Research in VeterinaryScience1991, 51, 254-257
A quantitative measurement of the effect of avian influenza virus on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract V. SIVANANDAN, K. V. N A G A R A J A , D. A. HALVORSON, J. A. NEWMAN, Department o f Veterinary Pathobiology, College o f Veterinary Medicine, University o f Minnesota, St Paul, Minnesota 55108, USA
The effect of avian influenza virus (AIV) infection on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract was evaluated. Four-week-old turkeys were experimentally infected with an apathogenic AIV subtype (H5N2) by the oculonasal route and subsequently superinfected with P multocida (Urbach strain) by the intranasal route three days after infection with AIr. Quantitative clearance of P multocida from the trachea and lung was determined using a pour plate technique on samples collected at intervals after infection. Samples from turkeys which had been infected with AIV were found to yield more P multocida than those from turkeys which had not been infected with AIV. The numbers of P multocida increased in infected birds to a greater extent than in birds which had not been infected with the virus. The present study suggests that AIV infection may contribute to the increased numbers and a decreased clearance of P multocida in turkeys.
IN general, viral infections of the respiratory tract increase the frequency and severity of bacterial pneumonia by altering pulmonary clearance processes. These secondary bacterial infections can increase the morbidity and mortality observed during viral epidemics. Influenza-infected humans have an increased susceptibility to secondary infection in the lung (Loosli 1973). It has been demonstrated that influenza virus infection established by intranasal inoculation suppresses bacterial killing in the lung and delays the clearance of bacteria from bronchoalveolar spaces (Nugent and Pesanti 1982). Studies in mice using an aerosol challenge technique (Rodriguez et a11985) in which modest numbers of staphylococci are deposited in the lower respiratory tract enabled evaluation of the early phase of bacterial clearance from the lung in mammalian species. Little is known about what effects environmental factors have on the ability of birds to clear inhaled bacteria. Although aerosolised pathogenic Escher-
ichia coli is rapidly cleared from the lungs and air sacs of normal turkeys (Arp et al 1979), studies in this laboratory have shown that retention of E coli in the lungs and air sacs is increased if birds are kept in an atmosphere containing ammonia (Nagaraja et al 1984). Recent studies haves indicated that clearance of E coli from air sacs was little affected in turkeys infected with Bordetella avium (Ficken et al 1986). Clearance of aerosolised E coli by the lungs and air sacs of turkeys previously exposed to Newcastle disease virus was shown to be depressed between five and nine days after such exposure (Ficken et al 1987). Mortality up to 30 per cent has been reported in turkey flocks simultaneously infected with non-pathogenic avian influenza virus (AW) and the cu strain of fowl cholera vaccine (Newman et al 1981). To date, there has been no study of the effect of AIr on the ability of the respiratory tract of turkeys to clear bacterial infections. This paper presents the results of experiments to determine the effect of an apathogenic AIr infection on pulmonary clearance of Pasteurella multocida in turkeys.
Materials and methods
Virus Avian influenza virus subtype H5N2 was used, This virus had been isolated from a turkey flock during a surveillance programme (Halvorson et al 1983). The virus was found to be non-pathogenic for chickens as reported by the National Veterinary Services Laboratory Science and Technology, Animal Plant Health Inspection Service, USDA, Ames, Iowa. The virus was propagated in 10-day-old embryonating chicken eggs. Infectious allantoic fluid was collected, clarified by centrifugation at 6000 g for 20 minutes at 4°C and haemagglutinating activity determined by making twofold dilutions in a microtest plate followed by the addition of 0- 5 per cent chicken erythrocytes.
254
255
Effect o f m y on P multocida in turkeys Bacteria Pasteurella multocida serotype A:3 was used. This strain of P multocida had been isolated from turkeys during an outbreak of fowl cholera in Minnesota. Lyophilised bacteria were suspended in tryptic soy broth and subcultured on blood agar plates and incubated for 24 hours at 37°C. Single colonies of bacteria were transferred to tryptic soy broth and incubated for 18 hours at 37°C. The pour plate technique (Nagaraja et al 1984) was used to enumerate viable bacteria. Before its use biochemical tests were conducted to confirm the isolate to be P multocida. Turkeys One hundred and sixty one-day-old turkeys were obtained from a commercial hatchery and raised in isolation units at the university research facilities. At four weeks of age, 20 turkeys were randomly selected and euthanased. The trachea and lungs were removed from each bird and screened for the presence of P multocida by plating on blood agar plates containing 2 mg m l - l clindamycin. The remaining 140 turkeys were divided equally into two groups and placed in two separate isolation rooms.
Experimental design The 70 birds in group 1 were infected with AW. Each bird was infected by the oculonasal route with 0.2 ml of inoculum containing 1024 haemagglutinating (HA) units. The 70 birds in group 2 each received 0" 2 ml of virus diluent by the same route. Three days after these procedures were performed all birds in both groups were superinfected with P multocida by the intranasal route. Each bird was infected using an inoculum of 0- 2 ml of an 18 hour broth culture containing 1-2 x 109 colony forming units (cfu) m1-1.
and 120 hours after exposure to P multocida, 10 birds were removed from each group and euthanased. The trachea and lungs were aseptically removed from each bird. The tissues from each bird were weighed, placed separately in sterile phosphate buffered saline (PBS) and homogenised to give a 20 per cent (w/v) suspension.
Enumeration of bacteria Sterile PBS was used to prepare 10-fold serial dilutions of each tissue suspension. Bacteria present in the suspensions were enumerated using standard plate counts to determine cfu g - t of tissue. Blood agar plates containing 2 izg m l - l clindamycin were used as a selective medium (Garlinghouse et al 1981). Each dilution was plated out onto blood agar in triplicate. Colonies were counted after 24 hours incubation at 37°C. The mean bacterial count was calculated for each tissue suspension.
Results The results are presented in Table 1 and Fig 1. The mean deposition of bacteria immediately following exposure was 1.97× 107 and 2 . 8 2 × 107 cfu g-] of tissue in AIV infected and uninfected groups, respectively. There was no apparent difference in the number of cfu of P multocida recovered from AIV infected and uninfected groups until four hours after exposure. However, at six, eight and 24 hours after exposure, the AIV infected birds had more P multocida than both the initial inoculum and the nonAIV infected (control) birds. One hundred and twenty hours after exposure, the non-Air infected group showed a 100-fold reduction of P multocida g i with 96 per cent of the initial peak
o
~= 10.0.
Sample collection
~,
Immediately (0 hour) and at four, six, eight, 24, 72 TABLE 1: Colony forming units of P multocida g - 1 of trachea and lung in AIV-infected and uninfected turkeys Hours after P rnultocida exposure O* 4 6 8 24 72 120
Non-AIV infected 2"82x107t 1'21x107 2.19x108 4 " 3 7 x 107 1 , 6 4 x 108 1 . 3 B x 10 e 1 . 1 4 x 106
* Immediately following exposure "P Mean of 1Obirds
AIV infected 1.97x1071 3"10x107 1 .53x109 1-29x109 1 . 9 0 x 109 8.10x108 1 . 8 7 x 108
Ratio AIV infected/ non-AIV infected 0.7 2.6 7.0 29-5 11.6 5.9 164-0
AtVinfected . . . . . Control
9,5.
.~
9-0.
~
8.5.
.~ o 7.0- I
4
~
6"5
~
5.5
~-
5.01
0
2'4
7'2
Hours following exposureto P multocida FIG 1 : Quantitative clearance of P multocida from trachea and lungs of AIV-infected and uninfected turkeys
256
V. Sivanandan, K. V. Nagaraja, D. A. Halvorson, J. A. Newman
load being cleared. The AIv-infected group showed only a corresponding fourfold decrease in cfu g - 1.
Discussion The present study indicates decreased clearance of P multocida from the respiratory tract o f AWinfected turkeys compared with birds not infected with AIV. This suggests an impairment of the mucociliary defence system in AIV infected birds. The mechanisms involved in the suppression o f pulmonary bactericidal activity during the experimental AIV infection remains to be elucidated. Severe influenza virus infections in mammals with pneumonic involvement have been shown to impair p u l m o n a r y antibacterial defence, resulting in bacterial infections (Jakab 1981). The majority of influenza virus infections, however, are limited to the upper respiratory tract (Douglas 1975) and the effect of infections with milder strains on lung defences is not known. In the present study the effect of a nonpathogenic strain of avian influenza virus infection on the clearance of P multocida from trachea and lungs of turkeys was examined. The A1V infected group showed an increase in numbers of P multocida in these tissues reaching a maximum 24 hours after exposure. Normally, in mammals, inhaled bacteria entering the lungs as small droplets or by aspiration of fluid from the upper respiratory tract will be cleared by the natural defence mechanism (Green et al 1977). Bacteria deposited in the distal part of the ltlng are rapidly engulfed and inactivated by resident phagocytes, the alveolar macrophages. These phagocytic cells in the terminal airways play the pivotal defensive role against bacterial infection (Goldstein et al 1974, Kim et al 1976, Goldstein 1983). In addition, when inflammatory processes are established, polymorphonuclear leucocytes migrate into the lungs and assist in phagocytosis (Pierce et al 1977, Rehm et al 1979). Thus the pulmonary antibacterial defence is dependent on a dual phagocytic system that involves both alveolar macrophages and polymorphonuclear leucocytes. In the avian species, the paucity of resident macrophages in the respiratory tract has been documented (Toth and Siegel 1986). This may contribute to the vulnerability of the avian respiratory tract to inhaled pathogens. Hence, the respiratory tract in the avian species largely depends on recruited phagocytic cells for pulmonary antibacterial defence. Impaired phagocytosis of bacteria by alveolar macrophages of mice with influenza viral infection has been reported (Warshauer et al 1977). However, Jakab et al (1980) reported impaired phagosomelysosome fusion while Nugent and Pesanti (1979) reported normal phagocytosis, phagosome-lysosome
fusion and killing with mouse alveolar macrophages. The authors speculate that the differences reported may be due to one or more variable factors such as the virus type, subtype, variant, strain, passage history, suspending medium, dose and method of inoculation. It has been demonstrated that spontaneous quantitative and qualitative changes in the tracheal flora, including the appearance of Gram-negative bacteria, occur during sublethal influenza infections in mice, and that they are temporarily associated with overt damage to the tracheal mucosa (Nugent and Pesanti 1983). This experiment with an aerosol challenge model demonstrated that the influenza infection reduced tracheal clearance. Currently, commercial turkeys are vaccinated with a live cu or M9 vaccine. Although these vaccines have helped to control fowl cholera, it is not uncommon to see post-vaccinal reactions in the field resulting in fowl cholera. Serological evidence in many of these cases indicates a prior exposure of these turkeys to AIV and non-pathogenic AIV subtypes have been isolated from such cases (unpublished observations). In fowl cholera vaccination the doses of the vaccines, the health status of the flock and the time of vaccination are important factors. In mammals, the loss of ciliary action during influenza virus infection (Kleinerman et al 1975), in addition to impairment of phagosome-lysosome fusion (Jakab et al 1980) has been reported. Whether there was any ciliary destruction or impaired phagosome-lysosome fusion in the macrophages of turkeys exposed to AIV was not investigated in this study and remains to be determined. The results of this study suggest that even nonpathogenic strains of influenza virus may predispose the host to secondary bacterial infections.
Acknowledgements This work was supported in part by grants from the Minnesota Agricultural Experiment Station (03024863-22) and the Minnesota Turkey Grower's Association.
References ARP, L. H., G R A H A M , C. L. ~ . & CHEVEILLE, N. F. (1979) Comparison of clearance rates of virulent Escherichia coli in turkeys after aerosol exposure. Avian Diseases 2 3 , 3 8 6 - 3 9 1 DOUGLAS, R. G. (1975) The Influenza Viruses and Influenza. Ed E. D. Kilbourne. London, Academic Press. pp 395-481 FICKEN, M. D., EDWARDS, J. F. & LAY, J. C. (1986) Clearance of bacteria in turkeys with Bordetella avium-induced tracheitis. Avian Diseases 30, 352-357 FICKEN, M. D., EDWARDS, J. F., LAY, J. C. & TVETER, D. E. (1987) Tracheal mucus transport rate and bacterial clearance in turkeys exposed by aerosol to La Sota strain of Newcastle disease virus. Avian Diseases 3 1 , 2 4 1 - 2 4 8
E f f e c t Of AIV on P multocida in turkeys GARLINGHOUSE Jr, L. E., DIGIACOMO, R. V., VAN HOOSER Jr, G. L. & CONDON, J. (1981) Selective media for Pasteurella multocida and Bordetella bronchiseptica. Laboratory of Animal Science 31, 39-42 GOLDSTEIN, E. (1983) Hydrolytic enzymes of alveolar macrophages. Review of Infectious Diseases 5, 1078-1092 GOLDSTEIN, E., LIPPERT, W. & WARSHAUER, D. (1974) Pulmonary alveolar macrophage. Defender against bacterial infection of the lung. Journal of Clinical Investigation 54, 519-528 GREEN, G. M., JAKAB, G. J., LOW, R. B. & DAVIS, G. S. (1977) Defense mechanisms of the respiratory membrane. American Review of Respiratory Disease 115, 479-514 HALVORSON, D. A., KARUNUKARAN, D., SENNE, D. A., KELLEHER, C. J., BAILEY, C., ABRAHAM, A., HINSHAW, D. & NEWMAN, J. A. (1983) Epizootiology of avian influenzasimultaneous monitoring of sentinel ducks and turkeys in Minnesota. Avian Diseases 27, 77-85 JAKAB, G. J., WARR, G. A. & SANNES, P. L. (1980) Alveolar macrophage ingestion and phagosome-lysosome fusion defect associated with virus pneumonia. Infection and Immunity 27, 960-968 JAKAB, G. J. (1981) Mechanisms of virus-induced bacterial super infections of the lung. Clinics in Chest Medicine 2, 59-66 KIM, M., GOLDSTEIN, E., LEWIS, J. P., LIPPERT, W. & WARSHAUER, D. (1976) Murine pulmonary alveolar macrophage: rates of bacterial ingestion, inactivation and destruction. Journal of Infectious Diseases 133, 310-320 KLEINERMAN, E. S., SNYDERMAN, R. & DANIELS, C. A. (1975) Depressed monocyte chemotaxis during acute influenza infection. Lancet ii, 1063-1066 LOOSLI, C. G. (1973) Influenza and the interaction of viruses and bacteria in respiratory infections. Medicine 52, 369 NAGARAJA, K. V., EMERY, D. A., JORDAN, K. A., SIVANANDAN, V., NEWMAN, J. A. & POMEROY, B. S. (1984) Effect of ammonia on the quantitative clearance of Escherichia eoli from lungs, air sacs and livers of turkeys aerosol vaccinated against Escherichia coli. American Journal of Veterinary Research 45, 392-395
257
NEWMAN, J. A., HALVORSON, D., KARUNAKARAN, D., POSS, P. E. & JOHNSON, J. (1981) Complications associated with avian influenza virus infections. Proceedings of the First International Symposium on Avian Influenza. Ed R. A. Bankowski. pp 8-11 NUGENT, K. M. & PESANTI, E. L. (1979) Effect of influenza infection on the phagocytic and bactericidal activities of pulmonary macrophages. Infection and Immunity 26, 651-657 NUGENT, K. M. & PESANTI, E. L. (1982) Staphylococcal clearance and pulmonary macrophage function during influenza infection. Infection and Immunity 38, 1256-1262 NUGENT, K. M. & PESANTI, E. L. (1983) Tracheal function during influenza infections. Infection and Immunity 42, 1102-1108 PIERCE, A. K., REYNOLDS, R. C. & HARRIS, G. D. (1977) Leukocytic response to inhaled bacteria. American Review of Respiratory Disease 116, 679-684 REHM, S. R., GROSS, G. N., HART, D. A. & PIERCE, A. K. (1979) Animal model of neutropenia suitable for the study of dualphagocyte systems. Infection and Immunity 25, 299-303 RODRIGUEZ, L. M., LOPEZ, A., MERINO-MONCADA, M., MARTINEZ-BURNES, J. & MONDRAGON, I. (1985) Tracheal versus pulmonary deposition and clearance of inhaled pasteurelta hemolyticao f Staphylococcus aureus in mice. Canadian Journal of Comparative Medicine 49, 323-326 TOTH, T. E. & SIEGEL, P. B. (1986) Cellular defense of the avian respiratory tract: paucity of free-residing macrophages in the normal chicken. Avian Diseases 30, 67-75 WARSHAUER, D., GOLDSTEIN, E., AKERS, T., LIPPERT, W. & KIM, M. (1977) Effect of influenza viral infection on the ingestion and killing of bacteria by alveolar macrophages. American Review of Respiratory Diseases 115,269-277
Received December 21, 1990 Accepted May 28, 1991
A quantitative measurement of the effect of avian influenza virus on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract V. SIVANANDAN, K. V. N A G A R A J A , D. A. HALVORSON, J. A. NEWMAN, Department o f Veterinary Pathobiology, College o f Veterinary Medicine, University o f Minnesota, St Paul, Minnesota 55108, USA
The effect of avian influenza virus (AIV) infection on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract was evaluated. Four-week-old turkeys were experimentally infected with an apathogenic AIV subtype (H5N2) by the oculonasal route and subsequently superinfected with P multocida (Urbach strain) by the intranasal route three days after infection with AIr. Quantitative clearance of P multocida from the trachea and lung was determined using a pour plate technique on samples collected at intervals after infection. Samples from turkeys which had been infected with AIV were found to yield more P multocida than those from turkeys which had not been infected with AIV. The numbers of P multocida increased in infected birds to a greater extent than in birds which had not been infected with the virus. The present study suggests that AIV infection may contribute to the increased numbers and a decreased clearance of P multocida in turkeys.
IN general, viral infections of the respiratory tract increase the frequency and severity of bacterial pneumonia by altering pulmonary clearance processes. These secondary bacterial infections can increase the morbidity and mortality observed during viral epidemics. Influenza-infected humans have an increased susceptibility to secondary infection in the lung (Loosli 1973). It has been demonstrated that influenza virus infection established by intranasal inoculation suppresses bacterial killing in the lung and delays the clearance of bacteria from bronchoalveolar spaces (Nugent and Pesanti 1982). Studies in mice using an aerosol challenge technique (Rodriguez et a11985) in which modest numbers of staphylococci are deposited in the lower respiratory tract enabled evaluation of the early phase of bacterial clearance from the lung in mammalian species. Little is known about what effects environmental factors have on the ability of birds to clear inhaled bacteria. Although aerosolised pathogenic Escher-
ichia coli is rapidly cleared from the lungs and air sacs of normal turkeys (Arp et al 1979), studies in this laboratory have shown that retention of E coli in the lungs and air sacs is increased if birds are kept in an atmosphere containing ammonia (Nagaraja et al 1984). Recent studies haves indicated that clearance of E coli from air sacs was little affected in turkeys infected with Bordetella avium (Ficken et al 1986). Clearance of aerosolised E coli by the lungs and air sacs of turkeys previously exposed to Newcastle disease virus was shown to be depressed between five and nine days after such exposure (Ficken et al 1987). Mortality up to 30 per cent has been reported in turkey flocks simultaneously infected with non-pathogenic avian influenza virus (AW) and the cu strain of fowl cholera vaccine (Newman et al 1981). To date, there has been no study of the effect of AIr on the ability of the respiratory tract of turkeys to clear bacterial infections. This paper presents the results of experiments to determine the effect of an apathogenic AIr infection on pulmonary clearance of Pasteurella multocida in turkeys.
Materials and methods
Virus Avian influenza virus subtype H5N2 was used, This virus had been isolated from a turkey flock during a surveillance programme (Halvorson et al 1983). The virus was found to be non-pathogenic for chickens as reported by the National Veterinary Services Laboratory Science and Technology, Animal Plant Health Inspection Service, USDA, Ames, Iowa. The virus was propagated in 10-day-old embryonating chicken eggs. Infectious allantoic fluid was collected, clarified by centrifugation at 6000 g for 20 minutes at 4°C and haemagglutinating activity determined by making twofold dilutions in a microtest plate followed by the addition of 0- 5 per cent chicken erythrocytes.
254
255
Effect o f m y on P multocida in turkeys Bacteria Pasteurella multocida serotype A:3 was used. This strain of P multocida had been isolated from turkeys during an outbreak of fowl cholera in Minnesota. Lyophilised bacteria were suspended in tryptic soy broth and subcultured on blood agar plates and incubated for 24 hours at 37°C. Single colonies of bacteria were transferred to tryptic soy broth and incubated for 18 hours at 37°C. The pour plate technique (Nagaraja et al 1984) was used to enumerate viable bacteria. Before its use biochemical tests were conducted to confirm the isolate to be P multocida. Turkeys One hundred and sixty one-day-old turkeys were obtained from a commercial hatchery and raised in isolation units at the university research facilities. At four weeks of age, 20 turkeys were randomly selected and euthanased. The trachea and lungs were removed from each bird and screened for the presence of P multocida by plating on blood agar plates containing 2 mg m l - l clindamycin. The remaining 140 turkeys were divided equally into two groups and placed in two separate isolation rooms.
Experimental design The 70 birds in group 1 were infected with AW. Each bird was infected by the oculonasal route with 0.2 ml of inoculum containing 1024 haemagglutinating (HA) units. The 70 birds in group 2 each received 0" 2 ml of virus diluent by the same route. Three days after these procedures were performed all birds in both groups were superinfected with P multocida by the intranasal route. Each bird was infected using an inoculum of 0- 2 ml of an 18 hour broth culture containing 1-2 x 109 colony forming units (cfu) m1-1.
and 120 hours after exposure to P multocida, 10 birds were removed from each group and euthanased. The trachea and lungs were aseptically removed from each bird. The tissues from each bird were weighed, placed separately in sterile phosphate buffered saline (PBS) and homogenised to give a 20 per cent (w/v) suspension.
Enumeration of bacteria Sterile PBS was used to prepare 10-fold serial dilutions of each tissue suspension. Bacteria present in the suspensions were enumerated using standard plate counts to determine cfu g - t of tissue. Blood agar plates containing 2 izg m l - l clindamycin were used as a selective medium (Garlinghouse et al 1981). Each dilution was plated out onto blood agar in triplicate. Colonies were counted after 24 hours incubation at 37°C. The mean bacterial count was calculated for each tissue suspension.
Results The results are presented in Table 1 and Fig 1. The mean deposition of bacteria immediately following exposure was 1.97× 107 and 2 . 8 2 × 107 cfu g-] of tissue in AIV infected and uninfected groups, respectively. There was no apparent difference in the number of cfu of P multocida recovered from AIV infected and uninfected groups until four hours after exposure. However, at six, eight and 24 hours after exposure, the AIV infected birds had more P multocida than both the initial inoculum and the nonAIV infected (control) birds. One hundred and twenty hours after exposure, the non-Air infected group showed a 100-fold reduction of P multocida g i with 96 per cent of the initial peak
o
~= 10.0.
Sample collection
~,
Immediately (0 hour) and at four, six, eight, 24, 72 TABLE 1: Colony forming units of P multocida g - 1 of trachea and lung in AIV-infected and uninfected turkeys Hours after P rnultocida exposure O* 4 6 8 24 72 120
Non-AIV infected 2"82x107t 1'21x107 2.19x108 4 " 3 7 x 107 1 , 6 4 x 108 1 . 3 B x 10 e 1 . 1 4 x 106
* Immediately following exposure "P Mean of 1Obirds
AIV infected 1.97x1071 3"10x107 1 .53x109 1-29x109 1 . 9 0 x 109 8.10x108 1 . 8 7 x 108
Ratio AIV infected/ non-AIV infected 0.7 2.6 7.0 29-5 11.6 5.9 164-0
AtVinfected . . . . . Control
9,5.
.~
9-0.
~
8.5.
.~ o 7.0- I
4
~
6"5
~
5.5
~-
5.01
0
2'4
7'2
Hours following exposureto P multocida FIG 1 : Quantitative clearance of P multocida from trachea and lungs of AIV-infected and uninfected turkeys
256
V. Sivanandan, K. V. Nagaraja, D. A. Halvorson, J. A. Newman
load being cleared. The AIv-infected group showed only a corresponding fourfold decrease in cfu g - 1.
Discussion The present study indicates decreased clearance of P multocida from the respiratory tract o f AWinfected turkeys compared with birds not infected with AIV. This suggests an impairment of the mucociliary defence system in AIV infected birds. The mechanisms involved in the suppression o f pulmonary bactericidal activity during the experimental AIV infection remains to be elucidated. Severe influenza virus infections in mammals with pneumonic involvement have been shown to impair p u l m o n a r y antibacterial defence, resulting in bacterial infections (Jakab 1981). The majority of influenza virus infections, however, are limited to the upper respiratory tract (Douglas 1975) and the effect of infections with milder strains on lung defences is not known. In the present study the effect of a nonpathogenic strain of avian influenza virus infection on the clearance of P multocida from trachea and lungs of turkeys was examined. The A1V infected group showed an increase in numbers of P multocida in these tissues reaching a maximum 24 hours after exposure. Normally, in mammals, inhaled bacteria entering the lungs as small droplets or by aspiration of fluid from the upper respiratory tract will be cleared by the natural defence mechanism (Green et al 1977). Bacteria deposited in the distal part of the ltlng are rapidly engulfed and inactivated by resident phagocytes, the alveolar macrophages. These phagocytic cells in the terminal airways play the pivotal defensive role against bacterial infection (Goldstein et al 1974, Kim et al 1976, Goldstein 1983). In addition, when inflammatory processes are established, polymorphonuclear leucocytes migrate into the lungs and assist in phagocytosis (Pierce et al 1977, Rehm et al 1979). Thus the pulmonary antibacterial defence is dependent on a dual phagocytic system that involves both alveolar macrophages and polymorphonuclear leucocytes. In the avian species, the paucity of resident macrophages in the respiratory tract has been documented (Toth and Siegel 1986). This may contribute to the vulnerability of the avian respiratory tract to inhaled pathogens. Hence, the respiratory tract in the avian species largely depends on recruited phagocytic cells for pulmonary antibacterial defence. Impaired phagocytosis of bacteria by alveolar macrophages of mice with influenza viral infection has been reported (Warshauer et al 1977). However, Jakab et al (1980) reported impaired phagosomelysosome fusion while Nugent and Pesanti (1979) reported normal phagocytosis, phagosome-lysosome
fusion and killing with mouse alveolar macrophages. The authors speculate that the differences reported may be due to one or more variable factors such as the virus type, subtype, variant, strain, passage history, suspending medium, dose and method of inoculation. It has been demonstrated that spontaneous quantitative and qualitative changes in the tracheal flora, including the appearance of Gram-negative bacteria, occur during sublethal influenza infections in mice, and that they are temporarily associated with overt damage to the tracheal mucosa (Nugent and Pesanti 1983). This experiment with an aerosol challenge model demonstrated that the influenza infection reduced tracheal clearance. Currently, commercial turkeys are vaccinated with a live cu or M9 vaccine. Although these vaccines have helped to control fowl cholera, it is not uncommon to see post-vaccinal reactions in the field resulting in fowl cholera. Serological evidence in many of these cases indicates a prior exposure of these turkeys to AIV and non-pathogenic AIV subtypes have been isolated from such cases (unpublished observations). In fowl cholera vaccination the doses of the vaccines, the health status of the flock and the time of vaccination are important factors. In mammals, the loss of ciliary action during influenza virus infection (Kleinerman et al 1975), in addition to impairment of phagosome-lysosome fusion (Jakab et al 1980) has been reported. Whether there was any ciliary destruction or impaired phagosome-lysosome fusion in the macrophages of turkeys exposed to AIV was not investigated in this study and remains to be determined. The results of this study suggest that even nonpathogenic strains of influenza virus may predispose the host to secondary bacterial infections.
Acknowledgements This work was supported in part by grants from the Minnesota Agricultural Experiment Station (03024863-22) and the Minnesota Turkey Grower's Association.
References ARP, L. H., G R A H A M , C. L. ~ . & CHEVEILLE, N. F. (1979) Comparison of clearance rates of virulent Escherichia coli in turkeys after aerosol exposure. Avian Diseases 2 3 , 3 8 6 - 3 9 1 DOUGLAS, R. G. (1975) The Influenza Viruses and Influenza. Ed E. D. Kilbourne. London, Academic Press. pp 395-481 FICKEN, M. D., EDWARDS, J. F. & LAY, J. C. (1986) Clearance of bacteria in turkeys with Bordetella avium-induced tracheitis. Avian Diseases 30, 352-357 FICKEN, M. D., EDWARDS, J. F., LAY, J. C. & TVETER, D. E. (1987) Tracheal mucus transport rate and bacterial clearance in turkeys exposed by aerosol to La Sota strain of Newcastle disease virus. Avian Diseases 3 1 , 2 4 1 - 2 4 8
E f f e c t Of AIV on P multocida in turkeys GARLINGHOUSE Jr, L. E., DIGIACOMO, R. V., VAN HOOSER Jr, G. L. & CONDON, J. (1981) Selective media for Pasteurella multocida and Bordetella bronchiseptica. Laboratory of Animal Science 31, 39-42 GOLDSTEIN, E. (1983) Hydrolytic enzymes of alveolar macrophages. Review of Infectious Diseases 5, 1078-1092 GOLDSTEIN, E., LIPPERT, W. & WARSHAUER, D. (1974) Pulmonary alveolar macrophage. Defender against bacterial infection of the lung. Journal of Clinical Investigation 54, 519-528 GREEN, G. M., JAKAB, G. J., LOW, R. B. & DAVIS, G. S. (1977) Defense mechanisms of the respiratory membrane. American Review of Respiratory Disease 115, 479-514 HALVORSON, D. A., KARUNUKARAN, D., SENNE, D. A., KELLEHER, C. J., BAILEY, C., ABRAHAM, A., HINSHAW, D. & NEWMAN, J. A. (1983) Epizootiology of avian influenzasimultaneous monitoring of sentinel ducks and turkeys in Minnesota. Avian Diseases 27, 77-85 JAKAB, G. J., WARR, G. A. & SANNES, P. L. (1980) Alveolar macrophage ingestion and phagosome-lysosome fusion defect associated with virus pneumonia. Infection and Immunity 27, 960-968 JAKAB, G. J. (1981) Mechanisms of virus-induced bacterial super infections of the lung. Clinics in Chest Medicine 2, 59-66 KIM, M., GOLDSTEIN, E., LEWIS, J. P., LIPPERT, W. & WARSHAUER, D. (1976) Murine pulmonary alveolar macrophage: rates of bacterial ingestion, inactivation and destruction. Journal of Infectious Diseases 133, 310-320 KLEINERMAN, E. S., SNYDERMAN, R. & DANIELS, C. A. (1975) Depressed monocyte chemotaxis during acute influenza infection. Lancet ii, 1063-1066 LOOSLI, C. G. (1973) Influenza and the interaction of viruses and bacteria in respiratory infections. Medicine 52, 369 NAGARAJA, K. V., EMERY, D. A., JORDAN, K. A., SIVANANDAN, V., NEWMAN, J. A. & POMEROY, B. S. (1984) Effect of ammonia on the quantitative clearance of Escherichia eoli from lungs, air sacs and livers of turkeys aerosol vaccinated against Escherichia coli. American Journal of Veterinary Research 45, 392-395
257
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Received December 21, 1990 Accepted May 28, 1991
