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 Julian W. Allen, Laurent Viel, Kenneth G. Bateman, Eva Nagy, Soren R0sendal and Patricia E. Shewen

ABSTRACT Acute and convalescent serum samples were taken from 59 calves with signs of respiratory disease (cases) and 60 clinically normal animals (controls) during their first month in the feedlot. Sera were analyzed for antibodies to bovine parainfluenza 3 (P13) virus by hemagglutination inhibition, to bovine viral diarrhea (BVD) virus, bovine respiratory syncytial (BRS) virus and bovine herpesvirus 1 (BHV1) by virus neutralization, and to Pasteurella haemolytica by indirect agglutination (PhIA) and cytotoxin neutralization (PhCN) tests. There was minimal evidence of serological activity to BHV1. Serological activity to the other agents occurred commonly and the prevalence of acute titers and their mean values was similar in case and control groups. Mean convalescent P13 and P. haemolytica (PhIA) titers were higher in controls than cases (p < 0.01) but, otherwise, convalescent titers did not differ between groups. The incidence of seroconversion was similar in both groups for all agents except for P13 virus which was more frequent in controls than cases (p < 0.0001). There was a positive association between PhIA and CN seroconversion and isolation of P. haemolytica from bronchoalveolar lavage (BAL) fluid (p < 0.1). The measure of agreement (kappa) between seroconversion with the P. haemolytica PhIA and PhCN tests was 0.51. Bacteriological and cytological evaluations of the respiratory tract were made using

BAL. No associations were evident between serological titers and pulmonary cytology. A multivariate logistic analysis was used to evaluate associations between disease status and serological, bacteriological and cytological data. Cases were positively associated with the presence of neutrophils and Pasteurella multocida in BAL fluid and negatively associated with P13 virus and PhIA seroconversion.

RESUME

tica (PhIA) etaient plus eleves chez les animaux temoins que chez les sujets ayant e't malades; dans les autres cas, ils furent similaires dans les deux groupes. L'incidence de seroconversion etait semblable dans les deux groupes pour tous les agents sauf contre le virus PI3 qui fut rencontre plus frequemment chez les temoins que chez les malades (P < 0,001). On observa une association positive entre le test de seroconversion a Pasteurella haemolytica, le test de neutralisation a la cytotoxine et l'isolement de P. haemolytica a partir des residus de lavages bronchoalveolaires (P < 0,1). La mesure du rapport des tests de seroconversion entre le P. haemolytica (PhIA) et le test de neutralisation de la cytotoxine (PhCN) fut de 0,51. Les evaluations bacteriologiques et cytologiques du tractus respiratoire furent faites en utilisant le liquide bronchoalveolaire (LBA). Aucune association evidente ne fut observee entre les titres serologiques et la cytologie pulmonaire. Une analyse multivariable fut utilisee pour evaluer les liens entre l'etat de la maladie et les donnees bacteriologiques et cytologiques. Les cas ciniques etaient positivement relies aIla presence de neutrophiles et de Pasteurella multocida dans le liquide bronchoalveolaire (LBA) et negativement associes avec le virus P13 et la seroconversion a Pasteurella haemolytica (PhIA). (Traduit par Andre C&cyre)

Des echantillons paires de serum furent preleves sur 59 veaux presentant des signes de maladie respiratoire ainsi que sur 60 animaux cliniquement sains (temoins) au cours de leur premier mois en parquet d'engraissement. Les serums etaient vrifiNs pour determiner les titres seriques contre le virus parainfluenza 3 bovin (P13) par la methode d'inhibition de l'hemagglutination, contre le virus de la diarrhee bovine (BVD), le virus syncytial bovin (BRS) et l'herpes I bovin par la methode de neutralisation du virus et contre Pasteurella haemolytica par la methode d'agglutination indirecte (PHIA) et par les tests de neutralisation de la cytotoxine (PhCN). II sembla n'y avoir que tres peu d'activite serologique contre le virus herpes I (BHVI). Une activite serologique a l'egard des autres agents fut constatee couramment et la prevalence de titres aigus ainsi que leurs valeurs moyennes etaient similaires tant chez les animaux INTRODUCTION malades que chez les sujets temoins. Les titres moyens des deuxiemes serums Several microbiological agents have en ce qui concerne P13 et P. haemoly- been implicated in the etiology of

Department of Clinical Studies (Allen, Viel), Department of Population Medicine (Bateman), Department of Veterinary Microbiology and Immunology (Nagy, Rosendal, Shewen), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. The work was supported by the Ontario Cattlemen's Association and the Ontario Ministry of Agriculture and Food. Submitted October 9, 1991.

Can J Vet Res 1992; 56: 281-288

281

respiratory disease in feedlot calves (1,2). For many of these, however, the causative associations with this complex syndrome are poorly understood. Recently, in an attempt to identify the important organisms involved, serum antibody titers to a number of putative respiratory pathogens were contrasted in groups of sick and healthy (with respect to respiratory disease) feedlot calves (3). Serological evidence of infection with potential pathogens was found to occur commonly, and although associations were evident between some of these agents and disease status, it was also apparent that many of the infected calves remained healthy. In this project, as well as using serological methods to study respiratory disease, nasopharyngeal swabs and bronchoalveolar lavage were used to perform bacteriological and cytological evaluations of the airways in feedlot calves. It was hoped that by performing this detailed examination of the respiratory tract it would be possible to further define the role of a variety of microorganisms in this disease syndrome. The bacteriological and cytological results have been reported elsewhere (4,5). This paper presents the serological findings for five putative respiratory pathogens together with a description and evaluation of their associations with the bacteriological and cytological data. A multivariable logistic analysis of data from all parts of the study is also described.

MATERIALS AND METHODS EXPERIMENTAL ANIMALS

A group of six to eight month old steer calves (n = 136) were observed for 28 days after their arrival at a feedlot. Animals showing clinical signs of respiratory disease were assigned to a group of cases. These were matched, at the same time, with apparently normal calves which formed a control group. The calves, their management, and the criteria used for selecting cases and controls are described elsewhere

(4). SAMPLING PROCEDURES

Calves were sampled on the same day that they were assigned to their 282

respective case and control groups, and before any treatment was given. Samples were taken from the upper and lower respiratory tracts using nasopharyngeal swabs (NPS) and bronchoalveolar lavage (BAL) respectively (4). These were cultured for bacteria and mycoplasmas (4). Bronchoalveolar lavage fluid was also evaluated cytologically and differential cell counts were performed (5). Calves were bled at the same time that the NPS and BAL were performed (acute sample), and again 12 days later (convalescent sample). Blood was collected into sterile vacutainers (Becton Dickinson, Mississauga, Ontario) by jugular venipuncture. Sera were separated and stored at -20°C until required for testing. SEROLOGICAL PROCEDURES

Acute and convalescent serum samples were analyzed blindly, i.e. the laboratory did not know the identity of the cases or controls, nor which two sera constituted a pair. The virus neutralization test was used to determine antibodies to bovine herpesvirus 1 (BHVl) (i.e. infectious bovine rhinotracheitis), bovine viral diarrhea (BVD) virus and bovine respiratory syncytial (BRS) virus in a microtiter system. The hemagglutination inhibition test was used for antibodies to bovine parainfluenza 3 (P13) virus according to standard protocol. The immune response to Pasteurella haemolytica biotype A, serotype 1 surface antigens was evaluated with the indirect (antiglobulin) microagglutination (PhIA) test using washed formalinized P. haemolytica Al as the antigen (6). Cytotoxin neutralizing activity (PhCN) was determined in a microplate colorimetric assay as the ability of serial twofold dilutions of serum to neutralize the toxic effect of P. haemolytica type 1 culture supernatant for BL-3 cells, the latter being a bovine leukemia B cell line (7). Toxicity was determined by the inability of cells to incorporate the vital dye, neutral red. Cell viability, measured as uptake of neutral red, was determined by reading the optical density of each well using a spectrophotometer. For all tests the titer of each sample was expressed as the highest dilution which yielded at least 50% agglutination or neutralization.

STATISTICAL PROCEDURES

The serological titers for all agents are reported as coded values 0, 1, 2, 3, ... for endpoint titers of 0, 1/2, 1/4,

1/8, etc. These codes correspond to the reciprocal log2 of the dilution factor

and thus calculation of mean titers using these values yields the geometric mean titer (GMT) for each group. Differences between acute and convalescent GMTs for case and control groups were evaluated using a two sample Student's t-test (8). The level at which a serological titer to a particular agent becomes biologically significant in terms of affording some protection from disease is difficult to determine. Therefore, in order to evaluate whether or not the acute serum titers to the various agents differed between case and control groups, several cutpoints were used to discriminate between biologically "significant" and "nonsignificant" acute titers. The coding used to describe the cutpoint titers is the same as that used for the endpoint titers, i.e. 0, 1, 2, 3 correspond to 0, 1/2, 1/4, 1/8, etc. All associations examined between disease status, acute titers, seroconversion and airway cultures were evaluated in 2 x 2 tables using odds ratio and chi-square techniques (9). Individuals were determined to have seroconverted if they showed at least a fourfold increase in titer (or a twofold increase in reciprocal log2 titers). Agreement between the different serological tests for P. haemolytica was measured using the kappa statistic (9). A multiple linear regression analysis was performed to evaluate possible associations between the observed BAL differential macrophage and neutrophil counts (5) and seroconversion with any of the agents evaluated in this study (8). Data was initially evaluated and found to be normally distributed using the Wilk-Shapiro test (Fig. 1). A multivariate logistic regression analysis was used to evaluate the associations between classification as a case or control and various independent variables thought to be possible predictors of respiratory disease (8). These variables included: positive cultures of P. haemolytica (BALPh), Pasteurella multocida (BALPm), Mycoplasma bovis (BALMb), Mycoplasma bovirhinis (BALMbr) or Haemophilus somnus (BALHs) from (BAL) fluid

30

RESULTS

25 Q) >

20 u C)

1 C5

za 1 0 0

5

0-20

21 -40

41 -60

61-80 81- 100

BAL Fluid Differential Macrophage Counts

(%)

30

25 (1) 0

0

20 15

I2)

CD

10

ID

z

5 0

0-20

21-40

41-60

61-80 81-100

BAL Fluid Differential Neutrophil Counts (%) Fig. 1. Differential alveolar macrophage and neutrophil counts in bronchoalveolar lavage (BAL) fluid in sick (case) and clinically healthy (control) feedlot calves. Cases (n = 59) solid bars Controls (n = 60) open bars -

-

(4); evidence of seroconversion (SC) to P13 virus, BVD virus, BRS virus or P. haemolytica [indirect agglutination (PhIA) and cytotoxin neutralizing (PhCN) tests]; and BAL differential neutrophil (BALNEUT) and macro-

phase (BALMAC) count data (5). Statistical analyses were performed using Statistical Analysis Systems (SAS Institute Inc., Cary, North Carolina) and Statistix (Analytical Software, St Paul, Minnesota).

The results of culturing NPS and BAL samples for bacteria and mycoplasmas, and the cytological findings from BAL fluid are presented elsewhere (4,5). There was evidence of serological activity to all the agents evaluated except BHVl, for which titers were only recorded in four animals (two cases and two controls). In all instances the BHV1 titers were low (mean coded value = 2), and there was no evidence of seroconversion. The mean acute and convalescent serum titers for the other agents are shown in Table I. The only significant differences found between cases and controls were for convalescent PI3 virus titers (p < 0.0001 and convalescent PhIA titers (p c 0.01) which in both instances were higher in the control group. The prevalence of "significant" acute titers to each organism varied depending on the cutpoint used, but in general the prevalence of acute titers in cases and controls remained similar at different cutpoints (Table II). For some organisms, (PI3 virus and BRS virus) there was a tendency at higher cutpoints for acute titers to be more prevalent among controls, however, in no instance were the differences

significant. The association between seroconversion to different agents and case or control status (expressed as odds ratios) is shown in Table III. Odds ratios greater than one reflect an increased risk of disease, odds ratios less than one imply a sparing effect of exposure. Seroconversion to P13 virus had a sparing effect with regard to respiratory disease (p c 0.05), but the incidence of seroconversion to the remaining agents was similar in cases and controls. An evaluation was made of the number of different agents to which each calf had seroconverted and a comparison was made between the case and control groups (Table IV). No significant difference was found between the groups. The associations between isolation of P. haemolytica from the respiratory tract and PhIA and PhCN seroconversion are shown in Table V. Seroconversion with PhIA titers was positively associated with NPS (p c 0.06) and 283

BAL (p < 0.06) isolations. Seroconversion with PhCN titers was also positively associated with NPS (p < 0.06) and BAL (p < 0.07 isolations. The measure of agreement (kappa) between seroconversion with the P. haemolytica PhIA and PhCN tests was 0.51 (Table VI). Kappas of less than 0.4, 0.4 to 0.6, and greater than 0.6 are considered to represent poor, moderate, and good agreement respectively (9). A multiple linear regression analysis was performed to evaluate possible associations between differential macrophage or neutrophil counts in BAL fluid and evidence of seroconversion to any of the agents tested. No significant associations were observed. Tables VII and VIII describe the results of a multivariable logistic analysis of the data to evaluate associations between respiratory disease and variables potentially predictive of respiratory disease. With the exception of BALNEUT and BALMAC, none of the independent variables were found to be strongly correlated with each other. Variables entered the model in a forward stepwise manner; entry into the model was allowed at the p c 0.1 level. All variables except BALNEUT and BALMAC were permitted to enter the first model (Table VII). Only BALPm, P13 SC and PhIA SC remained in this model (note that PhIA SC was only significant at the p = 0.09 level), and their final coefficients were 0.92, - 0.77 and - 0.73, respectively. Cases were positively associated with BALPm (Odds ratio = 2.5) and negatively associated with P13 SC and PhIA SC (Odds ratios 0.46 and 0.48 respectively). Based on the distribution of BALPm, P13 SC and PhIA SC and their associations with respiratory disease, the adjusted population attributable fractions for the final model were calculated (10). These were 41 o for BALPm and 52% and 26% for the absence of P13 SC and PhIA SC, respectively. When BALNEUT was allowed to enter the model it replaced BALPm as the variable most strongly associated with disease and PhIA SC became insignificant. Parainfluenza 3 SC, however, remained more or less unchanged (Table VIII). When BALMAC (but not BALNEUT) was allowed to enter, it did not remain in the final model. Bronchoalveolar-

284

TABLE I. Mean acute and convalescent serum titers of respiratory pathogens in feedlot calves

Antigen P13

Acute Convalescent BVD Acute Convalescent Acute BRS Convalescent PhIA Acute Convalescent PhCN Acute Convalescent Significant differences between cases and controls: ap c 0.001

Reciprocal log2 of mean titer (SEM) Cases Controls 3.47 (0.26) 3.03 (0.24) 6.47 (0.31)a 4.73 (0.26)a 1.81 (0.36) 2.01 (0.40) 4.85 (0.43) 4.93 (0.47) 3.68 (0.21) 3.87 (0.26 8.70 (0.26) 8.02 (0.26) 10.27 (0.19) 10.46 (0.18) 11.76 (0.16)b 11.05 (0.17)b 4.26 (0.30) 4.37 (0.25) 5.91 (0.19) 6.08 (0.28)

bp < 0.01

TABLE H. The assodation between cutpoints and acute titers in cases (n = 59) and controls (n = 60). Odds ratios comparing cases and controls

4 5 6

Odds ratiob 0.59 0.59 1.27 0.46 0.28 0.32

% cases/controls with acute titer 77/85 77/85 51/45 11/21 3/10 2/6

Chisquarec 1.59 1.59 0.50 3.01 2.96 1.17

1 2 3 4 5 6

1.17 1.00 1.00 1.00 0.91 1.23

44/41 28/28

23/23 18/18 12/13 12/10

0.16 0.00 0.00 0.00 0.05 0.05

BRS

1 2 3 4 5 6

1.50 0.86 1.20 0.53 0.49 0.34

96/93 83/85 62/58 27/41 11/20 5/13

0.38 0.03 0.18 3.76 2.44 2.99

PhIA

7 8 9 10 11 12 13

1.52 0.42 0.69 0.73 0.59 1.43 1.00

98/97 89/95 69/76 46/53 12/18 7/5 3/3

0.00 1.69 0.90 0.68 0.97 0.08 0.00

Agent P13

BVD

Cut pointa 1 2 3

PhCN

1 2.02 89/80 2.44 2 1.79 84/76 2.22 3 0.97 72/73 0.00 4 0.84 54/58 0.21 5 0.64 27/40 1.66 6 1.22 12/10 0.05 7 1.51 3/2 0.00 aAny individual having a titer greater than or equal to the cutpoint had an acute titer to that agent b If odds ratio > 1, more cases had titers than controls; if odds ratio < 1, more controls had titers than cases cp < 0.05 if x2 : 3.84

lavage macrophage and BALNEUT were not allowed to enter the same model because of the strong negative correlation between these two variables (r = - 0.0987). In order to detect the

presence of interaction, two way cross product terms were created involving all the variables in each of the final models. None of the interaction terms were significant.

TABLE III. The assoation between seroconversion to sdected respirtory pathogens and respiratory disease in feedlot calves. Odds ratios comparing cases (n = 59) with controls (n =60)

Odds Organism ratio PI3 0.42 BHV1 BVD 0.96 BRS 1.67 PhIA 0.46 PhCN 0.78 aAny fourfold or greater increase in titer bp < 0.05 if chi-square . 3.84

Seroconversiona 01 cases/controls 44/65 0/0 51/51 88/82

Chisquareb 5.26 -

0.01 0.97 3.62 0.43

23/40 42/48

TABLE V. Association between P. haemolyica isolation from NPS and BAL and PhIA and PhCN seroconversion Yes

No

TABLE IV. The frequency with which feedlot calves seroconverted to different numbers of P. haemolytica (PhIA and PhCN tests), P3-, BVD-, BRS- or BHV1-viruses

Number of Cases Controls seroconversionsa (n = 59) (n = 60) 0 2 1 1 10 8 2 14 13 3 13 14 4 13 13 5 7 11 aAny fourfold or greater increase in titer No significant difference between cases and controls

cent PhIA titer was significantly higher

Total

in the controls. Seroconversion with PhIA and PhCN titers occurred more p = 0.06 frequently in controls than cases but the differences were not significant. PhCN Yes 17 37 54 Odds ratio 2.25 Thomson et al (16) found that sick animals had lower PhIA titers on seroconversion No 11 54 65 p = 0.06 arrival and greater titer increases over Total 91 28 119 the first seven days than well animals. P. haemolytica cultured from BAL Frank and Smith found no difference PhIA Yes 8 30 38 Odds ratio 2.81 in either arrival PhIA titers or PhIA seroconversion No 7 74 81 p = 0.05 titer increases between calves that were Total 15 104 119 or were not treated for respiratory PhCN Yes 44 10 54 Odds ratio 2.73 disease (12). Bateman found no difseroconversion No 5 60 65 p = 0.07 ference in arrival PhIA titers but at Total 104 15 119 28 days postarrival PhIA titers were higher in treated calves than controls TABLE VI. Level of agreement between PhIA and PhCN seroconversion (13). The PhCN titers were higher in controls at arrival but by day 28 they PhCN were higher in treated animals. It seroconversion should be pointed out that these difYes No Total ferences were small and not statistically PhIA Yes 32 6 38 significant (p c 0.05). Martin et al (3), seroconversion No 22 59 81 in a three year study involving 569 calves Total 54 65 119 in 15 groups, showed that higher PhIA Level of agreement between PhCN and PhIA seroconversion: kappa = 0.51, x2 = 31.7 titers at arrival and PhCN seroconversion were significantly associated with In agreement with previous work treatment for respiratory disease. DISCUSSION (3, 11-13) there was evidence of wide- Shewen and Wilkie found that PhIA The design of the serological portion spread serological reactivity to P. hae- and PhCN titers of cattle dying of of this project is similar to that used molytica in the calves at initial sam- fibrinous pneumonia in the field were in a recent seroepidemiological study pling. This is likely to represent recent lower than those of cattle dying of of feedlot respiratory disease by or ongoing exposure since, although other causes (6,17). They proposed a Martin et al (3). In contrast to that calves frequently show titers to P. hae- protective role for PhIA and PhCN work, which used samples from multi- molytica at the farm of origin prior to titers in fibrinous pneumonia induced ple groups of calves over three years, weaning and transportation (14, 15), by P. haemolytica which was later supour data were obtained from one these titers usually increase by the time ported experimentally (18). group of animals. The results are of arrival at the feedlot (12). The relaIt becomes obvious that there are therefore less likely to be representative tionship between P. haemolytica titers conflicting data regarding the associof the general population. Neverthe- and health status has been evaluated ation between sickness and P. haemoless, the results were interesting and on several occasions. In this study, lytica titers in field studies of feedlot valuable, especially when compared to there was no significant difference respiratory disease. A possible reason those from previous work, as they between the mean acute PhIA and is that variations may exist in the time demonstrate the inconsistency found in PhCN titers or the mean convalescent at which acute and convalescent sera serological data sets from calves with PhCN titers in the case and control were collected in relation to the depargroups. However, the mean convales- ture of the calves from the home ranch respiratory disease. P. haemolytica cultured from NPS PhIA Yes No seroconversion Total

13 15 28

25 66 91

38 81 119

Odds ratio 2.28

285

and their arrival at the feedlot. As an example, animals taking several days to travel from ranch to feedlot could be exposed to P. haemolytica and already have experienced titer rises by the time the acute sample is taken at the feedlot. For calves travelling directly to the feedlot, however, there may not be time for this to occur. Another important consideration lies in the definition of sickness. In the studies of Shewen and Wilkie, sick animals were defined as those dying of fibrinous pneumonia (6,17). In our study, and others, however, the nature of the respiratory disease was undifferentiated and a sick animal was one that was treated because it was febrile and possibly depressed, anorexic or showing abnormal respiratory signs (3,12,13). Under theses circumstances, which are more typical of those encountered in the field, P. haemolytica titers and titer changes did not always follow the same pattern. One reason for this may be that only a proportion of P. haemolytica infections result in clinical disease (4) and that fibrinous pneumonia is not the only condition which causes clinical signs of respiratory disease in feedlot calves (5). It is interesting that Thomson et al (16), using an elevated fibrinogen as one of the criteria to define sick calves, found similar PhIA titer changes and associations with sickness as Shewen and Wilkie (7). It is possible that, by using this definition of sickness, calves with a bacterial pneumonia were more likely to be included in the sick category than calves with other conditions (e.g. upper respiratory viral infections), which in the feedlot would also be classified as respiratory disease. A positive correlation between seroconversion to P. haemolytica and isolation of this organism from the respiratory tract has been documented previously by Bateman (13), but not by others (12,14). The fact that some calves seroconverted but did not yield positive isolations shows either that the sampling techniques were not sensitive enough to detect the organism in the airways because only a few were present, or that the period when the animal was infected did not coincide with the timing of the NPS and BAL. In contrast, some calves showed positive isolations but did not seroconvert. This may have been because these 286

TABLE VII. Forward stepwise logistic regression analysis of classification as a case or control on bacteriological and serological variables in feedlot calves

Step Variables added number to model 0 1 BALPm 2 P13 SC PhIA SC 3 Regression coefficients and standard errors of variables in the

Variables available in order of significance, p c 0.1 BALPm, P13 SC, PhIA SC P13 SC, PhIA SC PhIA SC final model

Standard Odds p error ratio Variable Coefficient value 0.115 0.394 Constant 1.12 0.77 BALPm 0.921 0.393 2.51 0.02 P13 SC -0.768 0.392 0.46 0.05 PhIA SC -0.728 0.424 0.48 0.09 Adjusted population attributable fraction for BALPm = 41 %7o, for P13 SC = - 52/o and for PhIA SC = - 26%

TABLE VIII. Forward stepwise logistic regression analysis of classification as a case or control on bacteriological, cytological and serological variables in feedlot calves

Step number 0

Variables added to model

Variables available in order of significance, p c 0.1 BALNEUT BALPm, P13 SC, PhIA SC P13 SC, BALPm BALPm

1 BALNEUT 2 P13 SC 3 BALPm Regression coefficients and standard errors of variables in the final model Variable Constant BALNEUT P13 SC BALPm

Coefficient 0.450 0.014 -0.849 0.674

calves had already been exposed and shown a rise in titer before the NPS and BAL were performed or because there was insufficient stimulus by the organism to elicit seroconversion. It is not known whether nasal colonization alone will cause a systemic immune response to P. haemolytica or whether the organism must be present in the lungs for this to occur. Poor agreement between the PhIA and PhCN tests has been reported previously (3,13). This may be partly explained by the fact that the PhIA test is specific for P. haemolytica serotype Al (6) whereas the cytotoxin is produced by all serotypes of P. haemolytica. The PhCN test is therefore not serotype specific (19). The higher incidence of seroconversion with PhCN than PhIA titers may indicate that serotype Al was not the only one active in these calves. The data also show that calves can seroconvert to the PhIA test but not the PhCN test. In other words, animals may mount an

Standard error 0.400 0.006 0.398 0.412

Odds ratio 0.64 1.02 0.43 1.96

p value 0.26 0.03 0.03 0.10

immune response to bacterial surface antigens but not the cytotoxin. The toxin is produced primarily during the log phase of growth. Therefore, if multiplication of the organism is prevented by the respiratory defence mechanisms, exposure to the toxin may be limited. There is evidence that all of the viruses evaluated, except BHV1, were active in this group of calves. Only four calves showed detectable BHVI activity and in all instances the titers were low, indicating that the virus did not contribute significantly to the occurrence of disease. These titers may represent colostral antibodies rather than exposure to the virus through natural infection or vaccination. The low prevalence of BHVI activity is consistent with observations from previous work (3). Calves seroconverted frequently to BRS and BVD viruses, however, the frequency with which this occurred was similar in cases and controls.

Serological activity to these agents has been demonstrated previously in feedlot calves (15,20) and both, on occasion, have been found to be significantly associated with treatment for respiratory disease (3,21). Seroconversion to PI3 virus occurred more frequently in controls and, although not significant, control calves tended to have higher acute PI3 virus titers than cases. In previous work a significant protective effect of arrival titers to PI3 virus has been demonstrated (3,11). However, it was also found that seroconversion to PI3 virus was more frequent in cases, which is contradictory to the data from this study. In other work, no association was observed between PI3 virus titers and disease (16). Thus, although this virus has been shown experimentally to cause pathological changes in the respiratory tract and to act synergistically

with P. haemolytica to cause respiratory disease (22), it appears that in the field, serological evidence for the role of PI3 virus in respiratory disease is not consistent. It is possible that the contribution of this and other agents to the causation of disease may vary in different groups of calves. Changes in the BAL differential cell count, indicative of a pulmonary inflammatory response (5), were not associated with seroconversion to any of the organisms evaluated in this study, despite work which suggests

that both P. haemolytica and some viruses will affect the cellular constituents of BAL fluid. Experimental infection of calves with BRS virus (23)

and aerosol challenge with P. haemoly-

tica (24) both cause a rapid increase in the percentage of neutrophils in BAL fluid. Part of the early histological response to P13 virus is an accumulation of neutrophils and mononuclear cells in alveoli and bronchioles (25). Bovine viral diarrhea virus, however, does not alter the BAL differential cell count (26). Likely explanations for the lack of association between serological and cytological changes include the possibility that some infections could have been subclinical and confined to the upper respiratory tract. While this may result in seroconversion, BAL cytology would be unaffected. Another is that the viral infection and rise in antibody titer may have occurred subsequent to the HAL.

The multivariable logistic regression models bring together bacteriological, cytological and serological data and evaluate their associations with case/ control status. In the model of bacteriological and serological variables (Table VII), only the presence of P. multocida in BAL fluid was harmful and it accounted for 41 % of the occurrence of respiratory disease. This association was evident in the analysis of the bacteriological data and the implications of this finding have been discussed (4). Seroconversion to PI3 virus and PhIA were sparing, but it is difficult to explain why exposure to these agents should decrease the risk of respiratory disease. When information on the BAL fluid neutrophil differential count was allowed to enter the model (Table VIII), it was the variable most strongly associated with disease status. In addition, the strength of the association with BALPm was decreased, indicating that part of the effects of BALPm were likely mediated through BALNEUT. That BALNEUT appears to be acting in part as an intervening variable makes biological sense as one would expect that exposure of the lung to an infectious agent might cause an increase in neutrophil numbers which would subsequently result in clinical signs of disease. The fact that BALPm remains significant when BALNEUT is included in the model indicates that P. multocida exerts effects on disease status in addition to those caused by its effect on the neutrophil differential cell count. One of the most notable features of this study was the limited extent to which it was possible to differentiate between cases and controls, based on serological or bacteriological evidence of their exposure to various organisms. Indeed, of all the agents evaluated, only P. multocida was found to be associated with an increased risk of respiratory disease, and almost 600/ of disease occurrence was unexplained by an analysis of the data. Since the objective of the study was to evaluate the etiological role of various potential pathogens in feedlot respiratory disease, it is useful to examine why this was so. First, there are likely to have been factors determining disease status which were not assessed. These could include the presence of infectious agents for which no tests were per-

formed, and environmental or management factors which were not possible to measure. Second, if one assumes that in fact many of the organisms evaluated are important etiologically, it is necessary to look at the sampling methods or the study design for possible explanations. There are certain limitations to the bacteriological and serological methods that were used. Those relating to the techniques used to sample the respiratory tract for culture have been discussed previously (4). Some of the limitations of serological titers are mentioned in this discussion. Others, including those concerning the sensitivity and specificity of serological tests have been discussed by Pritchard et al (27) and Martin and Bohac (28). Useful information regarding the design of the study was provided by BAL fluid cytology. Cytological abnormalities indicative of pulmonary inflammation were prevalent in both cases and controls, and, likewise, there were calves in both groups which were cytologically normal (5). This indicates that the clinical criteria used to select cases and controls failed to discriminate between calves with and without some degree of lower respiratory tract disease. The presence of subclinical disease would help explain the high prevalence of infections observed in the control calves and hence, their similarity with the cases. This would contribute to the paucity of associations observed between potential pathogens and clinical disease. The clinical signs and criteria used to define cases were chosen as being similar to those often used in feedlots to select calves for treatment of respiratory disease (4). In this way it was hoped to study cases typical of those in the field. Unfortunately, some of these clinical signs are not necessarily referable to the respiratory system, and, with the exception of rectal temperature, they can be subjective and open to variable interpretation. This leads to a rather loose definition of respiratory disease which in turn has compromised the ability of this study, and others like it, to investigate its cause. Future work of this nature should take these factors into consideration and use more stringent methods to determine the presence of disease.

287

ACKNOWLEDGMENTS The authors wish to thank Allen McBurney and the staff at the Elora Beef Research Centre for their cooperation, Ivan Linjacki for technical assistance, the Clinical Microbiology Laboratory of the Ontario Veterinary College, Debbie Bateman and Sheila Watson for performing bacteriological cultures, and Eva Varady and Heather Edwards for performing serological tests. REFERENCES 1. FRANK GH. Bacteria as etiologic agents. In: Loan RW, ed. Symposium on Bovine Respiratory Disease. Texas A&M University Press, 1984: 347-362. 2. ROSENQUIST BD. Viruses as etiologic agents. In: Loan RW, ed. Symposium on Bovine Respiratory Disease. Texas A&M University Press, 1984: 364-375. 3. MARTIN SW, BATEMAN KG, SHEWEN PE, ROSENDAL S, BOHAC JE. The frequency, distribution and effects of antibodies, to seven putative respiratory pathogens, on respiratory disease and weight gain in feedlot calves in Ontario. Can J Vet Res 1989; 53: 355-362. 4. ALLEN JW, VIEL L, BATEMAN KG, ROSENDAL S, SHEWEN PE, PHYSICKSHEARD P. The microbial flora of the respiratory tract in feedlot calves: Associations between nasopharyngeal and bronchoalveolar lavage cultures. Can J Vet Res 1991; 55: 341-346. 5. ALLEN JW, VIEL L, BATEMAN KG, ROSENDAL S, SHEWEN PE. Cytological findings in bronchoalveolar lavage fluid from feedlot calves: Associations with pulmonary microbial flora. Can J Vet Res 1992; 56: 122-126. 6. SHEWEN PE, WlKXlE BN. Antibody titers to Pasteurella haemolytica Al in Ontario beef cattle. Can J Comp Med 1982; 46:

354-356.

288

7. GREER CN, SHEWEN PE. Automated colorimetric assay for the detection of Pasteurella haemolytica leukotoxin. Vet Microbiol 1986; 12: 33-42. 8. MATTHEWS DE, FAREWELL VT. Using and Understanding Medical Statistics. 2nd ed. Basel: Karger, 1988. 9. MARTIN SW, MEEK AH, WILLEBERG P. Veterinary Epidemiology. Ames: Iowa State University Press, 1987. 10. BRUZZI P, GREEN SB, BYAR DP, BRINTON LA, SCHAIRER C. Estimating the population attributable risk for multiple risk factors using case-control data. Am J Epidemiol 1985; 122: 904-914. 11. THOMSON RG, BENSON ML, SAVAN M. Pneumonic pasteurellosis of cattle: Microbiology and immunology. Can J Comp Med 1969; 33: 194-206. 12. FRANK GH, SMITH PC. Prevalence of Pasteurella haemolytica in transported calves. Am J Vet Res 1983; 44: 981-985. 13. BATEMAN KG. Undifferentiated bovine respiratory disease: an evaluation of antimicrobial therapy. MSc thesis, University of Guelph, 1988. 14. CONFER AW, CORSTVEr RE, PANCIERA RJ, RUMMAGE JA. Isolation of Pasteurella haemolytica and correlation with serum antibody response in clinically normal beef calves. Vet Microbiol 1983; 8: 601-610. 15. YATES WDG, KINGSCOTE BF, BRADLEY JA, MITCHELL D. The relationship of serology and nasal microbiology to pulmonary lesions in feedlot cattle. Can J Comp Med 1983; 47: 375-378. 16. THOMSON RG, CHANDLER S, SAVAN M, FOX ML. Investigation of factors of probable significance in the pathogenesis of pneumonic pasteurellosis in cattle. Can J Comp Med 1975; 39: 194-207. 17. SHEWEN PE, WILKIE BN. Pasteurella haemolytica cytotoxin neutralizing activity in sera from Ontario beef cattle. Can J Comp Med 1983; 47: 497498. 18. CONFER AW, PANCIERA RJ, FULTON RW. Effect of prior natural exposure to Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis. Am J Vet Res 1984; 45: 2622-2624. 19. SHEWEN PE, WILKIE BN. Pasteurella haemolytica cytotoxin: Production by

recognised serotypes and neutralization by type specific rabbit antisera. Am J Vet Res 1983; 44: 715-719. 20. GILLETTE KG, SMITH PC. Respiratory syncytial virus infection in transported calves. Am J Vet Res 1985; 46: 2596-2600. 21. REGGIARDO C. Role of BVD virus in shipping fever of feedlot cattle. Case studies and diagnostic considerations. Proc 22nd Am Assoc Vet Lab Diagnost 1979: 315-320. 22. JERICHO KWF, DARCEL CleQ, LANGFORD EV. Respiratory disease in calves produced with aerosols of parainfluenza-3 virus and Pasteurella haemolytica. Can J Comp Med 1982; 46: 293-301. 23. KIMMAN TG, ZIMNER GM, STRANER PJ, DE LEEUW PW. Diagnosis of bovine respiratory syncytial virus infections improved by virus detection in lung lavage samples. Am J Vet Res 1986; 47: 143-147. 24. WALKER RD, HOPKINS FM, SCHULTZ TW, McCRACKEN MD, MOORE RN. Changes in leukocyte populations in pulmonary lavage fluids of calves after inhalation of Pasteurella haemolytica. Am J Vet Res 1985; 46: 2429-2433. 25. BRYSON DG, McNULTY MS, BALL HJ. The experimental production of pneumonia in calves by intranasal inoculation of parainfluenza type 3 virus. Vet Rec 1979; 105: 566-573. 26. LOPEZ A, MAXIE MG, RUHNKE L, SAVAN M, THOMSON RG. Cellular inflammatory response in the lungs of calves exposed to bovine viral diarrhea virus. Mycoplasma bovis, and Pasteurella haemolytica. Am J Vet Res 1986; 47: 1283-1286. 27. PRITCHARD DG, EDWARD S, MORZARIA SP, ANDREWS AH, PEiIERS AR, GILMORE NJR. Case control studies in the evaluation of serological data from respiratory disease outbreaks in cattle. Proc Soc Vet Epidemiol Prev Med 1983. 28. MARTIN SW, BOHAC JG. The association between serological titers in infectious bovine rhinotracheitis virus, bovine virus diarrhea virus, parainfluenza-3 virus, respiratory syncytial virus and treatment for respiratory disease in Ontario feedlot calves. Can J Vet Res 1986; 50: 351-358.