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Some observations on the effects of age of calves on the phagocytosis and killing of Staphylococcus aureus by polymorphonuclear leucocytes
Br. vet.
1. (1990) . 146, 165
SOME OBSERVATIONS ON THE EFFECTS OF AGE OF CALVES ON THE PHAGOCYTOSIS AND KILLING OF STAPHYLOCOCCUS A UREUS BY POLYMORPHONUCLEAR LEUCOCYTES
Z . WOLDEHIWET and T . G . ROWAN* Department of Veterinary Pathology and *Department of Animal Husbandry, University of Liverpool, Leahurst, Neston, South Wirral L64 7TE, UK
SUMMARY 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 < 1 and 84 days . One group of seven calves was kept in a controlled environmental chamber with air temperature of 5 °C and 58% relative humidity (RH) and another group of eight calves was kept at 16 °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 °C and 16°C (P> 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< 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 < 1, 14, 28, 70 and 84 days of age (P<0 .001) .
INTRODUCTION Infectious diseases are common causes of morbidity and mortality in calves . Increased susceptibility to infection can result from hypogammaglobulinaemia, defective cellular immune responses, disorders of complement metabolism and functional defects in phagocytic cells (Renshaw et al., 1976) . The roles of polymorphonuclear cells (PMN) in inflammatory responses of domestic animals are well documented (Roth & Kaeberle, 1981 ; Jacobsen et al., 1982) . The phagocytosis and killing of bacteria requires the efficient function of PMN (Williams et al., 1985) . The higher incidence of infectious disease in calves compared with adult cattle has been attributed, in part at least, to lower resistance to infectious agents in calves . Improvements in resistance to infectious agents with age may be related to functional maturation of cellular defence mechanisms (Renshaw et al., 1976) . For example, some aspects of PMN function have been found to be significantly
166
BRITISH VETERINARY JOURNAL, 146 . 2
lower in human neonates (Miller, 1969), foals (Coignoul et al., 1984) and calves (Renshaw et al., 1976) than in the respective adult animals . Hauser, Koob & Roth (1986) found differences in PMN functions between calves of different ages . In several species, including calves, cold environments have been shown to impair immune responses (Kelley, 1980) . The aims of this study were to investigate the effects of age of calf and of environmental temperature on the capacity of calf PMN to phagocytose, and to kill, Staphylococcus aureus .
MATERIALS AND METHODS Calves Two groups of eight Friesian male calves were purchased directly from nearby farms of known histories . Within 24 hours of birth they were transported to the Department of Animal Husbandry and kept in a controlled environment chamber. Feeding In the first 36 hours each calf was given a total of 5 1 mixed-source colostrum and then was given, twice daily, a skimmed-milk based milk substitute diet (Easy-Mix, Volac Ltd, Royston) . The quantities of milk substitute diet given, twice daily, to each calf increased gradually from 200 g air dry matter (ADM) per day to 400 g ADM per day after 8 days of age . In addition, from 8 days of age the calves were offered ad libitum an early weaning, pelleted diet (Calfwena Quicklettes, BOCM Silcock Ltd, Basingstoke) and water . At 35 days of age, when each calf was consuming at least 0 . 75 kg ADM per day of the pelleted diet, the calves were weaned abruptly . No hay was offered . Environment The calves were housed on wood shavings in a controlled environment chamber with internal dimensions of 4 . 4 m by 3 . 2 m by 2 . 2 m . The air in the chamber was changed 50 times per hour without recycling and the air velocity was less than 0 . 1 m/s . Air temperature (± 0 . 5 °C) and relative humidity (RH ± 2%) were controlled continuously and recorded electronically (Humicap, Viasala Ltd, Cambridge) with twice weekly calibration using an aspirated hygrometer (Casella Ltd, London) . One group of calves was exposed to an air temperature of 5 °C and the other group to an air temperature of 16 ° C . For both groups of calves the relative humidity was approximately 58% . The data of Webster (1984) suggests that the thermal environment of the calves in the 16°C air was thermoneutral and that of calves in 5 ° C air was colder than thermoneutral until the calves were at least about 4 weeks of age . Twice daily, the calves were examined for signs of ill-health by a veterinary surgeon . Collection and preparation of leucocytes Jugular vein puncture was used to collect blood samples from the calves at less than 24 hours of age and thereafter at intervals of 14 days until the calves were 84 days of age . Peripheral blood leucocytes were obtained and processed as described previously (Woldehiwet, 1987) . Briefly, blood collected in EDTA-coated tubes was used to estimate total and differential white cell concentrations and blood collected in heparin-coated universal bottles was used to prepare leucocytes for bacterial phagocytosis and bacterial killing . The heparinized blood samples were treated with 0 . 83% ammonium chloride to
PHAGOCYTOSIS OF S. AUREUS BY CALF POLYMORPHS
1 67
lyse red blood cells, washed three times with cold phosphate buffered solution (PBS) at pH 7 . 2 and resuspended in minimum essential medium (MEM) (Gibco BRL, Paisley) with 0 . 5% inactivated fetal calf serum to give a final concentration of 10 8 viable PMN cells per ml . Staphylococcus aureus A strain of coagulase-positive Staphylococcus aureus isolated in this laboratory was used (Woldehiwet, 1987) . The bacteria were cultured in brain heart infusion broth (Oxoid Ltd) for 1 hour at 37 ° C and the optical density (OD) at 570 nm of the resulting suspension was determined . The number of colony forming units (CFU) of S. aureus in the suspension was estimated from a graph of CFU against OD at 570 nm established in subsidiary experiments . Then the bacteria were washed in PBS and resuspended in M EM to give a final concentration of 5X 10 8 bacteria per ml . Twenty 1ul of this suspension were then added to one ml of each of the PMN suspensions to give a ratio of bacteria to PMN of 1 to 1 . Assay of phagocytosis
The mixtures of bacteria (S. aureus) and leucocytes were incubated at 37°C for 2 hours with regular stirring and then the mixtures were centrifuged at 20 g for 5 min at 4 ° C . The supernatants were used to estimate the percentage of bacteria phagocytosed using the formula : Phagocytosis (%) =CFU before incubation-CFU after incubation X 100 CFU before incubation The viability of bacteria before incubation and after 2 hours of incubation were estimated by the method of Miles et al. (1938) . Assay of bacterial killing
Samples of MEM containing bacteria (S . aureus) alone and containing mixtures of bacteria (S. aureus) and leucocytes were incubated at 37 °C for 2 hours . Then the samples were treated with one cycle of freezing with liquid nitrogen and thawing . The number of CFU was then estimated by the method of Miles et al. (1938) . The percentage of bacteria killed was estimated according to the formula :
CFU in sample containing bacteria aloneBacterial kill (%)=CFU in sample containing bacteria & leucocyte mixture X 100 CFU in sample containing bacteria alone
Statistical analyses
The effects of environment were confounded with those for groups of calves . The significance of differences in the percentages of bacteria phagocytosed, and of bacteria killed, by leucocytes between environments (groups of calves) and age of calves were determined using analyses of variance (Snedecor & Cochran, 1980) and Duncan's multiple range test (Duncan, 1955) .
168
BRITISH VETERINARY JOURNAL, 146 . 2 RESULTS
Coughing was observed in both groups of calves . It was more marked in the group of calves in the 5 °C air than in calves in 16°C air . One calf in 5 ° C air was withdrawn from the experiment for treatment . None of the other calves required treatment . The total and differential WBC concentrations were within the ranges of values for normal calves suggested by Jain (1986) . There were no significant differences between environments (groups of calves) in the total and differential WBC concentrations (P> 0 . 05) (Table I) . The total WBC concentrations decreased with age until approximately 56 days of age . Most of the decreases were in PMN cells, the predominant phagocyte . The ratio of PMN to lymphocytes changed from 2 . 2 :1 at < 1 day of age to 0 . 7 : 1 at 56 days of age .
Table I Total, and differential, white blood cell concentrations (X109/1) in calves at various ages*
Age (days) Total WBC count Polymorphonuclear leucocytes Lymphocytes Monocytes No . of calves
<1
14
28
42
56
70
84
11 . 4t ±1 .3 7. 6 ±1 . 1 3. 5 ±0 .6 0. 3 ±0 . 08 15
8. 0 ±1 .3 3.5 ±0 . 7 3. 7 ±0 .5 0. 5 ±0 . 09 15
6.9 ±0 .5 3. 7 ±0 . 4 3.0 ±0 .3 0. 2 ±0 . 04 15
7. 5 ±0 .8 3.2 ±0 . 6 3. 7 ±0 .3 0. 5 ±0 . 09 15
5.2 ±0 .5 2. 1 ±0 . 3 2. 9 ±0 . 3 0. 3 ±0 . 04 15
5.6 ±0 .5 2.2 ±0 . 3 3.2 ±0 . 4 0.3 ±0 . 04 15
7 .1 ±0 .5 3.1 ±0 . 4 3 .2 ±0 . 3 0 .4 ±0 . 05
* Calves were weaned at 35 days of age . tMean ± SE of mean. There were no significant environment (group of calves)Xage interactions and no significant differences between environments (group of calves) (P> 0 . 05) .
For the percentages of S. aureus phagocytosed, and the percentages of S. aureus killed by peripheral blood leucocytes, there were no significant environment (group of calves)Xage interactions and there were no significant differences between environments (groups of calves) (P> 0 . 05) . The percentages of S . aureus phagocytosed at < 1 day of age were significantly greater than at all other ages (P<0 . 001) (Table II) . The lowest percentages of S. aureus phagocytosed, and of S. aureus killed, were in leucocytes obtained from calves of 42 days of age, that is 7 days after weaning . These percentages were significantly lower than those from the same calves at all other ages (P< 0 . 001) . The percentages of S. aureus phagocytosed, and of S. aureus killed by leucocytes obtained from calves of 56 days of age, that is 21 days after weaning, were significantly lower than those from the same calves at < 1, 14, 28, 70 and 84 days of age .
1 69
PHAGOCYTOSIS OF S. AUREUS BY CALF POLYMORPHS
Table II Percentages of bacteria (Staphylococcus aureus) phagocytosed, and of bacteria killed, by leucocytes from calves at various ages* Age (days)
Bacteria phagocytosed (%)t Bacteria killed (%) No . of calves
< 1
14
28
42
56
70
84
SE__
97 •l ' 92 . 8' 15
88 . 1b 82 . 3a 15
81 . 3 6 80 . 6' 15
43 . 5d 40 . 2° 15
65 . 3` 52 . 1" 15
82 . 1 b 79 . 1" 15
85 . 8`' 84 . 0' 15
3 . 11 4 . 38
*Calves were weaned at 35 days of age . tMeans with different letters in the same row are significantly different (P< 0 . 001) . There were no significant environment (group of calves)Xage interactions and no significant differences between environments (groups of calves) (P> 0 . 05) .
DISCUSSION There have been few controlled experiments on the effects of age of calf and thermal environment on immune function in young calves . The winter weather and housing conditions of young calves in the United Kingdom are likely to represent conditions which are at least intermittently below thermoneutral (Williams, 1977) . The effective lower critical temperature of newborn calves either standing in still air or lying on wooden slats is about 11 ° C and lying in a deep, dry straw bed is about 6 ° C (Webster, 1984) . The effects of environment were confounded with the effects of group of calves . However, there were no indications of significant differences in bacterial phagocytosis, and in bacterial killing, by leucocytes between environments (groups of calves) at any ages of calves. The authors are not aware of other reports on the effects of thermal environment of animals on the in-vitro functions of phagocytes . The results suggest that the in-vitro phagocytosis and killing of bacteria by peripheral blood leucocytes were very efficient, being > 90% in calves < 1 day of age and > 80% in calves 14, 28 and 84 days . Hauser et al. (1986) found that PMN from unweaned calves of 4-5 weeks of age were significantly more efficient in phagocytosing S aureus in vitro than those from weaned calves of 9 weeks to 14 months of age (P< 0 . 05) . Renshaw et al. (1976) found no differences in the opsonic capacity of serum against S. aureus between newborn calves and adult cattle but adult cattle serum was able to opsonize E . coli more efficiently than that of newborn calf serum . The in-vitro efficiencies of phagocytosis, and of killing, of S . aureus by peripheral blood leucocytes decreased greatly between samples taken from calves at 28 and 42 days of age . At these ages the blood concentrations of PMN, the principal phagocyte, were also decreasing from the high concentrations in newborn calves . These decreases in PMN concentration are normal physiological events (Jain, 1986) . The calves were weaned abruptly at 35 days of age . This is a commonly used method and age of weaning calves in the United Kingdom . As the experiment did not include calves not weaned at 35 days of age, then whether or not the weaning either caused or contributed to the decreases in the in-vitro efficiencies of bacterial phagocytosis and bacterial killing by leucocytes is a moot
1 70
BRITISH VETERINARY JOURNAL, 146 . 2
point . If these in-vitro decreases were reflected in similar in-vivo decreases, then the point is worthy of further study as it may have considerable practical relevance to the prevention of disease in young calves . The recovery of preweaning levels of efficiency of bacterial phagocytosis and bacterial killing by leucocytes did not occur until between 56 and 70 days of age . Therefore, between the ages of approximately 4 and 10 weeks the calves may have been susceptible to infections normally prevented by efficient bacterial phagocytosis and bacterial killing by leucocytes. Roy et al. (1971) noted that calves weaned at 5 weeks of age had a higher incidence and severity of pneumonic lung lesions than calves given both a higher level and longer duration of milk feeding . Gwazdauskas et al. (1978) found in calves that antibody syntheses to heterologous erythrocytes were decreased by weaning . Crookshank et al. (1979) and Gwazdauskas et al. (1978) found that weaning caused increased serum glucocorticoid concentrations . Possibly the effects of weaning on immune responses, including bacterial phagocytosis and bacterial killing by leucocytes, are mediated by increased glucocorticoid concentrations (Kelley, 1980) .
ACKNOWLEDGEMENTS The technical support of T . Houghton, C . Savage and M . Savage is gratefully acknowledged .
REFERENCES COIGNOUL, F . L ., BERTRAM, T . A ., ROTH, J . A . & CHEVILLE, N . F. (1984) . American Journal of Veterinary Research 45, 898 . CROOKSHANK, H . R ., ELISSALDE, M . H ., WHITE, R. G ., CLANTON, D . C . & SMALLEY, H . E . (1979) . Journal of Animal Science 48, 430 . DUNCAN, D . B . (1955) . Biometrics 11, 1 . GWAZDAUSKAS, F . C ., GROSS, W . B ., BIBB, T . L . & MCGILLIARD, M . L . (1978) . Canadian Veterinary Journal 19, 150 . HAUSER, M . A ., Koos, M . D. & ROTH, J . A . (1986) . American Journal of Veterinary Research 47, 152 . JACOBSEN, K ., GINIz, T., REED, S . M ., NEWBRY, J ., BAYLY, W . M ., PERRYMAN, L . E . & WES LEID, R . (1982) . American Journal of Veterinary Research 43, 1912. JAIN, N . C . (1986) . Schalm's Veterinary Haematology, 4th edn, p . 178. Philadelphia : Lea and Febiger. KELLEY, K. W . (1980) . Annales de Recherches Vétérinaires 11, 445 . MILES, R. A., MISRA, S . S. & IRWIN, J . O . (1938). Journal of Hygiene 38, 732 . MILLER, M . E . (1969) . Journal of Paediatrics 74, 255 . RENSHAW, H . W ., ECKBLAD, W . P ., THACKER, D . L . & FRANK, F . W (1976) . American Journal of Veterinary Research 37, 1267 . ROTH, J . A . & KAEBERLE, M . L . (1981) . Veterinary Immunology and Immunopathology 2, 157 . RoY, J . H . B ., STOBo, I . J . F ., GASTON, H . J ., GANDERTON, P., SHOT"ION, S . M . & OSTLER, D . C . (1971) . British Journal of .Nutrition 26, 363 . SNEDECOR, G . W . & COCHRAN, W . G . (1980) . Statistical Methods, 7th edn, p . 144 . Ames : Iowa State
University Press . WEBSTER, A . J. F . (1984) . Calf Husbandry, Health and Welfare, p . 71 . London : Granada . WILLIAMS, M . R ., CRAVIN, N ., FIELD, T . R . & BUNCH, K. J . (1985) . British Veterinary journal 141, 362 . WILLIAMS, P . E . V . (1977) . PhD Thesis, Glasgow University . WOLDEHIWET, Z . (1987) . Journal of Comparative Pathology 97, 481 . (Accepted for publication 27May 1989)
1. (1990) . 146, 165
SOME OBSERVATIONS ON THE EFFECTS OF AGE OF CALVES ON THE PHAGOCYTOSIS AND KILLING OF STAPHYLOCOCCUS A UREUS BY POLYMORPHONUCLEAR LEUCOCYTES
Z . WOLDEHIWET and T . G . ROWAN* Department of Veterinary Pathology and *Department of Animal Husbandry, University of Liverpool, Leahurst, Neston, South Wirral L64 7TE, UK
SUMMARY 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 < 1 and 84 days . One group of seven calves was kept in a controlled environmental chamber with air temperature of 5 °C and 58% relative humidity (RH) and another group of eight calves was kept at 16 °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 °C and 16°C (P> 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< 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 < 1, 14, 28, 70 and 84 days of age (P<0 .001) .
INTRODUCTION Infectious diseases are common causes of morbidity and mortality in calves . Increased susceptibility to infection can result from hypogammaglobulinaemia, defective cellular immune responses, disorders of complement metabolism and functional defects in phagocytic cells (Renshaw et al., 1976) . The roles of polymorphonuclear cells (PMN) in inflammatory responses of domestic animals are well documented (Roth & Kaeberle, 1981 ; Jacobsen et al., 1982) . The phagocytosis and killing of bacteria requires the efficient function of PMN (Williams et al., 1985) . The higher incidence of infectious disease in calves compared with adult cattle has been attributed, in part at least, to lower resistance to infectious agents in calves . Improvements in resistance to infectious agents with age may be related to functional maturation of cellular defence mechanisms (Renshaw et al., 1976) . For example, some aspects of PMN function have been found to be significantly
166
BRITISH VETERINARY JOURNAL, 146 . 2
lower in human neonates (Miller, 1969), foals (Coignoul et al., 1984) and calves (Renshaw et al., 1976) than in the respective adult animals . Hauser, Koob & Roth (1986) found differences in PMN functions between calves of different ages . In several species, including calves, cold environments have been shown to impair immune responses (Kelley, 1980) . The aims of this study were to investigate the effects of age of calf and of environmental temperature on the capacity of calf PMN to phagocytose, and to kill, Staphylococcus aureus .
MATERIALS AND METHODS Calves Two groups of eight Friesian male calves were purchased directly from nearby farms of known histories . Within 24 hours of birth they were transported to the Department of Animal Husbandry and kept in a controlled environment chamber. Feeding In the first 36 hours each calf was given a total of 5 1 mixed-source colostrum and then was given, twice daily, a skimmed-milk based milk substitute diet (Easy-Mix, Volac Ltd, Royston) . The quantities of milk substitute diet given, twice daily, to each calf increased gradually from 200 g air dry matter (ADM) per day to 400 g ADM per day after 8 days of age . In addition, from 8 days of age the calves were offered ad libitum an early weaning, pelleted diet (Calfwena Quicklettes, BOCM Silcock Ltd, Basingstoke) and water . At 35 days of age, when each calf was consuming at least 0 . 75 kg ADM per day of the pelleted diet, the calves were weaned abruptly . No hay was offered . Environment The calves were housed on wood shavings in a controlled environment chamber with internal dimensions of 4 . 4 m by 3 . 2 m by 2 . 2 m . The air in the chamber was changed 50 times per hour without recycling and the air velocity was less than 0 . 1 m/s . Air temperature (± 0 . 5 °C) and relative humidity (RH ± 2%) were controlled continuously and recorded electronically (Humicap, Viasala Ltd, Cambridge) with twice weekly calibration using an aspirated hygrometer (Casella Ltd, London) . One group of calves was exposed to an air temperature of 5 °C and the other group to an air temperature of 16 ° C . For both groups of calves the relative humidity was approximately 58% . The data of Webster (1984) suggests that the thermal environment of the calves in the 16°C air was thermoneutral and that of calves in 5 ° C air was colder than thermoneutral until the calves were at least about 4 weeks of age . Twice daily, the calves were examined for signs of ill-health by a veterinary surgeon . Collection and preparation of leucocytes Jugular vein puncture was used to collect blood samples from the calves at less than 24 hours of age and thereafter at intervals of 14 days until the calves were 84 days of age . Peripheral blood leucocytes were obtained and processed as described previously (Woldehiwet, 1987) . Briefly, blood collected in EDTA-coated tubes was used to estimate total and differential white cell concentrations and blood collected in heparin-coated universal bottles was used to prepare leucocytes for bacterial phagocytosis and bacterial killing . The heparinized blood samples were treated with 0 . 83% ammonium chloride to
PHAGOCYTOSIS OF S. AUREUS BY CALF POLYMORPHS
1 67
lyse red blood cells, washed three times with cold phosphate buffered solution (PBS) at pH 7 . 2 and resuspended in minimum essential medium (MEM) (Gibco BRL, Paisley) with 0 . 5% inactivated fetal calf serum to give a final concentration of 10 8 viable PMN cells per ml . Staphylococcus aureus A strain of coagulase-positive Staphylococcus aureus isolated in this laboratory was used (Woldehiwet, 1987) . The bacteria were cultured in brain heart infusion broth (Oxoid Ltd) for 1 hour at 37 ° C and the optical density (OD) at 570 nm of the resulting suspension was determined . The number of colony forming units (CFU) of S. aureus in the suspension was estimated from a graph of CFU against OD at 570 nm established in subsidiary experiments . Then the bacteria were washed in PBS and resuspended in M EM to give a final concentration of 5X 10 8 bacteria per ml . Twenty 1ul of this suspension were then added to one ml of each of the PMN suspensions to give a ratio of bacteria to PMN of 1 to 1 . Assay of phagocytosis
The mixtures of bacteria (S. aureus) and leucocytes were incubated at 37°C for 2 hours with regular stirring and then the mixtures were centrifuged at 20 g for 5 min at 4 ° C . The supernatants were used to estimate the percentage of bacteria phagocytosed using the formula : Phagocytosis (%) =CFU before incubation-CFU after incubation X 100 CFU before incubation The viability of bacteria before incubation and after 2 hours of incubation were estimated by the method of Miles et al. (1938) . Assay of bacterial killing
Samples of MEM containing bacteria (S . aureus) alone and containing mixtures of bacteria (S. aureus) and leucocytes were incubated at 37 °C for 2 hours . Then the samples were treated with one cycle of freezing with liquid nitrogen and thawing . The number of CFU was then estimated by the method of Miles et al. (1938) . The percentage of bacteria killed was estimated according to the formula :
CFU in sample containing bacteria aloneBacterial kill (%)=CFU in sample containing bacteria & leucocyte mixture X 100 CFU in sample containing bacteria alone
Statistical analyses
The effects of environment were confounded with those for groups of calves . The significance of differences in the percentages of bacteria phagocytosed, and of bacteria killed, by leucocytes between environments (groups of calves) and age of calves were determined using analyses of variance (Snedecor & Cochran, 1980) and Duncan's multiple range test (Duncan, 1955) .
168
BRITISH VETERINARY JOURNAL, 146 . 2 RESULTS
Coughing was observed in both groups of calves . It was more marked in the group of calves in the 5 °C air than in calves in 16°C air . One calf in 5 ° C air was withdrawn from the experiment for treatment . None of the other calves required treatment . The total and differential WBC concentrations were within the ranges of values for normal calves suggested by Jain (1986) . There were no significant differences between environments (groups of calves) in the total and differential WBC concentrations (P> 0 . 05) (Table I) . The total WBC concentrations decreased with age until approximately 56 days of age . Most of the decreases were in PMN cells, the predominant phagocyte . The ratio of PMN to lymphocytes changed from 2 . 2 :1 at < 1 day of age to 0 . 7 : 1 at 56 days of age .
Table I Total, and differential, white blood cell concentrations (X109/1) in calves at various ages*
Age (days) Total WBC count Polymorphonuclear leucocytes Lymphocytes Monocytes No . of calves
<1
14
28
42
56
70
84
11 . 4t ±1 .3 7. 6 ±1 . 1 3. 5 ±0 .6 0. 3 ±0 . 08 15
8. 0 ±1 .3 3.5 ±0 . 7 3. 7 ±0 .5 0. 5 ±0 . 09 15
6.9 ±0 .5 3. 7 ±0 . 4 3.0 ±0 .3 0. 2 ±0 . 04 15
7. 5 ±0 .8 3.2 ±0 . 6 3. 7 ±0 .3 0. 5 ±0 . 09 15
5.2 ±0 .5 2. 1 ±0 . 3 2. 9 ±0 . 3 0. 3 ±0 . 04 15
5.6 ±0 .5 2.2 ±0 . 3 3.2 ±0 . 4 0.3 ±0 . 04 15
7 .1 ±0 .5 3.1 ±0 . 4 3 .2 ±0 . 3 0 .4 ±0 . 05
* Calves were weaned at 35 days of age . tMean ± SE of mean. There were no significant environment (group of calves)Xage interactions and no significant differences between environments (group of calves) (P> 0 . 05) .
For the percentages of S. aureus phagocytosed, and the percentages of S. aureus killed by peripheral blood leucocytes, there were no significant environment (group of calves)Xage interactions and there were no significant differences between environments (groups of calves) (P> 0 . 05) . The percentages of S . aureus phagocytosed at < 1 day of age were significantly greater than at all other ages (P<0 . 001) (Table II) . The lowest percentages of S. aureus phagocytosed, and of S. aureus killed, were in leucocytes obtained from calves of 42 days of age, that is 7 days after weaning . These percentages were significantly lower than those from the same calves at all other ages (P< 0 . 001) . The percentages of S. aureus phagocytosed, and of S. aureus killed by leucocytes obtained from calves of 56 days of age, that is 21 days after weaning, were significantly lower than those from the same calves at < 1, 14, 28, 70 and 84 days of age .
1 69
PHAGOCYTOSIS OF S. AUREUS BY CALF POLYMORPHS
Table II Percentages of bacteria (Staphylococcus aureus) phagocytosed, and of bacteria killed, by leucocytes from calves at various ages* Age (days)
Bacteria phagocytosed (%)t Bacteria killed (%) No . of calves
< 1
14
28
42
56
70
84
SE__
97 •l ' 92 . 8' 15
88 . 1b 82 . 3a 15
81 . 3 6 80 . 6' 15
43 . 5d 40 . 2° 15
65 . 3` 52 . 1" 15
82 . 1 b 79 . 1" 15
85 . 8`' 84 . 0' 15
3 . 11 4 . 38
*Calves were weaned at 35 days of age . tMeans with different letters in the same row are significantly different (P< 0 . 001) . There were no significant environment (group of calves)Xage interactions and no significant differences between environments (groups of calves) (P> 0 . 05) .
DISCUSSION There have been few controlled experiments on the effects of age of calf and thermal environment on immune function in young calves . The winter weather and housing conditions of young calves in the United Kingdom are likely to represent conditions which are at least intermittently below thermoneutral (Williams, 1977) . The effective lower critical temperature of newborn calves either standing in still air or lying on wooden slats is about 11 ° C and lying in a deep, dry straw bed is about 6 ° C (Webster, 1984) . The effects of environment were confounded with the effects of group of calves . However, there were no indications of significant differences in bacterial phagocytosis, and in bacterial killing, by leucocytes between environments (groups of calves) at any ages of calves. The authors are not aware of other reports on the effects of thermal environment of animals on the in-vitro functions of phagocytes . The results suggest that the in-vitro phagocytosis and killing of bacteria by peripheral blood leucocytes were very efficient, being > 90% in calves < 1 day of age and > 80% in calves 14, 28 and 84 days . Hauser et al. (1986) found that PMN from unweaned calves of 4-5 weeks of age were significantly more efficient in phagocytosing S aureus in vitro than those from weaned calves of 9 weeks to 14 months of age (P< 0 . 05) . Renshaw et al. (1976) found no differences in the opsonic capacity of serum against S. aureus between newborn calves and adult cattle but adult cattle serum was able to opsonize E . coli more efficiently than that of newborn calf serum . The in-vitro efficiencies of phagocytosis, and of killing, of S . aureus by peripheral blood leucocytes decreased greatly between samples taken from calves at 28 and 42 days of age . At these ages the blood concentrations of PMN, the principal phagocyte, were also decreasing from the high concentrations in newborn calves . These decreases in PMN concentration are normal physiological events (Jain, 1986) . The calves were weaned abruptly at 35 days of age . This is a commonly used method and age of weaning calves in the United Kingdom . As the experiment did not include calves not weaned at 35 days of age, then whether or not the weaning either caused or contributed to the decreases in the in-vitro efficiencies of bacterial phagocytosis and bacterial killing by leucocytes is a moot
1 70
BRITISH VETERINARY JOURNAL, 146 . 2
point . If these in-vitro decreases were reflected in similar in-vivo decreases, then the point is worthy of further study as it may have considerable practical relevance to the prevention of disease in young calves . The recovery of preweaning levels of efficiency of bacterial phagocytosis and bacterial killing by leucocytes did not occur until between 56 and 70 days of age . Therefore, between the ages of approximately 4 and 10 weeks the calves may have been susceptible to infections normally prevented by efficient bacterial phagocytosis and bacterial killing by leucocytes. Roy et al. (1971) noted that calves weaned at 5 weeks of age had a higher incidence and severity of pneumonic lung lesions than calves given both a higher level and longer duration of milk feeding . Gwazdauskas et al. (1978) found in calves that antibody syntheses to heterologous erythrocytes were decreased by weaning . Crookshank et al. (1979) and Gwazdauskas et al. (1978) found that weaning caused increased serum glucocorticoid concentrations . Possibly the effects of weaning on immune responses, including bacterial phagocytosis and bacterial killing by leucocytes, are mediated by increased glucocorticoid concentrations (Kelley, 1980) .
ACKNOWLEDGEMENTS The technical support of T . Houghton, C . Savage and M . Savage is gratefully acknowledged .
REFERENCES COIGNOUL, F . L ., BERTRAM, T . A ., ROTH, J . A . & CHEVILLE, N . F. (1984) . American Journal of Veterinary Research 45, 898 . CROOKSHANK, H . R ., ELISSALDE, M . H ., WHITE, R. G ., CLANTON, D . C . & SMALLEY, H . E . (1979) . Journal of Animal Science 48, 430 . DUNCAN, D . B . (1955) . Biometrics 11, 1 . GWAZDAUSKAS, F . C ., GROSS, W . B ., BIBB, T . L . & MCGILLIARD, M . L . (1978) . Canadian Veterinary Journal 19, 150 . HAUSER, M . A ., Koos, M . D. & ROTH, J . A . (1986) . American Journal of Veterinary Research 47, 152 . JACOBSEN, K ., GINIz, T., REED, S . M ., NEWBRY, J ., BAYLY, W . M ., PERRYMAN, L . E . & WES LEID, R . (1982) . American Journal of Veterinary Research 43, 1912. JAIN, N . C . (1986) . Schalm's Veterinary Haematology, 4th edn, p . 178. Philadelphia : Lea and Febiger. KELLEY, K. W . (1980) . Annales de Recherches Vétérinaires 11, 445 . MILES, R. A., MISRA, S . S. & IRWIN, J . O . (1938). Journal of Hygiene 38, 732 . MILLER, M . E . (1969) . Journal of Paediatrics 74, 255 . RENSHAW, H . W ., ECKBLAD, W . P ., THACKER, D . L . & FRANK, F . W (1976) . American Journal of Veterinary Research 37, 1267 . ROTH, J . A . & KAEBERLE, M . L . (1981) . Veterinary Immunology and Immunopathology 2, 157 . RoY, J . H . B ., STOBo, I . J . F ., GASTON, H . J ., GANDERTON, P., SHOT"ION, S . M . & OSTLER, D . C . (1971) . British Journal of .Nutrition 26, 363 . SNEDECOR, G . W . & COCHRAN, W . G . (1980) . Statistical Methods, 7th edn, p . 144 . Ames : Iowa State
University Press . WEBSTER, A . J. F . (1984) . Calf Husbandry, Health and Welfare, p . 71 . London : Granada . WILLIAMS, M . R ., CRAVIN, N ., FIELD, T . R . & BUNCH, K. J . (1985) . British Veterinary journal 141, 362 . WILLIAMS, P . E . V . (1977) . PhD Thesis, Glasgow University . WOLDEHIWET, Z . (1987) . Journal of Comparative Pathology 97, 481 . (Accepted for publication 27May 1989)