Brief Communication Communication brève Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples Omid Nekouei, Jean Durocher, Greg Keefe Abstract — This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. Résumé — Performance diagnostique d’un test ELISA indirect pour détecter les anticorps contre le virus de la leucémie bovine dans des échantillons prélevés dans les réservoirs à lait. Cette étude a évalué la performance diagnostique d’un test ELISA commercial pour détecter les anticorps contre le virus de la leucémie bovine dans des échantillons prélevés dans des réservoirs à lait provenant de l’Est du Canada. La sensibilité et la spécificité du test ont été estimées à 97,2 % et à 100 %, respectivement. Le test a été recommandé comme un outil rentable pour les programmes de dépistage à grande échelle. Can Vet J 2016;57:778–780
E
nzootic bovine leukosis (EBL), caused by bovine leukemia virus (BLV), is an economically important disease of dairy cattle. The virus is transmitted through infected blood lymphocytes. Premature culling, death, and condemnation of carcasses at slaughter due to lymphosarcoma, as well as restrictions on international trade of infected cattle and their products are among the most significant economic losses attributed to the disease (1,2). Many European countries are now officially free from EBL, whereas prevalence of the infection in North America has been high and appears to have a rising trend (2,3). For instance, in the Maritime region of Canada, herd-level prevalence of the infection has increased from 70% in 1998 (4) to over 90% in 2013 (5). Using bulk-tank milk (BTM) samples has become one of the most convenient and economically efficient procedures for screening for important diseases in dairy herds, including EBL (6,7). Among available commercial tests for detection of BLV antibodies, milk enzyme-linked immunosorbent assay (ELISA) has been documented as a desirable method with good per-
Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, C1A 4P3 (Nekouei, Keefe); R&D Team, Valacta, Sainte-Anne-de-Belevue, Quebec H9X 3R4 (Durocher). Address all correspondence to Dr. Omid Nekouei; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 778
(Traduit par Isabelle Vallières)
formance in large-scale surveillance programs (8,9). However, applying commercial ELISA tests, particularly to pooled samples (e.g., BTM), could lead to variable levels of uncertainty in the results. Several factors, including study region, herd (pool) size, sampling procedures, and transferring process can potentially contribute to the variable test results. Therefore, it has been recommended that the validity of diagnostic tests should be evaluated in different populations before integrating the tests in large-scale control and eradication programs (10,11). The objective of this study was to assess the diagnostic performance (sensitivity and specificity) of a commercially available ELISA for detecting BLV antibodies in BTM samples from eastern Canada, in order to validate the routine application of this test to the BLV surveillance programs implemented in the region. The study population consisted of 133 dairy herd improvement (DHI)-registered dairy herds, including 8358 lactating cows, from 4 eastern provinces of Canada [Prince Edward Island (PE), New Brunswick (NB), Nova Scotia (NS), and Quebec (QC)]. During 2013, 30 farms were randomly selected from each of PE, NB, and NS (a total of 90 farms) based on a wide range of BLV within-herd prevalences (5). In 2014, a similar study was carried out on 43 purposively selected dairy farms in Quebec. These herds were selected from a pool of volunteer herds with a wide range of expected prevalences (according to the available data from historic BLV surveys in Quebec). One BTM sample was obtained from every selected farm, and on the same day of the BTM sampling, all lactating cows that contributed milk to the BTM were also individually sampled via the corresponding milk meters. Bulk-tank milk and individual cow milk samples (30 mL each) from PE, NB, and NS were transferred to the Maritime Quality Milk (MQM) laboratory in the University of Prince CVJ / VOL 57 / JULY 2016
Table 1. Descriptive summary for 133 study dairy herds that had all their lactating cows tested for bovine leukemia virus milk antibodies, from eastern provinces of Canada
New Brunswick Nova Scotia Prince Edward Island Quebec All 4 provinces
30 30 30 43 133
a Positive
23 30 28 14 14
51 65.5 52 56 52
Tested Positive Proportion cows cowsa (%)b
287 2232 214 2281 126 1598 145 2247 287 8358
845 460 555 801 2661
B R I E F C O M M U N I CAT I O N
Province
Lactating herd size Tested herds Min Median Max
37.8 20.2 34.7 35.6 32.1
in individual milk ELISA test (percent positivity . 45). of positive cows/number of tested cows.
b Number
[PP = (ODcorrected sample/ODcorrected positive control) 3 100] Where: OD = optical density. The statistical analyses were conducted in Stata 13.1 (StataCorp, College Station, Texas, USA). To determine the apparent within-herd prevalence of BLVantibodies (AP), number of positive cows (PP . 45) was divided by the number of lactating (tested) cows for every herd. True within-herd prevalence of BLV infection (TP) was then estimated for each of the selected herds using the following formula (12): TP = (AP 1 Sp 2 1)/(Se 1 Sp 2 1) Where: AP is the apparent prevalence of BLV infection within the study herds; sensitivity (Se) and specificity (Sp) of the individual ELISA test at the applied threshold of 45 were 95.5% and 98.4%, respectively (13). The true within-herd prevalence was regarded as the reference standard for evaluating the diagnostic performance of the pooled-level application of the BTM ELISA. If the true within herd prevalence was zero (i.e., all cows were negative), the herd was considered as uninfected; and if it was above zero (i.e., at least 1 positive cow was present), the herd was considered as infected with BLV. A two-graph receiver operating characteristic (TG-ROC) analysis was carried out to determine the optimal cut-point on BTM ELISA values using the defined dichotomized reference standard. Based on the true within-herd prevalence of BLV, 81.2% (108/133) of the study herds were found to be infected (i.e., true positive herds). Of 8358 cows tested (90% Holstein), 2661 (31.8%) were positive to BLV milk-antibodies in the individual cow tests. Descriptive statistics for the study herds, by province, are presented in Table 1. The mean of BLV true within-herd prevalence for the 108 infected herds was 0.39 (SD = 0.27). Figure 1 displays the distribution of BLV true within-herd prevalence for the study herds. From the TG-ROC analysis, maximum accuracy for BTM ELISA titers was achieved at 2.1 and 7.2 (percent positivity), CVJ / VOL 57 / JULY 2016
25
20
Number of herds
Edward Island, Charlottetown to be tested for BLV antibodies. All Quebec samples were submitted to the Valacta central laboratory (Sainte-Anne-de-Bellevue, Quebec) for BLV testing. All samples were preserved with BROTAB (Sierra Court, California, USA) and tested within a maximum of 7 d from the original sampling dates using a commercial indirect ELISA (Svanovir BLV gp51-Ab, Svanova, Uppsala, Sweden). The test results were reported as percent positivity (PP) values:
15
10
5
0
0
0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 ,
Within-herd prevalence (%)
Figure 1. Frequency distribution of the within-herd prevalence of infection with bovine leukemia virus for 133 study herds from the eastern provinces of Canada. The black bar represents uninfected herds (25/133).
respectively. Hence, the midpoint of 5 was considered as our practical cut-point value. At this cut-point, sensitivity and specificity of the BTM ELISA were estimated at 97.2% [95% confidence interval (CI): 92.1% to 99.4%] and 100% (95% CI: 86.3% to 100%), respectively. According to our established reference standard, 3 truly infected herds were negative by the BTM ELISA (defined as false negative herds). Each of those 3 herds harbored only 1 infected cow: 1 herd from NS (including 53 lactating cows), and 2 herds from QC (including 37 and 47 lactating cows). In stringent eradication programs, repeated sampling from bulk-tank over appropriate time intervals has been recommended in order to compensate for the imperfect sensitivity of the BTM tests, and to capture as many positive animals in a herd as possible (12). Addition or elimination of some cows can be influential on the BTM titers, such as those at advanced stages of BLV infection (e.g., cows with persistent lymphocytosis), because they often produce high levels of virus and circulating antibodies (14). However, it is generally believed that the prevalence of BLV in herds from endemic areas (e.g., North America) remains fairly steady over time (15); this characteristic supports the credibility of the current testing strategies (BTM ELISA) used in detecting BLV infection. We were not able to apply more sophisticated statistical analyses to our data in order to include some potentially important herd-level factors such as lactating herd size (representing the potential dilution effect of BTM) because there were only 3 false 779
C O M M U N I CAT I O N B R È V E
diagnoses by the BTM test. However, this issue should not lead to any substantial bias since the selected herds were fairly representative of the herds in eastern Canada with regards to the main characteristics, including herd size. For instance, the average lactating herd size in our study was 62.8, which was very close to the average of lactating herd size in all 4 study provinces (approximately 60). Applying a cut-point of 5 as the criterion for a negative herd test, when using the ELISA for BTM samples, generated reasonably valid results. Farmers whose farms are free from BLV and would desire to maintain their negative status or those who wish to monitor their decreasing trend of BLV prevalence on their farms (due to taking control measures) could efficiently adopt ongoing monitoring using the BTM ELISA test over appropriate time-intervals. Application of the BTM ELISA in other regions (particularly with large herds) should be validated before use in future surveillance programs.
Acknowledgments The authors gratefully acknowledge the contribution of all parties to this project, including study farmers, personnel at the MQM laboratory (Charlottetown, PE), Natasha Robinson, Shereen Hassan and other Valacta personnel (Sainte-Anne-de-Bellevue, QC). This study was funded by the MQM laboratory, Valacta, Dairy Farmers of Prince Edward Island, New Brunswick, and Nova Scotia, the New Brunswick Department of Agriculture, and Agriculture and Agri-Food Canada through the Agricultural Adaptation programs in Prince Edward Island and Nova Scotia. CVJ
References 1. Sandev N, Kostadinova N, Zarkov I. Economic problems concerning enzootic bovine leukosis. Agric Econ Manag 2000;45:38–41. 2. Bartlett PC, Sordillo LM, Byrem TM, et al. Options for the control of bovine leukemia virus in dairy cattle. J Am Vet Med Assoc 2014; 244:914–922.
780
3. Nekouei O, VanLeeuwen J, Sanchez J, Kelton D, Tiwari A, Keefe G. Herd-level risk factors for infection with bovine leukemia virus in canadian dairy herds. Prev Vet Med 2015;119:105–113. 4. VanLeeuwen JA, Keefe GP, Tremblay R, Power C, Wichtel JJ. Seroprevalence of infection with Mycobacterium avium subspecies paratuberculosis, bovine leukemia virus, and bovine viral diarrhea virus in maritime Canada dairy cattle. Can Vet J 2001;42:193–198. 5. Nekouei OA, Stryhn H, VanLeeuwen J, Kelton D, Hanna P, Keefe G. Predicting within-herd prevalence of infection with bovine leukemia virus using bulk-tank milk antibody levels. Prev Vet Med 2015; 122:53–60. 6. Sargeant JM, Kelton DF, Martin SW, Mann ED. Evaluation of a bulkmilk ELISA test for the classification of herd-level bovine leukemia virus status. Prev Vet Med 1997;31:211–221. 7. Sorge US, Lissemore K, Cantin R, Kelton DF. Milk ELISA status for bovine leukosis virus infection is not associated with milk production in dairy cows. J Dairy Sci 2011;94:5062–5064. 8. Gutierrez SE, Ferrer JF, Esteban EN, Rodriguez Dubra C, Dolcini GL, Arroyo GH. Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle. Am J Vet Res 2001;10:1571–1577. 9. Erskine RJ, Bartlett PC, Byrem TM, Render CL, Febvay C, Houseman JT. Using a herd profile to determine age-specific prevalence of bovine leukemia virus in Michigan dairy herds. Vet Med Int 2012;2012:350–374. 10. Christensen J, Gardner IA. Herd-level interpretation of test results for epidemiologic studies of animal diseases. Prev Vet Med 2000;45:83–106. 11. Greiner M, Gardner IA. Epidemiologic issues in the validation of veterinary diagnostic tests. Prev Vet Med 2000;45:3–22. 12. Dohoo IR, Martin SW, Stryhn H. Veterinary Epidemiologic Research. 2nd ed. Charlottetown, Prince Edward Island: VER Inc., 2009:91–133. 13. Nekouei OA, Sanchez J, Keefe GP, Carryover of bovine leukemia virus antibodies in samples from shared milk meters. J Dairy Sci 2015; 98:5274–5279. 14. Juliarena MA, Gutierrez SE, Ceriani C. Determination of proviral load in bovine leukemia virus-infected cattle with and without lymphocytosis. Am J Vet Res 2007;68:1220–1225. 15. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. 10th ed. New York, New York: Elsevier Saunders, 2006: 1209–1221.
CVJ / VOL 57 / JULY 2016
E
nzootic bovine leukosis (EBL), caused by bovine leukemia virus (BLV), is an economically important disease of dairy cattle. The virus is transmitted through infected blood lymphocytes. Premature culling, death, and condemnation of carcasses at slaughter due to lymphosarcoma, as well as restrictions on international trade of infected cattle and their products are among the most significant economic losses attributed to the disease (1,2). Many European countries are now officially free from EBL, whereas prevalence of the infection in North America has been high and appears to have a rising trend (2,3). For instance, in the Maritime region of Canada, herd-level prevalence of the infection has increased from 70% in 1998 (4) to over 90% in 2013 (5). Using bulk-tank milk (BTM) samples has become one of the most convenient and economically efficient procedures for screening for important diseases in dairy herds, including EBL (6,7). Among available commercial tests for detection of BLV antibodies, milk enzyme-linked immunosorbent assay (ELISA) has been documented as a desirable method with good per-
Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, C1A 4P3 (Nekouei, Keefe); R&D Team, Valacta, Sainte-Anne-de-Belevue, Quebec H9X 3R4 (Durocher). Address all correspondence to Dr. Omid Nekouei; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 778
(Traduit par Isabelle Vallières)
formance in large-scale surveillance programs (8,9). However, applying commercial ELISA tests, particularly to pooled samples (e.g., BTM), could lead to variable levels of uncertainty in the results. Several factors, including study region, herd (pool) size, sampling procedures, and transferring process can potentially contribute to the variable test results. Therefore, it has been recommended that the validity of diagnostic tests should be evaluated in different populations before integrating the tests in large-scale control and eradication programs (10,11). The objective of this study was to assess the diagnostic performance (sensitivity and specificity) of a commercially available ELISA for detecting BLV antibodies in BTM samples from eastern Canada, in order to validate the routine application of this test to the BLV surveillance programs implemented in the region. The study population consisted of 133 dairy herd improvement (DHI)-registered dairy herds, including 8358 lactating cows, from 4 eastern provinces of Canada [Prince Edward Island (PE), New Brunswick (NB), Nova Scotia (NS), and Quebec (QC)]. During 2013, 30 farms were randomly selected from each of PE, NB, and NS (a total of 90 farms) based on a wide range of BLV within-herd prevalences (5). In 2014, a similar study was carried out on 43 purposively selected dairy farms in Quebec. These herds were selected from a pool of volunteer herds with a wide range of expected prevalences (according to the available data from historic BLV surveys in Quebec). One BTM sample was obtained from every selected farm, and on the same day of the BTM sampling, all lactating cows that contributed milk to the BTM were also individually sampled via the corresponding milk meters. Bulk-tank milk and individual cow milk samples (30 mL each) from PE, NB, and NS were transferred to the Maritime Quality Milk (MQM) laboratory in the University of Prince CVJ / VOL 57 / JULY 2016
Table 1. Descriptive summary for 133 study dairy herds that had all their lactating cows tested for bovine leukemia virus milk antibodies, from eastern provinces of Canada
New Brunswick Nova Scotia Prince Edward Island Quebec All 4 provinces
30 30 30 43 133
a Positive
23 30 28 14 14
51 65.5 52 56 52
Tested Positive Proportion cows cowsa (%)b
287 2232 214 2281 126 1598 145 2247 287 8358
845 460 555 801 2661
B R I E F C O M M U N I CAT I O N
Province
Lactating herd size Tested herds Min Median Max
37.8 20.2 34.7 35.6 32.1
in individual milk ELISA test (percent positivity . 45). of positive cows/number of tested cows.
b Number
[PP = (ODcorrected sample/ODcorrected positive control) 3 100] Where: OD = optical density. The statistical analyses were conducted in Stata 13.1 (StataCorp, College Station, Texas, USA). To determine the apparent within-herd prevalence of BLVantibodies (AP), number of positive cows (PP . 45) was divided by the number of lactating (tested) cows for every herd. True within-herd prevalence of BLV infection (TP) was then estimated for each of the selected herds using the following formula (12): TP = (AP 1 Sp 2 1)/(Se 1 Sp 2 1) Where: AP is the apparent prevalence of BLV infection within the study herds; sensitivity (Se) and specificity (Sp) of the individual ELISA test at the applied threshold of 45 were 95.5% and 98.4%, respectively (13). The true within-herd prevalence was regarded as the reference standard for evaluating the diagnostic performance of the pooled-level application of the BTM ELISA. If the true within herd prevalence was zero (i.e., all cows were negative), the herd was considered as uninfected; and if it was above zero (i.e., at least 1 positive cow was present), the herd was considered as infected with BLV. A two-graph receiver operating characteristic (TG-ROC) analysis was carried out to determine the optimal cut-point on BTM ELISA values using the defined dichotomized reference standard. Based on the true within-herd prevalence of BLV, 81.2% (108/133) of the study herds were found to be infected (i.e., true positive herds). Of 8358 cows tested (90% Holstein), 2661 (31.8%) were positive to BLV milk-antibodies in the individual cow tests. Descriptive statistics for the study herds, by province, are presented in Table 1. The mean of BLV true within-herd prevalence for the 108 infected herds was 0.39 (SD = 0.27). Figure 1 displays the distribution of BLV true within-herd prevalence for the study herds. From the TG-ROC analysis, maximum accuracy for BTM ELISA titers was achieved at 2.1 and 7.2 (percent positivity), CVJ / VOL 57 / JULY 2016
25
20
Number of herds
Edward Island, Charlottetown to be tested for BLV antibodies. All Quebec samples were submitted to the Valacta central laboratory (Sainte-Anne-de-Bellevue, Quebec) for BLV testing. All samples were preserved with BROTAB (Sierra Court, California, USA) and tested within a maximum of 7 d from the original sampling dates using a commercial indirect ELISA (Svanovir BLV gp51-Ab, Svanova, Uppsala, Sweden). The test results were reported as percent positivity (PP) values:
15
10
5
0
0
0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 ,
Within-herd prevalence (%)
Figure 1. Frequency distribution of the within-herd prevalence of infection with bovine leukemia virus for 133 study herds from the eastern provinces of Canada. The black bar represents uninfected herds (25/133).
respectively. Hence, the midpoint of 5 was considered as our practical cut-point value. At this cut-point, sensitivity and specificity of the BTM ELISA were estimated at 97.2% [95% confidence interval (CI): 92.1% to 99.4%] and 100% (95% CI: 86.3% to 100%), respectively. According to our established reference standard, 3 truly infected herds were negative by the BTM ELISA (defined as false negative herds). Each of those 3 herds harbored only 1 infected cow: 1 herd from NS (including 53 lactating cows), and 2 herds from QC (including 37 and 47 lactating cows). In stringent eradication programs, repeated sampling from bulk-tank over appropriate time intervals has been recommended in order to compensate for the imperfect sensitivity of the BTM tests, and to capture as many positive animals in a herd as possible (12). Addition or elimination of some cows can be influential on the BTM titers, such as those at advanced stages of BLV infection (e.g., cows with persistent lymphocytosis), because they often produce high levels of virus and circulating antibodies (14). However, it is generally believed that the prevalence of BLV in herds from endemic areas (e.g., North America) remains fairly steady over time (15); this characteristic supports the credibility of the current testing strategies (BTM ELISA) used in detecting BLV infection. We were not able to apply more sophisticated statistical analyses to our data in order to include some potentially important herd-level factors such as lactating herd size (representing the potential dilution effect of BTM) because there were only 3 false 779
C O M M U N I CAT I O N B R È V E
diagnoses by the BTM test. However, this issue should not lead to any substantial bias since the selected herds were fairly representative of the herds in eastern Canada with regards to the main characteristics, including herd size. For instance, the average lactating herd size in our study was 62.8, which was very close to the average of lactating herd size in all 4 study provinces (approximately 60). Applying a cut-point of 5 as the criterion for a negative herd test, when using the ELISA for BTM samples, generated reasonably valid results. Farmers whose farms are free from BLV and would desire to maintain their negative status or those who wish to monitor their decreasing trend of BLV prevalence on their farms (due to taking control measures) could efficiently adopt ongoing monitoring using the BTM ELISA test over appropriate time-intervals. Application of the BTM ELISA in other regions (particularly with large herds) should be validated before use in future surveillance programs.
Acknowledgments The authors gratefully acknowledge the contribution of all parties to this project, including study farmers, personnel at the MQM laboratory (Charlottetown, PE), Natasha Robinson, Shereen Hassan and other Valacta personnel (Sainte-Anne-de-Bellevue, QC). This study was funded by the MQM laboratory, Valacta, Dairy Farmers of Prince Edward Island, New Brunswick, and Nova Scotia, the New Brunswick Department of Agriculture, and Agriculture and Agri-Food Canada through the Agricultural Adaptation programs in Prince Edward Island and Nova Scotia. CVJ
References 1. Sandev N, Kostadinova N, Zarkov I. Economic problems concerning enzootic bovine leukosis. Agric Econ Manag 2000;45:38–41. 2. Bartlett PC, Sordillo LM, Byrem TM, et al. Options for the control of bovine leukemia virus in dairy cattle. J Am Vet Med Assoc 2014; 244:914–922.
780
3. Nekouei O, VanLeeuwen J, Sanchez J, Kelton D, Tiwari A, Keefe G. Herd-level risk factors for infection with bovine leukemia virus in canadian dairy herds. Prev Vet Med 2015;119:105–113. 4. VanLeeuwen JA, Keefe GP, Tremblay R, Power C, Wichtel JJ. Seroprevalence of infection with Mycobacterium avium subspecies paratuberculosis, bovine leukemia virus, and bovine viral diarrhea virus in maritime Canada dairy cattle. Can Vet J 2001;42:193–198. 5. Nekouei OA, Stryhn H, VanLeeuwen J, Kelton D, Hanna P, Keefe G. Predicting within-herd prevalence of infection with bovine leukemia virus using bulk-tank milk antibody levels. Prev Vet Med 2015; 122:53–60. 6. Sargeant JM, Kelton DF, Martin SW, Mann ED. Evaluation of a bulkmilk ELISA test for the classification of herd-level bovine leukemia virus status. Prev Vet Med 1997;31:211–221. 7. Sorge US, Lissemore K, Cantin R, Kelton DF. Milk ELISA status for bovine leukosis virus infection is not associated with milk production in dairy cows. J Dairy Sci 2011;94:5062–5064. 8. Gutierrez SE, Ferrer JF, Esteban EN, Rodriguez Dubra C, Dolcini GL, Arroyo GH. Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle. Am J Vet Res 2001;10:1571–1577. 9. Erskine RJ, Bartlett PC, Byrem TM, Render CL, Febvay C, Houseman JT. Using a herd profile to determine age-specific prevalence of bovine leukemia virus in Michigan dairy herds. Vet Med Int 2012;2012:350–374. 10. Christensen J, Gardner IA. Herd-level interpretation of test results for epidemiologic studies of animal diseases. Prev Vet Med 2000;45:83–106. 11. Greiner M, Gardner IA. Epidemiologic issues in the validation of veterinary diagnostic tests. Prev Vet Med 2000;45:3–22. 12. Dohoo IR, Martin SW, Stryhn H. Veterinary Epidemiologic Research. 2nd ed. Charlottetown, Prince Edward Island: VER Inc., 2009:91–133. 13. Nekouei OA, Sanchez J, Keefe GP, Carryover of bovine leukemia virus antibodies in samples from shared milk meters. J Dairy Sci 2015; 98:5274–5279. 14. Juliarena MA, Gutierrez SE, Ceriani C. Determination of proviral load in bovine leukemia virus-infected cattle with and without lymphocytosis. Am J Vet Res 2007;68:1220–1225. 15. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. 10th ed. New York, New York: Elsevier Saunders, 2006: 1209–1221.
CVJ / VOL 57 / JULY 2016
