Evaluation of phagocytic function of canine peripheral polymorphonuclear leucocytes by whole blood chemiluminescence.

Zymosan-induced and luminol-aided chemiluminescence (CL) of whole blood from beagle dogs was estimated for the function of polymorphonuclear leucocytes (PMNs). Whole blood (0.1 ml) was examined directly and results were obtained within 20 min. A phagocytic function of PMNs can be estimated from the peak CL counts and the number of PMNs in a specimen, and the opsonic activity can also be estimated by the peak time showing peak CL after the addition of non-opsonized zymosan. The optimal temperatures to keep diluted whole blood for the CL measurement was around 13 degrees C. Thus, this method offers information concerning the functions of phagocytic cells in whole blood.     

The effects of glucocorticosteroids on the chemiluminescence response of bovine phagocytic cells

The effects of corticosteroids on the chemiluminescence response of bovine phagocytic cells were determined both in vitro and in vivo. The in vitro addition of hydrocortisone or dexamethasone had no significant effect on the chemiluminescence response of leukocytes in a whole blood or purified polymorphonuclear leukocyte (PMN) population. Cattle that received a single 20 mg dose of dexamethasone or three 20 mg doses of dexamethasone (given 24 hours apart) demonstrated the expected effects on the bovine leukogram (leukocytosis, neutrophilia, lymphopenia, eosinopenia, and monocytosis) and also demonstrated the expected suppressive effect on lymphocyte response to phytohemagglutinin (PHA). However, neither a single nor multiple dexamethasone treatment(s) had an effect on the chemiluminescence response of phagocytes in whole blood, but significantly enhanced the chemiluminescence response of the purified PMN leukocyte population. There was no significant difference between the two dexamethasone treatment groups in either the degree or duration of the effects observed in the chemiluminescence or lymphocyte response assays.     

Endotoxin activity in whole blood measured by neutrophil chemiluminescence is applicable to canine whole blood

The dog is widely used as a translational experimental model studying the host response and new treatments for human endotoxemia. The present study evaluated the applicability of a novel patient-near neutrophil chemiluminescence assay for the measurement of endotoxin activity in human blood when applied to canine blood samples. The assay was observed to be analytically sensitive and specific to endotoxin when tested in vitro, spiked with purified Escherichia coli lipopolysaccharide and live E. coli. The diagnostic sensitivity was sustained during Gram-positive contamination. Finally, it also demonstrated diagnostic potential when able to discriminate dogs with spontaneously occurring endotoxemia from both healthy dogs and diseased dogs without endotoxemia. The rapid patient-near assessment of endotoxin activity in canine blood should facilitate future studies on endotoxemia in both spontaneous disease and in experimental settings.     

A rapid microassay for measuring the luminol-dependent chemiluminescent response in canine whole blood

A microassay for the luminol-dependent chemiluminescence (CL) response in canine whole blood was developed to measure indirectly the oxidative metabolism of peripheral blood leukocytes. Fifty microliters of blood were mixed with 705 microliters of Hank's balanced salt solution containing 25 mM Hepes and 1.3 x 10(-4) M luminol. This mixture was allowed to equilibrate for 5 min after which 60 microliters of latex beads (0.801 microns diameter) were added as a stimulant, and the CL response was monitored continuously for 5 min at 37 degrees C using a luminometer. The whole blood CL response was significantly correlated (r = 0.784, P less than 0.01, n = 14) with the number of neutrophils in the peripheral blood. Further, the whole blood CL response was abolished by the depletion of neutrophils after passing the blood through an adherence column and by the addition of sodium azide. The relative chemiluminescent light unit (RCLU) was a reliable marker for comparing each peak value in different samples. The coefficient of variation (CV) of repetitive samples was 9.87%, and the CV of 14 normal dogs was 15.7%. This method is useful and applicable for screening the CL response in canine whole blood.     

Assessment of bovine mammary gland macrophage oxidative burst activity in a chemiluminescence assay

A major bactericidal mechanism of neutrophils and macrophages is the generation of toxic oxygen-free radicals upon phagocytosis of microbes. Studies were conducted to assess the oxidative metabolism of bovine mammary gland macrophages. Bovine mammary gland macrophages were challenge exposed with a variety of phagocytic stimuli in an in vitro, luminol-assisted chemiluminescence assay. A measurable oxidative burst was observed when macrophages were challenge exposed with heat-aggregated bovine immunoglobulin, opsonified zymosan, and nonosponified zymosan. Addition of superoxide dismutase decreased mammary gland macrophage chemiluminescence in a dose-dependent manner. Brucella abortus, when opsonified with antiserum, lacteal antibody, or normal serum, produced an oxidative event, whereas nonopsonified B abortus did not. When challenge exposed with phagocytic stimuli, mammary gland macrophages produced an oxidative burst similar to that produced by other phagocytes for which an oxidative event is known to be bactericidal.     

The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma-interferon in whole blood culture

The recently developed gamma-interferon (IFN-gamma) assay system for the diagnosis of bovine tuberculosis in cattle has been accredited by the Standing Committee on Agriculture for use in Australia. In this test system, whole blood is incubated with tuberculin purified protein derivative (PPD) antigens for 16 to 24 h. The plasma is then collected and assayed for IFN-gamma production using an enzyme immunoassay (EIA). The assay system has proven to be a rapid, sensitive and inexpensive method for measuring antigen specific cell-mediated reactivity when compared with the more traditional lymphocyte proliferation assay. The IFN-gamma assay is the first in-vitro cellular assay to be used as a routine diagnostic test in veterinary medicine. While the IFN-gamma EIA has been optimised, several conditions affecting the production of IFN-gamma in whole blood culture needed investigation. We determined that optimal IFN-gamma production required the use of heparinised blood, cultured with 20 micrograms/ml of PPD within 8 h of collection. The use of blood collected post mortem resulted in reduced sensitivity for the assay. The kinetics of IFN-gamma release were established as were the effects of intradermal tuberculin testing on the IFN-gamma assay.     

The loss of opsonic activity of bovine milk whey following depletion of IgA

The role of the IgA antibody to Streptococcus agalactiae found in the whey of milks 12 hours after the first intramammary infection of six Friesian first lactation heifers was assessed using an in vitro bactericidal assay. The mean percentage kill of the streptococci by neutrophils in the presence of these wheys was 36.2% while the equivalent figure for the non-infected quarter whey was 0%. When the IgA antibody was absorbed from the infected quarter wheys using class specific IgA antiserum cross linked with glutaraldehyde the percentage kill of the test system fell to 0%. Elution of the absorbed antibody partially restored the activity to a mean percentage kill of 18.2%. The results indicated that the IgA antibody found in infected quarter whey during the acute stages of intramammary infection with Streptococcus agalactiae was responsible for the opsonic activity which pertained at that time.     

Comparison of selenium levels and glutathione peroxidase activity in bovine whole blood.

Blood glutathione peroxidase activity and selenium levels were found to correlate well, indicating that glutathione peroxidase activity can be used to assess blood selenium levels in beef cattle. The glutathione peroxidase activity of blood is less stable than is the selenium concentration but when blood was stored at 4 degrees C, the glutathione peroxidase activity remained constant for seven days.     

Prostaglandin E2 as a read out for endotoxin detection in a bovine whole blood assay

The detection of endotoxin contamination is an essential part of drug safety testing. The rabbit pyrogen test (RPT), the limulus amoebocyte lysate (LAL) test, and the monocyte activation test (MAT) are established methods for the detection of pyrogens. However, the RPT is insufficiently standardized; the LAL test is solely capable of identifying the presence of endotoxins, whereas the use of the MAT is limited by the availability of human blood. Here, we introduce a new procedure for testing endotoxin contamination using prostaglandin E₂ (PGE₂) release from bovine whole blood. We incubated bovine whole blood overnight with lipopolysaccharide (LPS) from Escherichia coli 0111:B4, concentrations ranging from 1.56 to 12.5 pg/mL, and found significantly increased PGE₂ production for even the lowest LPS concentrations. Testing the possibility of storing the blood at 4 °C before use also yielded positive results as 1.56 pg/mL still significantly increased PGE₂ production, thus suggesting some flexibility of the assay regarding time. These results emphasize the potential of using bovine whole blood for highly sensitive endotoxin testing. As a perspective, currently ongoing research aims to show whether the assay is also capable of detecting nonendotoxin pyrogens.     

Effects of in vitro atmospheric ammonia exposure on recovery rate and luminol-dependent chemiluminescence of bovine neutrophils and bronchoalveolar macrophages.

The effects of atmospheric ammonia, a major pollutant in animal confinement facilities, on bovine neutrophils and bronchoalveolar macrophages were evaluated in vitro. Ammonia exposure at concentrations 50, 100 and 200 ppm for one hour impaired recovery rates of neutrophils dose-dependently but enhanced their chemiluminescence activity per cell at lower concentrations (50 and 100 ppm). Macrophages were resistant to the exposure. Their recovery rates and chemiluminescence remained unaffected even at 200 ppm exposure. The present results suggest that ammonia exposure is unfavorable for bovine neutrophils in vitro, and probably in vivo also, in light of causing cell damage and triggering wider inflammatory responses.     

Bovine pneumonic pasteurellosis: evaluation of interactions between bovine pulmonary lavage cells and Pasteurella haemolytica (biotype A, serotype 1), using a luminol-dependent chemiluminescence assay

In vitro interactions of bovine pulmonary lavage cells (PLC) and pathogenic isolates of Pasteurella haemolytica biotype A, serotype 1, were examined, using a luminol-dependent chemiluminescence (LDCL) assay. The PLC containing high concentrations of bovine alveolar macrophages were incubated with living and heat-killed P haemolytica at bacteria to PLC ratio of approximately 1:1. Kinetics of the mean LDCL response of bovine PLC to heat-killed P haemolytica cells were characterized by a gradual increase in the amount of light emitted over 150 minutes followed by a slight decrease at 180 minutes. In contrast, the LDCL responses of reaction mixtures containing living P haemolytica were characterized by the development of a maximal response at 60 minutes followed by a continued precipitous decrease in light emission to background values by 150 minutes. Differences were not noticed in the LDCL response of PLC suspensions from the same cow to 3 P haemolytica isolates. In each instance, reaction mixtures containing heat-killed bacteria had a similar LDCL profile that was characterized by continuous production of light over 180 minutes, whereas all reaction mixtures containing living bacteria underwent a precipitous decrease in light emission, which eventually resulted in a complete cessation of chemiluminescence. The PLC suspensions from different cattle did not respond to bacterial stimuli uniformly, with respect to the amplitude or detailed nature of the LDCL profile. The time that lapsed between the addition of living P haemolytica to PLC suspensions and the complete cessation of chemiluminescence varied for different cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

The opsonic activity of bovine milk whey for the phagocytosis and killing by neutrophils of encapsulated and non-encapsulated Escherichia coli

Encapsulated strains of Escherichia coli were found to be more resistant to phagocytosis and killing by bovine neutrophils; requiring in the order of 100 times more serum opsonins than non-encapsulated strains. Mid-lactation pooled whey from cows with no history of mastitis was opsonic for non-encapsulated strains, but had no effect on encapsulated organisms. In contrast, early lactation pooled whey (5-10 days post-partum) was opsonic for all strains of E. coli. It is concluded that since early lactation milk contains sufficient opsonins, severe E. coli mastitis at this stage of lactation is not due to opsonic deficiency.     

Effect of cephapirin and mecillinam on the phagocytic and respiratory burst activity of neutrophil leukocytes isolated from bovine blood

Antimicrobial therapy is the most commonly used treatment of bacterial infections in dairy cows. Polymorphonuclear neutrophil leukocytes (PMN) play an important role in the first line defence against invading bacteria and it is important that the function of PMN is not compromised by antibiotics. We investigated the in vitro effect of cephapirin, a first generation cephalosporin, and mecillinam, an amidinopenicillin with activity against mainly Gram-negative bacteria, on phagocytosis and respiratory burst activity of PMN isolated from bovine blood. After in vitro incubation of PMN with different concentrations of the antibiotics, phagocytosis was evaluated by flow cytometry and respiratory burst activity was evaluated by registration of chemiluminescence (CL) with a luminometer. None of the investigated concentrations of cephapirin and mecillinam had an effect in vitro on phagocytosis of Escherichia coli by PMN. At high concentrations (100 and 1000 μg/mL), cephapirin and mecillinam reduced the respiratory burst activity of PMN. Part of these suppressive effects could be ascribed to oxidant scavenging. Inhibitory effects of cephapirin were stronger than mecillinam. In conclusion, cephapirin and mecillinam did not seem to affect antibacterial activity of PMN isolated from bovine blood in vitro at therapeutic concentrations.     

A simple method for the in vitro determination of phagocytic activity of bovine polymorphonuclear leukocytes

A simple method for the in vitro determination of the phagocytic activity of bovine polymorphonuclear leukocytes (PMNL) is described. An enriched PMNL population was obtained from peripheral blood of healthy cattle and mixed with opsonized Candida utilis or with opsonized bovine red blood cells on BSA treated coverglass slides. The phagocytosis and killing of C. utilis was determined simultaneously on the coverglass slides stained with acridine orange. Phagocytosis of bovine erythrocytes was determined by microscopic examination of the Wright-Giemsa stained slides. This method is appropriate under limited laboratory conditions because it does not require highly sophisticated equipment and materials, it is easy and rapid to perform, reproducible and inexpensive. Therefore, it could be used as a first evaluation of the phagocytic activity of bovine PMNL.     

Neutrophil adherence,phagocytic-nitroblue tetrazolium reduction and chemiluminescence in canine whole blood

Methods for measuring neutrophil adherence, phagocytic-nitroblue tetrazolium (NBT) reducing activity and chemiluminescence were applied to canine whole blood as means for routine assessment of neutrophil functions. The phagocytic-NBT reduction test appeared to be useful for monitoring the NBT reducing activity of phagocytic cells associated with phagocytic functions. Ethylene diamine tetraacetic acid suppressed both the adherence and the phagocytic-NBT reducing activity of neutrophils. Increased phagocytic-NBT reduction and an enhanced chemiluminescence response were observed in dogs with neutrophilia. These methods provide a rapid and practical screening procedure for measuring selected phagocytic functions in canine whole blood.     

Evaluation of synthetic activity of lymphocyte nucleoli and phagocytic activity in peripheral blood leukocytes in heifers after infection with infectious bovine rhinotracheitis virus

The biosynthetic activity of the nucleoli of peripheral blood lymphocytes in calves, and the proportion of lymphocytes with the micronucleoli, with compact and ring-shaped nucleoli, were evaluated after experimental intranasal and intratracheal infections with the infectious bovine rhinotracheitis (IBR) virus. IBR virus belongs to the group of herpes viruses, the control of which is dominated by cellular immunity. The results are compared with the values for phagocytizing neutrophils, phagocytic activity and phagocytic index of the leucocytes of peripheral blood and with some basic haematological data.     

Serum opsonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake

Neutrophil (PMN) counts, immune complex (IC) uptake by PMN, and serum opsonising activity for promoting yeast uptake were used to evaluate infection clearing capacity in 16 lambs prior to colostrum feeding (two lambs fed bovine colostrum, 14 suckled lambs) and at 2 days of age. At 2 days of age lambs had more circulating PMN than they had prior to colostrum uptake (P less than 0.01). Colostrum feeding caused a significant increase in the percent of lamb PMN phagocytosing IC, although at Day 2 the percent phagocytosis was significantly lower (32.2%) than for adult controls (90%). Yeast opsonophagocytosis was greater when 24-36 h post-feeding serum was the source of opsonin than when pre-feeding serum was used (P less than 0.001). When adult serum was the opsonin, yeast opsonophagocytosis was approximately twice the phagocytosis mediated by 24-36 h post-feeding serum. The peripheral neutrocytosis and the enhancement of opsonophagocytosis generated by absorption of either ovine or bovine colostrum did not differ. The results of this study suggest that the parameters evaluated may be used for indicating the presence (or absence) of passively acquired protective immunity.     

The effect of estrogens on phagocytic activity of blood leukocytes in heifers and cows]

Phagocytic activity of peripheral leucocytes (PA) was measured in heifers during the luteal phase and oestrus and after administration of various doses of oestradiol, in ovariectomized heifers and cows in the early post-partum period. PA was demonstrated in 22.63 +/- 2.49% and 50.61 +/- 3.76% of phagocytes in the luteal phase and oestrus respectively (P less than 0.01, Fig. 1). The phagocytic index (PI) rose parallelly from 2.26 +/- 0.31 to 6.55 +/- 0.64 particles per cell (Fig. 2). Intramuscular administration of a single dose 3 mg of oestradiol dipropionate resulted in an increase of PA from 28.93 +/- 3.34 to 69.60 +/- 3.32 on post-treatment day 3 (P less than 0.05, Fig. 3). A nonsignificant increase of PA was observed in heifers treated with 10 mg oestradiol. Increases of PA and PI in postparturient cows, treated with various doses of oestradiol, were nonsignificant (Fig. 5, 6) owing to a wide variance of the values obtained, which might be due to individual differences in endocrine and metabolic status or to uterine bacterial contamination. The most marked of haematological changes was the increase of eosinophil counts from 317 to 525.10(6).l-1 in a group of cows treated with 10 mg oestradiol and a decrease of the lymphocyte: neutrophil ratio (Tab. I). Both endogenous and exogenous oestrogens stimulate PA of peripheral leucocytes and 3 mg of oestradiol is a sufficient dose to obtain the effect.