Protective effect of inactivated Pasteurella haemolytica bacterin challenged in bovine herpesvirus-1 experimentally infected calves K.W.F. Jericho, H.J. Cho and G.C. Kozub Formalin-inactivated and sonicated Pasteurella haemolytica bacterins were prepared from 1 h cultures of bacterial pellets in RPMI-1640 medium containing 7% fetal calf serum. The bacterial pellets were obtained from logarithmic phase growth of the organism by centrifugation. The protective effect of the vaccine was evaluated in 43 specific-pathogen-free Hereford crossbred calves and yearlings in three experiments. Cattle were either single vaccinated or boosted via three routes; intratracheally (i.t.), intranasally (i.n.) or intramuscularly (i.m.), using low or high doses. The two low-dose groups were also given supernatant by the same routes and volume as the bacterin. Cattle were challenged by P. haemolytica in aerosol at 24 or 39 days after" last vaccination. To enhance the susceptibility of the cattle to this challenge, the cattle were exposed to bovine herpesvirus-1 aerosol 4 days before the bacterial challenge. The extent of pneumonia was significantly less in three groups of cattle (i.n.-i.n., i.m.-i.n., Lm.-i.m.) boosted with high dose of the bacterin than in the controls. Protection was observed when challenge isolates were heterologous or homologous to the isolates used to prepare the bacterins. It was also observed that the level of complement fixing antibody or anticytotoxin activity to P. haemolytica did not correlate with the degree of protection.
Keywords:Pasteurella haemolytica; bacterin; infectiousbovine respiratory disease Introduction Pasteurella haemolytica is recognized as an important respiratory tract pathogen in cattle ~-a. Pasteurella spp. bacterins have been used for many years in an attempt to control infectious respiratory disease (IRD) 4'5. For example, a bacterin containing P. haemolytica and Pasteurella multocida with the addition of 0.25 % formalin was tried in farm livestock in 19576 . In one well designed field study 7 of Pasteurella bacterins, mono or bivalent bacterins were found to effectively reduce IRD in vaccinates as compared to natural controls s. However, similar to deficiencies in most field studies 7, the experimental demonstration of the protective effect of formalin inactivated P. haemolytica bacterins is also incomplete. The first experimental challenge of a formalininactivated P. haemolytica bacterin (polyvalent) was reported in 19649. However, it was not until pneumonic pasteurellosis was reproducible by large bacterial dose ~° or low viral-bacterial dose ~~ that a renewed interest was shown in experimental evaluations of P. haemolytica bacterins. Unfortunately, these studies differed not only in virus induced susceptibility of experimental calves, but also in challenge (dose, volume, route, mode of application), in bacterin (valency, bacterial source, method of Agriculture Canada, Animal Diseases Research Institute, PO Box 640, Lethbridge, Alberta, Canada T1J 3Z4 and Statistics Unit, Agriculture Canada Research Station, Agriculture Center, Lethbridge, Alberta, Canada T1J 4B1 (Kozub). (Received 7 October 1988; revised 2 October 1989; accepted 2 October 1989) 0264-410X/90/040315-06 © Canadian Crown Copyright 1990
preparation, adjuvants, type, concentration and dose frequency and route of application) and in cattle parameters (age, breed, microbial exposures, housing). It is, therefore, not surprising that formalin-inactivated bacterins have been shown to be effective 12-14, ineffective ls'~6 and even detrimentaP ~. In this report, three experiments are described in which the close relationship between bacterin, challenge and disease production is demonstrated. The low-dose viral-bacterial aerosol challenge was chosen because it is considered the best emulation of natural challenge available 18 against which a useful bacterin should protect. This is the first report of protection afforded by a formalin-inactivated and sonicated, adjuvant-free bacterin when challenged by bovine herpesvirus-1 (BHV-1) and P. haemolytica in aerosol.
Materials and methods Bacterin production Colonies of P. haemolytica A1 from experimental lung tissue stored at - 6 2 ° C were grown on blood agar plates and used to produce a 5 h culture in 750 ml BHI broth. The culture was centrifuged at 5000 rev. min- 1 for 10 min, and the pellet resuspended in RPMI-1640 with 7.% fetal calf serum (FCS). This suspension was incubated for 1 h in a shaker incubator. A sample of culture was taken for a biochemical profile of P. haemolytica and the culture was centrifuged at 7000rev. min-1 for 10min. The supernatant was filtered, lyophilized, reconstituted in distilled water, dialysed overnight at 4°C in RPMI-1640 isotonic medium and frozen at 62°C. The pellet was
Vaccine, Vol. 8, August 1990 315
Inactivated P. h a e m o l y t i c a bacterin and BHV-I: K.W.F. Jericho et al.
suspended in phosphate buffered saline pH 7.2 (PBS), inactivated by the addition of 0.3°,/0 formalin and let to stand overnight. The inactivated sample was centrifuged (8000rev. min -1 for 20min) and the cells were then resuspended and washed once in PBS. The final pellet was suspended in 95 ml of PBS, sonicated (Artek, Sonic 300 Dismembrator, medium probe at a setting of 55 for 5min) and frozen at - 6 2 ° C . Adjuvant was not added. Total particles of the bacterin of experiment 1 were counted by haemocytometry. The total protein content was determined by the Folin Lowry method for the bacterins of experiments 2 and 319, and cells of the bacterins were visualized by routine electron microscopy.
Animals Twenty-eight non-castrated male calves and 15 yearling heifers were derived from the Institute's specific pathogenfree Hereford crossbred herd. The herd is free of BHV-1, bovine virus diarrhoea virus, bovine leukosis virus, Brucella abortus, Mycobacterium tuberculosis, and has only a few reactors at low serum dilution to P. haemolytica, Leptospira spp., parainfluenza-3 virus, and bovine respiratory syncytial virus. This isolated herd has been free of respiratory disease during the last 17 years. Eight male calves were used in experiment 1, 15 heifers in experiment 2 and 20 male calves in experiment 3. Calves were born in concrete floored corrals (15 × 35 m) from March to April, and those of experiments 1 and 2 in the same year. Cows and calves of experiments 1 and 2 were kept in these corrals and then moved to a small pasture where they remained for 1 month until the day of weaning. Cows and calves were fed hay and hay cubes in the corrals, followed by 1-month exposure to native grass. Calves of experiment 3 were on native pasture for 4 months before weaning. Calves were weaned 3, 32 and 1.5 weeks before day 0 (day of first vaccination) in experiments 1, 2 and 3 respectively. After weaning, all calves of.the herd were grouped by sex, kept in corrals, and fed cubed hay. On day 0, the ranges in weight of calves were l10-155kg, 230-285kg and 160-240kg for experiments 1, 2 and 3 respectively. Experiments 1 and 3 were conducted in the fall of the year and experiment 2 in the spring. Calves were assigned randomly to control and vaccinate groups. The Guide to the Care and Use of Experimental Animals (Canadian Council of Animal Care 1984) was applied to the care of the animals.
Experiment 1 During the first 35 days of the experiment, control (n = 3) and vaccinate (n = 5) calves were kept separate in two outdoor concrete-floored pens with other herd Table 1
calves. From day 35 to the end of the experiment, vaccinates and controls were kept together in a third pen (10 x 10m). The ranges in age of calves on day 0, and days and levels of vaccinations, viral exposure, and bacterial challenge are summarized in Tables I and 2. Vaccination was begun on day 0 using 2.5 ml of bacterin and 2.5 ml of culture supernatant (see Bacterin production) intratracheally (i.t.). For this purpose, the cervical trachea was manually stabilized and a 20-gauge, 3 cm needle inserted into the trachea. Heads were held elevated for 1 min after inoculation. The animals were exposed to BHV1 and P. haemolytica cultures on days 35 and 39 respectively. The BHV1 (isolate 108) and P. haernolytica cultures (without FCS) were prepax:ed as described previously 2°'21. Briefly, the BHV1 culture (isolate 108) was grown in bovine foetal kidney cells, using roller bottles. FCS (10%) was used to grow the cell culture. Virus was absorbed onto the cells for l h at 37°C and M E M (with penicillinstreptomycin, gentamyocin, fungizone and sodium bicarbonate) was added. Maximum cytopathic effect (100%) was usually reached on day 3. The culture was not frozen before use. The bacterial culture was prepared by transferring P. haemolytica colonies from blood agar plates inoculated with lung swabs, into BH1 broth (75 ml). After incubation at 37°C for 16 h, this inoculum was added to 750ml of BH1 broth in a flask with a side arm, incubated and monitored regularly via colorimeter. The fresh culture was used as soon as two readings were similar (4.5-5.5 h). Heterologous isolates of P. haemolytica from two experimental lungs were used to produce the bacterin and the challenge culture. However, the bacterial isolates used in all experiments have, as their source, a field isolate of P. haemolytica passaged many times in calf lungs. Viral exposure and bacterial challenge were done by inhalation of aerosol for 5min is in order to expose as much respiratory tissue as possible to a low dose of infection. The concentrations of cultures aerosolized are given in Table 2. Each 5min aerosol production dispensed 1.5+0.2ml of culture. Shelter, straw bedding, hay, water and mineral mix were provided at each location in experiments 1 and 2.
Experiment 2 Fifteen heifers were moved to an outdoor concretefloored corral (160m 2) on day minus 76. Four calves received 2.5 ml (low dose) and four received 5 ml (high dose) of bacterin intranasally (i.n.) (Table 2). The low dose group also received 2.5 ml of supernatant via the same route. As with i.t. vaccinations, heads were held elevated for 1 min after inoculation. I.n. inoculations
Days of booster, viral exposure, and bacterial'~hallenge in treatment groups of experiments 1, 2 and 3 Calves Day a Vaccinates
Expt No. 1 2 3
Booster
BHV-1 exposure
28 29
35 48 49
P. haemolytica
Controls, n (days)
challenge
Expt end
Age (weeks)
n~
IC °
NC°
39 52 53
43 56 57
16-24 45-51 25-29
5 4, 4 5, 5, 5
3(35-43) 4(0-56) 5( 0-57)
3(48-56)
"Day 0 was first vaccination bFor experiment 2 there were four calves in each of low and high dose groups; in experiment 3 there were five calves in each of three vaccination route groups tiC, Controls in contact with vaccinates; NC, controls not in contact with vaccinates
316
V a c c i n e , Vol. 8, A u g u s t 1990
Inactivated P. h a e m o l y t i c a bacterin and BHV-I: K.W.F. Jericho et al. Table 2
Levels of viral exposure and bacterial challenge, vaccinations and pathological responses Bacterin
Suspensions aerosolized Expt No.
BHV-1 (TCID50 m l - ' )
P. haemolytica (cells m l - ' )
Dose (mg protein/100 kg BW)
Inoculum (ml)
1
1 x 10'.5
5 x 10"
Not done
2.5 b
2
3
1 x 107.9
1 x 106.3
5 x 10•
7 × 107
3.9-4.8 7.8-9.6
13-19
2.5 b 5
5 5 5
Purulent tonsillitis
Pneumonia = (GM %)
i.t. Control
3/5 2/3 p>0.05 ~
i.n.-i.n. i.n.-i.n. Control i.n.-i.n. i.m.-i.n. i.m.-i.m. Control
Route(s)
n1
n=
4.8=1- 3.1 41.2=1-34.1 p>0.05
1 1
1
4/4 1/4 5/7 p
Keywords:Pasteurella haemolytica; bacterin; infectiousbovine respiratory disease Introduction Pasteurella haemolytica is recognized as an important respiratory tract pathogen in cattle ~-a. Pasteurella spp. bacterins have been used for many years in an attempt to control infectious respiratory disease (IRD) 4'5. For example, a bacterin containing P. haemolytica and Pasteurella multocida with the addition of 0.25 % formalin was tried in farm livestock in 19576 . In one well designed field study 7 of Pasteurella bacterins, mono or bivalent bacterins were found to effectively reduce IRD in vaccinates as compared to natural controls s. However, similar to deficiencies in most field studies 7, the experimental demonstration of the protective effect of formalin inactivated P. haemolytica bacterins is also incomplete. The first experimental challenge of a formalininactivated P. haemolytica bacterin (polyvalent) was reported in 19649. However, it was not until pneumonic pasteurellosis was reproducible by large bacterial dose ~° or low viral-bacterial dose ~~ that a renewed interest was shown in experimental evaluations of P. haemolytica bacterins. Unfortunately, these studies differed not only in virus induced susceptibility of experimental calves, but also in challenge (dose, volume, route, mode of application), in bacterin (valency, bacterial source, method of Agriculture Canada, Animal Diseases Research Institute, PO Box 640, Lethbridge, Alberta, Canada T1J 3Z4 and Statistics Unit, Agriculture Canada Research Station, Agriculture Center, Lethbridge, Alberta, Canada T1J 4B1 (Kozub). (Received 7 October 1988; revised 2 October 1989; accepted 2 October 1989) 0264-410X/90/040315-06 © Canadian Crown Copyright 1990
preparation, adjuvants, type, concentration and dose frequency and route of application) and in cattle parameters (age, breed, microbial exposures, housing). It is, therefore, not surprising that formalin-inactivated bacterins have been shown to be effective 12-14, ineffective ls'~6 and even detrimentaP ~. In this report, three experiments are described in which the close relationship between bacterin, challenge and disease production is demonstrated. The low-dose viral-bacterial aerosol challenge was chosen because it is considered the best emulation of natural challenge available 18 against which a useful bacterin should protect. This is the first report of protection afforded by a formalin-inactivated and sonicated, adjuvant-free bacterin when challenged by bovine herpesvirus-1 (BHV-1) and P. haemolytica in aerosol.
Materials and methods Bacterin production Colonies of P. haemolytica A1 from experimental lung tissue stored at - 6 2 ° C were grown on blood agar plates and used to produce a 5 h culture in 750 ml BHI broth. The culture was centrifuged at 5000 rev. min- 1 for 10 min, and the pellet resuspended in RPMI-1640 with 7.% fetal calf serum (FCS). This suspension was incubated for 1 h in a shaker incubator. A sample of culture was taken for a biochemical profile of P. haemolytica and the culture was centrifuged at 7000rev. min-1 for 10min. The supernatant was filtered, lyophilized, reconstituted in distilled water, dialysed overnight at 4°C in RPMI-1640 isotonic medium and frozen at 62°C. The pellet was
Vaccine, Vol. 8, August 1990 315
Inactivated P. h a e m o l y t i c a bacterin and BHV-I: K.W.F. Jericho et al.
suspended in phosphate buffered saline pH 7.2 (PBS), inactivated by the addition of 0.3°,/0 formalin and let to stand overnight. The inactivated sample was centrifuged (8000rev. min -1 for 20min) and the cells were then resuspended and washed once in PBS. The final pellet was suspended in 95 ml of PBS, sonicated (Artek, Sonic 300 Dismembrator, medium probe at a setting of 55 for 5min) and frozen at - 6 2 ° C . Adjuvant was not added. Total particles of the bacterin of experiment 1 were counted by haemocytometry. The total protein content was determined by the Folin Lowry method for the bacterins of experiments 2 and 319, and cells of the bacterins were visualized by routine electron microscopy.
Animals Twenty-eight non-castrated male calves and 15 yearling heifers were derived from the Institute's specific pathogenfree Hereford crossbred herd. The herd is free of BHV-1, bovine virus diarrhoea virus, bovine leukosis virus, Brucella abortus, Mycobacterium tuberculosis, and has only a few reactors at low serum dilution to P. haemolytica, Leptospira spp., parainfluenza-3 virus, and bovine respiratory syncytial virus. This isolated herd has been free of respiratory disease during the last 17 years. Eight male calves were used in experiment 1, 15 heifers in experiment 2 and 20 male calves in experiment 3. Calves were born in concrete floored corrals (15 × 35 m) from March to April, and those of experiments 1 and 2 in the same year. Cows and calves of experiments 1 and 2 were kept in these corrals and then moved to a small pasture where they remained for 1 month until the day of weaning. Cows and calves were fed hay and hay cubes in the corrals, followed by 1-month exposure to native grass. Calves of experiment 3 were on native pasture for 4 months before weaning. Calves were weaned 3, 32 and 1.5 weeks before day 0 (day of first vaccination) in experiments 1, 2 and 3 respectively. After weaning, all calves of.the herd were grouped by sex, kept in corrals, and fed cubed hay. On day 0, the ranges in weight of calves were l10-155kg, 230-285kg and 160-240kg for experiments 1, 2 and 3 respectively. Experiments 1 and 3 were conducted in the fall of the year and experiment 2 in the spring. Calves were assigned randomly to control and vaccinate groups. The Guide to the Care and Use of Experimental Animals (Canadian Council of Animal Care 1984) was applied to the care of the animals.
Experiment 1 During the first 35 days of the experiment, control (n = 3) and vaccinate (n = 5) calves were kept separate in two outdoor concrete-floored pens with other herd Table 1
calves. From day 35 to the end of the experiment, vaccinates and controls were kept together in a third pen (10 x 10m). The ranges in age of calves on day 0, and days and levels of vaccinations, viral exposure, and bacterial challenge are summarized in Tables I and 2. Vaccination was begun on day 0 using 2.5 ml of bacterin and 2.5 ml of culture supernatant (see Bacterin production) intratracheally (i.t.). For this purpose, the cervical trachea was manually stabilized and a 20-gauge, 3 cm needle inserted into the trachea. Heads were held elevated for 1 min after inoculation. The animals were exposed to BHV1 and P. haemolytica cultures on days 35 and 39 respectively. The BHV1 (isolate 108) and P. haernolytica cultures (without FCS) were prepax:ed as described previously 2°'21. Briefly, the BHV1 culture (isolate 108) was grown in bovine foetal kidney cells, using roller bottles. FCS (10%) was used to grow the cell culture. Virus was absorbed onto the cells for l h at 37°C and M E M (with penicillinstreptomycin, gentamyocin, fungizone and sodium bicarbonate) was added. Maximum cytopathic effect (100%) was usually reached on day 3. The culture was not frozen before use. The bacterial culture was prepared by transferring P. haemolytica colonies from blood agar plates inoculated with lung swabs, into BH1 broth (75 ml). After incubation at 37°C for 16 h, this inoculum was added to 750ml of BH1 broth in a flask with a side arm, incubated and monitored regularly via colorimeter. The fresh culture was used as soon as two readings were similar (4.5-5.5 h). Heterologous isolates of P. haemolytica from two experimental lungs were used to produce the bacterin and the challenge culture. However, the bacterial isolates used in all experiments have, as their source, a field isolate of P. haemolytica passaged many times in calf lungs. Viral exposure and bacterial challenge were done by inhalation of aerosol for 5min is in order to expose as much respiratory tissue as possible to a low dose of infection. The concentrations of cultures aerosolized are given in Table 2. Each 5min aerosol production dispensed 1.5+0.2ml of culture. Shelter, straw bedding, hay, water and mineral mix were provided at each location in experiments 1 and 2.
Experiment 2 Fifteen heifers were moved to an outdoor concretefloored corral (160m 2) on day minus 76. Four calves received 2.5 ml (low dose) and four received 5 ml (high dose) of bacterin intranasally (i.n.) (Table 2). The low dose group also received 2.5 ml of supernatant via the same route. As with i.t. vaccinations, heads were held elevated for 1 min after inoculation. I.n. inoculations
Days of booster, viral exposure, and bacterial'~hallenge in treatment groups of experiments 1, 2 and 3 Calves Day a Vaccinates
Expt No. 1 2 3
Booster
BHV-1 exposure
28 29
35 48 49
P. haemolytica
Controls, n (days)
challenge
Expt end
Age (weeks)
n~
IC °
NC°
39 52 53
43 56 57
16-24 45-51 25-29
5 4, 4 5, 5, 5
3(35-43) 4(0-56) 5( 0-57)
3(48-56)
"Day 0 was first vaccination bFor experiment 2 there were four calves in each of low and high dose groups; in experiment 3 there were five calves in each of three vaccination route groups tiC, Controls in contact with vaccinates; NC, controls not in contact with vaccinates
316
V a c c i n e , Vol. 8, A u g u s t 1990
Inactivated P. h a e m o l y t i c a bacterin and BHV-I: K.W.F. Jericho et al. Table 2
Levels of viral exposure and bacterial challenge, vaccinations and pathological responses Bacterin
Suspensions aerosolized Expt No.
BHV-1 (TCID50 m l - ' )
P. haemolytica (cells m l - ' )
Dose (mg protein/100 kg BW)
Inoculum (ml)
1
1 x 10'.5
5 x 10"
Not done
2.5 b
2
3
1 x 107.9
1 x 106.3
5 x 10•
7 × 107
3.9-4.8 7.8-9.6
13-19
2.5 b 5
5 5 5
Purulent tonsillitis
Pneumonia = (GM %)
i.t. Control
3/5 2/3 p>0.05 ~
i.n.-i.n. i.n.-i.n. Control i.n.-i.n. i.m.-i.n. i.m.-i.m. Control
Route(s)
n1
n=
4.8=1- 3.1 41.2=1-34.1 p>0.05
1 1
1
4/4 1/4 5/7 p
