Researchin VeterinaryScience1991, 51, 209-214
Protective effect of inactivated bovine herpesvirus-1 in calves experimentally infected with bovine herpesvirus-1 and
Pasteurella haemolytica K. W. F. JERICHO, K. G. LOEWEN, S. E. SMITHSON, Agriculture Canada, AnimalDiseases Research Institute, PO Box 640, Lethbridge, Alberta TIJ 3Z4, Canada, G. C. KOZUB, Agriculture Canada, Research Station, PO Box 3000 Main, Lethbridge, Alberta TIJ 4B1, Canada
The protective effect of an inactivated whole-virion bovine herpesvirus-1 (ntiv-1) immunising inoculum, without adjuvant, against viral-bacterial respiratory disease was studied in three experimental treatment groups of five calves each. One group was boosted 14 days after the first vaccination and at this time the second group received their initial inoculation. Seven days later, calves were challenged with BI~V-1 in aerosol and four days after this challenge all calves were exposed to Pasteurella haemolytica A1 in aerosol. Among the three groups, differences in rectal temperature responses four days after viral challenge (P < 0.01) did not relate to protection. However the main response variable, viral-bacterial pneumonia, was reduced in boosted calves (P < 0.05).
BOVINE herpesvirus-1 (BHV-1) vaccines are commonly used to control infectious respiratory disease in cattle raised intensively. A model of this disease complex, produced in experimental calves by aerosols of BHV-1 and Pasteurella haemolytica A1, has been used to demonstrate the significant protection offered by live BHV-1 vaccines against this mixed infection (Jericho and Langford 1978, Stockdale et al 1979, Jericho et al 1982, Jericho and Babiuk 1983). This disease model has also been used to evaluate killed BHV-1 vaccines (Darcel and Jericho 1981, Babiuk et al 1987) and, in one study, BHV-I glycoproteins significantly reduced clinical disease and mortality in challenged calves. However, it could not be determined from the report how many calves were used in how many experiments, or the extent of disease control in all experimental calves (Babiuk et al 1987). Nasal infection and virus shedding were not prevented by glycoprotein vaccines (Babiuk et al 1987, Israel et al 1988). The protective effect of inactivated BHV-1 vaccine against viral/bacterial challenge is therefore still poorly defined. It is well recognised that BHV-1 has many effects on cell-mediated immune mechanisms (Filion et al 1983,
McGuire and Babiuk 1984, Bielefeldt Ohmann and Babiuk 1985, Ogunbiyi et al 1986, Briggs et al 1988, Noel et al 1988). However, the pathways of enhanced susceptibility to P haemolytica, as well as the relationship between clinical manifestations of BHV-1 infection and the associated immune reactions, are as yet poorly understood. Consequently, a vaccine which is intended to control BHV-1 clinical disease may be challenged with BHV-1 alone, but a vaccine which is intended to control BHV-1/bacterial infections should be challenged by mixed infection. The BHV-1/P haemolytica model provides such a challenge. This guideline is further supported by the premise that vaccination against BHV-1 must be judged on its ability to protect against the economically damaging feature of the disease which, in the case of feeder cattle, is viral-bacterial respiratory disease (Staub and Mawhinney 1988). To assist the search and evaluation of killed. BHV-1 vaccines, this report describes for the first time the level of protection offered by inactivated wholevirion BHV-1 when challenged by the BHV-1/P haemolytica disease model.
Materials and methods
Experimental animals and experimental design Fifteen male calves, six to eight months old, were derived from the institute's specific pathogen-free herd. The herd is free of BHV-1, bovine viral diarrhoea virus, bovine leukosis virus, Brucella abortus and Mycobacterium tuberculosis, and has only a few reactors at low serum dilution to Pasteurella haemolytica, Leptospira species, parainfluenza-3 virus, and bovine respiratory syncytial virus. This isolated herd has been free of respiratory disease for the last 19 years. The calves of this study were born in August to October of the same year in an open-air concrete-floored corral (group size 30, 25 m z per
209
K. W. F. Jericho, K. G. Loewen, S. E. Smithson, G. C. Kozub
210
calf) weaned, and then relocated to another corral on day - 30 (day 0 is the day of first inoculation). On day - 2 7 all calves were without complement fixing (CF) antibodies to P haemolytica and serum neutralising (SN) antibodies to BHV-I. On day 0 the weight range of the calves was 186 to 266 kg. The calves were randomly assigned to three experimental treatment groups (five calves per group) and calves of group 2 were vaccinated. On day 14 the calves were moved to three identical pens in an indoor facility, group 2 calves were boosted and group 1 calves received their single vaccination. Each pen had a volume of 405 m 3, an animal-occupied rubber surface of 32 m 2, an individual air supply, I0 air changes per hour, controlled temperature (12 to 16°C) and humidity (40 to 50 per cent relative humidity), and a chute and squeeze for restraint. No more than two of the five calves of each group were represented in any one pen. Alfalfa hay cubes and water were provided ad libitum. On day 21, control calves (group 3) were exposed and vaccinates (groups 1 and 2) were challenged with BHV-1 aerosol. On day 25 all calves were exposed to a P haemolytica aerosol. The experiment was terminated on day 29 (Table 1). Calves which survived until the end of the experiment and those which were unable to walk away from an approaching animal attendant were killed by captive bolt pistol (Lambooy and Spanjaard 1981). The Guide to the Care and Use of Experimental Animals (Canadian Council of Animal Care 1984) was applied to the care of the animals.
resuspended in 99-3 ml of Dulbecco's phosphate buffered saline (PBS) and 70 per cent glutaraldehyde was added to a final concentration of 0-5 per cent. Before the addition of glutaraldehyde, the virus preparation had a titre of 109.5 TCID50 ml-~. After being held for 4°C for four days, the virus preparation was ultracentrifuged for four hours at 100,000 g in a Beckman sw 28 rotor onto a 40 per cent sucrose cushion. The interface was harvested. Adjuvant was not added to the product. Five ml of inoculum was given intramuscularly at each inoculation which contained 0.5 mg of protein. Calves of the control group and group 1 were placebo-treated with 5 ml of PBS intramuscularly on day 0.
Microbiological studies BHV-I and P haernolytica isolations were attempted from pharyngeal tonsil and pneumonic tissue. SN antibodies to BHV-1 were determined on the days of vaccination and challenge, and one day before the end of the experiment (day 28) (Jericho and Darcel 1978). cv antibodies to P haemolytica were determined for sera taken on the day of exposure to P haemolytica aerosol (Jericho et al 1985).
Clinical studies The rectal temperature of each calf was taken each morning (09.00) starting on the day of viral aerosol until the final day of the experiment.
Virus preparation and inoculation schedule Inoculum was prepared with strain 108 of BHV-1 (Jericho and Darcel 1978) propagated in BK44 cells (bovine virus diarrhoea virus-free bovine kidney cells) which were grown in Earle's minimal essential medium with 10 per cent fetal calf serum and antibiotics. When cytopathic effect was complete, the cell suspension was frozen and thawed three times. Culture fluids and cell debris were then pelleted by ultracentrifugation at 35,000 g for 4.5 hours in a Beckman Type 21 rotor or at 15,000 g for 18 hours in a Beckman JA-20 rotor. Pellets were then pooled and
Aerosol exposures All calves were exposed to BHV-I and Phaemolytica A1 aerosols on days 21 and 25, respectively. The cultures used for aerosolisation were prepared as described previously (Jericho et al 1990). The source of P haemolytica was an experimental calf whose lung was stored at - 6 3 ° C . Five colonies from blood agar plates were used to inoculate brain heart infusion broth without fetal calf serum. A five-hour log phase bacterial culture was used. The method of aerosol exposure was as described previously (Jericho et al
TABLE 1: Experimental design used for vaccination, BHV-1 challenge, P haemolytica exposure and necropsy Day* Group 1 2 3
Number of calves
Vaccination
5 5 5
14 O and 14 --
BHV-1 challenge 21 . 21 21
P haemolytica exposure
Necropsyt
25 25 25
29 29 29
* Day O was day of first vaccination t" Calves which were not euthanased or did not die before the last day of the experiment
BHV-1
211
immunisation of calves
1986). Each calf was exposed to viral and bacterial aerosol for five minutes, during which time 1.2 to 1.6 ml of culture fluid was aerosolised. The titres of the viral and bacterial cultures were 1 x 10 7 TCID50 m l - l , and 5 x 107 cells m l - l , respectively.
Results
Microbiological studies
Pathological changes in the respiratory tract were recorded and the extent of pneumonia was determined (Jericho and Langford 1982) before identity of the'calves' group was known. Atelectic pulmonary lobules, associated with BHV-1 infection (Jericho and Darcel 1978) and visible on lung surface, were counted and macroscopically recognisable necrosis of larynx and trachea was recorded. Viral and, or, bacterial involvement in changes of respiratory tissue were verified by microscopic examination (Jericho and Darcel 1978, Jericho 1983). Necrosis of epithelium, associated with BHV-] infection (Jericho and Darcel 1978), was identified in pharyngeal tonsils.
BHV-I was isolated from the tonsil of one animal of each of groups 1 and 2, all tonsils of group 3 animals, and the lungs of one group 1 calf and two group 2 calves (Table 2). P haemolytica was isolated from all nasal swabs on day 29, all but one tonsil (group 2) and all but three lungs (one in group 1 and two in group 2). CF antibody titres (1 /8 to 1/ 16) to P haem olytica were detected in one calf of each of groups 1 and 2 on the day of P haemolytica exposure. SN antibodies to BHV-1were not detected on day 0 in any group, on day 14 in group 1, nor in group 3 during the entire experimental period. In group 1, two calves had a sy antibody titre (1/2) seven days (day 21) after their single vaccination, and seven days after (day 28) the BHV-1 challenge the range in titre in all calves of this group was 1/2 to 1/32 (Table 2). All group 2 calves were SN antibody positive (1/2) on day 14, on day 21 (1/4) and on day 28 (1/32 to 1/128).
Statistical analysis
Clinical studies
Analysis of variance (Steel and Torrie 1980) was carried out to examine the effect of immunisation on the percentage of pneumonic tissue, rectal temperature and number of atelectic pulmonary lobules. Variation due to immunisation group and pen were accounted for in the analyses and the effects of animal weight and age on the extent of pneumonia were examined by including them as covariates. A logarithmic transformation was applied to the percentage pneumonic tissue data. Since comparisons of each of the vaccinated groups with the control group were of interest, the least significant difference (LSD) was calculated. A repeat measures analysis (Winer 1962) was also carried out for the temperature data to determine if there was a temperature response over time and if it was consistent for the group. Fisher's exact test (Steel and Torrie 1980) was used to compare the control and vaccinated groups for proportions of animals that had BHV-1 or P haemolytica isolates, positive CF and sN antibodies, and necrosis.
One calf from group 1 and one from group 2 were euthanased and two calves died in group 3. The mean temperature of all calves on the day of viral challenge was 39- 1°C. For temperature, there was a significant ( P < 0 . 0 1 ) interaction between the group and days after viral challenge. The temperature of calves in the control group increased with days after viral challenge (Fig 1). The temperatures of calves in the singly and doubly immunised groups did not rise as rapidly, and at day 25 the mean temperature in these groups was significantly lower (P < 0.01) than that of the control group. The temperature in the immunised groups rose after P haemolytica challenge on day 25.
Tissue changes
Tissue changes The geometric means of the extent of viral-bacterial pneumonia for groups 1, 2 and 3 were 7 per cent (range 0 to 45), 3 per cent (0 to 35) and 27 per cent (0 to 55), respectively. The lower percentage of
TABLE 2: Percentages* of animals with viral and bacterial isolations and BHV-1 serum neutralising antibody titres Isolations
P haemolytica
BHV-1 Group 1 2 3
Tonsil 20 a 20 a 1O0
Lung
Nose
Tonsil
Lung
Day 0
0 20 40
1O0 1O0 1O0
1O0 80 1O0
80 60 1O0
0 0 0
* Five calves per experimental group a,b Significantly different from control (P < O- 05, P < O. 01, respectively)
Antibody SN (BHV-1) Day 14 Day 21 0 1O0b 0
40 1O0 b 0
Day 28 1O0 b 1O0 b 0
K. IF. F. Jericho, K. G. Loewen, S. E. Smithson, G. C. Kozub
212 42-
-Group 1 - - = - Group2 ...... Control(Group3) O ........
Discussion .0
The aim of the cattle industry is to vaccinate feeder cattle before they leave their ranch of origin and again ,.."" "' ~ 0 \ ~/0 on arrival at the feedlot. Unfortunately, this objective o~ ..-" j / O / I \\ J/-/" is rarely met and most calves receive their first BHV-1 ._-'~jJ • \\ y/~ 40-vaccination on arrival at the feedlot after transport E and market exposure. This vaccination is practised to 39-control BHV-1 associated respiratory disease which may peak as soon as seven days after arrival at large feedlots. Immunisations in this study were intended to 38 I I I I I I 21 22 23 24 25 26 mimic the above scenarios by immunising two groups Day after first vaccination on day 14 ('arrival at the feedlot') and one group also on day 0 (14 days before 'arrival at the feedlot'). The FIG 1: Mean rectal temperatures of the three experimental treatment groups between day of viral challenge (day 21) and long term and irregular low dose exposure of calves one day after bacterial exposure (day 25). The standard error of which is likely to occur after arrival at the feedlot was a group mean with 10 df is given by the length of the bar at each not mimicked; instead challenge was served by the day five minute aerosol seven days after 'arrival at the feedlot'. A more natural challenge may have been achieved by introducing BHV-1 infected challenge calves into each pen on day 14 (day of 'arrival at the pneumonic tissue observed in the immunised calves feedlot') or long term challenge with a constant viral compared to the control calves was significant cloud in a walk-in room (Ho 1988, Staub and ( P < 0 . 0 5 ) only for the doubly immunised group Mawhinney 1988). The pathological consequences of (Table 3). No effect of age or weight on the percentage these methods of exposure to BHV-1and P haemolytica of pneumonic tissue was evident (P>0.05). There have not been investigated and, therefore, the proven was no correlation between percentage of pneumonic methods of producing the main response variable of tissue and rectal temperature on the days following this study, namely BHV-t induced mixed infectious BHV-] challenge and before P haemolytica challenge respiratory disease, was used instead (Jericho and (P > 0.05). However, there was a significant correla- Langford 1978, Jericho et al 1986). tion (r = 0.80, P < 0" 01)between these variables on SN antibody response after single or double day 26 following P haemolytica exposure. The mean vaccination and challenge did not indicate that number of surface atelectic pulmonary lobules, which humoral immune responses relate closely to virusare associated with BHV-1 aerosol exposure, was five induced tissue changes in epithelium of the upper for the control group 3 and did not differ significantly respiratory tract or to protection from BHV-1/P from the immunised groups (P>0"05) (Table 3). haemolytica challenge. This confirms the results of BHV-1 induced epithelial necrosis of pharyngeal tonsil other studies (Babiuk et al 1987). Similarly, the low (microscopic) and macroscopic focal necrosis of the level of serum antibody to P haemolytica in calves of larynx and proximal trachea in all three groups this study had no effect on the outcome of this disease (Table 3). In one control calf, severe necropurulent model (Jericho et al 1985). Although the dose of changes were seen throughout the length of the inactivated BHVq vaccine has been shown to relate to the neutralisation titre produced in calves (Zuffa et al trachea.
~" 41 -
0 . . . . . . . . . . . 0'"
......... ?
~
I
I
I
TABLE 3: Macroscopic and microscopic pathological changes in experimental groups Mean* ± SE
Number# of animals with
Microscopic Group 1 2 3
% pneumonia
Pneumonic Iobules
6.5 ± 4.4 2.8 ± 2-2 a 27.1±16.4
5.6 ± 3.2 8.0 ± 3.2 5-4±3.2
Macroscopic necrosis Larynx Trachea 2 4 3
0 0 1
necrosis pharyngeal tonsil 2 3 4
* For percentage pneumonia the geometric mean and approximate standard error (SE) with 10 degrees of freedom are given since a Ioglo (X + 1) transformation was used ? Five calves per experimental group a Mean is significantly different from control (P
Protective effect of inactivated bovine herpesvirus-1 in calves experimentally infected with bovine herpesvirus-1 and
Pasteurella haemolytica K. W. F. JERICHO, K. G. LOEWEN, S. E. SMITHSON, Agriculture Canada, AnimalDiseases Research Institute, PO Box 640, Lethbridge, Alberta TIJ 3Z4, Canada, G. C. KOZUB, Agriculture Canada, Research Station, PO Box 3000 Main, Lethbridge, Alberta TIJ 4B1, Canada
The protective effect of an inactivated whole-virion bovine herpesvirus-1 (ntiv-1) immunising inoculum, without adjuvant, against viral-bacterial respiratory disease was studied in three experimental treatment groups of five calves each. One group was boosted 14 days after the first vaccination and at this time the second group received their initial inoculation. Seven days later, calves were challenged with BI~V-1 in aerosol and four days after this challenge all calves were exposed to Pasteurella haemolytica A1 in aerosol. Among the three groups, differences in rectal temperature responses four days after viral challenge (P < 0.01) did not relate to protection. However the main response variable, viral-bacterial pneumonia, was reduced in boosted calves (P < 0.05).
BOVINE herpesvirus-1 (BHV-1) vaccines are commonly used to control infectious respiratory disease in cattle raised intensively. A model of this disease complex, produced in experimental calves by aerosols of BHV-1 and Pasteurella haemolytica A1, has been used to demonstrate the significant protection offered by live BHV-1 vaccines against this mixed infection (Jericho and Langford 1978, Stockdale et al 1979, Jericho et al 1982, Jericho and Babiuk 1983). This disease model has also been used to evaluate killed BHV-1 vaccines (Darcel and Jericho 1981, Babiuk et al 1987) and, in one study, BHV-I glycoproteins significantly reduced clinical disease and mortality in challenged calves. However, it could not be determined from the report how many calves were used in how many experiments, or the extent of disease control in all experimental calves (Babiuk et al 1987). Nasal infection and virus shedding were not prevented by glycoprotein vaccines (Babiuk et al 1987, Israel et al 1988). The protective effect of inactivated BHV-1 vaccine against viral/bacterial challenge is therefore still poorly defined. It is well recognised that BHV-1 has many effects on cell-mediated immune mechanisms (Filion et al 1983,
McGuire and Babiuk 1984, Bielefeldt Ohmann and Babiuk 1985, Ogunbiyi et al 1986, Briggs et al 1988, Noel et al 1988). However, the pathways of enhanced susceptibility to P haemolytica, as well as the relationship between clinical manifestations of BHV-1 infection and the associated immune reactions, are as yet poorly understood. Consequently, a vaccine which is intended to control BHV-1 clinical disease may be challenged with BHV-1 alone, but a vaccine which is intended to control BHV-1/bacterial infections should be challenged by mixed infection. The BHV-1/P haemolytica model provides such a challenge. This guideline is further supported by the premise that vaccination against BHV-1 must be judged on its ability to protect against the economically damaging feature of the disease which, in the case of feeder cattle, is viral-bacterial respiratory disease (Staub and Mawhinney 1988). To assist the search and evaluation of killed. BHV-1 vaccines, this report describes for the first time the level of protection offered by inactivated wholevirion BHV-1 when challenged by the BHV-1/P haemolytica disease model.
Materials and methods
Experimental animals and experimental design Fifteen male calves, six to eight months old, were derived from the institute's specific pathogen-free herd. The herd is free of BHV-1, bovine viral diarrhoea virus, bovine leukosis virus, Brucella abortus and Mycobacterium tuberculosis, and has only a few reactors at low serum dilution to Pasteurella haemolytica, Leptospira species, parainfluenza-3 virus, and bovine respiratory syncytial virus. This isolated herd has been free of respiratory disease for the last 19 years. The calves of this study were born in August to October of the same year in an open-air concrete-floored corral (group size 30, 25 m z per
209
K. W. F. Jericho, K. G. Loewen, S. E. Smithson, G. C. Kozub
210
calf) weaned, and then relocated to another corral on day - 30 (day 0 is the day of first inoculation). On day - 2 7 all calves were without complement fixing (CF) antibodies to P haemolytica and serum neutralising (SN) antibodies to BHV-I. On day 0 the weight range of the calves was 186 to 266 kg. The calves were randomly assigned to three experimental treatment groups (five calves per group) and calves of group 2 were vaccinated. On day 14 the calves were moved to three identical pens in an indoor facility, group 2 calves were boosted and group 1 calves received their single vaccination. Each pen had a volume of 405 m 3, an animal-occupied rubber surface of 32 m 2, an individual air supply, I0 air changes per hour, controlled temperature (12 to 16°C) and humidity (40 to 50 per cent relative humidity), and a chute and squeeze for restraint. No more than two of the five calves of each group were represented in any one pen. Alfalfa hay cubes and water were provided ad libitum. On day 21, control calves (group 3) were exposed and vaccinates (groups 1 and 2) were challenged with BHV-1 aerosol. On day 25 all calves were exposed to a P haemolytica aerosol. The experiment was terminated on day 29 (Table 1). Calves which survived until the end of the experiment and those which were unable to walk away from an approaching animal attendant were killed by captive bolt pistol (Lambooy and Spanjaard 1981). The Guide to the Care and Use of Experimental Animals (Canadian Council of Animal Care 1984) was applied to the care of the animals.
resuspended in 99-3 ml of Dulbecco's phosphate buffered saline (PBS) and 70 per cent glutaraldehyde was added to a final concentration of 0-5 per cent. Before the addition of glutaraldehyde, the virus preparation had a titre of 109.5 TCID50 ml-~. After being held for 4°C for four days, the virus preparation was ultracentrifuged for four hours at 100,000 g in a Beckman sw 28 rotor onto a 40 per cent sucrose cushion. The interface was harvested. Adjuvant was not added to the product. Five ml of inoculum was given intramuscularly at each inoculation which contained 0.5 mg of protein. Calves of the control group and group 1 were placebo-treated with 5 ml of PBS intramuscularly on day 0.
Microbiological studies BHV-I and P haernolytica isolations were attempted from pharyngeal tonsil and pneumonic tissue. SN antibodies to BHV-1 were determined on the days of vaccination and challenge, and one day before the end of the experiment (day 28) (Jericho and Darcel 1978). cv antibodies to P haemolytica were determined for sera taken on the day of exposure to P haemolytica aerosol (Jericho et al 1985).
Clinical studies The rectal temperature of each calf was taken each morning (09.00) starting on the day of viral aerosol until the final day of the experiment.
Virus preparation and inoculation schedule Inoculum was prepared with strain 108 of BHV-1 (Jericho and Darcel 1978) propagated in BK44 cells (bovine virus diarrhoea virus-free bovine kidney cells) which were grown in Earle's minimal essential medium with 10 per cent fetal calf serum and antibiotics. When cytopathic effect was complete, the cell suspension was frozen and thawed three times. Culture fluids and cell debris were then pelleted by ultracentrifugation at 35,000 g for 4.5 hours in a Beckman Type 21 rotor or at 15,000 g for 18 hours in a Beckman JA-20 rotor. Pellets were then pooled and
Aerosol exposures All calves were exposed to BHV-I and Phaemolytica A1 aerosols on days 21 and 25, respectively. The cultures used for aerosolisation were prepared as described previously (Jericho et al 1990). The source of P haemolytica was an experimental calf whose lung was stored at - 6 3 ° C . Five colonies from blood agar plates were used to inoculate brain heart infusion broth without fetal calf serum. A five-hour log phase bacterial culture was used. The method of aerosol exposure was as described previously (Jericho et al
TABLE 1: Experimental design used for vaccination, BHV-1 challenge, P haemolytica exposure and necropsy Day* Group 1 2 3
Number of calves
Vaccination
5 5 5
14 O and 14 --
BHV-1 challenge 21 . 21 21
P haemolytica exposure
Necropsyt
25 25 25
29 29 29
* Day O was day of first vaccination t" Calves which were not euthanased or did not die before the last day of the experiment
BHV-1
211
immunisation of calves
1986). Each calf was exposed to viral and bacterial aerosol for five minutes, during which time 1.2 to 1.6 ml of culture fluid was aerosolised. The titres of the viral and bacterial cultures were 1 x 10 7 TCID50 m l - l , and 5 x 107 cells m l - l , respectively.
Results
Microbiological studies
Pathological changes in the respiratory tract were recorded and the extent of pneumonia was determined (Jericho and Langford 1982) before identity of the'calves' group was known. Atelectic pulmonary lobules, associated with BHV-1 infection (Jericho and Darcel 1978) and visible on lung surface, were counted and macroscopically recognisable necrosis of larynx and trachea was recorded. Viral and, or, bacterial involvement in changes of respiratory tissue were verified by microscopic examination (Jericho and Darcel 1978, Jericho 1983). Necrosis of epithelium, associated with BHV-] infection (Jericho and Darcel 1978), was identified in pharyngeal tonsils.
BHV-I was isolated from the tonsil of one animal of each of groups 1 and 2, all tonsils of group 3 animals, and the lungs of one group 1 calf and two group 2 calves (Table 2). P haemolytica was isolated from all nasal swabs on day 29, all but one tonsil (group 2) and all but three lungs (one in group 1 and two in group 2). CF antibody titres (1 /8 to 1/ 16) to P haem olytica were detected in one calf of each of groups 1 and 2 on the day of P haemolytica exposure. SN antibodies to BHV-1were not detected on day 0 in any group, on day 14 in group 1, nor in group 3 during the entire experimental period. In group 1, two calves had a sy antibody titre (1/2) seven days (day 21) after their single vaccination, and seven days after (day 28) the BHV-1 challenge the range in titre in all calves of this group was 1/2 to 1/32 (Table 2). All group 2 calves were SN antibody positive (1/2) on day 14, on day 21 (1/4) and on day 28 (1/32 to 1/128).
Statistical analysis
Clinical studies
Analysis of variance (Steel and Torrie 1980) was carried out to examine the effect of immunisation on the percentage of pneumonic tissue, rectal temperature and number of atelectic pulmonary lobules. Variation due to immunisation group and pen were accounted for in the analyses and the effects of animal weight and age on the extent of pneumonia were examined by including them as covariates. A logarithmic transformation was applied to the percentage pneumonic tissue data. Since comparisons of each of the vaccinated groups with the control group were of interest, the least significant difference (LSD) was calculated. A repeat measures analysis (Winer 1962) was also carried out for the temperature data to determine if there was a temperature response over time and if it was consistent for the group. Fisher's exact test (Steel and Torrie 1980) was used to compare the control and vaccinated groups for proportions of animals that had BHV-1 or P haemolytica isolates, positive CF and sN antibodies, and necrosis.
One calf from group 1 and one from group 2 were euthanased and two calves died in group 3. The mean temperature of all calves on the day of viral challenge was 39- 1°C. For temperature, there was a significant ( P < 0 . 0 1 ) interaction between the group and days after viral challenge. The temperature of calves in the control group increased with days after viral challenge (Fig 1). The temperatures of calves in the singly and doubly immunised groups did not rise as rapidly, and at day 25 the mean temperature in these groups was significantly lower (P < 0.01) than that of the control group. The temperature in the immunised groups rose after P haemolytica challenge on day 25.
Tissue changes
Tissue changes The geometric means of the extent of viral-bacterial pneumonia for groups 1, 2 and 3 were 7 per cent (range 0 to 45), 3 per cent (0 to 35) and 27 per cent (0 to 55), respectively. The lower percentage of
TABLE 2: Percentages* of animals with viral and bacterial isolations and BHV-1 serum neutralising antibody titres Isolations
P haemolytica
BHV-1 Group 1 2 3
Tonsil 20 a 20 a 1O0
Lung
Nose
Tonsil
Lung
Day 0
0 20 40
1O0 1O0 1O0
1O0 80 1O0
80 60 1O0
0 0 0
* Five calves per experimental group a,b Significantly different from control (P < O- 05, P < O. 01, respectively)
Antibody SN (BHV-1) Day 14 Day 21 0 1O0b 0
40 1O0 b 0
Day 28 1O0 b 1O0 b 0
K. IF. F. Jericho, K. G. Loewen, S. E. Smithson, G. C. Kozub
212 42-
-Group 1 - - = - Group2 ...... Control(Group3) O ........
Discussion .0
The aim of the cattle industry is to vaccinate feeder cattle before they leave their ranch of origin and again ,.."" "' ~ 0 \ ~/0 on arrival at the feedlot. Unfortunately, this objective o~ ..-" j / O / I \\ J/-/" is rarely met and most calves receive their first BHV-1 ._-'~jJ • \\ y/~ 40-vaccination on arrival at the feedlot after transport E and market exposure. This vaccination is practised to 39-control BHV-1 associated respiratory disease which may peak as soon as seven days after arrival at large feedlots. Immunisations in this study were intended to 38 I I I I I I 21 22 23 24 25 26 mimic the above scenarios by immunising two groups Day after first vaccination on day 14 ('arrival at the feedlot') and one group also on day 0 (14 days before 'arrival at the feedlot'). The FIG 1: Mean rectal temperatures of the three experimental treatment groups between day of viral challenge (day 21) and long term and irregular low dose exposure of calves one day after bacterial exposure (day 25). The standard error of which is likely to occur after arrival at the feedlot was a group mean with 10 df is given by the length of the bar at each not mimicked; instead challenge was served by the day five minute aerosol seven days after 'arrival at the feedlot'. A more natural challenge may have been achieved by introducing BHV-1 infected challenge calves into each pen on day 14 (day of 'arrival at the pneumonic tissue observed in the immunised calves feedlot') or long term challenge with a constant viral compared to the control calves was significant cloud in a walk-in room (Ho 1988, Staub and ( P < 0 . 0 5 ) only for the doubly immunised group Mawhinney 1988). The pathological consequences of (Table 3). No effect of age or weight on the percentage these methods of exposure to BHV-1and P haemolytica of pneumonic tissue was evident (P>0.05). There have not been investigated and, therefore, the proven was no correlation between percentage of pneumonic methods of producing the main response variable of tissue and rectal temperature on the days following this study, namely BHV-t induced mixed infectious BHV-] challenge and before P haemolytica challenge respiratory disease, was used instead (Jericho and (P > 0.05). However, there was a significant correla- Langford 1978, Jericho et al 1986). tion (r = 0.80, P < 0" 01)between these variables on SN antibody response after single or double day 26 following P haemolytica exposure. The mean vaccination and challenge did not indicate that number of surface atelectic pulmonary lobules, which humoral immune responses relate closely to virusare associated with BHV-1 aerosol exposure, was five induced tissue changes in epithelium of the upper for the control group 3 and did not differ significantly respiratory tract or to protection from BHV-1/P from the immunised groups (P>0"05) (Table 3). haemolytica challenge. This confirms the results of BHV-1 induced epithelial necrosis of pharyngeal tonsil other studies (Babiuk et al 1987). Similarly, the low (microscopic) and macroscopic focal necrosis of the level of serum antibody to P haemolytica in calves of larynx and proximal trachea in all three groups this study had no effect on the outcome of this disease (Table 3). In one control calf, severe necropurulent model (Jericho et al 1985). Although the dose of changes were seen throughout the length of the inactivated BHVq vaccine has been shown to relate to the neutralisation titre produced in calves (Zuffa et al trachea.
~" 41 -
0 . . . . . . . . . . . 0'"
......... ?
~
I
I
I
TABLE 3: Macroscopic and microscopic pathological changes in experimental groups Mean* ± SE
Number# of animals with
Microscopic Group 1 2 3
% pneumonia
Pneumonic Iobules
6.5 ± 4.4 2.8 ± 2-2 a 27.1±16.4
5.6 ± 3.2 8.0 ± 3.2 5-4±3.2
Macroscopic necrosis Larynx Trachea 2 4 3
0 0 1
necrosis pharyngeal tonsil 2 3 4
* For percentage pneumonia the geometric mean and approximate standard error (SE) with 10 degrees of freedom are given since a Ioglo (X + 1) transformation was used ? Five calves per experimental group a Mean is significantly different from control (P
