Seroprevalence of Maedi-Visna in Canadian Sheep Carole Simard and Randall S. Morley ABSTRACT A serological survey of Canadian sheep over one year of age was conducted to estimate the seroprevalence of maedi-visna. An indirect enzymelinked immunosorbent assay was used. An analysis of 14,047 sera from 286 randomly selected flocks provided an estimate of the seroprevalence of 19% and a mean flock prevalence of 12%. Sixty-three percent of the sampled flocks had one or more seropositive sheep. There appeared to be higher prevalences in sheep in Quebec (40%) and Nova Scotia (27%). An increased prevalence with increased age and flock size was noted. I
I
RESUME Une enquete seroepidemiologique a ete realisee afin de determiner la prevalence de maedi-visna chez les moutons canadiens Ages de plus d'un an. La technique ELISA (enzyme-linked immunosorbentassay) a 6te utilisee. L'analyse des 14 047 echantiflons sanguins provenant des 286 troupeaux de moutons selectionnes au hasard a demontre que la seroprevalence de l'infection au Canada serait de 19 % et que la prevalence moyenne par troupeau serait de 12 %. Parmi les troupeaux echantillonnes, 63 % se sont averes seropositifs. Une plus forte prevalence de moutons infectes a ete' observee au Quebec (40 No) et en Nouvelie-Ecosse (27 %O). Une augmentation de la prevalence associee a l'age des moutons et a la grosseur des troupeaux a aussi ete notee.
INTRODUCTION Maedi-visna (MV), a slow virus disease of sheep, is characterized by a lymphoproliferative pneumonia, meningeal arteritis with encephalitis, non-
suppurative arthritis and lymphocytic mastitis (1). The causal agent is a member of the Lentivirus genus of the Retroviridae. Once infected, the host is unable to eliminate the virus due to the integration of the provirus into the cellular genome (2). An insidious but continously advancing and eventually fatal pneumonitis, physical weakness, dyspnea and emaciation are the predominant clinical features (1). The disease occurs in most of the sheep-rearing areas of the world, with the exception of Australia and New Zealand (3). In Canada, the first cases were reported in the early 1970s (4-6). A serological survey in Quebec during 1976-1979 (7) showed that 35.7Olo of the sheep were seropositive by the complement fixation test. The authors suggested that MV was probably the most important respiratory infection in adult sheep because of its ability to predispose to other respiratory infections. In a recent survey of sheep diseases in Canada (8), pneumonia was the most important single cause of mortality in ewes. Sheep clinically affected with MV are often unthrifty and become emaciated during the course of the disease (9). Subclinical MV infection may have a detrimental effect on ewe productivity, through a reduction in conception rates, birth weight of lambs and lamb weights at 25-50 days of age (10). Poor preweaning lamb growth might be attributed to a reduction in milk production and poor milk quality associated with indurative or subclinical mastitis (11,12). Because MV is a lifelong infection which is easily transmitted via the milk from an infected dam to her progeny or horizontally via the aerosol route (13), the infection and clinical disease contribute to make this entity an important factor affecting the sheep industry. The purpose of this study was to determine the seroprevalence of MV anti-
bodies in Canadian sheep over one year of age. Most sheep develop a humoral response shortly after infection and the detection of specific antibodies is indicative of lifelong viral infection (14). MATERIALS AND METHODS The study was done by staff of the Health of Animals Laboratory (HAL) in Sackville, New Brunswick, the Regional and District Offices across Canada of the Operations Directorate (OPS) and the National Animal Health Information System (NAHIS), of the Food Production and Inspection Branch of Agriculture Canada. The District Office OPS staff carried out the data and sample collection during the period March 1988 to February 1989 while the serological testing was performed at HAL, Sackville. SAMPLE SIZE DETERMINATION
The target of this survey was the sheep population of Canada, that were one year of age or older. Cluster sampling was employed, in which flocks represented the initial sampling units. In flocks of more than 200 breeding sheep, only 200 were sampled. The individual ewe or ram corresponded to the unit of concern. Using a 95'70 level of confidence, setting 1% error limits (T = 0.01) and estimating the prevalence of MV antibodies in the breeding sheep population at 20O%o (p = 0.20), the desired sample size of randomly selected sheep over one year of age was n = 3.84 P(1-P)/T2 = 6,147 sheep (15). Since the initial sampling unit was the flock, the calculated sample size was doubled (12,294) to obtain the precision desired in the prevalence estimate (16).. This sample size requirement was divided according to the proportional sheep population of the ten Canadian provinces. The numbers of ewes and
Health of Animals Laboratory, Agriculture Canada, 4 College Street, Sackville, New Brunswick EOA 3C0 (Simard) and Animal Health Division, Agriculture Canada, 2255 Carling Avenue, Ottawa, Ontario KIA 0Y9 (Morley). Submitted July 3, 1990.
Can J Vet Res 1991; 55: 269-273
269
TABLE I. Distribution of ewes and wethers by province and the estimated number of flocks to sample
farmsa,b 1,921
Number of ewes and wethersa (¾ of total) 79,856(22.46)
Means flock sizec 42
1,411 428 168 126 355 3,494
25,858(7.28) 12,066(3.39) 4,707(1.32) 3,286(0.92) 18,227(5.13) 113,660(31.97)
18 28 28 26 51 33
103 1,144 931
3,507(0.99) 69,520(19.55) 24,846(6.99)
34 61 27
Number of
Province Alberta British Columbia Manitoba New Brunswick Newfoundland Nova Scotia Ontario Prince Edward Island Quebec Saskatchewan
Totals 10,081 355,543(100.00) aStatistics Canada, 1986 Census of Agriculture bAssumed farm equated to flock CRams ignored
Proportional no. of sheep to sample 2,761 895 417 162 113 631
3,930 122
Estimated no. of flocks to sample 66 50 15 6 5 13 119
859
4 40 32
12,294
350
2,404
wethers and numbers of farms for each number recorded on the laboratory test province (Statistics Canada, 1986 form. Each sample was centrifuged Census of Agriculture) provided the and the serum was decanted into proportions and average number of another vial similarly identified. The ewes and wethers per flock. These sera were held frozen or under allowed the determination of the pro- refrigeration until submission to the portional number of serum samples laboratory. and the approximate number of flocks to sample for each province. The SEROLOGICAL TEST All serum samples were tested using national total was 350 flocks (Table I). an indirect enzyme-linked immunosorLIST FRAMES bent assay (i-ELISA) with sodium Provincial sheep associations pro- dodecyl sulfate treated MV antigen vided lists of member producers from (17). Each serum sample was diluted which a random sample of flocks was to 1:50 in the ELISA buffer (0.34 M obtained. Numbers were assigned NaCl, 1.5 mM KH2PO4, 0.8 mM sequentially from the first member Na2HPO4.12H20, 3 mM KC1, 0.0507o name to the last for each list. Ten sets Tween 20, 7.5% fetal bovine serum) of computer generated random num- using an automatic diluter (Hamilton bers were used to identify the sample 1000, Fisher Scientific, Unionville, of flock owners for all provinces. Ontario). The samples were tested in A second set of random numbers was duplicate on antigen precoated microgenerated in case other flocks were titer plates, with a total of 44 samples required to meet the proportional per plate. Negative and positive refernumber of serum samples per province. ence sera obtained from specificpathogen-free (SPF) sheep and SPF DATA AND SERUM COLLECTION sheep experimentally inoculated with Data collected at the time of blood MV virus, respectively, were tested on sampling included the owner's name, each plate. A mean optical density address and flock location, eartag (OD) (X) was calculated for each number or name of sheep, age of sample and a predefined cutoff value animal (year of birth), sex, breed (the mean OD plus 3 or 4 standard description of animal and vial number. deviations (SD) of 330 serum samples This information was recorded on lab- from a known seronegative flock) was oratory test forms which were sub- used to classify the test results (17). mitted with the sera to the laboratory. Each sample was considered seroWhole blood was collected from the negative (less or equal to X + 3SD), jugular vein of each sheep and each suspicious (between X + 3SD and sample received the corresponding vial X + 4SD) or seropositive (equal or 270
greater than X + 4SD). A suspicious reaction was retested by the agar gel immunodiffusion (AGID) test (17). A positive reaction to the AGID test was seropositive whereas suspicious or negative sera remained suspicious. Serological testing was done during a 12 month period beginning two months after the start of sera collection. ANALYSIS OF DATA
All descriptive statistics and univariate analyses were obtained using the SAS computer software, FREQ and MEANS procedures (18). The breed descriptions were limited to groups which totalled more than 100 sheep. Breed groups of fewer sheep received the "miscellaneous" label. For cross-breeds the first breed recorded on the test form signified the breed category. The number of sheep sampled per flock was considered as a surrogate variable for the flock size. Chi-square tests of independence were used to portray any association between serological test result and province, breed, age, sex and flock size.
RESULTS The requirement of 350 flocks was not achieved although the number of serum samples exceeded the required number. For three provinces there was a shortfall in the proportional number of serum samples. This deficiency expressed as a percentage of the requirements was 4% for Nova Scotia, 130% for Saskatchewan and 35% for British Columbia. A total of 14,289 serum samples from 286 flocks was collected. Two hundred and twenty-nine gave suspicious reactions and 13 sera were unfit for testing. The remaining 14,047 sera reacted positively or negatively and represented the results presented. The apparent prevalence of antibodies to MV in Canadian sheep was 18.8% +/- 0.3% (standard error of the mean (SEM)). The 95% confidence limits based on 1%7o error limits were 17.8% and 19.8%. The mean flock prevalence was 11.7% while the percentage of flocks with at least one sheep seropositive was 62.9%. The percentage of seropositives, the percentage of flocks with at least one seropositive sheep and the mean flock
prevalence varied considerably among the provinces (Table II). The prevalence according to the breed of sheep varied from 5.6 to 39.307o (Table III). Breed descriptions were not recorded for 1,845 sheep. Sheep six and seven years old at the time of blood collection had the highest prevalence (Table IV). Age was not specified for 1,759 sheep. Of 13,179 sera originating from female sheep 2,495 (18.97o) reacted seropositively while 81 (14.507o) of 560 sera from male sheep were seropositive (p < 0.01). Sex was not specified for 308 sheep. When the test results were grouped according to the number of sheep sampled per flock the prevalence and mean flock prevalence increased with increasing flock size (Table V). The average number of sheep sampled per flock, representing here the mean flock sizes, (standard deviation (SD)) were as follows: Alberta 71.9 (66.9), British Columbia 18.3 (19.4), Manitoba 41.9 (29.6), New Brunswick 63.9 (52.2), Newfoundland 20.3 (15.5), Nova Scotia 46.4 (50.6), Ontario 41.2 (35.9), Prince Edward Island 40.5 (27.8), Quebec 80.2 (74.0) and Saskatchewan 49.9 (36.1). Further examination of the influence of province and breed through stratification of the test results by age and flock size was done. The percentage of seropositive sheep in Quebec when compared to all other provinces combined increased up to six years of age and was greater in all age groups. A higher percentage of seropositive sheep was noted in Quebec for results grouped according to the number of sheep sampled per flock, for all groups excepting 1-10 and 31-50 sheep. The percentage of seropositive sheep in the five breed descriptions exhibiting the highest prevalences (Finnsheep, Finnsheep cross-breed, Leicester, Dorset and Dorset cross-breed) versus all other breeds, increased up to six years of age and was higher in all age groups. This breed influence was also evident when test results were stratified by flock size. DISCUSSION
Although the sample size requirement of 350 flocks was not achieved and the proportional number of serum samples for three provinces was not
TABLE II. Enzyme-linked immunosorbent assay results for maedi-visna antibodies according to the province location of sheep Sera Number
Province Alberta British Columbia Manitoba New Brunswick Newfoundland Nova Scotia Ontario Prince Edward Island Quebec Saskatchewan
Totals ap
I
RESUME Une enquete seroepidemiologique a ete realisee afin de determiner la prevalence de maedi-visna chez les moutons canadiens Ages de plus d'un an. La technique ELISA (enzyme-linked immunosorbentassay) a 6te utilisee. L'analyse des 14 047 echantiflons sanguins provenant des 286 troupeaux de moutons selectionnes au hasard a demontre que la seroprevalence de l'infection au Canada serait de 19 % et que la prevalence moyenne par troupeau serait de 12 %. Parmi les troupeaux echantillonnes, 63 % se sont averes seropositifs. Une plus forte prevalence de moutons infectes a ete' observee au Quebec (40 No) et en Nouvelie-Ecosse (27 %O). Une augmentation de la prevalence associee a l'age des moutons et a la grosseur des troupeaux a aussi ete notee.
INTRODUCTION Maedi-visna (MV), a slow virus disease of sheep, is characterized by a lymphoproliferative pneumonia, meningeal arteritis with encephalitis, non-
suppurative arthritis and lymphocytic mastitis (1). The causal agent is a member of the Lentivirus genus of the Retroviridae. Once infected, the host is unable to eliminate the virus due to the integration of the provirus into the cellular genome (2). An insidious but continously advancing and eventually fatal pneumonitis, physical weakness, dyspnea and emaciation are the predominant clinical features (1). The disease occurs in most of the sheep-rearing areas of the world, with the exception of Australia and New Zealand (3). In Canada, the first cases were reported in the early 1970s (4-6). A serological survey in Quebec during 1976-1979 (7) showed that 35.7Olo of the sheep were seropositive by the complement fixation test. The authors suggested that MV was probably the most important respiratory infection in adult sheep because of its ability to predispose to other respiratory infections. In a recent survey of sheep diseases in Canada (8), pneumonia was the most important single cause of mortality in ewes. Sheep clinically affected with MV are often unthrifty and become emaciated during the course of the disease (9). Subclinical MV infection may have a detrimental effect on ewe productivity, through a reduction in conception rates, birth weight of lambs and lamb weights at 25-50 days of age (10). Poor preweaning lamb growth might be attributed to a reduction in milk production and poor milk quality associated with indurative or subclinical mastitis (11,12). Because MV is a lifelong infection which is easily transmitted via the milk from an infected dam to her progeny or horizontally via the aerosol route (13), the infection and clinical disease contribute to make this entity an important factor affecting the sheep industry. The purpose of this study was to determine the seroprevalence of MV anti-
bodies in Canadian sheep over one year of age. Most sheep develop a humoral response shortly after infection and the detection of specific antibodies is indicative of lifelong viral infection (14). MATERIALS AND METHODS The study was done by staff of the Health of Animals Laboratory (HAL) in Sackville, New Brunswick, the Regional and District Offices across Canada of the Operations Directorate (OPS) and the National Animal Health Information System (NAHIS), of the Food Production and Inspection Branch of Agriculture Canada. The District Office OPS staff carried out the data and sample collection during the period March 1988 to February 1989 while the serological testing was performed at HAL, Sackville. SAMPLE SIZE DETERMINATION
The target of this survey was the sheep population of Canada, that were one year of age or older. Cluster sampling was employed, in which flocks represented the initial sampling units. In flocks of more than 200 breeding sheep, only 200 were sampled. The individual ewe or ram corresponded to the unit of concern. Using a 95'70 level of confidence, setting 1% error limits (T = 0.01) and estimating the prevalence of MV antibodies in the breeding sheep population at 20O%o (p = 0.20), the desired sample size of randomly selected sheep over one year of age was n = 3.84 P(1-P)/T2 = 6,147 sheep (15). Since the initial sampling unit was the flock, the calculated sample size was doubled (12,294) to obtain the precision desired in the prevalence estimate (16).. This sample size requirement was divided according to the proportional sheep population of the ten Canadian provinces. The numbers of ewes and
Health of Animals Laboratory, Agriculture Canada, 4 College Street, Sackville, New Brunswick EOA 3C0 (Simard) and Animal Health Division, Agriculture Canada, 2255 Carling Avenue, Ottawa, Ontario KIA 0Y9 (Morley). Submitted July 3, 1990.
Can J Vet Res 1991; 55: 269-273
269
TABLE I. Distribution of ewes and wethers by province and the estimated number of flocks to sample
farmsa,b 1,921
Number of ewes and wethersa (¾ of total) 79,856(22.46)
Means flock sizec 42
1,411 428 168 126 355 3,494
25,858(7.28) 12,066(3.39) 4,707(1.32) 3,286(0.92) 18,227(5.13) 113,660(31.97)
18 28 28 26 51 33
103 1,144 931
3,507(0.99) 69,520(19.55) 24,846(6.99)
34 61 27
Number of
Province Alberta British Columbia Manitoba New Brunswick Newfoundland Nova Scotia Ontario Prince Edward Island Quebec Saskatchewan
Totals 10,081 355,543(100.00) aStatistics Canada, 1986 Census of Agriculture bAssumed farm equated to flock CRams ignored
Proportional no. of sheep to sample 2,761 895 417 162 113 631
3,930 122
Estimated no. of flocks to sample 66 50 15 6 5 13 119
859
4 40 32
12,294
350
2,404
wethers and numbers of farms for each number recorded on the laboratory test province (Statistics Canada, 1986 form. Each sample was centrifuged Census of Agriculture) provided the and the serum was decanted into proportions and average number of another vial similarly identified. The ewes and wethers per flock. These sera were held frozen or under allowed the determination of the pro- refrigeration until submission to the portional number of serum samples laboratory. and the approximate number of flocks to sample for each province. The SEROLOGICAL TEST All serum samples were tested using national total was 350 flocks (Table I). an indirect enzyme-linked immunosorLIST FRAMES bent assay (i-ELISA) with sodium Provincial sheep associations pro- dodecyl sulfate treated MV antigen vided lists of member producers from (17). Each serum sample was diluted which a random sample of flocks was to 1:50 in the ELISA buffer (0.34 M obtained. Numbers were assigned NaCl, 1.5 mM KH2PO4, 0.8 mM sequentially from the first member Na2HPO4.12H20, 3 mM KC1, 0.0507o name to the last for each list. Ten sets Tween 20, 7.5% fetal bovine serum) of computer generated random num- using an automatic diluter (Hamilton bers were used to identify the sample 1000, Fisher Scientific, Unionville, of flock owners for all provinces. Ontario). The samples were tested in A second set of random numbers was duplicate on antigen precoated microgenerated in case other flocks were titer plates, with a total of 44 samples required to meet the proportional per plate. Negative and positive refernumber of serum samples per province. ence sera obtained from specificpathogen-free (SPF) sheep and SPF DATA AND SERUM COLLECTION sheep experimentally inoculated with Data collected at the time of blood MV virus, respectively, were tested on sampling included the owner's name, each plate. A mean optical density address and flock location, eartag (OD) (X) was calculated for each number or name of sheep, age of sample and a predefined cutoff value animal (year of birth), sex, breed (the mean OD plus 3 or 4 standard description of animal and vial number. deviations (SD) of 330 serum samples This information was recorded on lab- from a known seronegative flock) was oratory test forms which were sub- used to classify the test results (17). mitted with the sera to the laboratory. Each sample was considered seroWhole blood was collected from the negative (less or equal to X + 3SD), jugular vein of each sheep and each suspicious (between X + 3SD and sample received the corresponding vial X + 4SD) or seropositive (equal or 270
greater than X + 4SD). A suspicious reaction was retested by the agar gel immunodiffusion (AGID) test (17). A positive reaction to the AGID test was seropositive whereas suspicious or negative sera remained suspicious. Serological testing was done during a 12 month period beginning two months after the start of sera collection. ANALYSIS OF DATA
All descriptive statistics and univariate analyses were obtained using the SAS computer software, FREQ and MEANS procedures (18). The breed descriptions were limited to groups which totalled more than 100 sheep. Breed groups of fewer sheep received the "miscellaneous" label. For cross-breeds the first breed recorded on the test form signified the breed category. The number of sheep sampled per flock was considered as a surrogate variable for the flock size. Chi-square tests of independence were used to portray any association between serological test result and province, breed, age, sex and flock size.
RESULTS The requirement of 350 flocks was not achieved although the number of serum samples exceeded the required number. For three provinces there was a shortfall in the proportional number of serum samples. This deficiency expressed as a percentage of the requirements was 4% for Nova Scotia, 130% for Saskatchewan and 35% for British Columbia. A total of 14,289 serum samples from 286 flocks was collected. Two hundred and twenty-nine gave suspicious reactions and 13 sera were unfit for testing. The remaining 14,047 sera reacted positively or negatively and represented the results presented. The apparent prevalence of antibodies to MV in Canadian sheep was 18.8% +/- 0.3% (standard error of the mean (SEM)). The 95% confidence limits based on 1%7o error limits were 17.8% and 19.8%. The mean flock prevalence was 11.7% while the percentage of flocks with at least one sheep seropositive was 62.9%. The percentage of seropositives, the percentage of flocks with at least one seropositive sheep and the mean flock
prevalence varied considerably among the provinces (Table II). The prevalence according to the breed of sheep varied from 5.6 to 39.307o (Table III). Breed descriptions were not recorded for 1,845 sheep. Sheep six and seven years old at the time of blood collection had the highest prevalence (Table IV). Age was not specified for 1,759 sheep. Of 13,179 sera originating from female sheep 2,495 (18.97o) reacted seropositively while 81 (14.507o) of 560 sera from male sheep were seropositive (p < 0.01). Sex was not specified for 308 sheep. When the test results were grouped according to the number of sheep sampled per flock the prevalence and mean flock prevalence increased with increasing flock size (Table V). The average number of sheep sampled per flock, representing here the mean flock sizes, (standard deviation (SD)) were as follows: Alberta 71.9 (66.9), British Columbia 18.3 (19.4), Manitoba 41.9 (29.6), New Brunswick 63.9 (52.2), Newfoundland 20.3 (15.5), Nova Scotia 46.4 (50.6), Ontario 41.2 (35.9), Prince Edward Island 40.5 (27.8), Quebec 80.2 (74.0) and Saskatchewan 49.9 (36.1). Further examination of the influence of province and breed through stratification of the test results by age and flock size was done. The percentage of seropositive sheep in Quebec when compared to all other provinces combined increased up to six years of age and was greater in all age groups. A higher percentage of seropositive sheep was noted in Quebec for results grouped according to the number of sheep sampled per flock, for all groups excepting 1-10 and 31-50 sheep. The percentage of seropositive sheep in the five breed descriptions exhibiting the highest prevalences (Finnsheep, Finnsheep cross-breed, Leicester, Dorset and Dorset cross-breed) versus all other breeds, increased up to six years of age and was higher in all age groups. This breed influence was also evident when test results were stratified by flock size. DISCUSSION
Although the sample size requirement of 350 flocks was not achieved and the proportional number of serum samples for three provinces was not
TABLE II. Enzyme-linked immunosorbent assay results for maedi-visna antibodies according to the province location of sheep Sera Number
Province Alberta British Columbia Manitoba New Brunswick Newfoundland Nova Scotia Ontario Prince Edward Island Quebec Saskatchewan
Totals ap
