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SUMMARY

Objective

To determine expression of the β2-integrin (CD18) family of adhesion molecules and L-selectin (CD62L) on neutrophils from periparturient cows and calves.

Animals

8 periparturient Holstein cows and 9 Holstein calves.

Procedure

Constitutive CD18 and CD62L expression on neutrophils was determined by flow cytometry, using specific monoclonal antibodies. Platelet-activating factor was used to activate neutrophils in vitro to measure down-regulation of CD62L and up-regulation of CD18 on activated neutrophils.

Results

Mean values for constitutive and platelet-activating factor-stimulated CD18 expression on neutrophils from cows and calves were highest at parturition, then decreased during the first 24 hours after parturition on calf neutrophils, whereas CD62L expression decreased markedly by 9 to 24 hours after parturition on cow and calf neutrophils. Constitutive amounts of CD18 and CD62L on cow neutrophils returned to prepartum values by day 3 after parturition. Amounts of CD18 expression on calves’ neutrophils recovered by 1 week of age, but did not reach original birth values, whereas CD62L expression exceeded birth values by day 3 after parturition. Cows had leukocytosis (neutrophilia), with a doubling of circulating neutrophils 9 hours after calving that was inversely correlated with CD62L expression on neutrophils.

Conclusions and Clinical Relevance

Low amounts of CD62L and CD18 on calf neutrophils and of CD62L on neutrophils from cows for several days after parturition may result in impaired inflammatory response. Low CD62L expression may contribute to increased susceptibility to disease. (Am J Vet Res 1998;59:37–43)

Free access
in American Journal of Veterinary Research

SUMMARY

The effects of 3 days of glucocorticoid administration on bovine blood neutrophil expression of l-selectin and CD18, and on the health status of mammary glands subclinically infected with Staphylococcus aureus were measured in 9 lactating Holsteins. The experiment was a 3 x 3 Latin square cross-over design, with 3 glucocorticoid treatments switched among groups of 3 cows/treatment during 3 periods. Treatments consisted of a vehicle (control, 10 ml of excipient/cow/d), cortisol (7.5,15, and 7.5 mg/cow on days 1, 2, and 3, respectively), and dexamethasone (0.04 mg/kg of body weight/cow/d for total daily dosages that ranged from 21.6 to 33.2 mg). Blood samples for immunostaining and flow cytometric analysis of l-selectin and CD18 and leukograms, as well as foremilk samples for determination of S aureus shedding, somatic cell counts, protein and fat percentages, and daily milk yields were collected repeatedly before, during, and after treatment days. Dexamethasone caused a profound, acute, short-lived down-regulation of l-selectin on neutrophils, which correlated in time to leukocytosis, mature and immature neutrophilias, increased shedding of S aureus in infected glands, and onset of high percentages of fat and protein and decreased milk yields. Dexamethasone also caused profound but delayed down-regulation of neutrophil CD18, which reached nadir simultaneously with reappearance of l-selectin-bearing neutrophils, normalized blood neutrophil counts, markedly high foremilk somatic cell counts and protein percentage, decreased S aureus shedding in milk, and finally, expression of clinical mastitis in some infected quarters. Each of these variables had returned to control (vehicle) values by the ninth (and last) sample collection day. Although cortisol treatment also decreased expression of l-selectin and CD18 on neutrophils, dosages used in this study were not sufficient to alter the number of circulating cells or to convert subclinical mammary gland infections to clinical mastitis. These results suggest that mammary gland health status can be altered by sudden exposure of blood neutrophils to glucocorticoids, because these steroid hormones caused profound down-regulation of the adhesion molecules that direct neutrophil margination and migration through the vascular endothelium. The results also reinforce the potential disease risk of treating infected animals with potent synthetic glucocorticoids, such as dexamethasone.

Free access
in American Journal of Veterinary Research

SUMMARY

The role of interleukin-1 (il-1), interleukin-6 (il- 6), and tumor necrosis factor α during endotoxin-induced mastitis in cows was characterized. Six cows had 10 μg of Escherichia coli lipopolysaccharide infused into 1 mammary gland. Three other cows served as nontreated controls. Within 1.5 to 2.5 hours after infusion, endotoxin caused obvious edema of the mammary gland and increased serum albumin concentration in milk of infused glands 6 times. Milk somatic cell count began to increase 3 to 5 hours after infusion in all treated glands. At 7 hours after infusion, somatic cell counts were increased >10 times, compared with counts in milk from control cows. Pyrexia of >1 C developed in only 1 cow, but all treated cows had serum cortisol concentrations >50 ng/ml in response to endotoxin treatment. High concentrations of il-1 (10 to 600 U/ml) and il-6 (2 to 22 U/ml) were detected in milk of infused glands beginning 2.5 to 4 hours after infusion. Endotoxin did not induce detectable amounts of tumor necrosis factor activity in milk or serum. Swelling and mammary gland permeability changes preceded any detectable increase in il-1 and il-6 activity, indicating that these clinical signs of inflammation were not mediated by these cytokines. Systemic responses and the leukocytic influx into endotoxin-infused glands developed after or concurrently with initial increases in il-1 and il-6 activities in milk. These results suggested that il-1 and il-6 may have a role in mammary gland defenses and in the pathophysiologic changes during endotoxin-induced mastitis.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To develop an efficient and reliable method that accurately differentiates bovine lymphocytes from monocytes in leukograms.

Sample Population—Blood samples from 30 healthy cows and 1 calf with bovine leukocyte adhesion deficiency.

Procedure—Flow cytometric analysis of intracellular complexity and CD45 expression on bovine leukocytes was compared with results for conventional light microscopy methods. Verification of leukocyte subpopulations determined by intracellular complexity and CD45 expression was conducted, using 2-color phenotypic analysis with selected monoclonal antibodies.

Results—The CD45 and side-scatter properties of bovine leukocytes clearly differentiated cell types, including neutrophils, eosinophils, monocytes, and lymphocytes.

Conclusions and Clinical Relevance—This is a rapid assay that is simple to use. More importantly, it is more accurate than the conventional method that involves the use of blood slides and light microscopy, because of the ability of the assay to readily distinguish bovine monocytes and lymphocytes. Rapid preparation of samples and short analysis times allow for efficient and reliable examination of a large number of samples, and the task of viewing slides by light microscopy is eliminated. The labor-savings benefit of this procedure is most apparent in research environments that require frequent processing of batches of blood samples. (Am J Vet Res 2001;62:1740–1744)

Full access
in American Journal of Veterinary Research

Summary

The immunomodulating polypeptide interleukin 1β (il-1β) has been shown to be homologous to osteoclast-activating factor and is capable of stimulating increased osteoclastic bone resorption. This effect prompted an investigation into the potential use of il-1β for prevention of parturient paresis, a disease of dairy cows characterized by hypocalcemia and poor osteoclastic resorption of bone. Six nonpregnant cows were treated with a high dosage of il-1β (166 ng/kg of body weight) every 8 hours for 4 days. The il-1β treatment significantly (P < 0.05) increased urinary hydroxyproline excretion, an index of osteoclast activity, indicating that bone calcium resorption might be stimulated by il-1β treatment of cows. However, il-1β treatment also caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis. The acute, but transient, effect of il-1β treatment was to cause a decrease in plasma calcium and phosphorus concentrations. The pleiotropic effects of il-1β administration negated the positive effects on osteoclastic bone resorption, and indicates that this cytokine may be of minimal benefit for prevention of parturient paresis.

Free access
in American Journal of Veterinary Research

SUMMARY

Six nonpregnant, nonlactating Jersey cows, averaging 4 to 6 years old, were used to evaluate the immunomodulatory effects of recombinant bovine interleukin 1β (rBoIL-1β). Cows were given 166 ng of rBoIL-1β/kg of body weight at 8-hour intervals for 96 hours. Persistent leukocytosis was observed within 3 hours of rBoIL-1 treatment, peaking 24 hours after the first IL-1β injection and returning to baseline values within 72 hours after cessation of IL-1β treatment. Injection of cows with rBoIL-1β stimulated lymphocyte blastogenesis and mitochondrial methyl-thiazoltetrazolium cleavage activity in resting cell cultures. Increases in the aforementioned lymphocyte activities were also observed in stimulated blood mononuclear cell cultures during IL-1β administration. Change in IgM production in cell cultures was not observed during IL-1β treatment. Within 24 hours of the first IL-1β injection, IL-1β mRNA transcription in stimulated blood mononuclear cell cultures was markedly increased, suggesting that IL-1β upregulates its own production in mononuclear cells. These data provide evidence that administration of cytokines, such as rBoIL-1β, enhances immune cell function and, therefore, may be useful in alleviating immunosuppression in cattle.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether numbers of coliform bacteria in feces of dairy cattle changed during the periparturient period and whether fluctuations were associated with changes in dry-matter intake.

Animals—12 healthy Holstein cows.

Procedure—Fecal samples were collected on a semiregular basis (ie, 3 to 7 times/wk) beginning 4 to 6 weeks before the anticipated parturition date and continuing through the third day (5 cows) or second week (7 cows) after parturition, and total numbers of fecal coliform bacteria were determined. Daily feed intake of 7 cows was monitored.

Results—For 11 cows, fecal coliform bacterial counts between 34 and 25 days prior to parturition were low and relatively constant (< 102 change in number of bacteria). Coliform bacteria were not detected in 4 to 8% of fecal samples from 10 cows. All cows had a 104 to 107 increase in number of colony forming units/g of feces near the time of parturition. Number of fecal coliform bacteria peaked within 7 days of parturition in 9 cows and within 12 days of parturition in 3. Number of fecal coliform bacteria was not correlated with feed intake.

Conclusions and Clinical Relevance—Cows may have large increases in fecal coliform bacteria count during the periparturient period; however, periparturient cows do not continually shed high numbers of coliform bacteria, and coliform bacteria may not always be detectable by conventional culture methods. Changes in fecal coliform bacteria count did not correlate with changes in dry-matter intake. (Am J Vet Res 2000;61:1636–1638)

Full access
in American Journal of Veterinary Research

Summary

Leukocyte adhesion deficiency was diagnosed in 4 Holstein calves from 1 to 4 months old. Calves had severe ulcers on oral mucous membranes, gingivitis, severe periodontitis, chronic pneumonia, and stunted growth associated with severe neutrophilia. Neutrophils from affected calves had function defect, characterized by severely decreased adherence, chemotactic movements, phagocytosis, luminol-dependent chemiluminescent response, and O- 2-producing activities. Deficient CD18 expression (0.1 to 1.7%) on neutrophils was clearly detected by use of flow cytometric analysis. These affected calves were linked to a common ancestral sire that has been documented to be a carrier. Clinical features, leukocyte functional abnormalities, deficient expression of CD18, and mode of inheritance indicated that affected calves had leukocyte adhesion deficiency. In vitro leukocyte functional abnormalities were associated with deficiency in the expression of CD11/CD18. Pathologic findings indicated possible increased susceptibility to infection associated with this disease.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To clone, sequence, and express porcine recombinant soluble tumor necrosis factor receptor 1 (sTNFR1).

Procedure

A polymerase chain reaction (PCR)-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a fusion protein. Protein product was purified by immunoaffinity chromatography, using a commercially available affinity gel specific for the marker peptide of the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay.

Results

A 927-base pair fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains, as well as 75 amino acids of the cytoplasmic domain, was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120 to 150 μg/L of culture. Affinity-purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in dose-dependent manner.

Conclusions

Porcine recombinant sTNFR1 inhibits TNF bioactivity in vitro. This recombinant protein will be useful for developing TNFR1 antibodies and studying the roles of TNF and TNFR1 in the pathogenesis of infectious diseases in swine. (Am J Vet Res 1998;59:1317–1322)

Free access
in American Journal of Veterinary Research

SUMMARY

Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence- related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (lad) on ligand binding and receptor expression.

We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from lad, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (cl) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of cl was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1.

A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from lad- affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and lad-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between lad-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from lad-affected calves. Receptor expression for aIgG was greater on neutrophils from lad-affected calves than on those from normal calves. Luminol-enhanced cl of neutrophils in response to IgG2 opsonized zymosan was not different between lad-affected and normal calves.

Results indicate increased binding and expression of Fc receptors for aIgG and decreased binding and expression for C3b and IgM on neutrophils from calves with lad. Leukocyte adhesion deficiency may be compounded by added defects in the expression and binding of receptors for opsonins, such as C3b and IgM.

Free access
in American Journal of Veterinary Research