Veterinary Research Communications, 16 (1992) 107-115
Copyright ~) Kluwer Academic Publishers by - Printed in the Netherlands
POLYMORPHONUCLEAR NEUTROPHIL LEUKOCYTE FUNCTION IN CLINICAL BOVINE PATIENTS AND IN COWS WITH OR WITHOUT S T A P H Y L O C O C C U S A UREUS MASTITIS K.L. A N D E R S O N , L.A. S M I T H , F.J. D e G R A V E S , E. H U N T A N D S.A. F L E M I N G D e p a r t m e n t of F o o d A n i m a l a n d E q u i n e Medicine, College of V e t e r i n a r y Medicine, N o r t h C a r o l i n a State University, 4700 Hillsborough Street, Raleigh, N C 27606, U S A
ABSTRACT Anderson, K.L., Smith, L.A., DeGraves, F.I., Hunt, E. and Fleming, S~,., 1992. Polymorphonuclear neutrophil leukocyte function in clinical bovine patients and in cows with or without Staphylococcus aureus mastitis. Veterinary Research Communications, 16 (2), 107-115 A fluorochrome microassay was used to investigate peripheral blood polymorphonuclear leukocyte (PMNL) function in cattle. Glass-adherent PMNL were reacted with Staphylococcus aureus preincubated in 20% bovine serum for 30, 60 and 90 rain. Coverslips were stained with acridine orange (AO) followed by crystal violet to quench extracellular bacterial fluorescence. PMNL function was evaluated by counting the number of dead (stained red with AO) and live (stained green with A O ) S. aureus contained within 100 PMNL. A phagocytic index was calculated as the average number of bacteria contained within PMNL. The percentage killing of S. aureus was calculated from the average proportion of S. aureus within PMNL that were dead. Six clinically normal Holstein calves, 3-4 months of age, were sampled on 6 consecutive days. PMNL phagocytosis and killing did not waD" significantly 09>0.05) among repeated samplings per calf. PMNL function increased with increasing time of incubation of PMNL with S. aureus. Means (_+ SD) for percentage killing were 46.7 _+ 13.1, 57.4 _+ 11.6, and 62.1 -+ 9.8% for 30, 60 and 90 min of reaction, respectively.Means (-+ SD) for the phagocytic index were 2.9 +_ 0.8, 3.6 -+ 1.0, and 4.2 _+ 1.1 bacteria/PMNL for 30, 60 and 90 min of reaction, respectively.PMNL function was determined in 30 normal cattle of various breeds, age and sex, and these values were pooled to provide normal values for PMNL function. When values for bovine clinical patients (n=25) with various diagnoses were compared with normal values (defined by the mean -+ 2SD for the 30 normal cattle) for PMNL function, only one patient was observed to exhibit PMNL hypofunction. A cow with disseminated intrawascular coagulation in association with peracute coliform mastitis exhibited decreased PMNL killing capacity. Abnormal PMNL function was uncommon in the hospital population studied. Peripheral blood PMNL function was evaluated in lactating Holstein cows with (n =15) or without (n = 15) chronic subclinicalS. aureus mastitis. There was no significant CO> 0.05) difference in PMNL function among these cows. Keywords: cattle, phagocytosis, polymorphonuclear leukocyte, Staphylococcus aureus
INTRODUCTION T h e activity of p o l y m o r p h o n u c l e a r leukocytes ( P M N L ) is critical for the m a i n t e n a n c e of host defence a n d in reaction to insult by infectious agents. Phagocytosis a n d killing of bacteria are a m o n g the most i m p o r t a n t functions of P M N L . O n e m e t h o d of evaluating P M N L function is the fluorochrome microassay (Pantazis and Kniker, 1979; Musclow e t a L , 1987; Pruzanski and Saito, 1988). I n one application of this method, glass-adherent P M N L suspensions exposed to a given n u m b e r of bacteria p r e t r e a t e d with s e r u m are subjected to staining with acridine orange (AO), which
108 stains dead bacteria red and live bacteria green. By counting the number of red and green bacteria contained within a given number of PMNL, the functional activity may be expressed as a phagocytic index and a killing index. Advantages of the method include its simplicity and the relatively short time required to evaluate PMNL phagocytosis and killing (Absolom, 1986; Moore and Humbert, 1986). Various modifications of a fluorochrome microassay have been used in clinical and research investigations in humans (Absolom, 1986; Hed, 1986; Moore and Humbert, 1986), including the detection of PMNL bactericidal defects (Pantazis and Kniker, 1979; Goldner et al., 1983; Musclow et al., 1987; Pruzanski and Saito, 1988). Bovine leukocyte phagocytosis and bacterial killing of Escherichia coli and Staphylococcus aureus were evaluated using a fluorochrome microassay in 22 normal bulls of four Swiss dairy breeds (Zanetti et al., 1987). Maximum PMNL uptake (approximately 12 bacteria/PMNL) and killing (80%) of serum-opsonized E. coli K12 and S. aureus SG511 were achieved within 60 min (Zanetti et aL, 1987). The purpose of this investigation was to investigate PMNL function in normal and diseased cattle using a fluorochrome microassay.
MATERIALS AND METHODS Animals
The investigation consisted of four studies. In the first of these, 6 clinically normal (4 male, 2 female) Holstein calves, 3-4 months of age, housed at one location, were sampled on 6 consecutive days (1-2 days between assays) to evaluate the variability in PMNL function among days within calves. In the second study, 30 clinically normal Holstein cattle of various ages, sexes, and production status were tested to determine normal values for PMNL function. The animals included 6 bull calves under 6 months of age, 6 males (4 castrated, 2 intact) 6-24 months of age, 6 non-pregnant adult cows, 6 heifer calves under 6 months of age and 6 pregnant females (3 adults and 3 heifers). In the third study, PMNL function was evaluated in 25 hospitalized bovine patients of various ages and both sexes with clinical disorders of both infectious and non-infectious nature. Finally, PMNL function was evaluated in adult lactating Holstein cattle with (n = 15) or without (n = 15) subclinical S. aureus mastitis. In all the studies, PMNL function was evaluated after 30, 60 and 90 min reaction with S. aureus.
Preparation of leukocyte monolayers
The procedure of Pantazis and Kniker (1979) was used with minor modifications. Whole blood (3 ml) was collected from the jugular or tail vein in sterile syringes. Monolayers of white blood cells were immediately prepared by applying about 0.2 ml of blood, without anticoagulant, onto 18-mm diameter circular glass microscope coverslips and allowing it to clot at 37°C. After 60 min at 37°C, the clots were gently removed from the coverslips using sterile tissue forceps. The coverslips were then carefully rinsed once with sterile Hank's balanced salt solution containing calcium and magnesium (Mediatech Inc., Herndon, VA, USA) at 37°C, and 25 #1 of a suspension of S. aureus, pretreated with bovine serum, was added to each coverslip. Coverslips
109 with bacteria were then placed on a rocker platform (Bellco Biotechnology, Vineland, NJ, USA) set at 20 cycles per minute in a 37°C incubator and allowed to react for 30, 60 or 90 min.
Preparation of Staphylococcus aureus
An 18 (-+ 2) hour culture of S. aureus (Newbould 305; coagulase positive, ATCC 29740, ATCC, Baltimore, MD, USA) was prepared fresh each day. Cultures were initiated by mixing 1-ml aliquots of frozen bacterial suspensions with tryptic soy broth (30 g/L) at 37°C. They were incubated at 37°C for 1 h and then transferred aseptically into 200 ml casein amino acid medium held in a 37°C shaker water bath (New Brunswick Scientific Company, Inc., NJ, USA). This medium contained the following components per litre: 0.5 g disodium citrate (Fisher Scientific, Pittsburg, PA, USA); 0.1 g hydrated magnesium sulphate (Cumberland Manufacturing Co., Smyrna, TN, USA); 0.2 g dextrose (Fisher Scientific, Pittsburg, PA), 10 g casein amino acid (Humko-Sheffield Chemical Company, Memphis, TN, USA) and 3.0 g glycine (Fisher Scientific, Pittsburg, PA). Bacteria were harvested by centrifugation at 1000 g for 12 min, washed twice in 0.85% sterile saline and resuspended in saline to give a final absorbance of 0.79 at 520 nm (Bausch and Lomb Spectronic 20 spectrophotometer, Milton Roy Analytical, USA). From growth curves, the relationship of absorbance at 520 nm to the number of colony-forming units (CFU)/ml was used to determine that the bacterial suspensions contained 3.1 × 106 CFU/ml S. aureus. S. aureus suspensions (1.0 ml) were combined with 1.0 ml 20% pooled bovine serum (Granite Diagnostics, Burlington, NC, USA) containing 67.7 mg D-(+)-glucose/100 ml PBS (3.5 mmol/L glucose) for 30 rain prior to inoculation of coverslips, giving a final concentration of 1.6 × 106 CFU/ml S. aureus.
Preparation of dyes
Acridine orange (AO) and crystal violet (CV) dyes (Sigma Chemical, St. Louis, MO, USA) were prepared fresh each day in sterile PBS at 0.15 mg/ml and 0.5 mg/ml, respectively.
Assay of phagocytosis and microbial killing
The method of Pantazis and Kniker (1979) was modified to include the use of CV to quench extracellular bacterial fluorescence (Musclow et aL, 1987; Pruzanski and Saito, 1988). Fixed slides were immediately viewed using an ultraviolet microscope with an oil-immersion objective and an unfiltered halogen light source (Nikon Labo Phot Fluorescent Microscope, Nippon Kogeku K.K., Tokyo, Japan). Phagocytic index (PI) was calculated as the average number of bacteria per leukocyte in the 100 PMNL evaluated. The percentage killing was then calculated as the number of bacterial cells observed as red or orange divided by the total number of ingested bacteria x 100%.
110 P M N L recovery on coverslips
The adequacy of PMNL numbers on the coverslips and the percentage of adherent cells that were PMNL were assessed by evaluation after staining with Wright'sGiemsa.
Staining of Staphylococcus aureus
Washed preparations of live and heat-killed S. aureus were viewed by UV microscopy after staining with AO. In six replicates, the proportions of green, yellow, orange and red bacteria in successive 100 x microscopic fields of a given slide were assessed by two individuals. The viability of the bacterial cultures was subsequently determined by plating onto 5% sheep blood agar (TSA II, BBL Media, Cockeysville, MD, USA).
P M N L function in cows with or without Staphylococcus aureus mastitis
Adult lactating Holstein cows were selected from a herd from which quarter milk samples of cows had been cultured at 60-day intervals during lactation. Cows with subclinical intramammary infection with S. aureus were selected and matched by age and stage of lactation with milk culture-negative cows. Prior to assay of PMNL function, the intramammary infection status was confirmed by repeated culture of aseptically collected milk samples.
Statistical analysis
Data were evaluated by analysis of variance (Proc GLM and Proc Anova; SAS Institute Inc., Cary, NC, USA).
RESULTS P M N L recovery on glass slides
In 11 initial trials, a mean of 85% of the glass-adherent cells (range 75.2-97.4%) were PMNL. In 20 trials, a range of 12-50 PMNL were found per high power (100x) microscopic field.
Staining of Staphylococcus aureus
Heat-killed S. aureus cultures stained entirely red and orange, while a mean of 91.2% of the bacteria in viable S. aureus cultures stained green or yellow.
111
Variation in P M N L function in normal calves
The PMNL functional indices varied significantly among calves and for reaction time (Table I). Bacterial killing by PMNL and phagocytic index increased significantly 09 0.05) among days within individual calves.
TABLE I Means (_+ SD) and range of PMNL functional indices for calves
Time a (min)
Percent killing b
PI c
30
46.7 _+ 13.1 (16.2-72.6)
2.85 _+ 0.75 (1.79-4.87)
60
57.4 _+ 11.6 (33.5-83.8)
3.60 _+ 0.96 (2.03-6.00)
90
62.1 _ 9.8 (33.3-78.7)
4.16 +_ 1.10 (2.37-7.38)
aTime of reaction of PMNL with S. aureus b Percent killing = number of ingested S. aureus stained red (dead)/total number of intracellular bacteria per PMNL x 100% CpI = phagocytic index; the average number of intracellular S. aureus contained within PMNL
Variation in P M N L function in cattle of differing age, sex and production status
The PMNL functional indices for 30 clinically normal Holstein cattle are contained in Table II. Significant differences (p 0.05) for PMNL function in cattle with or without S. aureus mastiffs.
DISCUSSION The results of this study support the findings of Zanetti et al. (1987) who used a similar method to evaluate bovine PMNL function, and extend their findings to cattle of varied ages, of both sexes and to diseased animals. The functional activity of PMNL from normal animals was reasonably similar in both studies, except for differences in maximal uptake (about 12 S. aureus/PMNL compared with about 5 S. aureus/PMNL in the present study). Phagocytic index values obtained from microscopic assays of phagocytosis vary with the type, number and ratio of bacteria to phagocytic cells, and with the conditions of incubation. Comparison of data from different laboratories is difficult at best (Absolom, 1986) and the most useful comparison is that with values within the given laboratory. The lack of significant variation among days within calves was important. If assay repeatability were poor in calves tested from day to day in the absence of disease, it would be difficult to use the assay to evaluate PMNL function among animals. The values obtained in the second study represented the expected values for PMNL function in Holstein cattle of various ages, sexes and production status under the conditions employed in this laboratory.
113
Copyright ~) Kluwer Academic Publishers by - Printed in the Netherlands
POLYMORPHONUCLEAR NEUTROPHIL LEUKOCYTE FUNCTION IN CLINICAL BOVINE PATIENTS AND IN COWS WITH OR WITHOUT S T A P H Y L O C O C C U S A UREUS MASTITIS K.L. A N D E R S O N , L.A. S M I T H , F.J. D e G R A V E S , E. H U N T A N D S.A. F L E M I N G D e p a r t m e n t of F o o d A n i m a l a n d E q u i n e Medicine, College of V e t e r i n a r y Medicine, N o r t h C a r o l i n a State University, 4700 Hillsborough Street, Raleigh, N C 27606, U S A
ABSTRACT Anderson, K.L., Smith, L.A., DeGraves, F.I., Hunt, E. and Fleming, S~,., 1992. Polymorphonuclear neutrophil leukocyte function in clinical bovine patients and in cows with or without Staphylococcus aureus mastitis. Veterinary Research Communications, 16 (2), 107-115 A fluorochrome microassay was used to investigate peripheral blood polymorphonuclear leukocyte (PMNL) function in cattle. Glass-adherent PMNL were reacted with Staphylococcus aureus preincubated in 20% bovine serum for 30, 60 and 90 rain. Coverslips were stained with acridine orange (AO) followed by crystal violet to quench extracellular bacterial fluorescence. PMNL function was evaluated by counting the number of dead (stained red with AO) and live (stained green with A O ) S. aureus contained within 100 PMNL. A phagocytic index was calculated as the average number of bacteria contained within PMNL. The percentage killing of S. aureus was calculated from the average proportion of S. aureus within PMNL that were dead. Six clinically normal Holstein calves, 3-4 months of age, were sampled on 6 consecutive days. PMNL phagocytosis and killing did not waD" significantly 09>0.05) among repeated samplings per calf. PMNL function increased with increasing time of incubation of PMNL with S. aureus. Means (_+ SD) for percentage killing were 46.7 _+ 13.1, 57.4 _+ 11.6, and 62.1 -+ 9.8% for 30, 60 and 90 min of reaction, respectively.Means (-+ SD) for the phagocytic index were 2.9 +_ 0.8, 3.6 -+ 1.0, and 4.2 _+ 1.1 bacteria/PMNL for 30, 60 and 90 min of reaction, respectively.PMNL function was determined in 30 normal cattle of various breeds, age and sex, and these values were pooled to provide normal values for PMNL function. When values for bovine clinical patients (n=25) with various diagnoses were compared with normal values (defined by the mean -+ 2SD for the 30 normal cattle) for PMNL function, only one patient was observed to exhibit PMNL hypofunction. A cow with disseminated intrawascular coagulation in association with peracute coliform mastitis exhibited decreased PMNL killing capacity. Abnormal PMNL function was uncommon in the hospital population studied. Peripheral blood PMNL function was evaluated in lactating Holstein cows with (n =15) or without (n = 15) chronic subclinicalS. aureus mastitis. There was no significant CO> 0.05) difference in PMNL function among these cows. Keywords: cattle, phagocytosis, polymorphonuclear leukocyte, Staphylococcus aureus
INTRODUCTION T h e activity of p o l y m o r p h o n u c l e a r leukocytes ( P M N L ) is critical for the m a i n t e n a n c e of host defence a n d in reaction to insult by infectious agents. Phagocytosis a n d killing of bacteria are a m o n g the most i m p o r t a n t functions of P M N L . O n e m e t h o d of evaluating P M N L function is the fluorochrome microassay (Pantazis and Kniker, 1979; Musclow e t a L , 1987; Pruzanski and Saito, 1988). I n one application of this method, glass-adherent P M N L suspensions exposed to a given n u m b e r of bacteria p r e t r e a t e d with s e r u m are subjected to staining with acridine orange (AO), which
108 stains dead bacteria red and live bacteria green. By counting the number of red and green bacteria contained within a given number of PMNL, the functional activity may be expressed as a phagocytic index and a killing index. Advantages of the method include its simplicity and the relatively short time required to evaluate PMNL phagocytosis and killing (Absolom, 1986; Moore and Humbert, 1986). Various modifications of a fluorochrome microassay have been used in clinical and research investigations in humans (Absolom, 1986; Hed, 1986; Moore and Humbert, 1986), including the detection of PMNL bactericidal defects (Pantazis and Kniker, 1979; Goldner et al., 1983; Musclow et al., 1987; Pruzanski and Saito, 1988). Bovine leukocyte phagocytosis and bacterial killing of Escherichia coli and Staphylococcus aureus were evaluated using a fluorochrome microassay in 22 normal bulls of four Swiss dairy breeds (Zanetti et al., 1987). Maximum PMNL uptake (approximately 12 bacteria/PMNL) and killing (80%) of serum-opsonized E. coli K12 and S. aureus SG511 were achieved within 60 min (Zanetti et aL, 1987). The purpose of this investigation was to investigate PMNL function in normal and diseased cattle using a fluorochrome microassay.
MATERIALS AND METHODS Animals
The investigation consisted of four studies. In the first of these, 6 clinically normal (4 male, 2 female) Holstein calves, 3-4 months of age, housed at one location, were sampled on 6 consecutive days (1-2 days between assays) to evaluate the variability in PMNL function among days within calves. In the second study, 30 clinically normal Holstein cattle of various ages, sexes, and production status were tested to determine normal values for PMNL function. The animals included 6 bull calves under 6 months of age, 6 males (4 castrated, 2 intact) 6-24 months of age, 6 non-pregnant adult cows, 6 heifer calves under 6 months of age and 6 pregnant females (3 adults and 3 heifers). In the third study, PMNL function was evaluated in 25 hospitalized bovine patients of various ages and both sexes with clinical disorders of both infectious and non-infectious nature. Finally, PMNL function was evaluated in adult lactating Holstein cattle with (n = 15) or without (n = 15) subclinical S. aureus mastitis. In all the studies, PMNL function was evaluated after 30, 60 and 90 min reaction with S. aureus.
Preparation of leukocyte monolayers
The procedure of Pantazis and Kniker (1979) was used with minor modifications. Whole blood (3 ml) was collected from the jugular or tail vein in sterile syringes. Monolayers of white blood cells were immediately prepared by applying about 0.2 ml of blood, without anticoagulant, onto 18-mm diameter circular glass microscope coverslips and allowing it to clot at 37°C. After 60 min at 37°C, the clots were gently removed from the coverslips using sterile tissue forceps. The coverslips were then carefully rinsed once with sterile Hank's balanced salt solution containing calcium and magnesium (Mediatech Inc., Herndon, VA, USA) at 37°C, and 25 #1 of a suspension of S. aureus, pretreated with bovine serum, was added to each coverslip. Coverslips
109 with bacteria were then placed on a rocker platform (Bellco Biotechnology, Vineland, NJ, USA) set at 20 cycles per minute in a 37°C incubator and allowed to react for 30, 60 or 90 min.
Preparation of Staphylococcus aureus
An 18 (-+ 2) hour culture of S. aureus (Newbould 305; coagulase positive, ATCC 29740, ATCC, Baltimore, MD, USA) was prepared fresh each day. Cultures were initiated by mixing 1-ml aliquots of frozen bacterial suspensions with tryptic soy broth (30 g/L) at 37°C. They were incubated at 37°C for 1 h and then transferred aseptically into 200 ml casein amino acid medium held in a 37°C shaker water bath (New Brunswick Scientific Company, Inc., NJ, USA). This medium contained the following components per litre: 0.5 g disodium citrate (Fisher Scientific, Pittsburg, PA, USA); 0.1 g hydrated magnesium sulphate (Cumberland Manufacturing Co., Smyrna, TN, USA); 0.2 g dextrose (Fisher Scientific, Pittsburg, PA), 10 g casein amino acid (Humko-Sheffield Chemical Company, Memphis, TN, USA) and 3.0 g glycine (Fisher Scientific, Pittsburg, PA). Bacteria were harvested by centrifugation at 1000 g for 12 min, washed twice in 0.85% sterile saline and resuspended in saline to give a final absorbance of 0.79 at 520 nm (Bausch and Lomb Spectronic 20 spectrophotometer, Milton Roy Analytical, USA). From growth curves, the relationship of absorbance at 520 nm to the number of colony-forming units (CFU)/ml was used to determine that the bacterial suspensions contained 3.1 × 106 CFU/ml S. aureus. S. aureus suspensions (1.0 ml) were combined with 1.0 ml 20% pooled bovine serum (Granite Diagnostics, Burlington, NC, USA) containing 67.7 mg D-(+)-glucose/100 ml PBS (3.5 mmol/L glucose) for 30 rain prior to inoculation of coverslips, giving a final concentration of 1.6 × 106 CFU/ml S. aureus.
Preparation of dyes
Acridine orange (AO) and crystal violet (CV) dyes (Sigma Chemical, St. Louis, MO, USA) were prepared fresh each day in sterile PBS at 0.15 mg/ml and 0.5 mg/ml, respectively.
Assay of phagocytosis and microbial killing
The method of Pantazis and Kniker (1979) was modified to include the use of CV to quench extracellular bacterial fluorescence (Musclow et aL, 1987; Pruzanski and Saito, 1988). Fixed slides were immediately viewed using an ultraviolet microscope with an oil-immersion objective and an unfiltered halogen light source (Nikon Labo Phot Fluorescent Microscope, Nippon Kogeku K.K., Tokyo, Japan). Phagocytic index (PI) was calculated as the average number of bacteria per leukocyte in the 100 PMNL evaluated. The percentage killing was then calculated as the number of bacterial cells observed as red or orange divided by the total number of ingested bacteria x 100%.
110 P M N L recovery on coverslips
The adequacy of PMNL numbers on the coverslips and the percentage of adherent cells that were PMNL were assessed by evaluation after staining with Wright'sGiemsa.
Staining of Staphylococcus aureus
Washed preparations of live and heat-killed S. aureus were viewed by UV microscopy after staining with AO. In six replicates, the proportions of green, yellow, orange and red bacteria in successive 100 x microscopic fields of a given slide were assessed by two individuals. The viability of the bacterial cultures was subsequently determined by plating onto 5% sheep blood agar (TSA II, BBL Media, Cockeysville, MD, USA).
P M N L function in cows with or without Staphylococcus aureus mastitis
Adult lactating Holstein cows were selected from a herd from which quarter milk samples of cows had been cultured at 60-day intervals during lactation. Cows with subclinical intramammary infection with S. aureus were selected and matched by age and stage of lactation with milk culture-negative cows. Prior to assay of PMNL function, the intramammary infection status was confirmed by repeated culture of aseptically collected milk samples.
Statistical analysis
Data were evaluated by analysis of variance (Proc GLM and Proc Anova; SAS Institute Inc., Cary, NC, USA).
RESULTS P M N L recovery on glass slides
In 11 initial trials, a mean of 85% of the glass-adherent cells (range 75.2-97.4%) were PMNL. In 20 trials, a range of 12-50 PMNL were found per high power (100x) microscopic field.
Staining of Staphylococcus aureus
Heat-killed S. aureus cultures stained entirely red and orange, while a mean of 91.2% of the bacteria in viable S. aureus cultures stained green or yellow.
111
Variation in P M N L function in normal calves
The PMNL functional indices varied significantly among calves and for reaction time (Table I). Bacterial killing by PMNL and phagocytic index increased significantly 09 0.05) among days within individual calves.
TABLE I Means (_+ SD) and range of PMNL functional indices for calves
Time a (min)
Percent killing b
PI c
30
46.7 _+ 13.1 (16.2-72.6)
2.85 _+ 0.75 (1.79-4.87)
60
57.4 _+ 11.6 (33.5-83.8)
3.60 _+ 0.96 (2.03-6.00)
90
62.1 _ 9.8 (33.3-78.7)
4.16 +_ 1.10 (2.37-7.38)
aTime of reaction of PMNL with S. aureus b Percent killing = number of ingested S. aureus stained red (dead)/total number of intracellular bacteria per PMNL x 100% CpI = phagocytic index; the average number of intracellular S. aureus contained within PMNL
Variation in P M N L function in cattle of differing age, sex and production status
The PMNL functional indices for 30 clinically normal Holstein cattle are contained in Table II. Significant differences (p 0.05) for PMNL function in cattle with or without S. aureus mastiffs.
DISCUSSION The results of this study support the findings of Zanetti et al. (1987) who used a similar method to evaluate bovine PMNL function, and extend their findings to cattle of varied ages, of both sexes and to diseased animals. The functional activity of PMNL from normal animals was reasonably similar in both studies, except for differences in maximal uptake (about 12 S. aureus/PMNL compared with about 5 S. aureus/PMNL in the present study). Phagocytic index values obtained from microscopic assays of phagocytosis vary with the type, number and ratio of bacteria to phagocytic cells, and with the conditions of incubation. Comparison of data from different laboratories is difficult at best (Absolom, 1986) and the most useful comparison is that with values within the given laboratory. The lack of significant variation among days within calves was important. If assay repeatability were poor in calves tested from day to day in the absence of disease, it would be difficult to use the assay to evaluate PMNL function among animals. The values obtained in the second study represented the expected values for PMNL function in Holstein cattle of various ages, sexes and production status under the conditions employed in this laboratory.
113
