arrival in the Alberta feedlot and. 82% of the calves had an antibody titer on arrival at the Ontario feed- lots; titers in calves in Alberta were almost twice as high ...
The Association of Titers to Bovine Coronavirus with Treatment for Bovine Respiratory Disease and Weight Gain in Feedlot Calves S.W. Martin, E. Nagy, P.E. Shewen, and R.J. Harland
ABSTRACT The association between bovine respiratory disease (BRD) and antibody titers to bovine coronavirus (BCV) was studied in 604 calves (19 different groups in 4 different feedlots from 2 provinces). Almost all calves had antibody titers on arrival in the Alberta feedlot and 82% of the calves had an antibody titer on arrival at the Ontario feedlots; titers in calves in Alberta were almost twice as high as those in calves in Ontario. The incidence of infection, in the first mo after arrival as judged by seroconversion, ranged from 61% to 100%; titer increases were much greater in calves in Ontario feedlots. Titer variables were not significantly related to BRD, except on a withingroup basis (group was a confounding variable for BCV-BRD associations). Given control of group effects, calves with an antibody titer on arrival appeared to be protected against BRD for the first 28 d in the feedlot, and the association was reasonably linear over the range of titers. Each titer unit on arrival decreased the risk of BRD by about 0.8x (odds ratio). Titer change was not strongly related to the risk of BRD and the relationship was not linear over the range of titer changes. Titer change was strongly and negatively correlated with titer on arrival, and titer change was not significantly related to BRD in the presence of arrival titers. Arrival titer retained its relationship with BRD in the presence of titer data for other putative pathogens. Each higher unit of titer to BCV on arrival increased the 28-day weight
gain (controlling for group, initial weight and the occurrence of BRD) by slightly more than 1 kg. Titer change was associated with decreased weight gain, when initial titer was not in the model. The lack of a linear or multivariable association between BCV titer change and BRD, and weight gain, may indicate that BCV is not a major pathogen; or, its lack of significance may merely be due to its strong correlation with arrival titer. Given the associations found in this study, particularly the interprovincial differences in arrival titers, more and different approaches to studying the possible effects of BCV on BRD are in order.
RESUME L'association entre la presence de maladie respiratoire bovine (MRB) et les titres d'anticorps envers le coronavirus bovin (CVB) a ete e'tudiee chez 604 veaux repartis en 19 groupes dans 4 parcs d'engraissement situes dans 2 provinces. Presque tous les animaux avaient des titres d'anticorps lors de leur arrivee dans les parcs d'engraissement en Alberta alors que le pourcentage etait de 82 % pour les animaux arrivant dans les parcs d'engraissment en Ontario; de plus, les titres d'anticorps des animaux en Alberta etaient presque le double de ceux en Ontario. L'incidence d'infection, tel qu'evalue par la seroconversion, variait de 61 % a 100 % durant le premier mois apres l'arrivee; I'augmentation des titres etaient beaucoup plus marquee chez les veaux dans les parcs d'engrais-
sement en Ontario. Les variables associees aux titres n'etaient pas significativement reliees aux MRB,
l'exception de l'effet intra-groupe (le groupe etant une variable confondante pour l'association CVBMRB). En controlant les effets de groupe, les veaux ayant 'a leur arrivee un titre d'anticorps sema
blaient proteges contre les MRB pour les 28 premiers j en parc d'engraissement, et cette association etait sensiblement lineaire sur toute l'etendue des titres. Chaque unite de titre a l'arrivee diminuait le risque de contracter une MRB par un facteur de 0,8 (odds ratio). Le changement de titre n'etait pas fortement relie au risque d'avoir une MRB et la relation n'etait pas lineaire sur l'etendue des changements de titre. Le changement de titre etait fortement correle negativement avec le titre 'a l'arrivee, et le changement de titre n'etait pas relie significativement 'a une MRB compte tenu des titres a l'arrivee. Le titre a l'arrivee a conserve sa relation avec une MRB en presence des donnees des titres envers des pathogenes reconnus. Chaque unite de titre plus eleve envers le CVB a l'arrivee se traduisait (en contr6lant pour le groupe, le poids initial et l'occurence de MRB) par une augmentation d'un peu plus de 1 kg en gain de poids au 28e jour apres l'arrivee. Lorsque le titre initial n'etait pas incorpore au modele, un changement de titre etait associe avec une diminution de gain de poids. Le manque d'association line'aire ou multivarie'e entre le changement de titre envers le CVB et une MRB, de meme qu'avec le gain de poids pourrait indiquer que
Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1 (Martin, Nagy, Shewen); Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan (Harland). Corresponding Author: Dr. Wayne Martin. Received March 10, 1997.
Can J Vet Res 1998; 62: 257-261
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le CVB n'est pas un pathogene majeur; ou que l'absence de signification serait causee par la forte correlation avec le titre a l'arrivee. Etant donnee les associations mises en evidence dans cette etude, plus particuli'erement les differences interprovinciales des titres a l'arrivee, des etudes supplementaires et differentes des effets possibles du CVB sur les MRB sont necessaires. (Traduit par docteur Serge Messier)
lots, and 9 groups were housed at a private cattle research facility. All of the calves in the Alberta feedlot were "western" calves; 2 calf groups (33 and 44) in Ontario were from western Canada sources, all others were calves from Ontario. Calves were weighed and bled on arrival and again approximately 28 d after arrival. Further details about calf processing and detection of BRD are available elsewhere (12). SEROLOGICAL METHODS
INTRODUCTION Bovine coronavirus (BCV) has been incriminated in cases of bovine respiratory disease (BRD) in Ontario, based on seroconversion in groups of cattle from which cadavers and/or sera had been submitted to a diagnostic laboratory (1). Seventy-two of 143 groups of cattle with diarrhea and/or respiratory disease had one or more animals with seroconversions. The numbers of animals per group averaged approximately 7, but were not stated exactly. The frequency of BCV seroconversions in "normal" cattle groups was not known; however the frequency of cattle with BCV seroconversions in diarrheic cattle groups (16 of 30) was similar to that in groups with respiratory disease (43 of 84). More recently, BCV has been implicated as a cause of BRD in U.S. feedlot cattle (2). The current study was conducted to estimate the prevalence of infection on arrival, and the incidence thereafter with BCV, and its relationship with BRD and weight gain in feedlot calves. Titers to other putative pathogens were also utilized to prevent confounding by these organisms.
MATERIALS AND METHODS CALF GROUPS AND DISEASE DEFINITION
Data were available from a total of 604 calves, aged 6 to 9 mo; 206 treated for BRD, and 398 calves not treated for BRD. Two hundred and forty-nine calves in 8 groups (Groups 60 to 67) were fed in an Alberta feedlot and 355 calves in 1 1 groups (Groups 11, 12, 15 to 21, 33, and 44) were fed in 3 feedlots in Ontario. Two of the groups in Ontario were housed at research station feed258
Serum samples were analysed for antibody titer on arrival (BCV-0) and at Day 28 (BCV-28) using the BCV virus-neutralization test adapted from Carman and Hazlett (1992). Serum samples also were analysed for titers to Mycoplasma bovis (Mbov) and M. dispar (Mdis), in addition to 4 other viruses: infectious bovine rhinotracheitis virus (IBRV), bovine virus diarrhea virus (BVDV), parainfluenza virus type 3 (PIV-3), and bovine respiratory syncytial virus (BRSV), according to previously documented methods (4). H. somnus (HS) titers were determined at the Veterinary Infectious Disease Organization using methods for the outer membrane protein (5). Serum samples were analysed for indirect agglutination titer (PhA), and leukotoxin neutralizing titer (PhL) to Pasteurella haemolytica (6). In the report by Carman and Hazlett (1992), seroconversion was deemed to have occurred if the sera went from negative to positive, or had a 4-fold or greater titer change in paired sera taken 2 to 3 wk apart. Here we pursue the formal analyses based on the actual titers on arrival (BCV-0) and titer difference between Day 28 and Day 0 (BCV-D), but also report on the percentage of calves with titers on arrival (BCV-POS), and the percentage that seroconverted (had a 4-fold or greater titer increase) within 28 d of arrival (BCV-SC). STATISTICAL METHODS
Statistical analyses were conducted at the calf level, with pneumonia occurrence within the first 28 d after arrival as the outcome. Initial models contained the BCV titer data on arrival, and/or the titer change data, and the location (province) of the feedlot. Interaction terms between
arrival titer and titer change and between these titers and feedlot site were evaluated for significance. Thereafter, because these models did not fit the data well and our concern over residual confounding within province, the provincial location of the feedlot was removed. Eighteen dummy variables, each denoting one group of calves, were forced into the logistic model; group 67 was the referent. Modelling was repeated using either or both of BCV titer data on arrival, and/or the titer change data. The titer and titer change variables were replaced by hierarchical dummies (called coded variables) denoting the 25th, 50th and 75th percentile cutpoints of each variable in order to identify points of significant association with the outcome and to assess the linearity of titer and BRD risk relationships controlling for group (7). Subsequently, models were constructed containing the titer change to each of the other putative causal organisms, one at a time, in addition to the BCV titer data. A similar approach to modelling the relationship of BCV titers with weight gain was conducted using multiple least squares regression. Statistical significance was set at P < 0.05, unless otherwise indicated.
RESULTS BCV PREVALENCE ON ARRIVAL
Calves in the Alberta feedlot had quite high antibody titers on arrival. The average titers in the 110 calves that later developed BRD (x = 7.5) differed (P = 0.05) from titers in the 139 calves not treated for BRD (x = 8.2) (Table I). Calves in Ontario feedlots had lower titers on arrival; titers in the 96 calves that were later treated for BRD were lower (x = 3.5) but did not differ from the titers in the 259 untreated calves (x = 4.0). In both instances, the trend was for treated calves to have lower titers on arrival; however, based on a chi-square test for trend, as well as visual inspection, the relationship was not linear. One of the calf groups (group 33) which originated from western Canada had the highest titers on arrival in the Ontario feedlots, but the other group originating in western Canada had only moderate titers. Overall, 500 calves (83%
prevalence) had BCV antibody titers > 1 on arrival, but having a BCV antibody titer on arrival was not related to subsequent BRD treatment. BCV TITER CHANGES
The average antibody titers, in both Alberta and Ontario calves, increased during the 1st mo postarrival. Antibody titers in calves from Alberta which were treated for BRD did not increase (x = 1.8) more than titers in calves which were not treated (x = 1.4). Titers in calves from Ontario which were treated for BRD did not increase (x = 4.9) more so than titers in non-treated calves (x = 4.3). The trend was for treated calves to have a greater titer increase than untreated calves, but this was not statistically significant. The titer on arrival was strongly and negatively correlated with subsequent titer increase (R = -0.84). Overall, 370 calves (61%) had a seroconversion to BCV (titer increase of 4-fold or greater; 23 animals had a 4-fold titer decrease) but neither seroconversion nor a 4-fold titer decrease was related, significantly, to BRD treatment. All animals seronegative on arrival seroconverted in the first mo after arrival at the feedlot. MULTIVARIABLE MODELS
In a multivariable model of the association of titer with risk of treatment for BRD, BCV-0 (b = -0.06) was significantly related to risk of BRD with PROVINCE in the model, and BCV-D was also significant (b = 0.05, P = 0.07) with PROVINCE in the model. Neither variable achieved significance when both were in the model. The interaction between arrival titer and titer change was not significant in the logistic model, nor was the interaction with feedlot site. When hierarchical dummies were entered into the logistic model, for the 25th (titer = 2), 50th (titer = 6), and 75th (titer = 8) percentiles of titer on arrival, the risk of BRD decreased most dramatically for calves with titers greater than 2 (b = -0.60). When hierarchical dummies were entered for the 25th (titer change = 0), 50th (titer change = 3), and 75th (titer change = 6) percentiles of titer change, the risk of BRD decreased for calves with titers between 3 and 6 (b = -0.53) and increased for calves
TABLE I. Antibody titers of calves to BCV on arrival and at 28 days post arrival in calves treated and not treated for BRD
Pneumonia status Location n Mean titera BCV titer on arrival (BCV-0) Not treated 398 5.50 Alberta 139 8.24 Ontario 259 4.03 Treated 206 5.62 Alberta 110 7.49 Ontario 96 3.47 Overall 604 5.54 BCV titer 28 d after arrival (BCV-28) Not treated 398 8.77 Alberta 139 9.61 Ontario 259 8.31 Treated 206 8.83 Alberta 110 9.26 Ontario 96 8.32 Overall 604 8.79 a Reciprocal log2 of mean antibody titer. The endpoint titer was the reciprocal dilution at complete inhibition of virus replication occurred in 50% of the wells
with titer changes greater than 6 (b = 0.49, P = 0.053). This coded titer change was not significant in a model containing the coded levels of arrival titer. When PROVINCE was removed and the group dummy identifiers added, BCV titer change, by itself, became significant (b = 0.10), and BCV-0, by itself, was significantly (b = -0.13) related to BRD risk. When both were in the model, only BCV-0 was significantly related (b = -0.16) to BRD risk. When the coded values for these two variables were entered, none were significant. When only coded values for arrival titer were entered, calves with titers equal to or higher than 2 had a significantly decreased (b = -0.6) risk of BRD. The coefficients for the 3 quartiles were -0.60, -0.13, and -0.40, respectively, indicating decreased risks (odds ratios) of between 0.55 and 0.88 at each of these levels. When only coded titers for titer change were entered, calves with changes equal to or great than 6 units had a significantly increased (b = 0.53; P = 0.06) BRD risk. The coefficients for the three quartiles were 0.39, -0.04, and 0.53, respectively. When titer variables for a 2nd organism were added to the model, the coefficient for BCV-0 was reasonably stable at about -0.16 which translates to an odds ratio of 0.84 for each unit titer increase on arrival (Table II). Increasing titers to BRSV, BVDV and PhL were significantly related to a higher BRD risk, whereas HS titer increases were significantly
SD
3.78 2.94 3.33 3.84 3.17 3.40 3.79 2.10 1.56 2.21 2.05 1.87 2.14 2.08 which
TABLE II. Logistic coefficients for the association of BCV titer on arrival with BRD adjusting for the association of arrival weight and titer change (D) to a 2nd putative pathogen Second BCV-0 Second Pathogen Pathogen Coefficient Coefficient None -0.15 BRSV-D -0.20 0.17 BVDV-D -0.17 0.16 IBRV-D -0.16 0.06 NS PIV-3D -0.16 0.05 NS Mbov-D -0.08 NS 0.02 NS Mdis-D -0.07 NS 0.07 NS PhA-D -0.15 0.04 NS PhL-D -0.12 0.18 Hs-D -0.17 -0.19 Coefficients for BCV-D varied from -0.01 to -0.07, but none were significant at P < 0.10 NS = Not significant at P < 0.05
associated with a decreased risk of BRD. WEIGHT DATA
The weight of calves on arrival and at 28 d, by feedlot location, and BRD status are shown in Table III. Calves in Alberta weighed more on arrival and gained more in the 1st 28 d than
calves in Ontario. Overall, the weight data were normally distributed, although there were large differences in weight gain by province. The average weight on arrival and weight gain in the first 28 d after arrival were: 278.5 kg and 26.9 kg; 216.0 kg and 6.14 kg in Alberta and Ontario feedlots, respectively. In a manual stepwise regression model, with weight gain as an outcome, and controlling for the effects of GROUP, a higher titer on arrival of one unit was related 259
TABLE III. Weight (kg) tion and BRD status Feedlot Location Alberta Ontario Overall Alberta Ontario
on
arrival and
BRD Status Positive
Negative Positive Negative Positive Negative
28-day weight gains, for feedlot calves by feedlot loca- these results. For reasons perhaps not related to BCV infection cattle in the Alberta feedlots had more BRD than
N 249 355 206 398 110 139 96 259
WT-0 (± SD) 277.39 ± 20.28 215.96 ± 34.76 263.80 ± 31.94 258.27 ± 38.30 277.58 ± 19.98 277.24 ± 20.58 215.92 ± 30.92 215.93 ± 36.12
WT-D (± SD) 26.84 ± 12.69 6.74 ± 15.60 12.31 ± 21.93 16.40 ± 18.42 22.23 ± 11.57 30.48 ± 12.40 0.82 ± 20.81 8.09 ± 12.68
calves in Ontario feedlots, thus distorting the interpretation of group
level responses. This was a major reason for relying on individual level analyses in this study, and also for . . controlling for tthe titers to other putative pathogens. Given that the association between arrival titer and BRD
persisted in the face of titer data to these other putative pathogens inferences about the possible causai role of BCV in BRD are strengthened. With respect to weight gain, higher antibody titers appeared to be slightly protective and promoted higher weight gains, even after removing the impact of BRD on weight gain. If we accept that higher titers are protective against BRD, and that BRD is likely to result in at least a short term weight gain decrease, the association is readily explainable. Even in the absence of clinical illness the same "protective" factors could be operating. Clearly, further research is needed on BCV and its association with BRD. occurrence
increased weight gain (0.99 kg). model controlling for arrival weight, higher titers on arrival were associated with increased weight gains (b = 1.08 kg), higher weight on arrival was associated with a slight decrease in weight gain (0.06 kg) and developing BRD was associated with a decrease of 7.49 kg weight gain. Change in titer to BCV was not related to weight gain in this model. If only titer change and not arrival titers were in the model, increased titers were associated with decreased to an
In
a
weight gains (-0.79 kg). DISCUSSION
Serologic evidence of active BCV
infection, as inferred from seroconversions, had been found in one or more cattle in a relatively large number of herds with respiratory disease. Therefore, it was logical to postulate that it had a role in causing BRD. Certainly, the agent appears to be widespread and animals respond to it immunologically during periods of respiratory disease. The previous
report on coronavirus infection in Ontario was based on samples submitted to diagnostic laboratories and it was not possible to ascertain how common BCV might be in normal cattle. This prevented the authors from making valid statistical associations and causal inferences about the role of BCV in respiratory disease (1) Based on serologic data from a study of beef cow-calf herds in Quebec, it appears that the BCV is very widespread there also (8). This is consistent with the high prevalence of isolations from nasal swab samples from feedlot cattle with signs of respiratory distress in one Kansas feedlot (32 of 50 calves) and one Arizona feedlot (6 of 50 calves) (2). Infer260
ences about were based on
causality in this study the frequency of isolation of BCV in cattle with respiratory
distress as no control cattle were sampled. Ganaba et al (1995) noted that calves 3-4 wk of age which developed pneumonia had lower BCV titers after the pneumonic event than those
developing pneumonia (as diagby the farmer). In the current study, there were no significant differences in Day 28 titers, before controlling for group, and a tendency for not
nosed
lower titers (P = 0.08) in calves treated for BRD, similar to the findings by Ganaba et al (1995). Other studies on BCV have been conducted in small numbers of young dairy calves up to 5 mo of age. These studies have indicated that BCV is a common and persistent inhabitant of the respiratory tract, that infected calves respond serologically to the infection, and that most of the infections are not related to respiratory disease episodes (9,10). In a related experimental exposure of 5 colostrumdeprived calves, no calves became ill after challenge (11). In the present study, it appears that most calves entering feedlots have been exposed to BCV prior to arrival, and that active infection (inference based on increasing titers) was occurring in the first 4 wk after arrival. The association between arrival titer and the risk of BRD in individual animals suggests that prior infection with BCV is protective against BRD. Therefore, we infer that there is evidence to link BCV to BRD in a causal sense. At the group level, there were associations between higher titers on arrival and increased risk of BRD, and between lower seroconversion rates and increased risk of BRD. However, given the findings at the individual calf level, it would seem that other confounding factors were distorting
ACKNOWLEDGMENTS This work was supported by a research grant from the Ontario Ministry of Agriculture, Food and Rural Affairs. The help of staff at the Elora Beef Cattle Research Station, Advanced Agricultural Testing Systems, and Animal Research International, Cattleland Feedyards Ltd. is gratefully acknowledged. The authors also thank Steve Duns, Tay Hwang and Victoria Edge for their excellent technical assistance.
REFERENCES 1. CARMAN PS, HAZLETT MJ. Bovine coronavirus infection in Ontario 1990-1991. Can Vet J 1992; 33: 812-814. 2. STORZ J, STINE L, LIEM A, ANDERSON GA. Coronavirus isolation from nasal swab samples in cattle with signs of respiratory tract disease after shipping. J Am Vet Med Assoc 1996; 208(9): 1452-1455.
3. MARTIN SW, HARLAND RJ, BATEMAN KG, NAGY E. The Association of Titers to Haemophilus somnus, and Other Putative Pathogens, with the Occurrence of Bovine Respiratory Disease and Weight
Gain in Feedlot Calves. Can J Vet Res 1998; 62: 262-267. 4. ALLEN JW, VIEL L, BATEMEN KG, NAGY E, ROSENDAL S, SHEWEN PE. Serological titers to bovine herpesvirus 1, bovine viral diarrhea virus, parainfluenza 3 virus, bovine respiratory syncytial virus and Pasteurella haemolytica in feedlot calves with respiratory disease: associations with bacteriological and pulmonary cytological variables. Can J Vet Res 1992; 56: 281-188. 5. HARLAND RJ, POTTER AA, VAN-DEN HURK-VANDRUNEN-LITTEL S, VAN DONKERSGOED J, PARKER MD, ZAMB TJ, JANZEN ED. The effect of subunit or modified live bovine herpesvirus-1 vaccines on the efficacy of a recombinant Pasteurella haemolytica vac-
6.
7.
8.
9.
cine for the prevention of respiratory disease in feedlot calves. Can Vet J 1992; 33: 734-741. SHEWEN PE, WILKIE BN. Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica. Can J Vet Res 1988; 52: 30-36. WALTER SD, FEINSTEIN AR, WELLS CK. Coding ordinal independent variables in multiple regression analyses. Am J Epidemiol 1987; 125(2): 319-323. GANABA R, BELANGER D, DEA S, BIGRAS-POULIN M. A seroepidemiological study of the importance in cow-calf pairs of respiratory and enteric viruses in beef operations from northwestern Quebec. Can J Vet Res 1995; 59: 26-33. HECKERT RA, SAIF LJ, HOBLET KH, AGNES AG. A longitudinal study of
bovine coronavirus enteric and respiratory infections in dairy calves in two herds in Ohio. Vet Microbiol 1990; 22(2-3): 187-201. 10. HECKERT RA, SAIF LJ, MYERS GW, AGNES AG. Epidemiologic factors and isotype-specific antibody responses in serum and mucosal secretions of dairy calves with bovine coronavirus respiratory tract and enteric tract infections. Am J Vet Res 1991; 52(6): 845-851. 11. HECKERT RA, SAIF LJ, MENGEL JP, MYERS GW. Isotype-specific antibody responses to bovine coronavirus structural proteins in serum, feces, and mucosal secretions from experimentally challengeexposed colostrum-deprived calves. Am J Vet Res 1991; 52(5): 692-6999.
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