT of chickens (n = 6) were 7.5–10.6 and 7.0–11.1 seconds for fresh and frozen plasma, respectively. The reference intervals for PT of Hispaniolan parrots (n = 6) were 7.5–13.4 and 9.0–11.3 seconds for fresh and frozen plasma, respectively. The reference intervals for PT of cockatoos (n = 14) differed significantly (P < ....

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% NaCl) solution or 0.25, 0.5, or 1.0 µg 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 µg/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 µg/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.Conclusion 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. (Am J Vet Res2002;63:1302–1308)

Previous reports about the nucleotide receptor P2X7, which exhibits ion channel and pore-forming activity and is known to promote IL-1β 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-1β release, we hypothesized that the P2X7 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 P2X7 receptor. Activation of P2X7 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 P2X7 receptor antagonists KN-62 and oATP. Evidence supporting the presence of the functional P2X7 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-κB 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 P2X7 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.

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.

Phagocytosis of particulate wear debris from arthroplasties by macrophages induces an inflammatory response that has been linked to implant loosening and premature failure of artificial joints. Inflammatory mediators released by phagocytic macrophages such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and prostaglandin E(2) (PGE(2)) are believed to play a central role in the pathogenesis of aseptic loosening. The objective of this study was to characterize titanium alloy particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by cultured monocytes. Peripheral blood monocytes were isolated from healthy human volunteers. Monocytes were cultured in 96-well plates for 24 h, washed, and exposed to three concentrations of titanium particulates and controls from 18-24 h. Supernatants were assayed for TNF-alpha, IL-1beta, IL-6, and PGE(2) activity. Energy dispersive X-ray spectroscopy (EDX) verified the titanium alloy to be Ti6A14V. Scanning electron microscopy (SEM) analysis showed significant titanium particulate heterogeneity with approximately 95% of the particles

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°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

Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has anti-inflammatory effects, we hypothesized that low molecular weight heparin (LMWH) would inhibit PAF-induced activation and chemotaxis in porcine neutrophils. Citrated blood was obtained from pentobarbital-anesthetized pigs, and neutrophils were isolated over a 55%/65% Percoll gradient. The effect of LMWH on basal phorbol myristate acetate (PMA)-induced superoxide (SO) release, as well as its effect on PAF priming for PMA-induced SO release, were investigated. Additionally, the effect of LMWH on PAF-induced chemotaxis of neutrophils across transwell membranes was evaluated. Baseline SO release in response to PMA was .351+/-.046 nmol/10(6) cells/min, and this was decreased to .289+/-.034 nmol/10(6) cells/min by pretreatment with 50 U/mL LMWH. PMA-induced SO production was increased by .240+/-.042 nmol/10(6) cells/min when cells were primed with 10 microM PAF. This priming effect of PAF was reduced significantly by pretreatment of neutrophils with LMWH at 10 and 50 U/mL. Chemotaxis of neutrophils in response to 100 microM PAF was significantly decreased to 70.02+/-6.4% (n = 8) of the control response by pretreatment of cells with 50 U/mL LMWH. We conclude that LMWH has anti-inflammatory effects on porcine neutrophils, which includes attenuation of cell activation and chemotaxis in response to the lipid-derived inflammatory mediator, PAF.

Naturally occurring and experimentally induced ascending colon volvulus (ACV) result in transmural compression, increased microvascular permeability and thrombosis and necrosis of the intestinal mucosa analysis of haemostatic variables in horses with naturally occurring ACV have identified changes in plasma antithrombin III (AT III) activity as the most reliable for diagnosing hypercoagulation and predicting survival. In an experimental pony model of no-flow ACV, which results in microvascular injury and thrombosis, systemic haemostasis was evaluated during ischaemia and reperfusion to determine whether selected haemostatic assays consistently yield results suggestive of altered haemostasis (hypercoagulation). Ten normal adult ponies were randomly divided into two equal groups (Group 1, control; Group 2, experimental volvulus). All experiments were performed under general anaesthesia and were terminal. Haemostatic variables (prothrombin time, PT; activated partial thromboplastin time, APTT, platelet count, fibrinogen concentration, and plasma AT III activity) were measured prior to general anaesthesia, immediately before colonic volvulus, at 60 and 120 min of ischaemia, and at 60 and 120 min of reperfusion. Mean plasma AT III activity decreased in both the control and experimental groups after induction of general anaesthesia. Mean plasma AT III activity continued to decrease after reperfusion in Group 2 ponies and differed significantly (p

The morphologic changes following ascending colon volvulus result from the interaction of inflammatory and coagulation mediators. The objective of this study was to establish a quantifiable histopathologic scoring system to evaluate the serial pathomorphologic changes during ischemia and reperfusion. Such a scoring system could then be applied to subsequent studies designed to attenuate bowel lesions by regulating activity of individual mediators. Ten normal, healthy adult ponies were randomly divided into two equal groups. Following anesthesia and a 30-minute stabilization period, the colon of ponies in the experimental group was twisted 720°: for 2 hours of ischemia and then untwisted for 2 hours of reperfusion. The control ponies underwent 4 hours of anesthesia. Full-thickness colonic biopsy specimens were obtained from the antimessenteric border of the pelvic flexure and prepared for light and transmission electron microscopy at 0, 1, 2, 3, and 4 hours after twisting. The mean histopathology score at 0 hours for control ponies was 1.2 and remained unchanged; the score for the experimental ponies was 1.8 and progressed to 4.2 by 4 hours. At 2 hours of ischemia, there was edema of the submucosa and lamina propria attributable to accumulation of platelets, fibrin, and neutrophils in capillaries of the lamina propria. The morphologic changes in experimental ponies progressed to complete destruction of the mucosa with congestion, hemorrhage, and microvascular thrombosis. Mean histopathology scores of control and experimental ponies were significantly different at 1 hour of ischemia, and lesions progressively worsened through the reperfusion period. The morphologic changes observed in this study accurately reflect those changes reported in horses with naturally occurring colonic volvulus and can serve as a reference for subsequent studies on attenuating bowel injury.

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.

Otitis media is an uncommon condition in South American camelids and is thought to occur more commonly as a consequence of upper respiratory disease rather than an extension of otitis externa as reported in dogs.1–3 Medical treatment of chronic otitis media in alpacas can be challenging because of the sigmoid shape of the external acoustic meatus, which prohibits evaluation of the tympanic membrane, and the multicompartmental tympanic bulla that does not allow for easy surgical drainage.2, 4, 5 Total ear canal ablation (TECA) with lateral bulla osteotomy (LBO) is a widely accepted surgical treatment for end-stage otitis externa, otitis media, and masses confined to the external or middle ear in dogs and cats.6–13 Although surgical treatment for otitis is well described in dogs and cats, there are only sparse reports describing surgical treatment of otitis media in South American camelids.2, 3 These reports document complications including hemorrhage, lack of improvement, and euthanasia2 or involve removal of the pinna,3 which may result in behavioral and social consequences in camelids.3 A modified technique (subtotal ear canal ablation) has been described for dogs and cats with otitis or neoplasia with minimal distal ear canal involvement.14 This technique would be ideal in camelids with otitis media, where the distal ear canal is typically not involved. We report a surgical technique for subtotal ear canal ablation and LBO in an alpaca, modified from that described in dogs. Computed tomography (CT) was used to confirm otitis media and provide relevant anatomy of the external and middle ear canal to guide surgical exploration.

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.

Background: Although adequate colostrum intake and properly used antibiotics can provide much protection for the bovine neonate, increased antibiotic scrutiny and consumer demand for organic products have prompted investigations of natural immunomodulators for enhancing calf health. One plant-based immunomodulator, Morinda citrifolia (noni) fruit, is a well-recognized natural product that has a broad range of immunomodulatory effects. Hypothesis: Neonatal calves fed noni puree would demonstrate whole blood phagocytic capacity in Gram-negative and Gram-positive in vitro assays. Animals: Blood samples from 18 neonatal Holstein bull calves. Methods: Calves were divided into 2 groups: Group 1 comprised control calves, whereas Group 2 received 30 mL of noni puree twice a day in milk replacer. Day 0 blood samples were obtained between 36 and 48 hours of age before the first feeding of puree. Ethylenediaminetetraacetic acid anticoagulated blood was collected from each calf on days 0, 3, 7, and 14. Bactericidal assays were performed to estimate the percentage killing of Escherichia coli and Staphylococcus epidermidis. Results: Blood samples from noni puree-fed calves displayed significantly more E. coli bacterial killing than did controls on day 14, and although differences were not significant on days 0, 3, and 7, bacterial killing progressively increased over time. There was no significant difference between the groups for S. epidermidis killing. Conclusions and Clinical Importance: The immunomodulatory effect of noni puree may prove valuable in the future as production animal antibiotic use becomes more restricted. Additional clinical trials are warranted to investigate the clinical application of noni puree in promoting calf health.

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.

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.

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.

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 technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmortem gravimetric analysis (wet:dry) ratio. To determine whether lung water can be quantitated morphometrically, as accurately as by the commonly used gravimetric analysis, perivascular edema (cuff) area to vessel area ratio was correlated to wet:dry ratio. Anesthetized pigs were given either oleic acid (20 mg/kg/h, intravenously) or physiologic saline. At 4 h, lungs were excised and cuff:vessel and wet:dry ratio analysis was performed. The intermediate lobe was clamped across its main stem bronchus to maintain peak inspiratory inflation, excised, frozen in liquid nitrogen, and stored at -70 degrees C until cryostat sectioning and quantification of perivascular interstitial edema (cuff) area. Gravimetric analysis (wet:dry ratio) was performed on the remaining lung. Mean cuff:vessel and wet:dry analyzes showed that lung water increased significantly (p < .01) in the oleic-acid treated group (4.9 +/- .22 and 6.78 +/- .47, respectively), compared with the saline group (.03 +/- .02 and 2.55 +/- .27, respectively). The correlation coefficient between mean cuff:vessel and wet:dry ratios was .86 (p = .0016). This study demonstrates that cuff:vessel ratio analysis can be used to identify the distribution of edema fluid versus vessel diameter, and seems to be as effective a technique as gravimetric analysis to quantitate lung water changes in acute lung injury models. Moreover cuff:vessel ratio analysis can differentiate modest changes in pulmonary edema by direct quantitation, an important end-point not provided by wet:dry analysis. Therefore, it may be a more sensitive technique when investigating therapeutic interventions in in vivo models of acute lung injury.

Cytokines and eicosanoids are well documented important mediators of endotoxemia. Bicyclic imidazoles are a novel class of nonsteroidal anti-inflammatory compounds that display unique pharmacological profiles by reducing cytokine production and arachidonic acid metabolism. In this study, we evaluated the ability of the bicyclic imidazole, SKF 86002, to attenuate endotoxin-induced cardiopulmonary dysfunction. Pigs were randomly assigned to one of four groups: LPS (n = 5), given .5 microgram/kg/h 055:B5 Escherichia coli lipopolysaccharide (LPS) intravenously (i.v.) for 6 h; saline (n = 5); SKF 86002 (n = 3), given 50 mg/kg SKF 86002 orally 30 min prior to anesthesia; and SKF 86002 + LPS (n = 5). Administration of LPS resulted in cardiopulmonary dysfunction characterized by decreased stroke volume and arterial oxygen tension, and increased room air alveolar-arterial oxygen gradient, pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure. Additionally, LPS administration was associated with leukopenia and increased pulmonary myeloperoxidase activity. Pretreatment with SKF 86002 attenuated LPS induced hypotension, hypoxemia and bronchoconstriction and blocked the pulmonary hypertension. SKF 86002 blocked the LPS-induced increase in myeloperoxidase activity, indicating a reduction in pulmonary neutrophil infiltration, but had no effect on systemic leukopenia. Pretreatment with SKF 86002 significantly attenuated LPS-induced increases in plasma thromboxane B2 and tumor necrosis factor-alpha. We hypothesize that ameliorating effects of SKF 86002 in this endotoxin model of cardiopulmonary dysfunction are related to inhibition of cytokine and eicosanoid biosynthesis.

Particulate wear debris found around joint arthroplasties has been linked to the premature failure of artificial joints. Cytokines and eicosanoids released by macrophages are known mediators of inflammation and osteolysis. Previous in vitro studies of wear debris-mediated inflammation have relied upon poorly characterized particulates and peripheral blood cell fractions of mixed cellular composition. The objective of this study was to utilize titanium particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by isolated monocytes.To mimic wear debris that surrounds artificial joints, titanium alloy particulates (1-3 μm in diameter) were obtained. A submicron fraction of these particulates was isolated by differential sedimentation. The morphology, size and concentration of the resulting population was characterized utilizing scanning electron microscopy. Titanium particulate size was estimated from photomicrographs of calibrated latex microspheres mixed with titanium particles (Figure 1). A known volume of titanium particulates in suspension was applied to a carbon planchette and allowed to dry.