In vitro determination of phagocytosis and intracellular killing of Brucella melitensis by goat's polymorphonuclear phagocytes

One hundred and forty samples of goats serum taken from animals suspected of having brucellosis, have been studied by trying to assess the phagocytosis in vitro of Brucella melitensis Rev-1 by polymorphonuclear neutrophil phagocytes opsonized with antibrucella serum. The serum agglutination test and the Coombs antiglobulin test were carried out with the serum samples. The opsonization ingestion, intracellular killing and germ killing power of the serum were also assessed. The results demonstrate that the phagocytic activity of the polymorphonuclear neutrophils opsonized by a particular serum can be of use to determine its titre; its opsonic activity varies according to whether the serums are inactivated or not. Likewise it shows the resistance of Brucella meilitensis Rev-1 to digestion by caprine polymorphonuclear neutrophils, this resistance being independent of the animal's degree of infection.     

Bovine pulmonary alveolar macrophages: antemortem recovery and in vitro evaluation of bacterial phagocytosis and killing

A system was developed to recover pulmonary alveolar macrophages (PAM) from living cattle and to evaluate the function of these cells by measuring bacterial phagocytosis and killing. For the collection of PAM, single-tube and telescoped double-tube pulmonary lavage devices were compared. The total recovery, using these systems, was 70 +/- 10.7% of infused fluid, yielding approximately 87% PAM. The total number of cells per collection was approximately 5 times higher with the single-tube device (6.87 +/- 0.78 x 10(7) cell/ml) than with the telescoped double-tube device (1.3 +/- 0.1 X 10(7) cells/ml). Phagocytosis and intracellular killing of Staphylococcus epidermidis and Staphylococcus aureus by PAM in media suspension and by plastic-adherent PAM were evaluated. In addition, different bacteria-to-macrophage ratios were assessed, as well as the intracellular killing of S epidermidis at periodic intervals. Results showed that over a 3-hour period, similar numbers of both bacteria were phagocytized, but intracellular killing of S epidermidis was more efficient than intracellular killing of S aureus. It also was found (i) that suspended PAM and adherent PAM phagocytized similar numbers of bacteria; (ii) that when the bacteria-to-cell ratio was 10:1, the numbers of phagocytized bacteria and intracellular killing were higher than when the ratio was 1:10; and (iii) that killing of S epidermidis by adherent PAM was directly proportional to incubation time. The time that PAM are in culture affects the phagocytosis and killing of intracellular bacteria, as shown by increased phagocytosis and by intracellular killing of S epidermidis by PAM in suspension for 48 hours or plastic adherent for 60 hours after collection.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

Inhibition of lacteal leukocyte phagocytosis by colostrum, nonlactating secretion, and mastitic milk

The Fc receptors on leukocytes obtained from normal milk, colostrum, nonlactating gland secretion, and mastitic milk were detected by rosette formation, using sensitized erythrocytes. The percentage of leukocytes bearing Fc receptors from normal milk was significantly (P less than 0.01) greater than that from other secretions. Fc receptors were found primarily on polymorphonuclear leukocytes from normal milk, mastitic milk, and colostrum. However, in nonlactating gland secretion obtained 6 weeks after milking was completed, Fc receptors were predominantly on macrophages. The low percentage of leukocytes bearing Fc receptors obtained from colostrum, nonlactating gland secretion obtained 7 days after milking was completed, and mastitic milk was associated with the presence of a blocking factor in these secretions, which specifically attached to Fc receptors. These secretions significantly (P less than 0.01) blocked the Fc receptors on leukocytes from normal milk and on other cells bearing FC receptors. The presence of Fc receptors on leukocytes obtained from normal milk was related to a high percentage of phagocytizing leukocytes through Fc receptors and a large number of phagocytized bacteria (phagocytic activities). In contrast, the low percentage of leukocytes bearing Fc receptors from colostrum, from nonlactating gland secretion (7 days after milking), and from mastitic milk was associated with depressed phagocytic activities. Preincubation of leukocytes from normal milk with whey from colostrum, nonlactating gland secretion (7 days after milking), and mastitic milk significantly (P less than 0.01) inhibited phagocytosis. This effect was associated with the blocking of Fc receptors by these secretions. Possible mechanisms for and implications of these findings are discussed.     

Phagocytosis and intracellular killing of Campylobacter jejuni by elicited chicken peritoneal macrophages.

In vitro phagocytosis and intracellular survival of Campylobacter jejuni (strains B540 and Clin 1) in chicken peritoneal macrophages were studied. Macrophages were induced with Sephadex G-50 and harvested 48 hr later by peritoneal lavage. The extent of phagocytosis over time was determined by enumerating the intracellular C. jejuni after removal of extracellular C. jejuni with gentamicin. Pre-incubation of C. jejuni with antiserum generally enabled the macrophages to ingest greater numbers of cells than when the organism was pre-incubated in phosphate-buffered saline. C. jejuni were exposed to macrophage uptake for 30 minutes in a 5% CO2 incubator at 42 C. This suspension was then exposed to 12.5 micrograms gentamicin/ml to eliminate extracellular bacteria. Subsequently, the intracellular survival of C. jejuni was examined by monitoring its number within the macrophage at 30 minutes, 3 hr, and 6 hr after phagocytosis. Macrophages from C. jejuni-colonized chickens and from uncolonized control chickens were able to almost destroy the organism within the experimental period.     

Effects of extracellular Ca2+ on phagocytosis and intracellular Ca2+ concentrations in polymorphonuclear leukocytes of postpartum dairy cows

The aim of this study was to determine the effects of extracellular Ca(2+) concentration ([Ca(2+)](e)) on phagocytosis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine polymorphonuclear leukocytes (PMNs). The experiments were performed by using blood samples from parturient paretic and clinically normal parturient cows and manipulating the [Ca(2+)](e) in vitro. Phagocytosis by PMNs (with and without stimulation with phorbol myristate acetate and inhibition with cytochalasin B) and resting [Ca(2+)](i) were significantly lower in parturient paretic cows. Repletion of Ca(2+) in the extracellular media for the samples from these animals increased phagocytosis and resting [Ca(2+)](i). In the blood of clinically normal parturient cows, decreasing the [Ca(2+)](e) decreased phagocytosis and resting [Ca(2+)](i) in PMNs, but increasing the [Ca(2+)](e) did not affect phagocytosis. These results suggest that the hypocalcemic condition of parturient paretic cows in vivo causes decreased phagocytosis and resting [Ca(2+)](i) in PMNs, which may partly contribute to greater susceptibility to infection.     

Bovine polymorphonuclear neutrophil-mediated phagocytosis and an immunoglobulin G2 protease produced by Porphyromonas levii.

Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine polymorphonuclear granulocytic neutrophils (PMN) from blood with bacterial species involved in AIP. Polymorphonuclear neutrophils were purified from whole bovine blood, exposed to one of the three putative etiologic agents of AIP and comparatively assessed for phagocytosis using light microscopy. Fusobacterium necrophorum and Prevotella intermedia were effectively phagocytosed by PMN, but Porphyromonas levii was phagocytosed significantly less effectively by PMN. The effect of high titre anti-P. levii bovine serum on antibody-mediated phagocytosis by PMN was also evaluated. High titre serum increased the efficiency of phagocytosis of P. levii by bovine PMN. This was independent of heat labile complement factors. Antibodies specific for P. levii were assessed for protease activity capable of cleaving bovine immunoglobulins (IgG, IgG1, IgG2, and IgM). Partially purified supernatant from broth cultures of P. levii were incubated with biotinylated immunoglobulins (Igs). Samples were taken from times 0 to 72 h and examined using SDS-PAGE followed by Western blot analysis. Streptavidin-alkaline phosphatase and NBT-BCIP were used to visualize the Igs for heavy and light chains as well as lower molecular weight fragments of these glycoproteins. Porphyromonas levii produced an immunoglobulin protease which readily cleaved bovine IgG into fragments, but did not act against IgM. Specifically, the enzyme may be a significant virulence factor as it may act to neutralize the antibodies demonstrated necessary for effective PMN-mediated phagocytosis.     

Enhancement of phagocytosis and bacterial killing by heterophils from neonatal chicks after administration of Salmonella enteritidis-immune lymphokines

During the first week post-hatch, chickens demonstrate an increased susceptibility to infection by bacteria such as Salmonella. The purpose of the present study was to evaluate the effects of immune lymphokines on phagocytosis and killing activities of heterophils in chicks during the first 1-7 days of life. Lymphokines isolated from chicken splenic T-cells harvested from Salmonella enteriditis (SE)-hyperimmunized hens (SE-ILK), have in past experiments, demonstrated augmentation of heterophil activity in day-of-hatch chicks resulting in protection from SE organ invasion. The present experiments reveal significant increases (p<0.05) in heterophil phagocytosis and killing when comparing chicks treated with SE-ILK to control groups in vitro. In SE-ILK-treated groups, a two-fold or greater increase is noted in heterophil phagocytosis within I h of incubation as compared to controls. Heterophils isolated from 1-day-old and 4-day-old chicks treated with SE-ILK killed significantly greater numbers (p<0.05) of SE than heterophils isolated from control groups. By Day 7 post-hatch, significance is not noted in the killing activity of heterophils from treated groups when compared to control groups. However, heterophils from SE-ILK groups continue to kill greater numbers of SE than control groups. These data support SE-ILK augmentation results in an enhanced heterophil function in chicks during the greatest period of susceptibility to Salmonella invasion.     

Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils

The interactions of 2 capsular serotype A and 4 serotype D strains of Pasteurella multocida with rabbit polymorphonuclear neutrophils (PMN) were compared in vitro, using a PMN phagocytic and bactericidal assay. Bacteria and rabbit PMN were incubated for 15 minutes. The suspensions were subjected to differential centrifugation and the percentage of phagocytosis (cell association) was determined from the number of viable noncell-associated bacteria. The cell pellets and the associated bacteria were resuspended and PMN bactericidal activity was calculated from the number of remaining viable cell-associated bacteria at 45 and 75 minutes after the start of the assay. Test bacteria were not opsonized or were opsonized with immune serum containing active complement. One type A strain was ingested and killed by PMN in the presence and absence of opsonins. The 5 remaining strains were resistant to PMN killing, but only the type A strain resisted phagocytosis. Resistance of the type A strain was attributed to the hyaluronic acid capsule, since pretreatment of the bacteria with hyaluronidase rendered opsonized bacteria susceptible to ingestion and killing. The pattern of resistance of the 4 type D strains was different from that of the resistant type A strain. Both opsonized and nonopsonized type D bacteria became cell associated, but none were killed by PMN. The mechanism of resistance of these 4 strains to PMN bactericidal activity is currently unknown.     

Phagocytosis and intracellular killing of Pasteurella multocida by porcine alveolar macrophages after infection with pseudorabies virus

The purpose of this study was to determine if pseudorabies virus (PrV) interfered with normal alveolar macrophage phagocytic functions. Porcine alveolar macrophages (PAM) obtained by pulmonary lavage were exposed to PrV. At 1 hour postinfection, cells were challenged with Pasteurella multocida labeled with 3[H] thymidine. The phagocytosis assay was performed by measuring total radioactivity 1 hour after Pm challenge in a soft-beta spectrophotometer. Intracellular killing was measured by counting viable bacteria 3 hours after P. multocida challenge. Phagocytic values of PrV-infected and control PAM ranged from 11% to 20%, a non-significant difference. Values for intracellular killing for PrV-infected PAM ranged from 7.1 X 10(5) to 1 X 10(6) in contrast to 5.1 X 10(1) to 1.8 X 10(2) for the control PAM. This difference in killing function was significantly lower in PrV-infected PAM than in control cells (P less than 0.01). This alteration of macrophage function may be a factor in the pathogenesis of PrV-Pm mediated pneumonia in pigs.     

Phagocytosis and intracellular killing of Staphylococcus aureus by bovine blood polymorphonuclear leukocytes after migration in vitro

Phagocytic activity and intracellular killing of opsonized staphylococci (Smith strain) by bovine blood polymorphonuclear leukocytes (PMNL) was studied after their migration in vitro in blind well chambers. The results indicate that migration of PMNL to RPMI-1640 medium without chemotactic factor significantly (P less than 0.01) increased the percentage of rosette-forming PMNL (from 11 +/- 2 to 49 +/- 4%), as well as phagocytic activity mediated through Fc receptors (FcRs) (from 25 +/- 3 to 81 +/- 4% phagocytizing PMNL and from 151 +/- 22 to 942 +/- 54 number of phagocytized staphylococci/100 PMNL), and intracellular killing of bacteria (from 13 +/- 2 to 59 +/- 7%). On the other hand, PMNL migration to RPMI-1640 medium with the chemotactic factor (serum activated with zymosan [AS] or lipopolysaccharide [LPS] or formyl-L-methionyl-L-leucyl-L-phenylalanine [fMLP]) did not significantly (p greater than 0.05) increase either the FcRs number on the PMNL surface or the phagocytic and bactericidal activity connected with these receptors. The possible mechanisms are discussed.     

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.     

Some observations on the effects of age of calves on the phagocytosis and killing of Staphylococcus aureus by polymorphonuclear leucocytes

The effects of age on bovine polymorphonuclear (PMN) cell function were investigated by comparing the efficiencies of phagocytosis and killing of Staphylococcus aureus by peripheral blood leucocytes sequentially obtained from 15 calves between the ages of less than 1 and 84 days. One group of seven calves was kept in a controlled environmental chamber with air temperature of 5 degrees C and 58% relative humidity (RH) and another group of eight calves was kept at 16 degrees C and 58% RH. The calves were given a diet a liquid milk substitute and dry food, and were weaned abruptly from the liquid diet at 35 days of age. The in-vitro efficiencies of phagocytosis, and of killing, Staphylococcus aureus by peripheral blood leucocytes were similar for calves in air temperatures of 5 degrees C and 16 degrees C (P greater than 0.05). Peripheral blood leucocytes obtained from calves of less than 1 day of age were more efficient in phagocytosing S. aureus than those obtained when the same calves were 14-84 days of age (P less than 0.001). Peripheral blood leucocytes obtained when the calves were 42 and 56 days of age were significantly less efficient in phagocytosing and killing S. aureus than those obtained when the same calves were less than 1, 14, 28, 70 and 84 days of age (P less than 0.001).     

Bovine chlamydial abortion: serodiagnosis by modified complement-fixation and indirect inclusion fluorescence tests and enzyme-linked immunosorbent assay

Sequential serum samples from 11 cows experimentally inoculated with different abortigenic strains of Chlamydia psittaci were tested by a modified complement-fixation (MoCF) test, an indirect inclusion fluorescence antibody (IIFA) test, and an enzyme-linked immunosorbent assay (ELISA). One of these cows was not pregnant, another gave birth at term to a healthy calf, and all the others prematurely delivered infected dead calves or weak live calves. The results achieved with these tests on sera of 3 of the cows were compared with those from the previously used standard complement-fixation (CF) test. Six of 11 cows had detectable preinoculation titers between 1:8 and 1:16 when tested by the MoCF test, yet preinoculation titers were not detected by CF. In contrast, 9 of 11 and 10 of 11 preinoculation samples had detectable chlamydia-specific antibodies when examined by the IIFA test and the ELISA, respectively. The preinoculation IIFA titers ranged from 1:8 to 1:64, and the ELISA optical density values varied from 0.150 to 0.450. All cows responded with significant increases in antibody levels detected by the MoCF test, the IIFA test, and ELISA after they were experimentally inoculated and after they aborted or delivered infected calves. Overall, the dynamics of the antibody responses were found to be similar with the 3 different serologic techniques. When cows aborted later than 36 days after they were inoculated, the antibody response was biphasic, whereby the more pronounced responses occurred after the abortion occurred. The nonpregnant cow and the cow that delivered a healthy calf at term had only one phase of increasing and decreasing titers directly after they were inoculated.