Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: I. Clinical, inflammatory, and metabolic response.

Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, ß-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and ß-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.

Hepatic effects of rumen-protected branched-chain amino acids with or without propylene glycol supplementation in dairy cows during early lactation.

Essential amino acids (EAA) are critical for multiple physiological processes. Branched-chain amino acid (BCAA) supplementation provides energy substrates, promotes protein synthesis, and stimulates insulin secretion in rodents and humans. Most dairy cows face a protein and energy deficit during the first weeks postpartum and utilize body reserves to counteract this shortage. The objective was to evaluate the effect of rumen-protected BCAA (RP-BCAA; 375 g of 27% l-leucine, 85 g of 48% l-isoleucine, and 91 g of 67% l-valine) with or without oral propylene glycol (PG) administration on markers of liver health status, concentrations of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB) in plasma, and liver triglycerides (TG) during the early postpartum period in dairy cows. Multiparous Holstein cows were enrolled in blocks of 3 and randomly assigned to either the control group or 1 of the 2 treatments from calving until 35 d postpartum. The control group (n = 16) received 200 g of dry molasses per cow/d; the RP-BCAA group (n = 14) received RP-BCAA mixed with 200 g of dry molasses per cow/d; the RP-BCAA plus PG (RP-BCAAPG) group (n = 16) received RP-BCAA mixed with 200 g of dry molasses per cow/d, plus 300 mL of PG, once daily from calving until 7 d in milk (DIM). The RP-BCAA and RP-BCAAGP groups, on average (± standard deviation), were predicted to receive a greater supply of metabolizable protein in the form of l-Leu 27.4 ± 3.5 g/d, l-Ile 15.2 ± 1.8 g/d, and l-Val 24.2 ± 2.4 g/d compared with the control cows. Liver biopsies were collected at d 9 ± 4 prepartum and at 5 ± 1 and 21 ± 1 DIM. Blood was sampled 3 times per week from calving until 21 DIM. Milk yield, dry matter intake, NEFA, BHB, EAA blood concentration, serum chemistry, insulin, glucagon, and liver TG and protein abundance of total and phosphorylated branched-chain ketoacid dehydrogenase E1α (p-BCKDH-E1α) were analyzed using repeated measures ANOVA. Cows in the RP-BCAA and RP-BCAAPG groups had lower liver TG and lower activities of aspartate aminotransferase and glutamate dehydrogenase during the first 21 DIM, compared with control. All cows, regardless of treatment, showed an upregulation of p-BCKDH-E1α at d 5 postpartum, compared with levels at 21 d postpartum. Insulin, Met, and Glu blood concentration were greater in RP-BCAA and RP-BCAAPG compared with control during the first 35 DIM. Therefore, the use of RP-BCAA in combination with PG might be a feasible option to reduce hepatic lipidosis in dairy cows during early lactation.

The effect of different treatments for early-lactation hyperketonemia on liver triglycerides, glycogen, and expression of key metabolic enzymes in dairy cattle.

Despite the widespread use of treatments for postpartum hyperketonemia in dairy cows, there is currently a lack of evidence comparing their effects on both the resolution of hyperketonemia and the potential effects on the liver of affected animals. The objective of our work was to investigate the effect of commonly used hyperketonemia treatments on hepatic triglyceride and glycogen content as well as on the mRNA and protein abundance of key enzymes involved in gluconeogenesis, ketogenesis, and lipid metabolism. Multiparous Holstein cows between 3 and 9 d in milk were screened 3 times per week and enrolled in the study when whole-blood ß-hydroxybutyrate concentrations measured ≥1.2 mmol/L. Cows were randomly allocated to 1 of 4 groups: (1) 500 mL of a 50% d-glucose solution intravenously once a day for 3 d (n = 8), (2) 300 mL of propylene glycol orally once a day for 3 d (n = 8), (3) 500 mL of a 50% d-glucose solution intravenously and 300 mL of propylene glycol orally once a day for 3 d (n = 8), or (4) an untreated control group (n = 8). Liver biopsies were taken on the day of enrollment as well as on the day following completion of treatments. Liver triglyceride and glycogen content were determined by colorimetric and fluorometric methods, respectively. Gene and protein expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase 1, glucose-6-phosphatase, 3-hydroxy-3-methylglutaryl-CoA synthase 2, acetyl-CoA carboxylase, and carnitine palmitoyltransferase 1A were compared between groups and time points using quantitative reverse transcriptase PCR and Western blotting techniques, respectively. In addition, the ratio of light chain 3B II:I was determined by Western blotting. Plasma samples from both time points for each enrolled cow were submitted for chemistry analysis. Data were analyzed using a repeated-measures ANOVA taking into account the paired nature of the data, and differences between all groups and time points were controlled for multiple comparisons using the Tukey procedure. No difference was found in triglyceride or glycogen concentration between treatment groups. The gene expression of pyruvate carboxylase decreased in the group receiving both treatments, whereas protein expression of this enzyme increased in all groups over time. The autophagy marker light chain 3B II:I decreased in the group receiving both glucose and propylene glycol. No other changes in gene or protein expression of key hepatic enzymes were associated with treatments. We conclude that intravenous glucose and oral propylene glycol, commonly used treatments for ketosis in postpartum dairy cows, administered alone or in combination for a duration of 3 d did not have important beneficial or detrimental effects on selected indicators of liver composition and function in cows with hyperketonemia.

Diagnostic performance of cytology for assessment of hepatic lipid content in dairy cattle.

The objective of our study was to characterize the diagnostic performance of cytology for assessing hepatic lipid content (HLC) in dairy cows by comparing microscopic evaluation of lipid vacuolation in touch imprint slide preparations of liver biopsies with quantitative measurement of triglyceride concentration ([TG]; mg/mg of wet weight) in paired biopsy samples. Our study also sought to compare the diagnostic performance of liver cytology, plasma nonesterified fatty acid concentration ([NEFA]), and plasma ß-hydroxybutyrate concentration ([BHB]) derived from a measurement performed on whole blood, for assessing HLC. Chemical extraction of TG from liver tissue remains the gold standard for quantifying HLC, largely because available blood tests, although useful for detecting some types of pathology, such as increased lipid mobilization, ketosis, or hepatocellular injury, are nonspecific as to etiology. Veterinary practitioners can sample bovine liver for cytological evaluation in a fast, minimally invasive, and inexpensive manner. Thus, if highly predictive of HLC, cytology would be a practical diagnostic tool for dairy veterinarians. In our study, liver biopsy samples from Holstein cows (219 samples from 105 cows: 52 from cows 2 to 20 d prepartum, 105 from cows 0 to 10 d in milk, 62 from cows 18 to 25 d in milk) were used to prepare cytology slides and to quantify [TG] using the Folch extraction method followed by the Hantzch condensation reaction and spectrophotometric measurement. An ordinal scale (0-4) based on amount of hepatocellular cytoplasm occupied by discrete clear vacuoles was used by 3 blinded, independent observers to rank HLC in Wright-Giemsa-stained slides. Interobserver agreement in cytology scoring was good. Corresponding plasma [NEFA] and [BHB] measurements were available for 187 and 195 of the 219 samples, respectively. Liver [TG] correlated more strongly with cytology score than with NEFA or BHB, and receiver operating characteristic curve analysis showed that cytology had better diagnostic performance than either NEFA or BHB for correctly categorizing [TG] at thresholds of 5, 10, and 15%. Hepatic lipidosis in high-producing dairy cows is of major clinical and economic importance, and this study demonstrates that cytology is an accurate means of assessing HLC. Additional work is indicated to evaluate the diagnostic utility of liver cytology.

The effect of different treatments for early-lactation hyperketonemia on blood ß-hydroxybutyrate, plasma nonesterified fatty acids, glucose, insulin, and glucagon in dairy cattle.

Despite increased efforts in preventing the occurrence of metabolic disorders in transition cows, hyperketonemia remains a frequent early-lactation metabolic disease affecting an average of 40% of cows in herds in the United States. Despite the demonstrated economic effect of this disorder, controlled clinical trials comparing different treatment strategies in affected cows are lacking. The objective of our study was to investigate the effect of treatment with intravenous glucose, oral propylene glycol, or a combination of both on the reduction in blood ß-hydroxybutyrate (BHB) concentrations of early-lactation hyperketonemic dairy cows. Multiparous Holstein cows between 3 to 9 d in milk were screened for hyperketonemia using a handheld meter 3 times per week, and enrolled at whole blood BHB concentration ≥1.2 mmol/L to 1 of 4 treatment groups: (1) 500 mL of a 50% dextrose solution i.v. once daily for 3 d (GLU, n = 9), (2) 300 mL of propylene glycol as a drench once daily for 3 d (PG, n = 9), (3) a combination treatment of a 500 mL of 50% dextrose solution i.v. and 300 mL of propylene glycol orally once daily for 3 d (GLU+PG, n = 8), or (4) an untreated control group (CTRL, n = 8). Blood samples were collected immediately before as well as at 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h after administration of the first treatment through a jugular catheter and 3 times per week thereafter from coccygeal vessels. Concentrations of BHB were measured in whole blood, and plasma samples were analyzed for glucose, fatty acid (NEFA), insulin, glucagon, and electrolyte concentrations. The EDTA-anticoagulated blood samples were assessed for red blood cell indices, and smears were made for evaluation of red blood cell morphology. Outcomes were analyzed using repeated measures analysis. Overall least squares means (95% CI) of whole blood BHB concentrations between 1 h and d 11 relative to first treatment were 1.11 (0.95 to 1.30), 1.26 (1.07 to 1.47), 0.96 (0.81 to 1.13), and 1.53 (1.30 to 1.80) mmol/L for the GLU, PG, GLU+PG, and CTRL groups, respectively. Treatment with both glucose and propylene glycol led to a greater magnitude and more prolonged decrease in BHB concentrations compared with individual treatments. The NEFA and glucagon concentrations were lower immediately after treatment in GLU and GLU+PG groups compared with CTRL, and treatment with both glucose and propylene glycol was associated with a greater increase in glucose and insulin concentrations immediately after treatment compared with CTRL and GLU treatment alone. Treatments did not lead to differences in plasma mineral concentrations. We conclude that treatments varied in the magnitude of decreasing blood BHB concentrations in hyperketonemic postpartum cows, with the greatest decline after treatment with a combination of intravenous glucose and oral propylene glycol.

Impact of dietary plane of energy during the dry period on lipoprotein parameters in the transition period in dairy cattle.

The high energy demands of dairy cows during the transition period from late gestation into early lactation can place them at an increased risk for the development of metabolic and infectious diseases. Modification of the dry period diet has been investigated as a preventive means to minimize the detrimental aspects of metabolic shifts during the transition period. Studies investigating the impact of dry period diet on lipid parameters during the transition period have largely focused on markers of lipolysis and ketogenesis. Total cholesterol declines during the periparturient period and increases in early lactation. The impact total energy in the dry period diet has on the ability of the cow to maintain total serum cholesterol, as well as its natural high-density lipoprotein-rich status, during this metabolically challenging window is not clear. The impact of lipoproteins on inflammation and immune function may have a clinical impact on the cow's ability to ward off production-related diseases. In this study, we hypothesized that the provision of adequate, but not excessive, total metabolizable energy, would better allow the cow to maintain total cholesterol and a higher relative proportion of HDL throughout the transition period. Cows were allocated to one of three dry period dietary treatment groups following a randomized block design. Total serum triglycerides, cholesterol and lipoprotein fractions were measured on a weekly basis from approximately 7 weeks pre-calving to 6 weeks post-calving. The cows on the high energy diet maintained total serum cholesterol as compared to the cows provided a lower energy diet, but there was no significant increase in the LDL fraction of lipoproteins between diet treatment groups.

Evaluation of a point-of-care calcium device in bovine plasma and serum.

Hypocalcemia is a common metabolic disease in dairy cows, and it is defined as total calcium (tCa) blood concentration <2.0 mmol/L. The alternatives for the gold standard test to measure tCa in bovine blood are limited. Therefore, our objective was to compare the performance of the calcium (Ca) point-of-care compact analyzer (POC; ARKRAY Inc.) device with the gold standard method to measure bovine blood tCa concentration. Blood samples (n = 151) from dairy cows were collected within 24 h postpartum from multiparous and primiparous dairy cows for serum and plasma. Then, serum and plasma were stored at -80°C until further analyses with the gold standard method on an automatic analyzer (Cobas C501 analyzer; Roche Diagnostics) and the POC device. The tCa blood concentration was measured in the laboratory in plasma and serum samples using both methods within 10 mo of sample collection. Correlation coefficients (Spearman), coefficients of variation (CV, %), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, Passing and Bablok regression, and Bland-Altman agreement test were performed between the gold standard and the POC device. The range and median tCa plasma concentrations measured with the POC device were 1.1 to 2.8 mmol/L and 2.4 mmol/L, respectively. The range and median tCa serum concentrations measured with the POC device were 1.1 to 2.7 mmol/L and 2.3 mmol/L, respectively. The tCa blood concentrations range and median with the gold standard were 1.1 to 2.6 mmol/L and 2.3 mmol/L. The hypocalcemia prevalence of our study population was 11.2%. The CV were 1.89% and 0.55% for low and high tCa in plasma samples measured with the POC, respectively. The CV were 2.57% and 1.58% for low and high tCa in serum, respectively. The Spearman correlation coefficient showed a strong correlation between the gold standard and the POC device for both serum and plasma tCa concentration. The sensitivity of the POC device for both plasma (41.1%) and serum (64.7%) Ca was poor. However, the specificity of the POC device was perfect in plasma (99.2%) and serum (99.2%). The PPV in plasma and serum were 87.5% and 91.6%, respectively. Negative predicted values were 93.0% and 95.6% in plasma and serum. The mean (95% CI) difference between the gold standard and the POC device in plasma and serum were 0.35 (-0.52, 1.23) mmol/L and 0.19 (-0.53, 0.92) mmol/L, respectively. Finally, we observed a strong correlation between the POC device and the gold standard method for tCa plasma and serum. However, the clinical application of the POC device should be carefully considered because its ability to detect cows with hypocalcemia in serum or plasma samples was poor. However, the device performed better than previously analyzed POC devices and needs further improvement to be a valuable tool for the dairy industry.

Agarose gel electrophoresis determination of bovine lipoproteins compared with a wet chemistry method.

Quantification of serum lipoproteins provides information relative to the overall metabolic health, degree of lipid mobilization, and hepatic function of dairy cattle. Automated assays performed on benchtop chemistry analyzers and commercially available kits use reagents developed for human lipoproteins. The substantial physical and chemical differences between bovine and human lipoproteins potentially confounds the use of these assays in evaluating bovine lipoproteins. In this study, we prospectively analyzed serum lipoproteins from 56 Holstein cows using horizontal slab agarose gel electrophoresis to semi-quantify the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions by optical densitometry. Ultracentrifugation was used to confirm the electrophoretic separation pattern of the lipoproteins. The values obtained using the electrophoretic method were compared with values obtained by direct measure of HDL cholesterol, total cholesterol, and triglyceride (TG) concentrations on a Roche chemistry analyzer, and calculated LDL cholesterol. Correlation between these methods was poor for HDL (Passing-Bablok regression line: y = 30.31 + 0.853x) and could not be calculated for LDL. Automated HDL values were equal to, or higher than, the total cholesterol concentration in 25 of the 56 samples. The TG concentrations were above the reference interval in 18 samples, and these samples had an average of 96% of the cholesterol measured as HDL by the automated method, and 78% HDL by electrophoresis. Given that it is physiologically impossible to have more cholesterol within the HDL fraction than in the total serum fraction, and the increased proportion of TG found in LDL and very-low-density lipoprotein, our results draw into question the accuracy of the Roche automated assay in quantifying bovine lipoprotein fractions.

In vitro Inhibition of Canine Complement-Mediated Hemolysis.

BACKGROUND: Immune-mediated hemolytic anemia (IMHA) is the most common hematologic immune-mediated disease in dogs. Complement fixation on erythrocytes causes hemolysis. Complement inhibition decreases hemolysis in people with the hemolytic disease and also may prove effective in treating IMHA in dogs. HYPOTHESIS/OBJECTIVES: Evaluate the in vitro efficacy of 2 complement inhibitors used in humans against canine complement. METHODS: The inhibitory activity of the C3-inhibitor compstatin and recombinant human C1-esterase inhibitor (C1-INH) was evaluated using an in vitro hemolytic assay and spectrophotometric measurement of released hemoglobin. Dose-response curves for each inhibitor were generated. RESULTS: Compstatin decreased approximately 50% of canine complement-mediated hemolysis in initial experiments. This inhibition largely was lost when a new lot of drug was purchased. C1-INH showed a dose-dependent inhibition. The highest concentration of C1-INH tested (500 µg/mL) decreased >80% of canine complement-mediated hemolysis, and the lowest concentration tested (31.25 µg/mL) decreased hemolysis >60%. CONCLUSIONS AND CLINICAL IMPORTANCE: Human C1-INH is a robust inhibitor of canine complement-mediated hemolysis, whereas compstatin was minimally and variably effective. Human C1-INH may substantially decrease complement-mediated hemolysis in dogs with IMHA and warrants further investigation.

Endothelial cells as active participants in veterinary infections and inflammatory disorders.

Endothelial cells were once viewed as relatively inert cells lining the vasculature. They are now recognized as active and responsive regulators of coagulation, platelet adhesion, fluid homeostasis, wound healing, leukocyte extravasation and vascular tone. Endothelial cells play a key role in the host response to infectious agents by regulating leukocyte trafficking, producing inflammatory cytokines and presenting antigen in association with major histocompatibility class II (MHC II) molecules. A number of infectious agents have a tropism for endothelial cells. Infection of endothelial cells can promote thrombosis, vascular leakage, and increased adherence and emigration of leukocytes. Furthermore, activation of a systemic inflammatory response, in the absence of direct endothelial cell infection, can also lead to endothelial cell dysfunction. The purpose of this review is to highlight the interactions between endothelial cells and infectious or inflammatory agents that contribute to coagulation disturbances, vasculitis and edema. A select group of viral and bacterial pathogens will be used as examples to demonstrate how endothelial cell dysfunction contributes to the pathogenesis of infectious and inflammatory disorders.

Serum lipoprotein changes in dogs with renal disease.

BACKGROUND: People with renal disease develop a dyslipidemia that contributes to progression of renal injury and development of cardiovascular disease. Lipoproteins in dogs with renal disease have not been investigated. HYPOTHESIS: Dogs with chronic kidney disease (CKD) have dyslipidemia characterized by increased lower density lipoproteins and decreased high-density lipoproteins (HDLs). The degree of dyslipidemia is positively correlated with severity of disease, as reflected by serum creatinine concentration. ANIMALS: Prospective study of client-owned dogs presented to the Cornell University Hospital for Animals: 29 dogs with confirmed CKD, 5 dogs with nephrotic syndrome (NS), and 12 healthy control dogs presented for routine vaccinations, dental cleaning, or owned by students. METHODS: Lipoprotein electrophoresis was used to quantify relative proportions of the 3 main classes of lipoproteins in canine serum: low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), and HDL. Serum cholesterol and creatinine concentrations; urinalysis and urine protein-to-creatinine ratio were measured by standard methods. RESULTS: Dyslipidemia was consistently found in dogs with CKD and NS and was characterized by a decrease in HDL and variable increases in LDL and VLDL. Dogs with NS had a proportionately greater increase in the VLDL fraction, as compared with dogs with CKD. CONCLUSION AND CLINICAL IMPORTANCE: Dyslipidemia similar to that documented in people with renal disease occurs in dogs with CKD, despite serum cholesterol concentrations often being within the reference interval. The contribution of altered lipoproteins to the pathogenesis of renal disease in dogs warrants additional study.

Viable "Haemophilus somnus" induces myosin light-chain kinase-dependent decrease in brain endothelial cell monolayer resistance.

"Haemophilus somnus" causes thrombotic meningoencephalitis in cattle. Our laboratory has previously reported that H. somnus has the ability to adhere to, but not invade, bovine brain endothelial cells (BBEC) in vitro. The goal of this study was to determine if H. somnus alters brain endothelial cell monolayer integrity in vitro, in a manner that would be expected to contribute to inflammation of the central nervous system (CNS). Monolayer integrity was monitored by measuring transendothelial electrical resistance (TEER) and albumin flux. BBEC incubated with H. somnus underwent rapid cytoskeletal rearrangement, significant increases in albumin flux, and reductions in TEER. Decreased monolayer TEER was preceded by phosphorylation of the myosin regulatory light chain and was partially dependent on tumor necrosis factor alpha and myosin light-chain kinase but not interleukin-1beta. Neither heat-killed H. somnus, formalin-fixed H. somnus, nor purified lipooligosaccharide altered monolayer integrity within a 2-h incubation period, whereas conditioned medium from H. somnus-treated BBEC caused a modest reduction in TEER. The data from this study support the hypothesis that viable H. somnus alters integrity of the blood-brain barrier by promoting contraction of BBEC and increasing paracellular permeability of the CNS vasculature.

Roles of cellular activation and sulfated glycans in Haemophilus somnus adherence to bovine brain microvascular endothelial cells.

Haemophilus somnus can cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used by H. somnus to migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate that H. somnus adheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherent H. somnus was significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-alpha). Addition of exogenous glycosaminoglycans significantly reduced H. somnus adherence to resting and TNF-alpha-activated BBEC. Heparinase digestion of the endothelial cell's glycocalyx or sodium chlorate inhibition of endothelial cell sulfated glycan synthesis significantly reduced the number of adherent H. somnus. In contrast, addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no inhibitory effect. These findings suggest a critical role for both cellular activation and sulfated glycosaminoglycans in adherence of H. somnus to BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed by H. somnus. Heparin was also shown to bind H. somnus in a 4 degrees C binding assay. These data suggest that heparin-binding proteins on H. somnus could serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface, thus contributing to the ability of H. somnus to infect the bovine CNS.

Localization of astrovirus in experimentally infected turkeys as determined by in situ hybridization.

Twenty-one 3-day-old turkey poults from British United Turkeys of America were orally inoculated with a recently characterized astrovirus, TAstV-2, isolated from turkeys with poult enteritis and mortality syndrome. At 1, 2, 3, 4, 5, 7, and 9 days postinfection (dpi), three inoculated birds were euthanatized, and tissues (intestines, spleen, bursa, and thymus) were collected immediately into 10% neutral buffered formalin. Inoculated birds were diarrheic by 3 dpi, and frothy feces persisted throughout the experimental period. Histologically, there was only slight evidence of enteric damage, which was characterized by mild epithelial necrosis, lamina propria infiltrates, minimal villus atrophy, and mild crypt hyperplasia. In situ hybridization, using a negative sense digoxigenin-labeled riboprobe to the capsid gene of TAstV-2, revealed viral RNA in intestinal epithelial cells at the basal margins of the villi, in distal small intestine, and in cecum at 2 dpi, with subsequent extension to epithelium of the large intestine and proximal small intestine (3-5 dpi). Minimal virus remained by 9 dpi.