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Isolation of Pasteurella haemolytica and correlation with serum antibody response in clinically normal beef calves
Veterinary Microbiology, 8 (1983) 601--610 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
601
ISOLATION OF P A S T E U R E L L A H A E M O L Y T I C A AND CORRELATION WITH SERUM ANTIBODY RESPONSE IN CLINICALLY NORMAL BEEF CALVES
A.W. CONFER, R.E. CORSTVET, R.J. PANCIERA and J.A. RUMMAGE
Department of Veterinary Pathology and Department of Veterinary Parasitology, Microbiology and Public Health, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078 (U.S.A.) Journal article 4249 o f the Agricultural Experiment Station, Oklahoma State University, Stillwater, OK. Address correspondence to: Dr. A.W. Confer, Department of Pathology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, U.S.A. (Accepted 6 June 1983)
ABSTRACT Confer, A.W., Corstvet, R.E., Panciera, R.J. and Rummage, J.A., 1983. Isolation of PasteurelIa haemoly tics and correlation with serum antibody response in clinically normal beef calves. Vet. Microbiol., 8: 601--610. Bacteria from the nasal cavity and trachea were cultured, and serum antibody titers determined for Pasteurella haemolytica serotype 1 in 164 beef calves obtained from a closed herd on range pasture. At the first sampling, P. haemolytica serotype 1 was cultured from 16.4% of the calves. Antibody titers were determined by a quantitative fluorimetric m e t h o d and the mean titer was 9.5 -+ 5.8. Fifty-seven randomly selected calves were used to study the correlation of serum antibody response and positive culture of P. haemolytica under natural conditions. Clinical signs of respiratory disease were not observed in those calves. During the observation periods, there was a two-fold increase in the percentage o f calves that were culture positive. There was no significant difference between mean serum antibody titers or frequency distribution of antibody titers from the two samplings. Comparisons between serum antibody titers, rise in titers, and P. haemolyrics isolation failed to reveal any significant correlation. Of the 9 calves that had a decline in antibody titer to P. haemolytica, none was culture positive. Seroconversion to respiratory viruses did not correlate with P. haemolytica related variables.
INTRODUCTION
Bovine pneumonic pasteurellosis caused by Pasteurella haemolytica biotype A serotype 1 represents a severe respiratory disease in feedlot cattle (Rehmtulla and Thomson, 1981). Pasteurella haemolytica can be isolated commonly from the nasal cavities and tracheas of pastured or feedlot cattle and transmission appears to be by direct contact among animals (Carter,
0378-1135/83/$03.00
© 1983 Elsevier Science Publishers B.V.
602 1967; Magwood et al., 1969; Thomson et al., 1969; Pass and Thomson~ 1971; Corstvet et al., 1973). The frequency of isolation o f P . haemolytica is dependent u p o n factors such as exposure to sick animals, overcrowding, shipment, and severe weather (Horlein et al., 1961; H a m d y and Trapp, 1967; Thomson et al., 1969). In general, P. haemolytica has been isolated more frequently and in greater numbers from sick than healthy feedlot cattle (Corstvet et al., 1973; T h o m s o n et al., 1969, 1975). The prevalence of antibodies and the antibody response to P. haemolytica have not been studied to a great extent in cattle under range or feedlot conditions. Passive transfer of antibodies via colostrum was demonstrated in dairy calves and shown to persist until 5--6 weeks of age (Wray and Thompson, 1973). Rice et al. (1955) using a complement fixation assay for P. haemolytica f o u n d that prior to shipment to Canadian feedlots less than 1% of cattle tested had positive serum antibody titers t o P . haemolytica. One to two months later, approximately 22% of cattle had positive titers indicating active infection. T h o m s o n et al. (1975) found that feedlot calves which developed respiratory disease had lower hemagglutinating antibody titers to P. haemolytica at the time of arrival than those that failed to become sick. There was also a greater rise in mean serum antibody titers in the diseased group of cattle compared to the healthy group. There have been no previous studies reported that compared the serum antibody response and frequency of isolation of P. haemoly tica in range cattle. The objective of this study was, retrospectively, to evaluate the prevalence of nasal and tracheal P. haemolytica and to correlate this with the serum antibody response in healthy beef calves obtained from a closed herd. The purpose was to a t t e m p t to understand the bacteria--host interaction under conditions typical of those for cattle prior to shipment to a feedlot. MATERIALS AND METHODS
A nima Is A total of 164 Hereford, Angus, and Hereford--Angus crossbred female and castrated male calves, 6--8 months old, was obtained from a closed herd during a period of 6 years. During that period, the herd had consisted of between 180 and 240 cows. Calving rate approached 90% and clinical evidence of respiratory disease was n o t reported among those cattle. Cows and calves grazed on approximately 1500 acres of native pasture. A winter supplement was provided that consisted o f alfalfa and prairie hays as well as a commercial protein supplement. Calves were born and raised on pasture until weaned. After weaning, calves were placed in holding pens 64 X 160 ft. in size and fed on hay and protein supplement from feed bunks for a period of 1--3 weeks before sale. Calves were purchased from the farm for use in 10 different vaccination and challenge experiments. The number of animals per experiment varied
603
from 6 to 30. Calves were moved by trailer directly from the farm to the research facilities (approximately 10 miles) and u p o n arrival, were placed in pens 40 X 94 ft. in size at no more than 6 calves per pen. Calves were fed prairie hay and protein supplement from feed bunks. Serum samples and nasal and tracheal swabs were collected within 48 h after arrival (Day 0 = first sampling). One-hundred-and-seven calves (Group A) were exposed to aerosol or parenteral live or killed P. haemolytica vaccines several weeks after the D a y 0 samples were obtained. Bacterial isolation and serological data obtained after the initial sampling are n o t included in this report. Fifty-seven calves (Group B) were randomly selected and held as non-exposed control animals. In each experiment, Group B calves were held together in pens that were separated from the pens containing Group A calves by either a 12 ft. wide walkway or a single pen width (40 ft.). Calves were held for periods ranging from 5 to 8 weeks and during that time, were observed and rectal temperatures recorded daily. Four to five days before slaughter, serum samples and nasal and tracheal swabs were collected (second sampling). Final nasal and tracheal swabs were n o t collected from 2 calves in one experiment. All calves were challenged subsequently b y a transthoracic injection into the caudal lung lobes with live P. haemolytica b i o t y p e A serotype 1, 4 days before slaughter, as previously described (Newman et al.1 1982).
Serology Serum samples were kept at - 7 0 ° C until use. A n t i b o d y to P. haemolytica somatic antigens was determined by a rapid quantitative fluorimetric immunoassay (FIAX) that compares to the indirect bacterial agglutination test (Confer et al., 1983). Antigen was a formalin-killed P. haemolytica serotype 1, obtained from a 22 h culture. Titer values from samples were determined by extrapolation of a quantitative fluorescence value from a regression curve established from four calibrator samples that have pre-determined endpoint titers. Variations in titers in replicate samples are minimal by this technique and so a two-fold or greater rise in antibody titer is considered to indicate active infection. A n t i b o d y titers to bovine virus diarrhea and bovine respiratory syncytial viruses were determined by an indirect fluorescent antibody test. A n t i b o d y titers to parainfluenza-3 and infectious bovine rhinotracheitis viruses were determined by microtiter hemagglutination-inhibition and microtiter virus neutralization tests, respectively. 1 All tests for antibody to viruses were performed with serial two-fold dilutions of serum. A four-fold or greater rise in antibody titer was considered to indicate active infection.
Bacteriology Nasal swabs were taken using sterile c o t t o n swabs and those from the left nasal cavity were cultured. Swabs were placed in sterile tubes and held at 1Viral serology performed by the Oklahoma Animal Disease Diagnostic Laboratory.
604 4°C until returned to the laboratory (approximately 1 h or less). Tracheal samples were obtained by passage of a guarded culture instrument (24 in. long) through the oropharynx and laryngeal aperture as described previously (Corstvet et al., 1973). Nasal swabs were streaked on to supplemented brain heart infusion agar (Corstvet et al., 1973). Tracheal swabs were incubated in supplemented trypticase--soy broth as well as streaked on to solid medium. Cultures were incubated at 37°C with reduced oxygen tension and examined for bacterial growth after 24 h. Pasteurella haemolytica was identified by colony characteristics and biochemical tests (Wessman and Hilkes, 1968). Only those isolates identified immunologically as serotype 1 were reported in this study (Carter, 1967).
Statistical analysis The data were analyzed by Student's t and chi-square tests (Bailey, 1981). RESULTS Group B calves remained healthy t h r o u g h o u t the observation periods. Prior to transthoracic challenge, the only signs of disease infrequently observed included temperature elevations of a single day's duration, and an occasional dry cough. Lung lesions characteristic of naturally-occurring pneumonic pasteurellosis were n o t observed at slaughter. In most calves, experimental challenge lesions in the caudal lung lobes were extensive indicating susceptibility to the organism. On Day 0, the antibody titers for all 164 calves ranged from 0 to 201 with a geometric mean value of 9.5 -+ 5.8 (standard deviation) (Table I). Geometric mean titers varied among experiments ranging from 3.2 + 2.5 to 25.5 -+ 4.5. The percentage frequency distribution of antibody to P. haemolytica on Day 0 was similar between Groups A and B (Table I). Geometric mean titers for Groups A and B on Day 0 were 11.2 + 3.1 and 8.1 -+ 5.9; respectively. These differences were n o t significant (P > 0.05). There was little change in the percentage frequency distribution for Group B calves at the end of the observation period (Table I). The geometric mean titer for Group B calves at that time was 11.4 _+ 6.1 and was n o t significantly different (P > 0.05) from the mean titer on Day 0. Sixteen calves had a rise in titer of twofold or greater and eleven of these calves had a rise in titer of four-fold or greater. Nine calves had a decline in titer such that their antibody titers were in a lower range in the second sampling than in the first sampling. U p o n arrival, cultures of the nasal cavity and trachea were obtained from all calves and of these 18 (11.0%) and 7 (4.3%), respectively, were positive for P. haemolytica serotype 1 (Table II). Organisms were isolated from b o t h sites from 2 calves (1.2%). Organisms were isolated from Group B calves in 4 of 10 experiments on Day 0, and in 7 o f 9 experiments 5--8 weeks later.
38 (66.6%) 34 (59.7%)
67 (62.6%)
105 (64%)
< 25
8 (14%) 9 (15.8%)
15 (14%)
23 (15%)
26--50
(8.4%)
7 (12.3%) 6 (10.5%)
9
16 ((9.8%)
51--75
2 (3.5%) 2 (3.5%)
5 (4.7%)
7 (4.3%)
76--100
0 1 (1.8%)
5 (4.7%)
5 (3.0%)
101--125
Number (percentage) of calves per antibody titer range
aCalves subsequently vaccinated for P. haemoly tica. bCaives not vaccinated for P. haemoly tica.
57 57
107
Group A a Day 0
Group Bb Day 0 Weeks 5--8
164
All calves Day 0
No. of calves
Frequency distribution of serum antibody titers to Pasteurella haemoly tica
TABLE I
1 (1.8%) 1 (1.8%)
2 (1.9%)
3 (1.8%)
126--150
1 (1.8%) 0
3 (2.8%)
4 (2.4%)
151--175
1 (0.9%)
1 (0.6%)
0 0 1 (1.8%) 1 (1.8%)
0
0
176--200 201--225
o ¢.n
606 TABLE II Summary of isolations of Pasteurella haemolytica from calves No. of calves
Total isolations (%)
Nasal cavity (%)
Trachea (%)
Both (%)
All calves Day 0
164
27 (16.4)
18 (11.0)
7 (4.3)
2 (1.2)
Group A a Day 0
107
18 (16.8)
11 (10.3)
6 (5.6)
1 (0.9)
9 (15.8) 19 (34.5) c
7 (12.3) 10 (17.5)
1 (1.8) 5 (9.1)
1 (1.8) 4 (7.3)
Group B b Day 0 Weeks 5--8
57 55
aCalves subsequently vaccinated for P. haemolytica. bCalves not vaccinated for P. haemoly tica. cp < 0.02 compared to total isolations on Day 0.
There was a significant increase (P < 0.02) in the total isolations between the two sample periods (Table II). Of the 9 Group B calves from which P. haemolytica was isolated upon arrival, only 1 (11.1%) later experienced a rise in antibody titer of two-fold to four-fold (Table III). Of the 48 Group B calves from which the organism was not isolated on Day 0, 15 (31.3%) subsequently had a rise in antibody titer of two-fold or greater. Of all the 16 calves t h a t experienced a rise in antibody titer during the observation periods, 11 (68.8%) had titers of less than 25 at the start of the experiment. Of the 41 t h a t did n o t experience a rise in antibody titer, 27 (65.9%) had titers o f less than 25 at the start of the experiment. The difference between the percentage of calves with titers of less than 25 t h a t subsequently had a rise in a n t i b o d y titer and those that did not have a rise in titer was n o t significant (P > 0.05). Six out of the 19 (31.6%) calves from which P. haemolytica was isolated at the second sampling had experienced a two-fold or greater rise in titer during the observation period. Ten o u t of 36 (27.8%) of the calves from which P. haemolytica was not isolated at the second sampling had experienced a two-fold or greater rise in titer during t h a t period. These differences were n o t significant (P > 0.05). The number of calves from which the organism was isolated was compared to the number of negative calves with respect to range of antibody titers at the second sampling (Table IV). There were no significant differences (P > 0.05) between the percentage of positive calves and negative calves at the different a n t i b o d y titer ranges. In 6 out of 9 experiments where serology to respiratory viruses was performed, significant rises in titer were seen in one or more calves. A significant rise in titer to PI3 virus was seen in 40.8% of the Group B calves. None of these calves had a rise in titer to IBR. Significant rises in titer to RSV and BVD viruses was seen in 19.2% and 8.2% of the Group B calves, respectively.
607 TABLE III C o m p a r i s o n o f Pasteurella haemolytica i s o l a t i o n , s e r u m a n t i b o d y body titer in Group B calves
titers, a n d rise in anti-
Isolation Positive Rise intiter
0
2--4x
Negative 4--6x
6x
0
2--4x
4--6x
6x
8
Day 0 Antibody
titers
Ranges
<25
7a
1
26--50
51--75 76--100 101--125 126--150 151--175
1
--
--
--
20
2
--
--
--
6
1
--
1
--. ---
--. ---
4 2 . -1
2 --
---
---
1 --
---
---
.
Weeks 5--8 Antibody titers <25
Ranges
--
--
1
18
2
--
2
26--50
II
1
1
--
--
3
--
--
4
51--75
1
--
--
--
4
1
--
--
76--100
--
1
--
1
--
--
101--125
--
--
1
--
--
--
--
--
126--150
.
.
151--175
.
.
176--200
--
201--225
--
--
1
1
.
.
.
.
.
.
1
.
.
aNo. of calves within range of antibody titer and corresponding rise in titer.
TABLE
IV
Comparison of antibody titer ranges with isolation of ond sampling a of Group B calves
Pasteurella haemolytica
P. haemolytica
Antibody titer range
<25 26 or greater 26--100 > 101
aWeeks 5--8.
Isolated (N = 19)
Not isolated (N = 36)
12 (63.2%) 7 (36.8%) 4 (21.1%)
22 (61.1%) 14 (38.8%) 13 ( 3 6 . 1 % )
3 (15.8%)
1
(2.8%)
a t t h e sec-
608
There did n o t appear to be an association among antibody response to viruses, frequency of isolation of P. haemolytica, and antibody response to
P. haemolytica. DISCUSSION
The results of this study corroborate the findings of Magwood et al. (1969) in t h a t P. haemolytica was isolated from the upper respiratory tract in low frequency from pastured calves t h a t were n o t subjected to severe stresses such as overcrowding, long shipments, or exposure to cattle from other locations. In the present study, P. haemolytica was isolated even less frequently from the trachea than from the nasal cavity. Within the Group B calves, there was a significantly greater frequency of isolation of P. haemolytica at the second sampling as compared to the first sampling. This increase may have been due to transmission among calves or merely to expression of the organism in calves from which it was n o t isolated initially. Periodic dominance of the bovine nasal flora by P. haemolytica for several days at a time has been reported (Pass and Thomson, 1971). It was suggested t h a t Pasteurella spp. could be isolated most readily during periods when the organisms were actively colonizing the mucosa. Therefore, variations in the expression of the organism may be a more reasonable explanation for increased isolation than that of transmission among calves because P. haemolytica isolations occurred at the second sampling in groups where the organism was n o t isolated at the first sampling. Transmission from experimentally-infected (Group A) calves seems unlikely because of precautions taken to prevent direct contact of Group B with Group A once they were experimentally exposed to the organism. On Day 0, 64% of the 164 cattle had antibody titers of 25 or less as determined by the FIAX test. Based on our studies of serum antibody response after aerosol exposure to P. haemolytica, a titer of 25 or less would be a low titer and probably indicate minimal exposure (Confer et al., 1983). On the other hand, titers were nearly always at least 50 in animals that had received two aerosol exposures to live P. haemolytica. Equivalent titers in the calves in the present studies would most likely indicate a substantial exposure to the organism. In other experiments, we have f o u n d that most calves with titers less than 25 are susceptible to experimental challenge while those with titers of greater than 100 are mostly resistant (Panciera et al., 1983). During the observation period, the geometric mean antibody titer failed to rise significantly for Group B calves. The frequency of animals with titers of 25 or less decreased insignificantly. Therefore, examination of titers for the calves as a whole did n o t indicate active infection with P. haemolytica. Antibody titer increases of up to six-fold in sixteen calves, however, suggested that active infection a n d / o r transmission occurred to a limited extent among the calves.
609
Isolation of P. haemolytica from the upper respiratory tract did not correlate with serum antibody response to the organism. One possible explanation for this is that colonization of the organism in the upper respiratory tract m a y n o t induce a significant serum antibody response because P. haemolytica normally inhabits the surface of the nasal epithelium only {Pass and Thomson, 1971). Instead, ulceration o f the upper respiratory tract due to trauma or viral infection m a y be necessary to allow organisms to invade the lamina propria, be phagocytized and carried to regional lymph nodes and thus stimulate a serum antibody response. Another mechanism could relate to the extent of lung exposure to organisms via inhalation from the nasal cavity (Grey and Thomson, 1971). We have demonstrated significant serum antibody responses to P. haemolytica following aerosol exposure to the organisms (Confer et al., 1983). In clinically normal cattle, however, p u l m ~ nary exposure to the organism and subsequent serum antibody response may be minimal. In conclusion, the prevalence of P. haemolytica serotype 1 in the nasal cavity and trachea as detected by cultures obtained from live animals was low in beef calves from a closed herd that were maintained on range pastures: Serum antibody levels were also low. With time, there appeared to be an increase in the frequency of isolation from healthy calves that were moved to pens, held in low concentrations, and maintained under low stress conditions. There was no correlation between isolation of P. haemolytica from the upper respiratory tract at t w o samplings and any increase in serum antibody titer. Interestingly, P. haemolytica was n o t isolated from the 9 calves that experienced a decline in serum antibody titers. A n t i b o d y responses to respiratory viruses did n o t appear to affect isolations of P. haemolytica or serum antibody titers to the bacterium. ACKNOWLEDGEMENTS
The authors thank Carolyn Gresham for excellent technical assistance.
REFERENCES Bailey, N.T.J., 1981. Statistical Methods in Biology, 2nd edn., Wiley, New York, pp. 43-66. Carter, G.R., 1967. Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Adv. Vet. Sci., 11: 321--379. Confer, A.W., Fox, J.C., Newman, P.R., Lawson, G.W. and Corstvet, R.E., 1983. A quantitative fluorometric assay for the measurements o f antibody to Pasteurella haemolytica in cattle. Can. J. Comp. Med., 47: 37--42. Corstvet, R.E., Panciera, R.J., Rinker, H.B., Starks, B.L. and Howard, C., 1973. Survey of tracheas of feedlot cattle for Haemophilus somnes and other selected bacteria. J. Am. Vet. ivied. Assoc., 163: 870--873. Grey, C.L. and Thomson, R.G., 1971. Pasteurella haemolytiea in the tracheal air of calves. Can. J. Comp. Med., 35: 121--128.
610 Hamdy, A.H. and Trapp, A.L., 1967. Investigation of nasal microflora of feedlot calves before and after weaning. Am. J. Vet. Res., 28: 1019--1025. Horlein, A.B., Saxena, S.P. and Mansfield, M.E., 1961. Studies of shipping fever of cattle. II. Prevalence of Pasteurella species in nasal secretions from normal calves and calves with shipping fever. Am. J. Vet. Res., 22: 470--472. Magwood, S.E., Barnum, D.A. and Thomson, R.G., 1969. Nasal bacterial flora of calves in healthy and in pneumonia-prone herds. Can. J. Comp. Med., 33: 237--243. Newman, P.R., Corstvet, R.E. and Panciera, R.J., 1982. Distribution of Pasteurella haemolytica and Pasteurella multocida in the bovine lung following vaccination and challenge exposure as an indicator of lung resistance. Am. J. Vet. Res., 43: 417--422. Panciera, R.J., Corstvet, R.E., Confer, A.W. and Gresham, C.N., 1983. Bovine pneumonic pasteurellosis: Effect of vaccination with live Pasteurella species. Am. J. Vet. Res., submitted. Pass, D.A. and Thomson, R.G., 1971. Wide distribution of Pasteurella haemoly tica type 1 over the nasal mucosa of cattle. Can. J. Comp. Med., 35: 181--186. Rehmtulla, A.J. and Thomson, R.G., 1981. A review of the lesions of shipping fever of cattle. Can. Vet. J., 22: 1--8. Rice, C.E., Beauregard, M. and Maybee, T.K., 1955. Survey of shipping fever in Canada: Serological studies. Can. J. Comp. Med., 19: 329--349. Thomson, R.G., Benson, M.L. and Savan, M., 1969. Pneumonic pasteurellosis of cattle: Microbiology and immunology. Can. J. Comp. Med., 33 : 194--206. Thomson, R.G., Chander, S., Savan, M. and Fox, M.L., 1975. Investigation of factors of probable significance in the pathogenesis of pneumonic pasteurellosis in cattle. Can. J. Comp. Med., 39: 194--207. Wessman, G.E. and Hilkes, G., 1968. Characterization ofPasteurella haemolytica isolated from the respiratory tract of cattle. Can. J. Comp. Med., 32: 498--504. Wray, C. and Thompson, D.A., 1973. An epidemiological study ofPasteureUa haemolytica in calves. Br. Vet. J., 129: 116--123.
601
ISOLATION OF P A S T E U R E L L A H A E M O L Y T I C A AND CORRELATION WITH SERUM ANTIBODY RESPONSE IN CLINICALLY NORMAL BEEF CALVES
A.W. CONFER, R.E. CORSTVET, R.J. PANCIERA and J.A. RUMMAGE
Department of Veterinary Pathology and Department of Veterinary Parasitology, Microbiology and Public Health, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078 (U.S.A.) Journal article 4249 o f the Agricultural Experiment Station, Oklahoma State University, Stillwater, OK. Address correspondence to: Dr. A.W. Confer, Department of Pathology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, U.S.A. (Accepted 6 June 1983)
ABSTRACT Confer, A.W., Corstvet, R.E., Panciera, R.J. and Rummage, J.A., 1983. Isolation of PasteurelIa haemoly tics and correlation with serum antibody response in clinically normal beef calves. Vet. Microbiol., 8: 601--610. Bacteria from the nasal cavity and trachea were cultured, and serum antibody titers determined for Pasteurella haemolytica serotype 1 in 164 beef calves obtained from a closed herd on range pasture. At the first sampling, P. haemolytica serotype 1 was cultured from 16.4% of the calves. Antibody titers were determined by a quantitative fluorimetric m e t h o d and the mean titer was 9.5 -+ 5.8. Fifty-seven randomly selected calves were used to study the correlation of serum antibody response and positive culture of P. haemolytica under natural conditions. Clinical signs of respiratory disease were not observed in those calves. During the observation periods, there was a two-fold increase in the percentage o f calves that were culture positive. There was no significant difference between mean serum antibody titers or frequency distribution of antibody titers from the two samplings. Comparisons between serum antibody titers, rise in titers, and P. haemolyrics isolation failed to reveal any significant correlation. Of the 9 calves that had a decline in antibody titer to P. haemolytica, none was culture positive. Seroconversion to respiratory viruses did not correlate with P. haemolytica related variables.
INTRODUCTION
Bovine pneumonic pasteurellosis caused by Pasteurella haemolytica biotype A serotype 1 represents a severe respiratory disease in feedlot cattle (Rehmtulla and Thomson, 1981). Pasteurella haemolytica can be isolated commonly from the nasal cavities and tracheas of pastured or feedlot cattle and transmission appears to be by direct contact among animals (Carter,
0378-1135/83/$03.00
© 1983 Elsevier Science Publishers B.V.
602 1967; Magwood et al., 1969; Thomson et al., 1969; Pass and Thomson~ 1971; Corstvet et al., 1973). The frequency of isolation o f P . haemolytica is dependent u p o n factors such as exposure to sick animals, overcrowding, shipment, and severe weather (Horlein et al., 1961; H a m d y and Trapp, 1967; Thomson et al., 1969). In general, P. haemolytica has been isolated more frequently and in greater numbers from sick than healthy feedlot cattle (Corstvet et al., 1973; T h o m s o n et al., 1969, 1975). The prevalence of antibodies and the antibody response to P. haemolytica have not been studied to a great extent in cattle under range or feedlot conditions. Passive transfer of antibodies via colostrum was demonstrated in dairy calves and shown to persist until 5--6 weeks of age (Wray and Thompson, 1973). Rice et al. (1955) using a complement fixation assay for P. haemolytica f o u n d that prior to shipment to Canadian feedlots less than 1% of cattle tested had positive serum antibody titers t o P . haemolytica. One to two months later, approximately 22% of cattle had positive titers indicating active infection. T h o m s o n et al. (1975) found that feedlot calves which developed respiratory disease had lower hemagglutinating antibody titers to P. haemolytica at the time of arrival than those that failed to become sick. There was also a greater rise in mean serum antibody titers in the diseased group of cattle compared to the healthy group. There have been no previous studies reported that compared the serum antibody response and frequency of isolation of P. haemoly tica in range cattle. The objective of this study was, retrospectively, to evaluate the prevalence of nasal and tracheal P. haemolytica and to correlate this with the serum antibody response in healthy beef calves obtained from a closed herd. The purpose was to a t t e m p t to understand the bacteria--host interaction under conditions typical of those for cattle prior to shipment to a feedlot. MATERIALS AND METHODS
A nima Is A total of 164 Hereford, Angus, and Hereford--Angus crossbred female and castrated male calves, 6--8 months old, was obtained from a closed herd during a period of 6 years. During that period, the herd had consisted of between 180 and 240 cows. Calving rate approached 90% and clinical evidence of respiratory disease was n o t reported among those cattle. Cows and calves grazed on approximately 1500 acres of native pasture. A winter supplement was provided that consisted o f alfalfa and prairie hays as well as a commercial protein supplement. Calves were born and raised on pasture until weaned. After weaning, calves were placed in holding pens 64 X 160 ft. in size and fed on hay and protein supplement from feed bunks for a period of 1--3 weeks before sale. Calves were purchased from the farm for use in 10 different vaccination and challenge experiments. The number of animals per experiment varied
603
from 6 to 30. Calves were moved by trailer directly from the farm to the research facilities (approximately 10 miles) and u p o n arrival, were placed in pens 40 X 94 ft. in size at no more than 6 calves per pen. Calves were fed prairie hay and protein supplement from feed bunks. Serum samples and nasal and tracheal swabs were collected within 48 h after arrival (Day 0 = first sampling). One-hundred-and-seven calves (Group A) were exposed to aerosol or parenteral live or killed P. haemolytica vaccines several weeks after the D a y 0 samples were obtained. Bacterial isolation and serological data obtained after the initial sampling are n o t included in this report. Fifty-seven calves (Group B) were randomly selected and held as non-exposed control animals. In each experiment, Group B calves were held together in pens that were separated from the pens containing Group A calves by either a 12 ft. wide walkway or a single pen width (40 ft.). Calves were held for periods ranging from 5 to 8 weeks and during that time, were observed and rectal temperatures recorded daily. Four to five days before slaughter, serum samples and nasal and tracheal swabs were collected (second sampling). Final nasal and tracheal swabs were n o t collected from 2 calves in one experiment. All calves were challenged subsequently b y a transthoracic injection into the caudal lung lobes with live P. haemolytica b i o t y p e A serotype 1, 4 days before slaughter, as previously described (Newman et al.1 1982).
Serology Serum samples were kept at - 7 0 ° C until use. A n t i b o d y to P. haemolytica somatic antigens was determined by a rapid quantitative fluorimetric immunoassay (FIAX) that compares to the indirect bacterial agglutination test (Confer et al., 1983). Antigen was a formalin-killed P. haemolytica serotype 1, obtained from a 22 h culture. Titer values from samples were determined by extrapolation of a quantitative fluorescence value from a regression curve established from four calibrator samples that have pre-determined endpoint titers. Variations in titers in replicate samples are minimal by this technique and so a two-fold or greater rise in antibody titer is considered to indicate active infection. A n t i b o d y titers to bovine virus diarrhea and bovine respiratory syncytial viruses were determined by an indirect fluorescent antibody test. A n t i b o d y titers to parainfluenza-3 and infectious bovine rhinotracheitis viruses were determined by microtiter hemagglutination-inhibition and microtiter virus neutralization tests, respectively. 1 All tests for antibody to viruses were performed with serial two-fold dilutions of serum. A four-fold or greater rise in antibody titer was considered to indicate active infection.
Bacteriology Nasal swabs were taken using sterile c o t t o n swabs and those from the left nasal cavity were cultured. Swabs were placed in sterile tubes and held at 1Viral serology performed by the Oklahoma Animal Disease Diagnostic Laboratory.
604 4°C until returned to the laboratory (approximately 1 h or less). Tracheal samples were obtained by passage of a guarded culture instrument (24 in. long) through the oropharynx and laryngeal aperture as described previously (Corstvet et al., 1973). Nasal swabs were streaked on to supplemented brain heart infusion agar (Corstvet et al., 1973). Tracheal swabs were incubated in supplemented trypticase--soy broth as well as streaked on to solid medium. Cultures were incubated at 37°C with reduced oxygen tension and examined for bacterial growth after 24 h. Pasteurella haemolytica was identified by colony characteristics and biochemical tests (Wessman and Hilkes, 1968). Only those isolates identified immunologically as serotype 1 were reported in this study (Carter, 1967).
Statistical analysis The data were analyzed by Student's t and chi-square tests (Bailey, 1981). RESULTS Group B calves remained healthy t h r o u g h o u t the observation periods. Prior to transthoracic challenge, the only signs of disease infrequently observed included temperature elevations of a single day's duration, and an occasional dry cough. Lung lesions characteristic of naturally-occurring pneumonic pasteurellosis were n o t observed at slaughter. In most calves, experimental challenge lesions in the caudal lung lobes were extensive indicating susceptibility to the organism. On Day 0, the antibody titers for all 164 calves ranged from 0 to 201 with a geometric mean value of 9.5 -+ 5.8 (standard deviation) (Table I). Geometric mean titers varied among experiments ranging from 3.2 + 2.5 to 25.5 -+ 4.5. The percentage frequency distribution of antibody to P. haemolytica on Day 0 was similar between Groups A and B (Table I). Geometric mean titers for Groups A and B on Day 0 were 11.2 + 3.1 and 8.1 -+ 5.9; respectively. These differences were n o t significant (P > 0.05). There was little change in the percentage frequency distribution for Group B calves at the end of the observation period (Table I). The geometric mean titer for Group B calves at that time was 11.4 _+ 6.1 and was n o t significantly different (P > 0.05) from the mean titer on Day 0. Sixteen calves had a rise in titer of twofold or greater and eleven of these calves had a rise in titer of four-fold or greater. Nine calves had a decline in titer such that their antibody titers were in a lower range in the second sampling than in the first sampling. U p o n arrival, cultures of the nasal cavity and trachea were obtained from all calves and of these 18 (11.0%) and 7 (4.3%), respectively, were positive for P. haemolytica serotype 1 (Table II). Organisms were isolated from b o t h sites from 2 calves (1.2%). Organisms were isolated from Group B calves in 4 of 10 experiments on Day 0, and in 7 o f 9 experiments 5--8 weeks later.
38 (66.6%) 34 (59.7%)
67 (62.6%)
105 (64%)
< 25
8 (14%) 9 (15.8%)
15 (14%)
23 (15%)
26--50
(8.4%)
7 (12.3%) 6 (10.5%)
9
16 ((9.8%)
51--75
2 (3.5%) 2 (3.5%)
5 (4.7%)
7 (4.3%)
76--100
0 1 (1.8%)
5 (4.7%)
5 (3.0%)
101--125
Number (percentage) of calves per antibody titer range
aCalves subsequently vaccinated for P. haemoly tica. bCaives not vaccinated for P. haemoly tica.
57 57
107
Group A a Day 0
Group Bb Day 0 Weeks 5--8
164
All calves Day 0
No. of calves
Frequency distribution of serum antibody titers to Pasteurella haemoly tica
TABLE I
1 (1.8%) 1 (1.8%)
2 (1.9%)
3 (1.8%)
126--150
1 (1.8%) 0
3 (2.8%)
4 (2.4%)
151--175
1 (0.9%)
1 (0.6%)
0 0 1 (1.8%) 1 (1.8%)
0
0
176--200 201--225
o ¢.n
606 TABLE II Summary of isolations of Pasteurella haemolytica from calves No. of calves
Total isolations (%)
Nasal cavity (%)
Trachea (%)
Both (%)
All calves Day 0
164
27 (16.4)
18 (11.0)
7 (4.3)
2 (1.2)
Group A a Day 0
107
18 (16.8)
11 (10.3)
6 (5.6)
1 (0.9)
9 (15.8) 19 (34.5) c
7 (12.3) 10 (17.5)
1 (1.8) 5 (9.1)
1 (1.8) 4 (7.3)
Group B b Day 0 Weeks 5--8
57 55
aCalves subsequently vaccinated for P. haemolytica. bCalves not vaccinated for P. haemoly tica. cp < 0.02 compared to total isolations on Day 0.
There was a significant increase (P < 0.02) in the total isolations between the two sample periods (Table II). Of the 9 Group B calves from which P. haemolytica was isolated upon arrival, only 1 (11.1%) later experienced a rise in antibody titer of two-fold to four-fold (Table III). Of the 48 Group B calves from which the organism was not isolated on Day 0, 15 (31.3%) subsequently had a rise in antibody titer of two-fold or greater. Of all the 16 calves t h a t experienced a rise in antibody titer during the observation periods, 11 (68.8%) had titers of less than 25 at the start of the experiment. Of the 41 t h a t did n o t experience a rise in antibody titer, 27 (65.9%) had titers o f less than 25 at the start of the experiment. The difference between the percentage of calves with titers of less than 25 t h a t subsequently had a rise in a n t i b o d y titer and those that did not have a rise in titer was n o t significant (P > 0.05). Six out of the 19 (31.6%) calves from which P. haemolytica was isolated at the second sampling had experienced a two-fold or greater rise in titer during the observation period. Ten o u t of 36 (27.8%) of the calves from which P. haemolytica was not isolated at the second sampling had experienced a two-fold or greater rise in titer during t h a t period. These differences were n o t significant (P > 0.05). The number of calves from which the organism was isolated was compared to the number of negative calves with respect to range of antibody titers at the second sampling (Table IV). There were no significant differences (P > 0.05) between the percentage of positive calves and negative calves at the different a n t i b o d y titer ranges. In 6 out of 9 experiments where serology to respiratory viruses was performed, significant rises in titer were seen in one or more calves. A significant rise in titer to PI3 virus was seen in 40.8% of the Group B calves. None of these calves had a rise in titer to IBR. Significant rises in titer to RSV and BVD viruses was seen in 19.2% and 8.2% of the Group B calves, respectively.
607 TABLE III C o m p a r i s o n o f Pasteurella haemolytica i s o l a t i o n , s e r u m a n t i b o d y body titer in Group B calves
titers, a n d rise in anti-
Isolation Positive Rise intiter
0
2--4x
Negative 4--6x
6x
0
2--4x
4--6x
6x
8
Day 0 Antibody
titers
Ranges
<25
7a
1
26--50
51--75 76--100 101--125 126--150 151--175
1
--
--
--
20
2
--
--
--
6
1
--
1
--. ---
--. ---
4 2 . -1
2 --
---
---
1 --
---
---
.
Weeks 5--8 Antibody titers <25
Ranges
--
--
1
18
2
--
2
26--50
II
1
1
--
--
3
--
--
4
51--75
1
--
--
--
4
1
--
--
76--100
--
1
--
1
--
--
101--125
--
--
1
--
--
--
--
--
126--150
.
.
151--175
.
.
176--200
--
201--225
--
--
1
1
.
.
.
.
.
.
1
.
.
aNo. of calves within range of antibody titer and corresponding rise in titer.
TABLE
IV
Comparison of antibody titer ranges with isolation of ond sampling a of Group B calves
Pasteurella haemolytica
P. haemolytica
Antibody titer range
<25 26 or greater 26--100 > 101
aWeeks 5--8.
Isolated (N = 19)
Not isolated (N = 36)
12 (63.2%) 7 (36.8%) 4 (21.1%)
22 (61.1%) 14 (38.8%) 13 ( 3 6 . 1 % )
3 (15.8%)
1
(2.8%)
a t t h e sec-
608
There did n o t appear to be an association among antibody response to viruses, frequency of isolation of P. haemolytica, and antibody response to
P. haemolytica. DISCUSSION
The results of this study corroborate the findings of Magwood et al. (1969) in t h a t P. haemolytica was isolated from the upper respiratory tract in low frequency from pastured calves t h a t were n o t subjected to severe stresses such as overcrowding, long shipments, or exposure to cattle from other locations. In the present study, P. haemolytica was isolated even less frequently from the trachea than from the nasal cavity. Within the Group B calves, there was a significantly greater frequency of isolation of P. haemolytica at the second sampling as compared to the first sampling. This increase may have been due to transmission among calves or merely to expression of the organism in calves from which it was n o t isolated initially. Periodic dominance of the bovine nasal flora by P. haemolytica for several days at a time has been reported (Pass and Thomson, 1971). It was suggested t h a t Pasteurella spp. could be isolated most readily during periods when the organisms were actively colonizing the mucosa. Therefore, variations in the expression of the organism may be a more reasonable explanation for increased isolation than that of transmission among calves because P. haemolytica isolations occurred at the second sampling in groups where the organism was n o t isolated at the first sampling. Transmission from experimentally-infected (Group A) calves seems unlikely because of precautions taken to prevent direct contact of Group B with Group A once they were experimentally exposed to the organism. On Day 0, 64% of the 164 cattle had antibody titers of 25 or less as determined by the FIAX test. Based on our studies of serum antibody response after aerosol exposure to P. haemolytica, a titer of 25 or less would be a low titer and probably indicate minimal exposure (Confer et al., 1983). On the other hand, titers were nearly always at least 50 in animals that had received two aerosol exposures to live P. haemolytica. Equivalent titers in the calves in the present studies would most likely indicate a substantial exposure to the organism. In other experiments, we have f o u n d that most calves with titers less than 25 are susceptible to experimental challenge while those with titers of greater than 100 are mostly resistant (Panciera et al., 1983). During the observation period, the geometric mean antibody titer failed to rise significantly for Group B calves. The frequency of animals with titers of 25 or less decreased insignificantly. Therefore, examination of titers for the calves as a whole did n o t indicate active infection with P. haemolytica. Antibody titer increases of up to six-fold in sixteen calves, however, suggested that active infection a n d / o r transmission occurred to a limited extent among the calves.
609
Isolation of P. haemolytica from the upper respiratory tract did not correlate with serum antibody response to the organism. One possible explanation for this is that colonization of the organism in the upper respiratory tract m a y n o t induce a significant serum antibody response because P. haemolytica normally inhabits the surface of the nasal epithelium only {Pass and Thomson, 1971). Instead, ulceration o f the upper respiratory tract due to trauma or viral infection m a y be necessary to allow organisms to invade the lamina propria, be phagocytized and carried to regional lymph nodes and thus stimulate a serum antibody response. Another mechanism could relate to the extent of lung exposure to organisms via inhalation from the nasal cavity (Grey and Thomson, 1971). We have demonstrated significant serum antibody responses to P. haemolytica following aerosol exposure to the organisms (Confer et al., 1983). In clinically normal cattle, however, p u l m ~ nary exposure to the organism and subsequent serum antibody response may be minimal. In conclusion, the prevalence of P. haemolytica serotype 1 in the nasal cavity and trachea as detected by cultures obtained from live animals was low in beef calves from a closed herd that were maintained on range pastures: Serum antibody levels were also low. With time, there appeared to be an increase in the frequency of isolation from healthy calves that were moved to pens, held in low concentrations, and maintained under low stress conditions. There was no correlation between isolation of P. haemolytica from the upper respiratory tract at t w o samplings and any increase in serum antibody titer. Interestingly, P. haemolytica was n o t isolated from the 9 calves that experienced a decline in serum antibody titers. A n t i b o d y responses to respiratory viruses did n o t appear to affect isolations of P. haemolytica or serum antibody titers to the bacterium. ACKNOWLEDGEMENTS
The authors thank Carolyn Gresham for excellent technical assistance.
REFERENCES Bailey, N.T.J., 1981. Statistical Methods in Biology, 2nd edn., Wiley, New York, pp. 43-66. Carter, G.R., 1967. Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Adv. Vet. Sci., 11: 321--379. Confer, A.W., Fox, J.C., Newman, P.R., Lawson, G.W. and Corstvet, R.E., 1983. A quantitative fluorometric assay for the measurements o f antibody to Pasteurella haemolytica in cattle. Can. J. Comp. Med., 47: 37--42. Corstvet, R.E., Panciera, R.J., Rinker, H.B., Starks, B.L. and Howard, C., 1973. Survey of tracheas of feedlot cattle for Haemophilus somnes and other selected bacteria. J. Am. Vet. ivied. Assoc., 163: 870--873. Grey, C.L. and Thomson, R.G., 1971. Pasteurella haemolytiea in the tracheal air of calves. Can. J. Comp. Med., 35: 121--128.
610 Hamdy, A.H. and Trapp, A.L., 1967. Investigation of nasal microflora of feedlot calves before and after weaning. Am. J. Vet. Res., 28: 1019--1025. Horlein, A.B., Saxena, S.P. and Mansfield, M.E., 1961. Studies of shipping fever of cattle. II. Prevalence of Pasteurella species in nasal secretions from normal calves and calves with shipping fever. Am. J. Vet. Res., 22: 470--472. Magwood, S.E., Barnum, D.A. and Thomson, R.G., 1969. Nasal bacterial flora of calves in healthy and in pneumonia-prone herds. Can. J. Comp. Med., 33: 237--243. Newman, P.R., Corstvet, R.E. and Panciera, R.J., 1982. Distribution of Pasteurella haemolytica and Pasteurella multocida in the bovine lung following vaccination and challenge exposure as an indicator of lung resistance. Am. J. Vet. Res., 43: 417--422. Panciera, R.J., Corstvet, R.E., Confer, A.W. and Gresham, C.N., 1983. Bovine pneumonic pasteurellosis: Effect of vaccination with live Pasteurella species. Am. J. Vet. Res., submitted. Pass, D.A. and Thomson, R.G., 1971. Wide distribution of Pasteurella haemoly tica type 1 over the nasal mucosa of cattle. Can. J. Comp. Med., 35: 181--186. Rehmtulla, A.J. and Thomson, R.G., 1981. A review of the lesions of shipping fever of cattle. Can. Vet. J., 22: 1--8. Rice, C.E., Beauregard, M. and Maybee, T.K., 1955. Survey of shipping fever in Canada: Serological studies. Can. J. Comp. Med., 19: 329--349. Thomson, R.G., Benson, M.L. and Savan, M., 1969. Pneumonic pasteurellosis of cattle: Microbiology and immunology. Can. J. Comp. Med., 33 : 194--206. Thomson, R.G., Chander, S., Savan, M. and Fox, M.L., 1975. Investigation of factors of probable significance in the pathogenesis of pneumonic pasteurellosis in cattle. Can. J. Comp. Med., 39: 194--207. Wessman, G.E. and Hilkes, G., 1968. Characterization ofPasteurella haemolytica isolated from the respiratory tract of cattle. Can. J. Comp. Med., 32: 498--504. Wray, C. and Thompson, D.A., 1973. An epidemiological study ofPasteureUa haemolytica in calves. Br. Vet. J., 129: 116--123.