Small ruminant lentiviruses (SRLV) are a group of viruses of the
The main target cells for SRLV are monocytes and macrophages. Following the transformation of its RNA to DNA by the viral reverse transcriptase, the SRLV genome integrates as a provirus into the monocyte and macrophage genomes. The SRLV genome is comprised of three structural genes and three accessory genes. The structure is formed by
Infection with SRLV persists throughout life and there is no treatment for it or vaccine against it. Therefore, the use of control programmes is the only way to avoid the spread of SRLV infection. These programmes rely on diagnostic tools to identify positive animals so that they may be eliminated. Therefore, the use of an appropriate test is crucial for the effective prevention and control of SRLV infections. Detection of SRLV infections is most commonly achieved using serological methods that detect antibodies to SRLV, while detection of the integrated provirus in infected monocytes and macrophages can be accomplished using PCR strategies. The two most commonly used tests for detecting specific antibodies against SRLV are agar gel immunodiffusion and enzyme-linked immunosorbent assays (ELISA). Agar gel immunodiffusion is highly specific, but its sensitivity is low, so it is increasingly being replaced by ELISA because of the immunosorbent assay’s good sensitivity, objectivity and ability to be automated. In addition, it is easy to perform and results are obtained quickly. Most diagnostic ELISAs use recombinant capsid and/or transmembrane proteins as antigens (27). More recently, real-time PCR assays have been developed for quantitative, sensitive, rapid and large-scale detection of SRLV (3, 5, 9, 10, 18, 20). However, despite advances in diagnostic techniques for detecting SRLV, there is no test capable of detecting all strains. This is mainly due to the high genetic and antigenic heterogeneity of these viruses. Many of the currently available diagnostic tests are still based on a monostrain format (genotype A or B), as it was assumed that the antigens of either strain can detect antibodies against both MVV and CAEV in infected animals’ sera (14, 38). However, it has been observed that ELISAs are more sensitive and specific when homologous antigens are used rather than heterologous ones (21, 37). The low cross-reactivity between genotype-mismatched SRLV antigen and antibody pairs starkly limits the diagnostic performance of monostrain ELISAs in a population where animals are infected with an SRLV genotype different from the one used in the test (6, 21, 37, 39). The high heterogeneity of the SRLV genome also hinders the usefulness of PCR to detect all SRLV strains, so it is suggested that PCRs should rather be developed based on strains circulating in a given area. Therefore, information on circulating genotypes would be helpful in selecting appropriate tests, especially in areas where genetic testing has not been conducted.
Current tools for SRLV characterisation include partial region sequencing and heteroduplex mobility assays, but these methods are quite complicated, take a long time and are not suitable for routine diagnosis (11, 28). Recently, the initial classification of A and B SRLV genotypes has become achievable by ELISA. To date, two such ELISAs have been developed, one based on the matrix and the other on the capsid epitope (17, 25). In addition, ELISAs based on the variable SU5 protein can be used as serotyping tools to provide information on SRLV subtypes, because the SU5 epitope is considered subtype specific (7, 25). Unfortunately, almost all of these ELISA tests have been developed in-house by non-commercial organisations and are not available worldwide. There is only one commercial test (the Eradikit SRLV Genotyping ELISA offered by In3 Diagnostic, Turin, Italy) that can distinguish between the A, B and E SRLV genotypes. However, its serotyping efficiency has not been well defined. In addition to serological tests, PCR protocols have been recently developed to detect the A and B genotypes and distinguish between them. This method uses genotype-specific primers and probes to detect genotype-specific nucleotide sequences (1, 10, 20, 40).
With the intention of simplifying the serotyping of SRLV field isolates and thereby improving the effectiveness of control programmes, this study investigated the serotyping efficiency of the Eradikit SRLV Genotyping ELISA test and the molecular typing efficiency of the newly developed real-time PCR targeting the LTR-
A panel of 183 fully typed samples was included in this study. This panel comprised 86 peripheral blood leukocyte (PBL) pellet samples originating from 31 sheep and 55 goats infected with SRLV, and 97 SRLV-positive serum samples originating from 34 sheep and 63 goats. All 183 samples were retrieved from a frozen collection at the National Veterinary Research Institute (Pulawy, Poland). The genotype of each of the 183 samples was assigned by genetic analysis based on
The DNA extracted from the 86 PBL samples was tested using a nested real-time PCR. The reaction was performed as previously described by Schaer
A reference plasmid encompassing the target LTR-gag region was used to generate a standard curve based on 10-fold serial dilutions of plasmid DNA from 109 to 101. The templates were obtained after amplification of samples originating from animals naturally infected with subtype A5 of genotype A or subtype B1 of genotype B of SRLV using primers designed for the real-time PCR as described above. After amplification, PCR products were analysed by electrophoresis on 2% agarose gel, and after purification were cloned into the pDRIVE vector with a TA cloning kit (Qiagen). Following transformation, the plasmid was isolated and linearised with
The 97 serum samples were tested using the Eradikit SRLV Genotyping kit. In this ELISA, separate strips of wells in the plates are coated with immunodominant epitopes of capsid antigens specific for genotypes A, B or E. Briefly, samples were diluted 1 : 20 in sample diluent and incubated for 60 min at 37°C. Following three washes, a peroxidase-labelled anti sheep/goat IgG antibody was added and the plate was incubated for 60 min at 37°C. After washing, the substrate was added, the mixture was incubated for 15 min and the colorimetric reaction was read at 405 nm. The results were calculated using an Excel file for automatic calculations downloadable from the manufacturer’s website. The results were given as inconclusive, positive for one (A, B or E) or indeterminate.
Of the PBL samples, 73 were from animals infected with known SRLV strains representing subtypes A1, A5, A12, A13, A16, A17, A18, A23, A24 and A27 of genotype A, and 13 were from animals infected with subtypes B1 and B2 of genotype B. All samples were tested separately with the primers and probe specific for MVV (MVV assay) and separately with the primers and probe specific for CAEV (CAEV assay). Of the 86 samples tested, 69 gave positive results by nested real-time PCR; however, in the remaining 17 (19.8%) proviral DNA of SRLV was not detected. Therefore, the diagnostic sensitivity of this PCR was 80.2% (95% confidence interval (CI) 71.6%–88.8%). The samples that yielded negative results represented subtypes A1 (n = 3), A12 (n = 3), A13 (n = 1), A17 (n = 3), A27 (n = 1), B1 (n = 2) and B2 (n = 4). Among the 73 genotype A (MVV) samples, 62 (84.9%) gave positive results with primers and a probe specific for MVV. In the other 11 (15.1%), neither MVV nor CAEV was detected. None of the 73 genotype A samples tested positive using the primers and probe specific for CAEV. Subtypes A5, A16, A18, A23 and A24 were detected without fail, while 86.0%, 81.2%, 77.0% and 75.0% of the instances of subtypes A13, A12, A17 and A27 were detected, respectively. Only 25% of the occurrences of subtype A1 were detected. Regarding samples representing subtype B, 7 out of 13 (53.8%) were positive with the primers and probe specific for CAEV, but in the remaining 6 (46.2%), neither CAEV nor MVV was detected. None of the samples tested positive using the primers and probe specific for MVV (Table 1). The detection rate of subtype B1 was 60.0% and that of subtype B2 was 50.0%. Agreement between the nested real-time PCR and the prior phylogenetic analysis was assessed by calculating the kappa coefficient. When all 86 samples were analysed, the kappa coefficient was estimated as 0.47 (95% CI 0.31–0.63), indicating moderate concordance. When only PCR-positive samples (69) were analysed, the kappa was 1.00 (95% CI 1.00–1.00), indicating that the differentiation between MVV (genotype A) and CAEV (genotype B) by real-time PCR was 100% concordant with the phylogenetic analysis.
Nested real-time PCR and ELISA SRLV genotyping results for classification of peripheral blood lymphocyte and serum samples of sheep and goats
Test type | Samples (n) | Sample genotype (n) | Positive for genotype A (MVV) (n) | Positive for genotype B (CAEV) (n) | Positive for genotype E (n) | Inconclusive or indeterminate (n) | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
A | B | Genotyped previously as A | Genotyped previously as B | Genotyped previously as A | Genotyped previously as B | Genotyped previously as A | Genotyped previously as B | Genotyped previously as A | Genotyped previously as B | ||
Nested realtime PCR | 86 | 73 | — | 62 (84.9%) | — | 0 | — | n/a | — | 11 (15.1%) | — |
— | 13 | — | 0 | — | 7 (53.8%) | — | n/a | — | 6 (46.2%) | ||
Eradikit SRLV Genotyping ELISA | 97 | 82 | — | 30 (36.6%) | — | 14 (17.1%) | — | 1 (1.2%) | — | 37 (45.1%) | — |
— | 15 | — | 2 (13.3%) | — | 11 (73.3%) | — | 0 | — | 2 (13.3%) |
The analytical sensitivity of the MVV and CAEV assays was evaluated using plasmid DNA carrying MVV-like (subtype A5) and CAEV-like (subtype B1) DNA, respectively. Both assays were able to detect fewer than five copies per reaction. The reaction efficiencies of the MVV assay ranged from 88.0% to 100% and its R2 was 0.986–0.996. The CAEV assay showed reaction efficiency in a 73.5%–95.5% range and an R2 of 0.981–0.994.
The total number of serum samples with a concordant genotype result in the phylogenetic analysis and the present serological test was 41 (out of 97 – 42.3%). As many as 17 out of 97 (17.5%) samples were incorrectly classified. The kappa value of the agreement between the previous phylogenetic analysis and the Eradikit test results was 0.15 (95% CI 0.05–0.28), indicating poor agreement. Samples totaling 30 out of the 82 (36.6%) of genotype A were correctly classified as MVV infected, but 14 (17.1%) and 1 out of 82 (1.2%) of the genotype A-infected sera were misclassified as genotype B-infected and genotype E-infected sera, respectively. The sera of genotype B in 11 out of 15 (73.3%) instances were correctly classified as CAEV infected, but 2 out of 13 (13.3%) genotype B-infected sera were misclassified as MVV infected (Table 1). The test was unable to classify 39 out of 97 (40.2%) of samples, which were categorised as inconclusive or indeterminate. The inconclusive results were those for sera of which the optical density (OD) values did not exceed 0.4 for at least one antigen. The indeterminate results were those for samples that showed reactivity to more than one antigen and yielded differences between OD values of <40%. Most of the indeterminate samples (60%) showed high reactivity to all antigens of genotypes A, B and E. Detailed information on inconclusive and indeterminate results is shown in Table 2.
Inconclusive and indeterminate results obtained using the Eradikit SRLV Genotyping kit for classification of peripheral blood lymphocyte and serum samples of sheep and goats
Inconclusive OD | Indeterminate OD | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
No. | Subtypes | Host | Genotype A | Genotype B | Genotype E | Subtypes | Host | Genotype A | Genotype B | Genotype E | |
1. | A1 | sheep | 0.163 | 0.33 | 0.26 | A1 | goat | 1.111 | 0.859 | 0.716 | |
2. | A5 | goat | 0.251 | 0.202 | 0.172 | A5 | goat | 0.589 | 0.777 | 0.634 | |
3. | A5 | goat | 0.291 | 0.359 | 0.255 | A5 | goat | 2.325 | 2.471 | 1.023 | |
4. | A5 | goat | 0.323 | 0.262 | 0.21 | A5 | goat | 1.806 | 1.726 | 0.265 | |
5. | A5 | goat | 0.144 | 0.246 | 0.142 | A12 | goat | 1.028 | 0.296 | 0.836 | |
6. | A12 | sheep | 0.169 | 0.384 | 0.206 | A12 | goat | 1.017 | 1.334 | 0.83 | |
7. | A12 | sheep | 0.193 | 0.347 | 0.152 | A12 | goat | 1.012 | 0.981 | 0.952 | |
8. | A12 | sheep | 0.178 | 0.236 | 0.29 | A12 | goat | 2.561 | 2.494 | 1.142 | |
9. | A13 | sheep | 0.191 | 0.162 | 0.297 | A13 | sheep | 1.256 | 1.026 | 1.159 | |
10. | A17 | goat | 0.393 | 0.344 | 0.297 | A13 | sheep | 0.775 | 0.82 | 0.701 | |
11. | A17 | goat | 0.336 | 0.341 | 0.18 | A13 | sheep | 0.151 | 0.547 | 0.687 | |
12. | A18 | sheep | 0.349 | 0.325 | 0.374 | A16 | goat | 0.944 | 0.763 | 0.314 | |
13. | A18 | sheep | 0.206 | 0.255 | 0.355 | A17 | goat | 2.559 | 2.433 | 2.797 | |
14. | A18 | sheep | 0.395 | 0.209 | 0.348 | A17 | goat | 2.456 | 2.438 | 0.434 | |
15. | A23 | sheep | 0.183 | 0.262 | 0.298 | A17 | goat | 1.181 | 1.378 | 0.736 | |
16. | A23 | sheep | 0.17 | 0.216 | 0.241 | A17 | goat | 0.912 | 0.921 | 0.344 | |
17. | A27 | goat | 0.251 | 0.228 | 0.317 | A17 | goat | 0.468 | 0.457 | 0.158 | |
18. | A27 | goat | 0.211 | 0.168 | 0.119 | A23 | sheep | 0.478 | 0.267 | 0.355 | |
19. | B2 | sheep | 0.17 | 0.272 | 0.177 | A27 | goat | 0.795 | 1.045 | 0.233 | |
20. | B2 | sheep | 0.553 | 0.606 | 0.473 |
OD – optical density
To evaluate the agreement between the Eradikit SRLV Genotyping kit and the real-time PCR, the results were compared for the samples of 78 animals which were a PBL and serum sample pair and therefore possible to test with both methods. We found that the results for 36 (46.2%) out 78 sample pairs concurred. Agreement based on kappa was poor (0.16; 95% CI 0.02–0.30). The classification of 28 out of the 36 samples was MVV or CAEV infected, but the remaining 8 samples gave inconclusive results in both the real-time PCR and ELISA. Divergent results were obtained for 42 out of 78 (53.8%) samples. Of these 42 samples, 25 were classified as MVV by real-time PCR and as inconclusive or indeterminate by the ELISA. Seven samples classified as MVV infected by real-time PCR were classified as CAEV infected using the ELISA test, and one sample was classified as genotype E infected. Six and three samples which were negative by real-time PCR were respectively classified as CAEV and MVV infected by the ELISA.
It has been shown that the amino acid sequences of one immunodominant epitope of the capsid antigen are not conserved and have a variable region specific for each SRLV genotype. Therefore, ELISAs based on this region can be tools for classifying SRLV genotypes circulating in the field (13, 17). In this study, we evaluated the serotyping efficiency of the Eradikit SRLV Genotyping ELISA kit, the only commercial test based on capsid antigens able to distinguish between the A, B and E SRLV genotypes. For this purpose, a panel of 97 serum samples from animals infected in Poland with known SRLV isolates representing subtypes A1, A5, A12, A13, A16, A17, A18, A23, A24, A27, B1 and B2 was tested.
Our results revealed that the percentage of samples with their genotype assignment by sequencing in agreement with the assignment by serology was 42.3. This value is higher than the one obtained by Acevedo Jiménez
It was observed that 20.6% of the tested sera gave indeterminate genotype results because they contained antibodies which reacted highly with all test antigens, and the reaction strengths with individual antigens differed too little to permit discrimination. This result was similar to that obtained by Nogarol
Nogarol
The differences in the sequence of the
In addition, Schaer
Our results showed that the Eradikit SRLV genotyping kit is not a reliable method for predicting SRLV genotype. Cross-reactivity was noted, many samples could not be serotyped, and some were misclassified. Unlike this ELISA, the nested real-time PCR based on the LTR-