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Factors associated with decay of colostral antibodies to bovine leukemia virus
Preventive Veterinary Medicine, 9 ( 1 9 9 0 ) 4 5 - 5 8
45
Elsevier Science Publishers B.V., A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
Factors associated with decay of colostral antibodies to bovine leukemia virus M-L. Lassauzet ~, W.O. Johnson 2, M.C. T h u r m o n d ~* a n d J.P. P i c a n s o 1 Veterinary Medicine Teaching and Research Center, Department of Medicine, School of Veterinary Medicine, University of California, 18830 Road 112, Tulare, CA 93274 (U.S.A.) 2Division of Statistics, University of California, Davis, CA 95616 (U.S.A.) (Accepted for publication 29 September 1989 )
ABSTRACT Lassauzet, M-L., Johnson, W.O., Thurmond, M.C. and Picanso, J.P., 1990. Factors associated with decay of colostral antibodies to bovine leukemia virus. Prey. Vet. Med., 9: 45-58. One hundred and sixty-one calves that had received colostrum from bovine leukemia virus (BLV)infected cows were followed to evaluate factors associated with the decay of colostral antibodies to BLV. Three survival parametric models (Weibull, log-normal and log-logistic ) were fitted to data on the age at which non-infected calves converted from a seropositive to a seronegative status (retroconversion). For 97 non-infected calves, the fit of the Weibull model for the age at retroconversion was better than those of the log-logistic and log-normal models; values for 2. and a for the Weibull model were 0.014 and 1.59. Non-infected calves with high initial levels of BLV colostral antibodies became seronegative at an older age than calves with low initial levels ( P < 0.001 ). Dam BLV infection status was not associated with age at retroconversion ( P = 0 . 3 2 8 ) after adjusting for the initial level of colostral antibodies. The rate of antibody decay in most calves that seroconverted later because of active infection was within the range of that in non-infected calves. Six calves that did not exhibit any decay could have been identified as infected as early as 80 days of age. Results indicate that the use of initial BLV colostral antibody conceniration in models depicting decay of colostral antibodies will improve the detectability of early BLV calf hood infection. For initial colostral antibody concentration scored from 1 to 4 (1 lowest, 4 highest), no detectable antibodies were predicted in non-infected calves after 39, 67, 115 and 196 days of age for scores l, 2, 3 and 4 respectively.
INTRODUCTION
In a program aimed at control or eradication of bovine leukemia virus (BLV)-infected cattle, early detection of infected calves is difficult because colostral antibodies to BLV cannot be differentiated from antibodies which are the result of infection (Crespeau et al., 1986; Kono et al., 1983; Oshima *Author to w h o m c o r r e s p o n d e n c e should be addressed.
0167-5877/90/$03.50
© 1990 Elsevier Science Publishers B.V.
46
M-L. LASSAUZET ET AL.
et al., 1984; T h u r m o n d et al., 1982; Van Der Maaten et al., 1981 ). In an effort to estimate the time at which a seropositive calf could be considered infected, the decay of colostral antibodies in infected and non-infected calves has been studied (Kono et al., 1983; Oshima et al., 1984; T h u r m o n d et al., 1982). Generally, results suggest that infected calves can be identified between 5 and 7 months of age. It is not known, however, whether the age at which a calf can be expected to become seronegative is a function of the a m o u n t of BLV-antibodies absorbed a n d / o r of the infection status of the dam. Knowledge of the existence of such associations would improve our estimates of the expected age at which colostral antibodies would no longer be detectable. The use of improved estimates that permit more accurate, and possibly earlier, detection of BLV-infected calves would allow for more efficient control of BLV transmission through segregation of infected livestock. The objectives of the study were: ( 1 ) to determine whether an association existed between the decay of BLV colostral antibodies in non-infected calves and two factors namely, serum BLV antibody titer during the first week of life and d a m BLV-infection status; (2) to determine whether the rate of decay adjusted for the effects of the factors could identify infected calves before they were 6-7 months old. MATERIALS AND METHODS
Population studied Calves studied were born between January 1984 and June 1987 on a 210cow dairy located in the Central San Joaquin Valley of California and managed like a typical California feedlot dairy (Wiersma et al., 1983 ). Within 12 h after birth, calves were fed 2 1 of colostrum from their own d a m or administered commercial immunoglobulins per os (Genecol 99 R) and fed 1-2 1 of pooled colostrum. Female calves were retained in the herd to provide replacements and most bull calves were sold. Within 12 h of birth, calves were put into individual hutches where they were fed 2 I of pooled colostrum twice a day for 2 days, after which time they were fed twice daily 21 of a combination of bulk-tank milk and powdered milk at a ratio of 1 : 1. Calves were placed in group pens at about 90 days of age and were weaned at about 6 months of age.
Blood collection and examination Whole blood was collected by jugular venipuncture from calves usually within 1 week of age. Precolostral samples were taken from calves born during bi-weekly visits to the herd. Blood was collected from calves every 2-3 weeks until they left the hutch, after which time blood was collected every 3 months from the jugular or coccygeal vein. Cows were bled quarterly and when
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
47
examined by the herd veterinarian during weekly visits for reproduction monitoring. Serum was separated by centrifugation and stored at - 8 0 oC. Agar gel immunodiffusion ( A G I D ) was used to detect gp-51 antibodies (Leukassay-B kit, Pitman-Moore Inc., Washington Crossing, N J). The test procedure was slightly different from the protocol described by Miller and Van Der Maaten ( 1976 ) and Nakajima et al. ( 1982 ). The agar-gel was made with 1% agarose and 8.5% NaC1 using distilled water. Glass plates measuring 100 × 80 m m and containing 21 ml of agar were used. Wells were 4 m m in diameter and at a distance of 5 m m from each other. Antigen was placed in the central well, the reference positive serum in two opposing wells and sera to be tested in the four remaining wells. Plates were incubated 48 h at 25°C in a humified chamber before being viewed. A line of identity between antigen and antibody was interpreted as a positive test result. The concentration of antibodies present in the serum was approximated by categorizing from 1 (lowest concentration) to 4 (highest concentration) according to the shape of the precipitating line and its distance to the perimeter of the antigen well (National Veterinary Laboratories Services, Ames, IA).
Determination of infection status A cow was considered infected if AGID-positive before its pregnancy started or at the time the study began. For cows that seroconverted during pregnancy or after parturition, the most likely time of infection was estimated as previously described (Lassauzet et al., 1989). If that time was during pregnancy, then the cow was classified as infected. Cows that were AGID-negative and removed from the herd within 2 months post-partum were classified as noninfected. Determination of infection status for calves with colostral antibodies was more difficult because colostrum-derived antibodies cannot be differentiated from infection-induced antibodies. Moreover, if a calf is infected while having colostral antibodies, those antibodies might have to decay below a certain threshold before infection-induced antibodies can be produced. In one study, the time between the last colostrum-related positive test and the first infection-related positive test was 2 months, using the AGID test for a calf experimentally infected after birth (Van Der Maaten et al., 1981 ). Because that calf was bled monthly, it could have been seronegative at the most for 60 days. It was decided, therefore, to classify as 'non-infected' calves that became seronegative and were observed to remain so for at least 2 months. All other calves that is, those that were not seronegative for at least 2 months and those that could not be observed (but could have been) seronegative for at least 2 months - were referred to as possibly-infected (PI) calves. Among PI calves, five categories were created to help differentiate calves for which no decay was observed (category 1 ) from those for which a decay -
48 Z
M-L. LASSAUZET ET AL.
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Fig. 1. Patterns of decay of colostral antibodies used to identify non-infected calves and five categories of PI calves. was observed (categories 2 - 5 ) , calves that were not observed seronegative (categories 1-3 ) from those that became seronegative (categories 4 and 5 ), and calves that were very likely infected (categories 1, 2 and 4) from those that could have been classified as non-infected if bled at shorter intervals (categories 3 and 5 ) (Fig. 1 ). Such categories were made in order to evaluate whether patterns could be observed in calves that were probably infected while having colostral antibodies. Precise definitions of categories were: ( 1 ) calves that had a colostral antibody concentration (CAC) score of 4 during the first and subsequent weeks; (2) calves for which an antibody decay was observed but which were never observed seronegative, where the time between the last test with the lowest CAC and the next test was < 2 months; (3) calves for which a decay was observed but which were never observed seronegative, where the time between the last test with the lowest CAC and the next test was >/2 months; (4) calves that were observed to be seronegative for < 2 months; (5) calves that were observed to be seronegative, but the duration of seronegativity was unknown.
Estimation of decay of colostral antibodies Our general approach was to model ( 1 ) the age at which non-infected calves with colostral antibodies converted to a seronegative status (referred to as age at retroconversion) and (2) the duration of stay at each level of CAC for non-infected calves, using survival techniques. The decay of colostral antibodies for each PI calf was then compared with that for non-infected calves to determine if some PI calves could be identified as having been infected while they had colostral BLV antibodies. When modelling the age at retroconversion for non-infected calves receiv-
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
49
ing colostral antibodies, non-parametric probability functions were first calculated using methods previously described (Lassauzet et al., 1989; Turnbull, 1976). The covariates (CAC during the first week of life (CAC1) and d a m infection status) were examined using a parametric model to determine if they were associated with the age at retroconversion (Kalbfleisch and Prentice, 1980; Lassauzet et al., 1989).Three survival parametric models (Weibull, log-normal and log-logistic) were fitted to the ages at retroconversion of all non-infected calves and the best-fitting model was used to examine the effect of the above factors. The loss corresponding to a particular model with a particular set of factors is defined as loss = - 2 log (maximized likelihood function ). Smaller losses or bigger maximized log-likelihood functions correspond to better fitting models. Differences between appropriate losses constitute likelihood ratio statistics for testing that particular regression coefficients are zero. These were used to perform the model selection procedures. The usual z statistics were also calculated. A negative regression coefficient indicated that the factor level coded 0 would delay retroconversion relative to the factor coded 1. For continuous variables, a negative regression coefficient indicated that, as that variable increased, the probability of retroconversion decreased. Interpretation of a positive coefficient was the reverse. Computations were done using B M D P A R (Dixon, 1981 ). The 50% (medians), 95% and 99% quantiles of age at retroconversion were computed for each CAC 1. For the Weibull model, those quantiles are equal to exp { [ln( - l n c) - b X - a In 2 ] / a } , where c equals 0.50, 0.05 and 0.01, respectively, X is the vector of covariates found to be significantly associated with age at retroconversion, b the vector of regression coefficients of those covariates. The parameters 2 and a define the Weibull model (Kalbfleisch and Prentice, 1980) and are a function of the mean and the coefficient of variation of the age at retroconversion respectively. The middle value between the age at the last lowest CAC and the age at the first negative test for each PI calf was compared with the 95% quantile of the age at retroconversion for non-infected calves with the same CAC 1. If the middle value was above this quantile, the age at infection was estimated to be between birth and the age at the first negative test. The duration of stay at a given antibody concentration was estimated for non-infected calves that had received colostral antibodies. This was because antibody concentrations were estimated on an ordinal scale from 1 to 4, making the use of regression methods inappropriate. Non-parametric probability functions for the duration of stay (Tj, j = 1,2,3,4) at a given level of antibody concentration were estimated as described previously (Lassauzet et al., 1989; Turnbull, 1976). I f a was the age of a calf at its last test with a CAC of j + l, b was the age at its first test with a CAC of j, c was the age at its last test with a CAC o f j and d was the age at its first test with a CAC of j - l, then Tj was
50
M-L. LASSAUZET
E T A L.
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A G E IN D A Y S
Fig. 2. Schematic diagram illustrating notation used to describe non-parametric function for duration of stay at antibody concentration at l e v e l j ( ~ ) . The last test at level j + 1 was done at age a, the first test at l e v e l j was done at age b, the last test at l e v e l j was done at age c and the first test at level j - 1 was done at age d. Tj was estimated to be at least equal to (c-b) and not more than (d-a) days.
considered to be > c - b and < d - a (Fig. 2 ). For calves that had a CAC 1 o f j, a and b were set to 0. If c was equal to b, then c - b was chosen to be 7 because the duration o f stay at a given antibody concentration was assumed to be at least 1 week. For instance, if a calf with a CAC 1 o f 4 was tested at 20 ( a ) , 40 (b), 60 (c), and 80 (d) days o f age and if the test results were 4 ( j + 1 ), 3 (j), 3 (j), and 2 ( j - 1 ) respectively, then, the duration o f stay at level 3 ( T3 or Tj) would be at least 20 days ( 6 0 - 40 or c - b) and at the most 60 days ( 80 - 20 or d - a ). The duration o f stay at level 4 (which was the level o f the CACI ) would be at least 20 days ( 2 0 - 0 ) and at the most 40 days ( 4 0 - 0 ) . Finally, if the calf could not be tested at 60 days 7'3 would be, by convention, at least 7 days and at the most 60 days. Because each level represents a range o f continuous values, the duration o f stay at a given level was c o m p u t e d separately for calves with CAC I equal to that same given level and for calves with a greater CAC 1. For example, if a calf had a CAC 1 of 2, its initial antibody level measured on a continuous scale could be anywhere within the range o f values scored as a 2. When the CAC o f calves which had a CAC 1 o f 3 or 4 is scored as a 2 for the first time, it is more likely that the antibody level is in the upper range of values scored as a 2 than in the lower range. Consequently, it was anticipated that a calf with a CAC 1 o f 2 would have had a CAC o f 2 for a lesser time, on the average, than calves that had had a CAC 1 o f 3 or 4. The duration o f stay at levelj for all PI calves was then compared with that obtained for non-infected calves after adjusting for the level of its CAC 1. If a PI calf had a CAC at level j longer than the 95% quantile computed for non-
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
51
infected calves for the same CAC 1, the PI calf was designated as having been infected between birth and the age at the first test with CAC of j - 1 - - the upper bound of the duration of stay at level j. The examination of the duration of stay at a given CAC was done for all PI calves. However, the examination of the age at retroconversion could be done only for calves that were eventually observed seronegative. RESULTS
Between 1 January 1984 and 30 June 1987 colostral antibodies to BLV were detected in 161 calves of which 109 were classified as not infected, based on the criteria set out above. Among the remaining 52 calves, 30 were censored and 22 were classified as PI calves. Information on all the PI calves that belonged to categories 2-5 is presented in Table 1. Information on CAC 1 (levels 1,2, 3, 4) was available for 97 non-infected calves. The non-parametric probability distribution of the age at retroconversion revealed a multimodal distribution with most non-infected calves becoming seronegative within 130 days (Fig. 3 ). When non-parametric probability distributions were estimated for each level of CAC 1, distributions tended to be more homogeneous and concentrated (Fig. 4). Values of the maximized log-likelihood functions for the log-normal, loglogistic and Weibull models for the age at retroconversion without covariates were - 209.7, - 209.3 and - 197.3 respectively, indicating that the Weibull model fit was better than those of the other two models. Values for 2 and a for the Weibull model were 0.014 and 1.59 respectively. The Weibull model was used to examine the effect of d a m infection status and CAC 1 on the age at retroconversion. D a m infection status was not associated with the age at retroconversion after adjusting for CAC 1 ( P = 0 . 3 2 8 ) (Table 2 ). High CAC 1, however, was associated with a delay in retroconversion ( P < 0 . 0 0 1 ) (Table 2). Medians, 95% and 99% quantiles of the age at retroconversion, computed using the Weibull model for non-infected calves at each CAC 1 level, indicated that non-infected calves with a CAC 1 of 1, 2, 3 or 4 would be expected to be seronegative no later than 46, 79, 135 and 230 days of age respectively (Table 3 ). For PI calves that were observed at some time to be seronegative (categories 4 and 5 ), the age midway between the age at the last lowest CAC and the age at the first negative test was compared with the 95% quantile of the age at retroconversion for non-infected calves with the same CAC 1 to determine when PI calves could have become infected (Tables 1 and 3). For calf 369, as an example, the age midway between the age at the test with the last lowest CAC and the age at the first negative test was 51 days [ (30 + 72 ) / 2 ] (Table 1 ); this was less than 115 days which is the 95% quantile of the age at retroconversion for non-infected calves with a CAC1 of 3 (Table 3). Thus,
52
M-L.LASSAUZETETAL.
TABLE 1
Information relating to 16 calves (PI calves of categories 2, 3, 4 and 5 ) for which a negative test was not observed for at least 60 days following the last test with lowest colostral antibody concentration (CAC) Calf
Precolostral sample a
First CAC
Age at test (days) (CAC at test )
203
NA
4
369
NA
3
377
NA
3
388
NEG
3
393
NEG
2
397
NEG
4
398
NEG
1-2
428
NA
4
788
NA
4
795
NA
3
804
NEG b
4
841
NA
4
869
NA
4
106 (1) 30 (2) 57 (2) 71 (1) 33 (1) 117 (1) 78 (1) 69 (4) 138 (1) 98 (1) 71 (2) 62 (1) 83
184 (4) 72 (0) 78 (0) 119 (0) 53 (0) 200 (4) 103 (0) 135 (3) 233 (4) 192 (4) 103 (0) 141 (4) 97
112
128
(1)
(o)
(o)
(2)
78 (1) 70 (1) 36 (1)
92 (0) 84 (0) 51 (0)
108 (0) 115 (0) 67 (1)
124 (4) 130 (2)
871
NEG b
3
874
mEG
1-2
886
NA
3
147 (3) 155 (4) 188 (4) 67 (0)
170 (4)
181 (4) 205 (2)
212 (3)
181 (4)
aNA, not available; NEG, negative. bNon-infected dam.
this calf was not identified as having become seronegative at an older age than non-infected calves. In fact, the age at retroconversion o f PI calves from category 4 or 5 was comparable with that o f non-infected calves. Medians and 95% and 99% quantiles were estimated from non-parametric probability functions for the duration o f stay for non-infected calves at given
COLOSTRAL ANTIBODIES TO BOVINE L E U K E M I A VIRUS
53
0.02 o
o
=- 0.01 -
e~ o a_ 0-
14 28 42 56 70 84 98 112126 A g e in d a y s
Fig. 3. Non-parametric probability function for the age at which non-infected calves became seronegative after consuming colostrum with BLV antibodies.
0.06 0.05 0.04 0.03 0.02
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0.020.01014 28 42 56 70 84 98 112 126
Age in days
Fig. 4. Non-parametric probability functions for the age at which non-infected calves became seronegative, given their colostrum BLV antibody concentration during the first week of life (CAC1).
54
M-L.LASSAUZETETAL.
TABLE 2 Effects of dam BLV infection status and colostral BLV antibody concentration during the first week of life (CAC 1 ) on age at retroconversion for non-infected calves receiving colostral antibodies, as determined using a Weibull regression model Factors in model
Loss
None CAC1 Dam status CAC1 (adjusted for dam status) Dam status (adjusted for CAC1 )
357.98 262.93 350.81 262.00
fl a
z-value
P-value
- 1.397 -0.648 - 1.377 -0.240
-8.767 -2.440 -8.107 -0.979
<0.001 0.014 <0.001 0.328
aFactor's regression coefficient.
TABLE 3 Medians (50%) and 95% and 99% quantiles of age at retroconversion for non-infected calves receiving BLV colostral antibodies Colostral antibody concentration during first week of life
Quantiles 50%
95%
99%
Age (days) 1
2 3 4
22 38 65 111
39 67 115 196
46 79 135 230
TABLE 4 Medians (50%) and 95% and 99% quantiles of duration of stay with a colostral antibody concentration of 4, 3, 2 and 1 for non-infected calves that received BLV colostral antibodies Colostrai BLV antibody concentration during first week of life
Colostral antibody concentration 4
3
2
1
Quantiles of duration of stay (days) 4 3 2 1
a50%, 95% and 99% quantile.
23,68,75 a
27,41,42 12,47,49
30,48,52 30,48,52 20,56,64
26,50,55 26,50,55 26,50,55 15,49,55
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
0.06
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Fig. 5. Non-parametric probability function of duration of stay at a colostral antibody concentration level of 4 for non-infected calves.
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Fig. 6. Survival curves for age at which non-infected calves became seronegative after consuming colostrum with BLV antibodies in studies by Thurmond et al., 1982 ( ), by Crespeau et al., 1986 ( - - - - ) and in the present study ( . . . . ) for calves with colostral antibody concentration during the first week of life of 4 using the Weibull model. C A C levels 4, 3, 2 a n d 1 ( T a b l e 4 ) . T h e d u r a t i o n o f stay at a given C A C for each o f the PI calves ( d a t a n o t s h o w n ) was c o m p a r e d with the estimated quantiles for n o n - i n f e c t e d calves. For PI calves b e l o n g i n g to categories 2, 3, 4 a n d 5, a longer d u r a t i o n o f stay at a given level was n o t f o u n d except for calf 428. I f a calf w i t h a CAC1 o f 4 was n o t infected, it w o u l d h a v e h a d a C A C o f 2 by 117 days ( 7 5 + 4 2 ) ( T a b l e 4 ) . B e c a u s e c a l f 4 2 8 had a C A C o f 3 at 135
56
M-L. LASSAUZET ET AL.
days of age (Table 1 ), the age at infection for that calf was estimated to be between birth and 135 days of age. It could have been identified as infected prior to 196 days, which is the 95% quantile of the age at retroconversion for non-infected calves with a CAC1 of 4 (Table 3 ). Six PI calves had antibodies constantly detected at a level of 4 and, therefore, belonged to category 1. One of those calves was sampled precolostrally and was found to be positive. Because all non-infected calves with a CAC 1 of 4 had a CAC of less than 4 within 80 days of age (Fig. 5), PI calves from category 1 could be detected as infected with certainty between 2 and 3 months of age. The results of this and two other studies were compared visually using plots of survival curves for the age at which non-infected calves became seronegative (Fig. 6). DISCUSSION
This study compared the decay of BLV colostral antibodies of each possibly-infected calf with that of non-infected calves after adjusting for the effect of the initial a m o u n t of colostral antibodies. The fact that the age at retroconversion was associated significantly with antibody concentration during the first week of life indicates that identification of infected calves with colostral antibodies would be possible at an earlier age if serologic results were available for the first week of life. The upper 99% limits of the age at retroconversion for non-infected calves varied considerably (from 46 to 230 days). Because colostral concentration during the first week of life was associated with the age at retroconversion, it is likely that variations in CAC 1 would explain the variations in the age at retroconversion which have been observed in previous studies. In those studies, retroconversion occurred between 2 and 6 months of age (Oshima et al., 1984 ), within 5 months of age (Kono et al., 1983 ), between 122 and 238 days (Crespeau et al., 1986 ) and between 51 and 187 days after birth ( T h u r m o n d et al., 1982). Data presented in Fig. 6 suggest that in the latter two studies, the age at retroconversion was comparable with that observed in our study for calves with a CAC 1 of 4. In a previous report (Kono et al., 1983 ), the m e t h o d of examining serologic results from repeated bleedings was proposed as a means of identifying calves that were infected in utero. In that study all calves with persistent antibody titers were found to be infected in utero. By extending those results to the present study, the six PI calves belonging to the first category could be classified as infected in utero. If no precolostral samples were available, such infected calves could be detected with certainty at 3 months of age (Fig. 5 ). In the same report (Kono et al., 1983 ), some calves with a positive precolostral sample had postcolostral titers that decreased and then either increased
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
57
or remained negative. One possible explanation is that 'precolostral' samples for those calves were obtained after colostrum intake. Others have reported that, in field studies, such an error can occur (Crespeau et al., 1986; Thurm o n d et al., 1983). The rate of decay of colostral antibodies was not associated with the subsequent infection status of calves except for PI calves belonging to the first category. This result agrees with previous observations (Kono et al., 1983) and indicates that virus isolation or the detection of viral antigen might be the only means of identifying infected calves that received BLV colostral antibodies. It should be noted that some calves classified as PI had long intervals between bleedings, which might have biased results by failing to detect a seronegative status that lasted 2 months. In future studies, the bleeding scheme for each calf could be determined according to its CAC 1: narrow bleeding intervals at the time the calf is expected to become seronegative and then a bleeding 2 months after the first negative test. Because the a m o u n t of colostral antibodies was determined using an ordinal scale instead of a continuous scale, some errors in identifying the score levels using the AGID might have occurred. This error inherent in ordinal data could account for some outliers which made frequency distributions for each CAC 1 overlap (Fig. 4). For instance, some calves with a CAC 1 of 3 that became seronegative after 105 days might in fact have had a CAC 1 of 4, which would have explained the prolonged duration of colostral antibodies. If feasible, therefore, the measurement of antibody concentration using a continuous scale (e.g. ELISA tests) or at least an ordinal scale with more intervals (e.g. endpoint titers ) would increase the accuracy of predicting infection. In epidemiological studies it is c o m m o n that the exact time of an event is not known, only the time interval during which it occurred. In this study only the time interval during which the animal's serological status changed from positive to negative (or changed from levelj to level j - 1 ) was known. Interval censoring of the data, however, has not always been accounted for in other studies. Possible reasons why the non-parametric estimation of interval censored data has not been used elsewhere, were that the underlying theory was not easily understood by non-statisticians and that no software was available. It is hoped that the methodology presented in our study will stimulate its use when appropriate. The practical application of these results is that, by using measures of colostral antibody concentration, calves infected in utero could be identified by 80 days of age, whether or not a precolostral sample was available. In addition, these results could be used to estimate the probability of infection (given their initial colostral antibody titers) in calves when they are removed from hutches. Calves could then be sorted into group pens according to their probability of infection; calves with a high probability of infection would be placed in one pen and calves with a low probability of infection would be placed in
58
M-L. LASSAUZET ET AL.
a n o t h e r . T h i s s t r a t e g y w o u l d f u r t h e r r e d u c e t h e e x p o s u r e o f s u s c e p t i b l e calves to B L V i n f e c t i o n . ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d in p a r t b y f u n d s p r o v i d e d b y t h e U.S. D e p a r t m e n t o f A g r i c u l t u r e u n d e r the A n i m a l H e a l t h A c t o f 1977, P u b l i c L a w 95I 13, b y the L i v e s t o c k D i s e a s e R e s e a r c h L a b o r a t o r y , School o f V e t e r i n a r y Medicine, University of California, Davis and by the French Ministry of Ind u s t r y a n d R e s e a r c h . W e t h a n k Drs. R a l p h a n d N a n c y W a l t o n a n d Mr. a n d M r s . R i c h a r d O r i s i o f o r h e l p i n g us to collect d a t a a n d f o r letting us w o r k o n t h e i r h e r d , M r s . D e n i s e W e n t k e r for t e c h n i c a l a s s i s t a n c e , a n d D r . R o g e r R u p p a n n e r for h e l p f u l d i s c u s s i o n s .
REFERENCES Crespeau, F., Manet, G., Vuillaume, A., Levy, D. and Parodi, A-L., 1986. Infection des veaux par le virus leuc6mog6ne bovin (BLV) - Etude en 61evage laitier au cours de la premi6re ann6e de vie. Rec. Med. Vet., 162: 989-997. Dixon, W.J. (Editor), 1981. BMDP Statistical Software. University of California Press, Los Angeles, CA, pp. 305-325. Kalbfleisch, J. and Prentice, R., 1980. The Statistical Analysis of Failure Time Data. Wiley, New York, pp. 23-24. Kono, Y., Sentsui, H., Arai, K., Fujigaki, A., Enomoto, C., Iwasaki, H. and Ishida, H., 1983. Serological methods to detect calves infected in utero with bovine leukemia virus. Jpn. J. Vet. Sci., 45: 453-461. Lassauzet, M-L., Johnson, W.O. and Thurmond, M.C., 1989. Regression models for time-toseroconversion after experimental bovine leukemia virus infection. Star. Med., 8:725-741. Miller, J.M. and Van der Maaten, M.J., 1976. Serological detection of bovine leukemia virus infection. Vet. Microbiol., l: 195-202. Nakajima, H., Oiakawa, H., Inumaru, S. and Sugiura, T., 1982. Immunodiffusion studies in bovine leukosis. JARQ, 16: 136-143. Oshima, K.I., Morimoto, N., Kagawa, Y., Numakunai, S., Hirano, T. and Kayano, H., 1984. A survey for maternal antibodies to bovine leukemia virus (BLV) in calves born to infected cows with BLV. Jpn. J. Vet. Sci., 46" 583-586. Thurmond, M.C., Carter, R.L., Puhr, D.M. and Burridge, M.J., 1982. Decay of colostral antibodies with application to detection of calfhood infection. Am. J. Vet. Res., 43" 1152-1155. Thurmond, M.C., Carter, R.L., Puhr, D.M., Burridge, M.J., Miller, J.M., Schmerr, M-J.F. and Van der Maaten, M.J., 1983. An epidemiological study of natural in utero infection with bovine leukemia virus. Can. J. Comp. Med., 47: 316-319. Turnbull, B.W., 1976. The empirical distribution function with arbitrarily grouped, censored and truncated data. R. Stat. Soc. B, 3: 290-295. Van der Maaten, M.J., Miller, J.M. and Schmerr, M-J.F., 1981. Effect of colostral antibody on bovine leukemia virus infection on neonatal calves. Am. J. Vet. Res., 42" 1498-1500. Wiersma, F., Armstrong, D.V. and Welchert, W.T., 1983. Housing systems for dairy production under warm, semi-arid conditions. In" Dairy Housing II. Proceedings of the 2nd National Dairy Housing Conference, 14-16 March, 1983, Madison, WI, pp. 307-314.
45
Elsevier Science Publishers B.V., A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
Factors associated with decay of colostral antibodies to bovine leukemia virus M-L. Lassauzet ~, W.O. Johnson 2, M.C. T h u r m o n d ~* a n d J.P. P i c a n s o 1 Veterinary Medicine Teaching and Research Center, Department of Medicine, School of Veterinary Medicine, University of California, 18830 Road 112, Tulare, CA 93274 (U.S.A.) 2Division of Statistics, University of California, Davis, CA 95616 (U.S.A.) (Accepted for publication 29 September 1989 )
ABSTRACT Lassauzet, M-L., Johnson, W.O., Thurmond, M.C. and Picanso, J.P., 1990. Factors associated with decay of colostral antibodies to bovine leukemia virus. Prey. Vet. Med., 9: 45-58. One hundred and sixty-one calves that had received colostrum from bovine leukemia virus (BLV)infected cows were followed to evaluate factors associated with the decay of colostral antibodies to BLV. Three survival parametric models (Weibull, log-normal and log-logistic ) were fitted to data on the age at which non-infected calves converted from a seropositive to a seronegative status (retroconversion). For 97 non-infected calves, the fit of the Weibull model for the age at retroconversion was better than those of the log-logistic and log-normal models; values for 2. and a for the Weibull model were 0.014 and 1.59. Non-infected calves with high initial levels of BLV colostral antibodies became seronegative at an older age than calves with low initial levels ( P < 0.001 ). Dam BLV infection status was not associated with age at retroconversion ( P = 0 . 3 2 8 ) after adjusting for the initial level of colostral antibodies. The rate of antibody decay in most calves that seroconverted later because of active infection was within the range of that in non-infected calves. Six calves that did not exhibit any decay could have been identified as infected as early as 80 days of age. Results indicate that the use of initial BLV colostral antibody conceniration in models depicting decay of colostral antibodies will improve the detectability of early BLV calf hood infection. For initial colostral antibody concentration scored from 1 to 4 (1 lowest, 4 highest), no detectable antibodies were predicted in non-infected calves after 39, 67, 115 and 196 days of age for scores l, 2, 3 and 4 respectively.
INTRODUCTION
In a program aimed at control or eradication of bovine leukemia virus (BLV)-infected cattle, early detection of infected calves is difficult because colostral antibodies to BLV cannot be differentiated from antibodies which are the result of infection (Crespeau et al., 1986; Kono et al., 1983; Oshima *Author to w h o m c o r r e s p o n d e n c e should be addressed.
0167-5877/90/$03.50
© 1990 Elsevier Science Publishers B.V.
46
M-L. LASSAUZET ET AL.
et al., 1984; T h u r m o n d et al., 1982; Van Der Maaten et al., 1981 ). In an effort to estimate the time at which a seropositive calf could be considered infected, the decay of colostral antibodies in infected and non-infected calves has been studied (Kono et al., 1983; Oshima et al., 1984; T h u r m o n d et al., 1982). Generally, results suggest that infected calves can be identified between 5 and 7 months of age. It is not known, however, whether the age at which a calf can be expected to become seronegative is a function of the a m o u n t of BLV-antibodies absorbed a n d / o r of the infection status of the dam. Knowledge of the existence of such associations would improve our estimates of the expected age at which colostral antibodies would no longer be detectable. The use of improved estimates that permit more accurate, and possibly earlier, detection of BLV-infected calves would allow for more efficient control of BLV transmission through segregation of infected livestock. The objectives of the study were: ( 1 ) to determine whether an association existed between the decay of BLV colostral antibodies in non-infected calves and two factors namely, serum BLV antibody titer during the first week of life and d a m BLV-infection status; (2) to determine whether the rate of decay adjusted for the effects of the factors could identify infected calves before they were 6-7 months old. MATERIALS AND METHODS
Population studied Calves studied were born between January 1984 and June 1987 on a 210cow dairy located in the Central San Joaquin Valley of California and managed like a typical California feedlot dairy (Wiersma et al., 1983 ). Within 12 h after birth, calves were fed 2 1 of colostrum from their own d a m or administered commercial immunoglobulins per os (Genecol 99 R) and fed 1-2 1 of pooled colostrum. Female calves were retained in the herd to provide replacements and most bull calves were sold. Within 12 h of birth, calves were put into individual hutches where they were fed 2 I of pooled colostrum twice a day for 2 days, after which time they were fed twice daily 21 of a combination of bulk-tank milk and powdered milk at a ratio of 1 : 1. Calves were placed in group pens at about 90 days of age and were weaned at about 6 months of age.
Blood collection and examination Whole blood was collected by jugular venipuncture from calves usually within 1 week of age. Precolostral samples were taken from calves born during bi-weekly visits to the herd. Blood was collected from calves every 2-3 weeks until they left the hutch, after which time blood was collected every 3 months from the jugular or coccygeal vein. Cows were bled quarterly and when
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
47
examined by the herd veterinarian during weekly visits for reproduction monitoring. Serum was separated by centrifugation and stored at - 8 0 oC. Agar gel immunodiffusion ( A G I D ) was used to detect gp-51 antibodies (Leukassay-B kit, Pitman-Moore Inc., Washington Crossing, N J). The test procedure was slightly different from the protocol described by Miller and Van Der Maaten ( 1976 ) and Nakajima et al. ( 1982 ). The agar-gel was made with 1% agarose and 8.5% NaC1 using distilled water. Glass plates measuring 100 × 80 m m and containing 21 ml of agar were used. Wells were 4 m m in diameter and at a distance of 5 m m from each other. Antigen was placed in the central well, the reference positive serum in two opposing wells and sera to be tested in the four remaining wells. Plates were incubated 48 h at 25°C in a humified chamber before being viewed. A line of identity between antigen and antibody was interpreted as a positive test result. The concentration of antibodies present in the serum was approximated by categorizing from 1 (lowest concentration) to 4 (highest concentration) according to the shape of the precipitating line and its distance to the perimeter of the antigen well (National Veterinary Laboratories Services, Ames, IA).
Determination of infection status A cow was considered infected if AGID-positive before its pregnancy started or at the time the study began. For cows that seroconverted during pregnancy or after parturition, the most likely time of infection was estimated as previously described (Lassauzet et al., 1989). If that time was during pregnancy, then the cow was classified as infected. Cows that were AGID-negative and removed from the herd within 2 months post-partum were classified as noninfected. Determination of infection status for calves with colostral antibodies was more difficult because colostrum-derived antibodies cannot be differentiated from infection-induced antibodies. Moreover, if a calf is infected while having colostral antibodies, those antibodies might have to decay below a certain threshold before infection-induced antibodies can be produced. In one study, the time between the last colostrum-related positive test and the first infection-related positive test was 2 months, using the AGID test for a calf experimentally infected after birth (Van Der Maaten et al., 1981 ). Because that calf was bled monthly, it could have been seronegative at the most for 60 days. It was decided, therefore, to classify as 'non-infected' calves that became seronegative and were observed to remain so for at least 2 months. All other calves that is, those that were not seronegative for at least 2 months and those that could not be observed (but could have been) seronegative for at least 2 months - were referred to as possibly-infected (PI) calves. Among PI calves, five categories were created to help differentiate calves for which no decay was observed (category 1 ) from those for which a decay -
48 Z
M-L. LASSAUZET ET AL.
4 .
:
.-
_-
/
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z
ILl .3C..)
\ \ • ~ ....
/
~--+---~,
\
Z
.
/ I I I
\
>.a 2" O
~/
acatego~ 5
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4
/
rn I-Z
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,/
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,_-category
/
I 1
I 2
I ~ 3 4
I \\\1 5 6
*. . . . .
~cotego~
I
I
I
I
7
8
9
10
3
TEST NUMBER
Fig. 1. Patterns of decay of colostral antibodies used to identify non-infected calves and five categories of PI calves. was observed (categories 2 - 5 ) , calves that were not observed seronegative (categories 1-3 ) from those that became seronegative (categories 4 and 5 ), and calves that were very likely infected (categories 1, 2 and 4) from those that could have been classified as non-infected if bled at shorter intervals (categories 3 and 5 ) (Fig. 1 ). Such categories were made in order to evaluate whether patterns could be observed in calves that were probably infected while having colostral antibodies. Precise definitions of categories were: ( 1 ) calves that had a colostral antibody concentration (CAC) score of 4 during the first and subsequent weeks; (2) calves for which an antibody decay was observed but which were never observed seronegative, where the time between the last test with the lowest CAC and the next test was < 2 months; (3) calves for which a decay was observed but which were never observed seronegative, where the time between the last test with the lowest CAC and the next test was >/2 months; (4) calves that were observed to be seronegative for < 2 months; (5) calves that were observed to be seronegative, but the duration of seronegativity was unknown.
Estimation of decay of colostral antibodies Our general approach was to model ( 1 ) the age at which non-infected calves with colostral antibodies converted to a seronegative status (referred to as age at retroconversion) and (2) the duration of stay at each level of CAC for non-infected calves, using survival techniques. The decay of colostral antibodies for each PI calf was then compared with that for non-infected calves to determine if some PI calves could be identified as having been infected while they had colostral BLV antibodies. When modelling the age at retroconversion for non-infected calves receiv-
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
49
ing colostral antibodies, non-parametric probability functions were first calculated using methods previously described (Lassauzet et al., 1989; Turnbull, 1976). The covariates (CAC during the first week of life (CAC1) and d a m infection status) were examined using a parametric model to determine if they were associated with the age at retroconversion (Kalbfleisch and Prentice, 1980; Lassauzet et al., 1989).Three survival parametric models (Weibull, log-normal and log-logistic) were fitted to the ages at retroconversion of all non-infected calves and the best-fitting model was used to examine the effect of the above factors. The loss corresponding to a particular model with a particular set of factors is defined as loss = - 2 log (maximized likelihood function ). Smaller losses or bigger maximized log-likelihood functions correspond to better fitting models. Differences between appropriate losses constitute likelihood ratio statistics for testing that particular regression coefficients are zero. These were used to perform the model selection procedures. The usual z statistics were also calculated. A negative regression coefficient indicated that the factor level coded 0 would delay retroconversion relative to the factor coded 1. For continuous variables, a negative regression coefficient indicated that, as that variable increased, the probability of retroconversion decreased. Interpretation of a positive coefficient was the reverse. Computations were done using B M D P A R (Dixon, 1981 ). The 50% (medians), 95% and 99% quantiles of age at retroconversion were computed for each CAC 1. For the Weibull model, those quantiles are equal to exp { [ln( - l n c) - b X - a In 2 ] / a } , where c equals 0.50, 0.05 and 0.01, respectively, X is the vector of covariates found to be significantly associated with age at retroconversion, b the vector of regression coefficients of those covariates. The parameters 2 and a define the Weibull model (Kalbfleisch and Prentice, 1980) and are a function of the mean and the coefficient of variation of the age at retroconversion respectively. The middle value between the age at the last lowest CAC and the age at the first negative test for each PI calf was compared with the 95% quantile of the age at retroconversion for non-infected calves with the same CAC 1. If the middle value was above this quantile, the age at infection was estimated to be between birth and the age at the first negative test. The duration of stay at a given antibody concentration was estimated for non-infected calves that had received colostral antibodies. This was because antibody concentrations were estimated on an ordinal scale from 1 to 4, making the use of regression methods inappropriate. Non-parametric probability functions for the duration of stay (Tj, j = 1,2,3,4) at a given level of antibody concentration were estimated as described previously (Lassauzet et al., 1989; Turnbull, 1976). I f a was the age of a calf at its last test with a CAC of j + l, b was the age at its first test with a CAC of j, c was the age at its last test with a CAC o f j and d was the age at its first test with a CAC of j - l, then Tj was
50
M-L. LASSAUZET
E T A L.
Z 0 n," l-Z
j+l
•
.a
z o >.+ o
0 n,'l
.b
j
.c
Z
d
I.---
q 0
I
I
I
I
i
a
b
I
I
I c
I
I
I
d
A G E IN D A Y S
Fig. 2. Schematic diagram illustrating notation used to describe non-parametric function for duration of stay at antibody concentration at l e v e l j ( ~ ) . The last test at level j + 1 was done at age a, the first test at l e v e l j was done at age b, the last test at l e v e l j was done at age c and the first test at level j - 1 was done at age d. Tj was estimated to be at least equal to (c-b) and not more than (d-a) days.
considered to be > c - b and < d - a (Fig. 2 ). For calves that had a CAC 1 o f j, a and b were set to 0. If c was equal to b, then c - b was chosen to be 7 because the duration o f stay at a given antibody concentration was assumed to be at least 1 week. For instance, if a calf with a CAC 1 o f 4 was tested at 20 ( a ) , 40 (b), 60 (c), and 80 (d) days o f age and if the test results were 4 ( j + 1 ), 3 (j), 3 (j), and 2 ( j - 1 ) respectively, then, the duration o f stay at level 3 ( T3 or Tj) would be at least 20 days ( 6 0 - 40 or c - b) and at the most 60 days ( 80 - 20 or d - a ). The duration o f stay at level 4 (which was the level o f the CACI ) would be at least 20 days ( 2 0 - 0 ) and at the most 40 days ( 4 0 - 0 ) . Finally, if the calf could not be tested at 60 days 7'3 would be, by convention, at least 7 days and at the most 60 days. Because each level represents a range o f continuous values, the duration o f stay at a given level was c o m p u t e d separately for calves with CAC I equal to that same given level and for calves with a greater CAC 1. For example, if a calf had a CAC 1 of 2, its initial antibody level measured on a continuous scale could be anywhere within the range o f values scored as a 2. When the CAC o f calves which had a CAC 1 o f 3 or 4 is scored as a 2 for the first time, it is more likely that the antibody level is in the upper range of values scored as a 2 than in the lower range. Consequently, it was anticipated that a calf with a CAC 1 o f 2 would have had a CAC o f 2 for a lesser time, on the average, than calves that had had a CAC 1 o f 3 or 4. The duration o f stay at levelj for all PI calves was then compared with that obtained for non-infected calves after adjusting for the level of its CAC 1. If a PI calf had a CAC at level j longer than the 95% quantile computed for non-
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
51
infected calves for the same CAC 1, the PI calf was designated as having been infected between birth and the age at the first test with CAC of j - 1 - - the upper bound of the duration of stay at level j. The examination of the duration of stay at a given CAC was done for all PI calves. However, the examination of the age at retroconversion could be done only for calves that were eventually observed seronegative. RESULTS
Between 1 January 1984 and 30 June 1987 colostral antibodies to BLV were detected in 161 calves of which 109 were classified as not infected, based on the criteria set out above. Among the remaining 52 calves, 30 were censored and 22 were classified as PI calves. Information on all the PI calves that belonged to categories 2-5 is presented in Table 1. Information on CAC 1 (levels 1,2, 3, 4) was available for 97 non-infected calves. The non-parametric probability distribution of the age at retroconversion revealed a multimodal distribution with most non-infected calves becoming seronegative within 130 days (Fig. 3 ). When non-parametric probability distributions were estimated for each level of CAC 1, distributions tended to be more homogeneous and concentrated (Fig. 4). Values of the maximized log-likelihood functions for the log-normal, loglogistic and Weibull models for the age at retroconversion without covariates were - 209.7, - 209.3 and - 197.3 respectively, indicating that the Weibull model fit was better than those of the other two models. Values for 2 and a for the Weibull model were 0.014 and 1.59 respectively. The Weibull model was used to examine the effect of d a m infection status and CAC 1 on the age at retroconversion. D a m infection status was not associated with the age at retroconversion after adjusting for CAC 1 ( P = 0 . 3 2 8 ) (Table 2 ). High CAC 1, however, was associated with a delay in retroconversion ( P < 0 . 0 0 1 ) (Table 2). Medians, 95% and 99% quantiles of the age at retroconversion, computed using the Weibull model for non-infected calves at each CAC 1 level, indicated that non-infected calves with a CAC 1 of 1, 2, 3 or 4 would be expected to be seronegative no later than 46, 79, 135 and 230 days of age respectively (Table 3 ). For PI calves that were observed at some time to be seronegative (categories 4 and 5 ), the age midway between the age at the last lowest CAC and the age at the first negative test was compared with the 95% quantile of the age at retroconversion for non-infected calves with the same CAC 1 to determine when PI calves could have become infected (Tables 1 and 3). For calf 369, as an example, the age midway between the age at the test with the last lowest CAC and the age at the first negative test was 51 days [ (30 + 72 ) / 2 ] (Table 1 ); this was less than 115 days which is the 95% quantile of the age at retroconversion for non-infected calves with a CAC1 of 3 (Table 3). Thus,
52
M-L.LASSAUZETETAL.
TABLE 1
Information relating to 16 calves (PI calves of categories 2, 3, 4 and 5 ) for which a negative test was not observed for at least 60 days following the last test with lowest colostral antibody concentration (CAC) Calf
Precolostral sample a
First CAC
Age at test (days) (CAC at test )
203
NA
4
369
NA
3
377
NA
3
388
NEG
3
393
NEG
2
397
NEG
4
398
NEG
1-2
428
NA
4
788
NA
4
795
NA
3
804
NEG b
4
841
NA
4
869
NA
4
106 (1) 30 (2) 57 (2) 71 (1) 33 (1) 117 (1) 78 (1) 69 (4) 138 (1) 98 (1) 71 (2) 62 (1) 83
184 (4) 72 (0) 78 (0) 119 (0) 53 (0) 200 (4) 103 (0) 135 (3) 233 (4) 192 (4) 103 (0) 141 (4) 97
112
128
(1)
(o)
(o)
(2)
78 (1) 70 (1) 36 (1)
92 (0) 84 (0) 51 (0)
108 (0) 115 (0) 67 (1)
124 (4) 130 (2)
871
NEG b
3
874
mEG
1-2
886
NA
3
147 (3) 155 (4) 188 (4) 67 (0)
170 (4)
181 (4) 205 (2)
212 (3)
181 (4)
aNA, not available; NEG, negative. bNon-infected dam.
this calf was not identified as having become seronegative at an older age than non-infected calves. In fact, the age at retroconversion o f PI calves from category 4 or 5 was comparable with that o f non-infected calves. Medians and 95% and 99% quantiles were estimated from non-parametric probability functions for the duration o f stay for non-infected calves at given
COLOSTRAL ANTIBODIES TO BOVINE L E U K E M I A VIRUS
53
0.02 o
o
=- 0.01 -
e~ o a_ 0-
14 28 42 56 70 84 98 112126 A g e in d a y s
Fig. 3. Non-parametric probability function for the age at which non-infected calves became seronegative after consuming colostrum with BLV antibodies.
0.06 0.05 0.04 0.03 0.02
CACl = 1
-
O.Ol
•
-
0
i o.o4t . °°31 I J I
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8 0.01 °°2~ t L o
0
CAC1 = 3
>, 0.041 =o
0.03 1 0.02
1 CACl = 4
0.04 0.03-
0.020.01014 28 42 56 70 84 98 112 126
Age in days
Fig. 4. Non-parametric probability functions for the age at which non-infected calves became seronegative, given their colostrum BLV antibody concentration during the first week of life (CAC1).
54
M-L.LASSAUZETETAL.
TABLE 2 Effects of dam BLV infection status and colostral BLV antibody concentration during the first week of life (CAC 1 ) on age at retroconversion for non-infected calves receiving colostral antibodies, as determined using a Weibull regression model Factors in model
Loss
None CAC1 Dam status CAC1 (adjusted for dam status) Dam status (adjusted for CAC1 )
357.98 262.93 350.81 262.00
fl a
z-value
P-value
- 1.397 -0.648 - 1.377 -0.240
-8.767 -2.440 -8.107 -0.979
<0.001 0.014 <0.001 0.328
aFactor's regression coefficient.
TABLE 3 Medians (50%) and 95% and 99% quantiles of age at retroconversion for non-infected calves receiving BLV colostral antibodies Colostral antibody concentration during first week of life
Quantiles 50%
95%
99%
Age (days) 1
2 3 4
22 38 65 111
39 67 115 196
46 79 135 230
TABLE 4 Medians (50%) and 95% and 99% quantiles of duration of stay with a colostral antibody concentration of 4, 3, 2 and 1 for non-infected calves that received BLV colostral antibodies Colostrai BLV antibody concentration during first week of life
Colostral antibody concentration 4
3
2
1
Quantiles of duration of stay (days) 4 3 2 1
a50%, 95% and 99% quantile.
23,68,75 a
27,41,42 12,47,49
30,48,52 30,48,52 20,56,64
26,50,55 26,50,55 26,50,55 15,49,55
COLOSTRAL ANTIBODIES TO BOVINE LEUKEMIA VIRUS
0.06
-
0.05
-
55
0 <
0 ._c ¢~ c t~
0.04 -
"~ o
0.03 -
,.Q
£
Q.
0.02
-
o.ol0 14
28
42
70
56
84
A g e in d a y s
Fig. 5. Non-parametric probability function of duration of stay at a colostral antibody concentration level of 4 for non-infected calves.
1 .0
.,,
.9
i n-
.8 .7
~
.6
~_~ o
~\-........ . ~. ""-.
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Fig. 6. Survival curves for age at which non-infected calves became seronegative after consuming colostrum with BLV antibodies in studies by Thurmond et al., 1982 ( ), by Crespeau et al., 1986 ( - - - - ) and in the present study ( . . . . ) for calves with colostral antibody concentration during the first week of life of 4 using the Weibull model. C A C levels 4, 3, 2 a n d 1 ( T a b l e 4 ) . T h e d u r a t i o n o f stay at a given C A C for each o f the PI calves ( d a t a n o t s h o w n ) was c o m p a r e d with the estimated quantiles for n o n - i n f e c t e d calves. For PI calves b e l o n g i n g to categories 2, 3, 4 a n d 5, a longer d u r a t i o n o f stay at a given level was n o t f o u n d except for calf 428. I f a calf w i t h a CAC1 o f 4 was n o t infected, it w o u l d h a v e h a d a C A C o f 2 by 117 days ( 7 5 + 4 2 ) ( T a b l e 4 ) . B e c a u s e c a l f 4 2 8 had a C A C o f 3 at 135
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M-L. LASSAUZET ET AL.
days of age (Table 1 ), the age at infection for that calf was estimated to be between birth and 135 days of age. It could have been identified as infected prior to 196 days, which is the 95% quantile of the age at retroconversion for non-infected calves with a CAC1 of 4 (Table 3 ). Six PI calves had antibodies constantly detected at a level of 4 and, therefore, belonged to category 1. One of those calves was sampled precolostrally and was found to be positive. Because all non-infected calves with a CAC 1 of 4 had a CAC of less than 4 within 80 days of age (Fig. 5), PI calves from category 1 could be detected as infected with certainty between 2 and 3 months of age. The results of this and two other studies were compared visually using plots of survival curves for the age at which non-infected calves became seronegative (Fig. 6). DISCUSSION
This study compared the decay of BLV colostral antibodies of each possibly-infected calf with that of non-infected calves after adjusting for the effect of the initial a m o u n t of colostral antibodies. The fact that the age at retroconversion was associated significantly with antibody concentration during the first week of life indicates that identification of infected calves with colostral antibodies would be possible at an earlier age if serologic results were available for the first week of life. The upper 99% limits of the age at retroconversion for non-infected calves varied considerably (from 46 to 230 days). Because colostral concentration during the first week of life was associated with the age at retroconversion, it is likely that variations in CAC 1 would explain the variations in the age at retroconversion which have been observed in previous studies. In those studies, retroconversion occurred between 2 and 6 months of age (Oshima et al., 1984 ), within 5 months of age (Kono et al., 1983 ), between 122 and 238 days (Crespeau et al., 1986 ) and between 51 and 187 days after birth ( T h u r m o n d et al., 1982). Data presented in Fig. 6 suggest that in the latter two studies, the age at retroconversion was comparable with that observed in our study for calves with a CAC 1 of 4. In a previous report (Kono et al., 1983 ), the m e t h o d of examining serologic results from repeated bleedings was proposed as a means of identifying calves that were infected in utero. In that study all calves with persistent antibody titers were found to be infected in utero. By extending those results to the present study, the six PI calves belonging to the first category could be classified as infected in utero. If no precolostral samples were available, such infected calves could be detected with certainty at 3 months of age (Fig. 5 ). In the same report (Kono et al., 1983 ), some calves with a positive precolostral sample had postcolostral titers that decreased and then either increased
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or remained negative. One possible explanation is that 'precolostral' samples for those calves were obtained after colostrum intake. Others have reported that, in field studies, such an error can occur (Crespeau et al., 1986; Thurm o n d et al., 1983). The rate of decay of colostral antibodies was not associated with the subsequent infection status of calves except for PI calves belonging to the first category. This result agrees with previous observations (Kono et al., 1983) and indicates that virus isolation or the detection of viral antigen might be the only means of identifying infected calves that received BLV colostral antibodies. It should be noted that some calves classified as PI had long intervals between bleedings, which might have biased results by failing to detect a seronegative status that lasted 2 months. In future studies, the bleeding scheme for each calf could be determined according to its CAC 1: narrow bleeding intervals at the time the calf is expected to become seronegative and then a bleeding 2 months after the first negative test. Because the a m o u n t of colostral antibodies was determined using an ordinal scale instead of a continuous scale, some errors in identifying the score levels using the AGID might have occurred. This error inherent in ordinal data could account for some outliers which made frequency distributions for each CAC 1 overlap (Fig. 4). For instance, some calves with a CAC 1 of 3 that became seronegative after 105 days might in fact have had a CAC 1 of 4, which would have explained the prolonged duration of colostral antibodies. If feasible, therefore, the measurement of antibody concentration using a continuous scale (e.g. ELISA tests) or at least an ordinal scale with more intervals (e.g. endpoint titers ) would increase the accuracy of predicting infection. In epidemiological studies it is c o m m o n that the exact time of an event is not known, only the time interval during which it occurred. In this study only the time interval during which the animal's serological status changed from positive to negative (or changed from levelj to level j - 1 ) was known. Interval censoring of the data, however, has not always been accounted for in other studies. Possible reasons why the non-parametric estimation of interval censored data has not been used elsewhere, were that the underlying theory was not easily understood by non-statisticians and that no software was available. It is hoped that the methodology presented in our study will stimulate its use when appropriate. The practical application of these results is that, by using measures of colostral antibody concentration, calves infected in utero could be identified by 80 days of age, whether or not a precolostral sample was available. In addition, these results could be used to estimate the probability of infection (given their initial colostral antibody titers) in calves when they are removed from hutches. Calves could then be sorted into group pens according to their probability of infection; calves with a high probability of infection would be placed in one pen and calves with a low probability of infection would be placed in
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a n o t h e r . T h i s s t r a t e g y w o u l d f u r t h e r r e d u c e t h e e x p o s u r e o f s u s c e p t i b l e calves to B L V i n f e c t i o n . ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d in p a r t b y f u n d s p r o v i d e d b y t h e U.S. D e p a r t m e n t o f A g r i c u l t u r e u n d e r the A n i m a l H e a l t h A c t o f 1977, P u b l i c L a w 95I 13, b y the L i v e s t o c k D i s e a s e R e s e a r c h L a b o r a t o r y , School o f V e t e r i n a r y Medicine, University of California, Davis and by the French Ministry of Ind u s t r y a n d R e s e a r c h . W e t h a n k Drs. R a l p h a n d N a n c y W a l t o n a n d Mr. a n d M r s . R i c h a r d O r i s i o f o r h e l p i n g us to collect d a t a a n d f o r letting us w o r k o n t h e i r h e r d , M r s . D e n i s e W e n t k e r for t e c h n i c a l a s s i s t a n c e , a n d D r . R o g e r R u p p a n n e r for h e l p f u l d i s c u s s i o n s .
REFERENCES Crespeau, F., Manet, G., Vuillaume, A., Levy, D. and Parodi, A-L., 1986. Infection des veaux par le virus leuc6mog6ne bovin (BLV) - Etude en 61evage laitier au cours de la premi6re ann6e de vie. Rec. Med. Vet., 162: 989-997. Dixon, W.J. (Editor), 1981. BMDP Statistical Software. University of California Press, Los Angeles, CA, pp. 305-325. Kalbfleisch, J. and Prentice, R., 1980. The Statistical Analysis of Failure Time Data. Wiley, New York, pp. 23-24. Kono, Y., Sentsui, H., Arai, K., Fujigaki, A., Enomoto, C., Iwasaki, H. and Ishida, H., 1983. Serological methods to detect calves infected in utero with bovine leukemia virus. Jpn. J. Vet. Sci., 45: 453-461. Lassauzet, M-L., Johnson, W.O. and Thurmond, M.C., 1989. Regression models for time-toseroconversion after experimental bovine leukemia virus infection. Star. Med., 8:725-741. Miller, J.M. and Van der Maaten, M.J., 1976. Serological detection of bovine leukemia virus infection. Vet. Microbiol., l: 195-202. Nakajima, H., Oiakawa, H., Inumaru, S. and Sugiura, T., 1982. Immunodiffusion studies in bovine leukosis. JARQ, 16: 136-143. Oshima, K.I., Morimoto, N., Kagawa, Y., Numakunai, S., Hirano, T. and Kayano, H., 1984. A survey for maternal antibodies to bovine leukemia virus (BLV) in calves born to infected cows with BLV. Jpn. J. Vet. Sci., 46" 583-586. Thurmond, M.C., Carter, R.L., Puhr, D.M. and Burridge, M.J., 1982. Decay of colostral antibodies with application to detection of calfhood infection. Am. J. Vet. Res., 43" 1152-1155. Thurmond, M.C., Carter, R.L., Puhr, D.M., Burridge, M.J., Miller, J.M., Schmerr, M-J.F. and Van der Maaten, M.J., 1983. An epidemiological study of natural in utero infection with bovine leukemia virus. Can. J. Comp. Med., 47: 316-319. Turnbull, B.W., 1976. The empirical distribution function with arbitrarily grouped, censored and truncated data. R. Stat. Soc. B, 3: 290-295. Van der Maaten, M.J., Miller, J.M. and Schmerr, M-J.F., 1981. Effect of colostral antibody on bovine leukemia virus infection on neonatal calves. Am. J. Vet. Res., 42" 1498-1500. Wiersma, F., Armstrong, D.V. and Welchert, W.T., 1983. Housing systems for dairy production under warm, semi-arid conditions. In" Dairy Housing II. Proceedings of the 2nd National Dairy Housing Conference, 14-16 March, 1983, Madison, WI, pp. 307-314.