Comparison of the validity of different serological and molecular tests for Bovine Leukemia Virus in the Xth region of Chile Universidad Austral de Chile, Medicina Preventiva de Ciencias Veterinarias, October-December 2007. Carolien Amperse Summary Bovine leukemia virus (BLV) is a retrovirus that causes economic losses in the livestock sector. Transmission occurs mainly horizontally via infected blood cells. Infected animals can be healthy carriers, have persistent leucocytosis, or have lymphosarcomas. The objective of the study was to compare the validity of different serological and molecular tests to find out which test could best be used in different stages of a BLV controlling and eradication program in Chile. Milk, serum and EDTA blood were collected from 400 dairy cows, originating from 72 different farms in the Xth region of Chile. An agar gel immunodiffusion (AGID) test, enzyme-linked immunosorbent assay (ELISA) serum test and ELISA milk test were compared to a PCR test, which was chosen as the golden standard. Sensitivity (SE), specificity (SP) and, for the ELISA tests, ROC curves and optimal cut-off points were calculated. The SE and SP for ELISA serum were respectively 90,8 (87,6; 94,0) and 78,3 (71,2; 85,4), for ELISA milk 88,0 (84,4; 91,6) and 77,5 (70,3; 84,7) and for AGID 85,2 (81,1; 89,2) and 86,1 (79,9; 92,2). The area under the curve was for the ELISA serum 0,884 (0,845; 0,922) and for ELISA milk 0.862 (0,821; 0,902). Statistically the test were comparable to each other, with slight differences between them. The tests that were validated in this study were not optimal for a complete eradication program, because sensitivity and specificity are not one hundred percent. Therefore, complementary management measures are critical to reduce transmission. 1. Introduction Bovine Leukemia Virus (BLV) is a retrovirus that causes economic losses in the livestock sector. Transmission occurs mainly horizontally via infected blood lymphocytes, but vertical transmission also takes place. There are three possible pathological forms after infection: asymptomatic course, persistent leucocytosis and lymphosarcomas. These three forms can follow each other, or occur without preceding other symptoms. The virus is distributed worldwide and under natural conditions only cattle, zebus, buffaloes and capybaras have been found to be infected (Miller 1990). The virus produces a large economical impact on the livestock sector of many countries in the world. These costs can be classified in direct and indirect costs. Direct costs are those costs associated with infection, including treatment and diagnostic costs, reduced performance, losses from dead animals or early replacement and veterinary costs due to treatment and diagnosis. Indirect costs are costs associated with: losses of revenues attributable to restrictions of export of cattle and cattle products (semen and embryos) (Thurmond 1987), blood collection, laboratory testing to fulfill export regulations, loss of genetic potential, an extended generation interval and costs to society for maintenance of regulatory agencies and research (Pelzer 1997). Milk production in Chile is an important economic activity and is for many smallholder dairy farmers an important mean of subsistence. Although dairy farmers are established all over the country, more than 80 % of the milk is produced in the southern regions (IXth and Xth) where 73.2 % of total dairy cows are concentrated (ODEPA 2003). Thirty per cent of the national production and forty per cent of the national dairy product exportation are produced in Xth Province (Anrique 1999). Although there are some non random prevalence studies (studies carried out in a specific population), the true prevalence of BLV in the Xth region remains unknown. There is also no published field validation of BLV tests in Chile. An accurate estimate of the true prevalence is essential information for future testing schemes, estimate differences between farms, regions or countries and to estimate changes in BLV prevalence over time, for example after applying mitigation measures. Validation of a diagnostic test has important implications for determination of true prevalence or probabilities of being infected for a given individual from a population, disease surveillance and monitoring, decision making and risk analysis. For those reasons reliable estimates of test performance are required. The objective of the study was to compare the validity of different serological and molecular test to find out which test could best be used in different stages of a BLV controlling and eradication program in Chile. 2. Material and Methods Study population Milk, serum and EDTA blood were collected of 400 adult cows originating from 72 different farms in the Xth region of Chile. The aim was to select the farms randomly, however because of practical reasons this was not possible. It was tried to get the best representative sample of farms as they exist in the Xth region. On a farm, the cows were randomly selected, excluding calfs younger than 6 months and oxen. Then the blood was tested for Bovine Leukosis with PCR, and the serum and milk were tested with two different kits of enzyme-linked immunosorbent assay (ELISA) and with agar gel immunodiffusion (AGID). PCR-primers Oligonucleotide primers for PCR were designed from published sequence data. Primers corresponding to the env and ltr genes were selected because this region is highly conserved among different BLV provirus isolates (Rice 1984). Forward primers were env5032 5’ – TCT GTG CCA AGT CTC CCA GAT A – 3’, and env5099 5’ – CCC ACA AGG GCG GCG CCG GTT T – 3’. The reverse primers were env5521r 5’ – GCG AGG CCG GGT CCA GAG CTG G – 3’ and env5608r 5’ – AAC AAC AAC CTC TGG GAA GGG T – 3’. The set env5099 and env5521 was established and described previously. The ltr primer sequences were: ltrB1 5’ – TGT ATG AAA GAT CAT GCC GAC CTA G – 3’, ltrB506 5’ – GGT CTC TCC TGG CCG CTA GA-3’, ltrB42 5’ – GTA AAC CAG ACA GAG ACG TCA GCT – 3;. All primers are commercially available (Monti 2005). PCR DNA was obtained from frozen blood collected with EDTA. The DNA was extracted by using the NucleoSpin Blood kit. The initial round of nested-PCRs was performed using (env5032 / env5608r) as first primers; incubation of samples was at 72˚C for 2 minutes; denaturation at 94˚C for 2 minutes; then 50 amplification cycles consisting of denaturation at 95˚C, 30 seconds, primer annealing at 58˚C, 30 seconds and extension at 72˚C for 1 minute and final extension for 4 minutes. For ltr primers, conditions for first round (ltrB1 / ltrB506) were set similarly to those for env primers except that primer annealing was done at 62˚C. The second round of amplifications was carried out using the second pair of primers (env5099 / env5521r). The second round conditions were set as the first round except for primer annealing temperature that was switched to 72˚C. Conditions in the second round (ltrB42 / ltrB478) for annealing were set to 62˚C as well. A sample was considered as positive when any of the two PCR assays (env or ltr primers) tested positive (Monti 2005). Known positive and negative control DNA samples were included in each test run and samples that showed a band migrating at 444 base pairs (bp) were considered as positive. PCR was performed in the laboratory at the Microbiology Department of the Universidad Austral de Chile. Serological tests Agar gel immunodiffusion (AGID) test A one per cent agar gel was prepared and mounted on a glass slide (13 x 10 cm) with a punching design that allows 48 samples to be tested simultaneously. Slides were incubated in a humidified chamber, and were read at 24 and 48 hours. Two experienced technicians interpreted the test reactions under a light beam in a dark room. To avoid verification bias, the readings were made independently by both technicians and samples in which disagreement was observed were not re-tested to avoid review bias and they were considered as incoherent. Test results were reported as positive, negative, and doubtful. There were some samples where the technicians were unsure about the result and these results were scored as doubtful. The incoherent and doubtful results were excluded from the calculations. Enzyme-linked immunosorbent assay (ELISA) test For serum and milk, two different types of ELISA were used. Idexx CHEKIT Leucose Serum Antibody Test Kit was used for the serum and CHEKIT Leukotest Verification Milk Antibody ELISA was used for the milk. Microtiter plates were supplied precoated with inactivated antigen. Dilutions of the samples to be tested were incubated in the wells of these plates. Any antibody specific for BLV bound to the antigen in the wells and formed an antigen/antibody complex on the plate well surface. Unbound material was removed from the wells by washing. A peroxidase-labeled anti-ruminant IgG conjugate was added, which bound to the antibodies complexed with the BLV antigen. Unbound conjugate was removed by washing, and the TMB containing substrate was added to the wells. The degree of colour that developed (optical density measured at 450 nm), was directly proportional to the amount of antibody specific for BLV present in the sample. The diagnostic relevance of the result was obtained by comparing the optical density (OD) of the samples with the OD of the positive control (protocol IDEXX Checkit Leucose) After two months of research, a big fire took place in one of the buildings of the UACH. In this fire all of the kits and a lot of the samples were completely burned. Due to this, the data used in this study were data from a comparable study in Argentina. Statistical analysis The data of the different tests were entered in Microsoft Excel. Then, with use of SPSS 15.0 for Windows, a receiver operating characteristic (ROC) curve was plotted. The definitive status of an animal was confirmed with a PCR test. The results of the different ELISA tests were compared with the PCR test and the ROC curve was plotted. Also the sensitivity and specificity were calculated with 2-by-2 tables. 3. Results Part of the results are summarized in table 1. The sensitivity (SE) and specificity (SP) of the ELISA serum test are respectively 90,8 and 78,3 when they are compared with the PCR (which was used as a gold standard). For the ELISA milk test the SE and SP are 88,0 and 77,5 and for the AGID test they are 85, 2 and 86,1. T able 1. Sensitivity and specificity of different tests for the detection of BLV, based on PCR as a gold standard SE 95% C.I. SP 95% C.I. ELISA serum 90,8 87,6-94,0 78,3 71,2-85,4 ELISA milk 88,0 84,4-91,6 77,5 70,3-84,7 AGID 85,2 81,1-89,2 86,1 79,9-92,2 In figure 1a and 1b the ROC Curves of ELISA serum and ELISA milk are shown. In table 2 the area under the curve of both ELISA’s is shown, including a 95% confidence interval. For the serum ELISA the area under the curve is 0,884 (95% confidence interval ranges from 0,845 until 0.922) and for the milk ELISA 0,862 (0,821; 0,902) In table 3a and 3b part of the coordinates of the ROC curves are shown. The complete tables can be found in the Appendix. Figure 1. ROC curves for ELISA serum (left) and ELISA milk (right). Table 2. Area under the curve of the ELISA serum and ELISA milk test and a 95% confidence interval Asymptotic 95% Confidence Interval Area Upper Bound Lower Bound ELISA serum 0,884 0,845 0,922 ELISA milk 0,862 0,821 0,902 Table 3a. Coordinates of the Curve of ELISA serum OD: Positive if Greater Than or Equal To(a) Sensitivity 1 - Specificity 73,2850 0,850 0,163 74,6075 0,850 0,155 78,7765 0,838 0,155 84,2055 0,825 0,155 86,8715 0,815 0,147 95,7915 0,790 0,147 97,4635 0,780 0,140 112,4625 0,701 0,140 113,8885 0,691 0,132 116,0855 0,682 0,124 118,6515 0,669 0,124 122,3735 0,653 0,116 123,3490 0,640 0,116 124,7015 0,631 0,109 126,1580 0,621 0,101 127,6050 0,611 0,093 130,3405 0,586 0,093 132,8725 0,576 0,085 134,1375 0,564 0,085 136,5065 0,554 0,078 139,2755 0,541 0,078 141,3095 0,532 0,070 151,3065 0,439 0,070 152,8525 0,430 0,062 157,7790 0,366 0,062 159,1370 0,360 0,047 (a) All cutoff values are the averages of two consecutive ordered observed test values. Table 3b. Coordinates of the Curve of ELISA milk Positive if Greater Than or Equal To(a) Sensitivity 1 - Specificity 168,8570 0,748 0,163 169,8945 0,744 0,155 175,3295 0,735 0,147 176,4680 0,722 0,147 180,3160 0,712 0,140 184,0720 0,702 0,132 186,6570 0,693 0,116 208,6940 0,641 0,116 214,5395 0,628 0,116 217,2635 0,618 0,109 220,8400 0,608 0,101 225,5100 0,583 0,101 226,7915 0,573 0,093 229,2765 0,560 0,093 231,4525 0,550 0,085 232,6155 0,537 0,085 236,8515 0,528 0,078 239,2070 0,518 0,070 242,4335 0,508 0,062 254,4110 0,469 0,062 266,7720 0,430 0,062 270,4890 0,421 0,054 274,6555 0,408 0,054 277,0995 0,405 0,047 (a) All cutoff values are the averages of two consecutive ordered observed test values. 4. Discussion The first thing to address here of course is that the results are not from Chile but from Argentina. There are a lot of differences between the two countries in the way they manage their farms and in characteristics of the farms. The way they manage their farms is of big influence of the spread of BLV. Also the characteristics of the farm are of big influence, for example if different farmers use the same bull and the bull is BLV positive, the disease can spread faster. Besides that, there are different types of BLV and Chile and Argentina may not have the same types. In addition, it is not known whether the test will have the same sensitivity and specificity for different types of the virus. For the sampling in Chile, the farms were not randomly selected because of practical reasons. We tried to get a good representation for the different kind of farms (number of cows, way of managing, different areas etcetera) but especially the small farm holders were difficult to reach. They are not registered, sometimes they did not have a phone and the distances in Chile did not make it possible to reach them personally. PCR was chosen to be the gold standard because it is an extremely specific test. We did not use different kind of mathematical models to estimate the conditional independence and conditional dependence between tests because it would have taken to much time. Unfortunately, the PCR test in this study was not as sensitive as it was hoped for. One of the reasons is, that the sensitivity of the PCR is highly dependent upon the design and execution of the primers in the PCR (Marsolais 1994). Another reason is that less than 10% of the lymphocytes of BLV infected cattle has been found infected (Mirsky 1996). These levels can be below the detection level of PCR but still might elicit a detectable immune response, so the ELISA tests can be positive. And in this study they will be named false positive, which is not true. In this study, the ELISA serum was found to be the most sensitive test with a sensitivity of 90,8 per cent. The ELISA milk had a sensitivity of 88,0 per cent. This is probably because the antibody level in milk is lower than in serum. The AGID had a sensitivity of 85,2 per cent. But the 95 % confidence intervals showed that the differences between de three test are very small and non-significant. Because the prevalence of BLV infection in Chile is not known, the first thing that is needed is a cheap test, which is easily usable. The sensitivity and specificity are less important because the outcome of the test is the apparent prevalence, and it is possible to correct the outcome for the sensitivity and specificity to get the true prevalence. The ELISA milk is a good test for this stage, because it is a cheap test and it is possible to screen bulk milk samples. The second stage of the controlling and eradication program is to control the infection. Therefore the sensitivity and the specificity have to be high. The AGID test can be used for this stage of the program, because both the sensitivity and the specificity are quite high. Another possibility is to change the cut-off point of the ELISA serum or the ELISA milk test to improve the specificity (table 3a and 3b). For ELISA serum test the cut-off point could be an OD of 126, then the sensitivity is 62,1 per cent and the specificity is 89,9 per cent. If the cut-off point of ELISA milk test is changed the sensitivity and specificity are slightly poorer than the ELISA serum (sensitivity 60,8 per cent and specificity 89,9 per cent at an OD of 221) but considering the practical advantages it would be a good option to use this test for screening as well. An other option is to first use the ELISA serum test when the cut-off point is changed to an optical density with a high sensitivity and a lower specificity, and after that, to test the positive animals again with the PCR test. In this study the quantitative characteristics of the PCR test are not known, but it is known that a PCR normally has a high specificity, while the sensitivity is not always high. Because none of the tests performed very well in this study, it is always the question whether the sensitivity or the specificity is more important. If the sensitivity is high, in this study it means that there is a poor specificity and vice versa. A poor specificity will lead to a lot of false-positive animals, thus the farmer will exclude a lot of healthy animals from his herd. If there is a poor sensitivity there will be a lot of false-negative tests results. That means that there will remain a lot of BLV infected animals in the herd. In case of BLV in Chile it has to be considered that BLV is a disease that spreads slowly if the management factors are optimal. It also has to be considered that in Chile the economic impact of the culling of a cow is very high and that it is not clear if the government will help financially to get the country free of BLV. So in this case it is possible to choose a high specificity with a lower sensitivity, and hopefully the falsenegative animals that stayed in the herd will be found in the next test round. In the third phase, when the infection is almost controlled, the ELISA serum can be used for certifying BLV-free status because of its high sensitivity. The specificity is not really high (78,3 per cent) but animals that are tested positive with this test should be tested again with another kind of test to increase the specificity. An advantage of the ELISA milk is that it needs a non-invasive technique of sampling. In an infected herd this could be preferred as in Chile it is not common practice to change needles between every animal. Another advantage of the ELISA milk is that it is possible to use it for a bulk milk sample. The test can find 1 positive animal out of a pool of 20 seronegative samples (test and results are not described in this article). Therefore it is a cheap way of screening in large epidemiological studies or monitoring programs. The most critical part of the eradication program is the last step, where almost every positive animal is traced. Because the results of the tests in this study are not optimal for the last part of eradication, complementary measures will make the difference for the program to succeed or not. If a farmer wants to get his herd free of BLV, he has to take hygienic measures like changing needles between every animal and feed calves with milk from cows that are free of BLV. He also has to separate the newborn calves until they can be tested for BLV. Only then the herd will eventually be free because the last infected animals will be replaced in time. 5. Acknowledgements First, I would like to thank Gustavo Monti for his hospitality and for making us part of his family for three months. 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