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Article

Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses

by
Jessica Schaer
1,
Zeljko Cvetnic
2,
Tomislav Sukalic
2,
Sven Dörig
3,
Martin Grisiger
4,
Carmen Iscaro
5,
Francesco Feliziani
5,
Folke Pfeifer
6,
Francesco Origgi
7,
Reto Giacomo Zanoni
1 and
Carlos Eduardo Abril
1,*
1
Institute of Virology and Immunology IVI, in Cooperation with the Vetsuisse-Faculty of the University of Bern, 3012 Bern, Switzerland
2
Regional Veterinary Department Križevci, Croatian Veterinary Institute, Zakmandijeva 10, 48260 Križevci, Croatia
3
Beratungs-und Gesundheitsdienst für Kleinwiederkäuer (BGK/SSPR), 3362 Niederoenz, Switzerland
4
Veterinaerdienst der Urkantone, 6440 Brunnen, Switzerland
5
National Reference Laboratory for Ruminant Retroviruses, Istituto Zooprofilattico, Sperimentale dell’Umbria e delle Marche Togo Rosati, 06126 Perugia, Italy
6
Tierseuchenkasse/Tiergesundheitsdienst Sachsen-Anhalt, 39116 Magdeburg, Germany
7
Institute of Veterinary Pathology, Vetsuisse-Faculty of the University of Bern, 3012 Bern, Switzerland
*
Author to whom correspondence should be addressed.
Pathogens 2022, 11(2), 129; https://doi.org/10.3390/pathogens11020129
Submission received: 9 December 2021 / Revised: 5 January 2022 / Accepted: 14 January 2022 / Published: 21 January 2022
(This article belongs to the Special Issue Small Ruminant Lentiviruses (SRLVs): Genetic Diversity, Pathogenicity and Diagnostic Approach)
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Abstract

:
Small ruminant lentiviruses (SRLVs), i.e., CAEV and MVV, cause insidious infections with life-long persistence and a slowly progressive disease, impairing both animal welfare and productivity in affected herds. The complex diagnosis of SRLVs currently combines serological methods including whole-virus and peptide-based ELISAs and Immunoblot. To improve the current diagnostic protocol, we analyzed 290 sera of animals originating from different European countries in parallel with three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation. A newly developed nested real-time PCR was carried out for the detection and genotype differentiation of the virus. Using a heat-map display of the combined results, the drawbacks of the current techniques were graphically visualized and quantified. The immunoblot and the SU5-ELISAs exhibited either unsatisfactory sensitivity or insufficient reliability in the differentiation of the causative viral genotype, respectively. The new truth standard was the concordance of the results of two out of three screening ELISAs and the PCR results for serologically false negative samples along with genotype differentiation. Whole-virus antigen-based ELISA showed the highest sensitivity (92.2%) and specificity (98.9%) among the screening tests, whereas PCR exhibited a sensitivity of 75%.

1. Introduction

Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) belong to the family Retroviridae, and the genus Lentivirus. They are members of a heterogeneous group called the small ruminant lentiviruses (SRLVs) comprising five different genotypes (A, B, C, D and E), infecting goats and sheep, causing cross- and superinfections [1,2,3,4,5]. MVV-like and CAEV-like strains belong to genotype A and B, respectively, with a worldwide distribution. The transmission routes for SRLVs are principally vertically through colostrum or milk but also horizontally through respiratory secretions, favored by overcrowding [6]. SRLV infections persist for life, whereby the mounted specific immune response does not protect against disease or superinfection [7]. Only one-third of the infected animals show clinical signs [8,9]. Still, SRLVs have a significant economic impact on animal production and affect animal welfare [10]. The presence of specific antibodies is useful as an indicator of an infection and therefore, eradication and surveillance programs are based on the serological detection of infected animals. The most frequently used assays are the agar gel immunodiffusion (AGID) test and the enzyme-linked immunosorbent assay (ELISA) (OIE Terrestrial Manual, https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.07.02_CAE_MV.pdf, accessed on 6 December 2021). For the AGID test, the two major SRLV antigens used routinely are the glycoprotein 135 (gp135) and the capsid protein (p28), respectively. The specificity of the AGID test is high, but its sensitivity has been reported as being low [11,12,13]. Still, a recent study carried out on Belgian sheep and goats [13] reported a sensitivity and specificity of 100% by combining the results of two AGID kits. Several ELISA tests have been developed and described in the literature using whole virus preparations, recombinant proteins or synthetic peptides, mostly designed as indirect but also as competitive assays (according to the OIE Terrestrial Manual). The high sensitivity, efficient handling and ease of interpretation of the results are the major advantages of the ELISA compared to AGID tests. For this reason, the ELISA tests are preferred for surveillance and eradication programs. A major drawback of serology is the high genetic variability of the SRLVs, translating into high antigenic diversity [14,15]. Therefore, the serological diagnosis of SRLV remains quite challenging [7,12,16] in the absence of a “gold standard” [17]. The high genomic variability also complicates the design of tools for the molecular detection of SRLV. Furthermore, apart from low viral load, no free virus is detectable in the blood of naturally infected animals using PCR methods. Therefore, these methods mainly target the provirus in peripheral blood leukocytes [18,19].
The current SRLV diagnostic procedure in Switzerland is based on a protocol consisting of an array of four sequentially used serological tests [20] performed on individual seropositive—not herd—samples. The sampling follows field criteria, e.g., animal purchase, animal transport or clinical suspicion. The serum samples are initially screened with the IDEXX CAEV/MVV Total Ab Test (Idexx Laboratories, Liebefeld, Switzerland), a whole-virus antigen-based indirect ELISA [21] and the Small Ruminant Lentivirus Antibody Test Kit VMRD (VMRD, Pullman, WA, USA), which is a gp135-based competitive ELISA [22]. Subsequently, the immunoblot based on whole virus antigen is performed as a confirmatory assay for seropositive samples [23] followed by the SU5-ELISA set as a serotyping differentiation test into the subtypes A1, A3, A4, B1 and B2 [24]. As far as the voluntary MVV control program in sheep is concerned, the sera are screened initially for SRLV antibodies using the Eradikit® SRLV Screening Kit (In3 diagnostic, Via B.S. Valfrè, 18, 10,121 Torino, Italia) before confirmation as described above. This thorough diagnostic procedure is expensive, laborious and time-consuming, and still quite frequently leads to inconclusive or contradictory results. Therefore, the aim of this work was to evaluate more closely the performance of the single and combined tests used, with the goal of optimizing the overall procedure. Additionally, we designed a nested real-time PCR targeting highly conserved genomic regions [25] and evaluated the benefit of the inclusion of this tool in the current diagnostic strategy.

2. Results

2.1. Determination of SRLV True Positive Standard

The results obtained for the 290 samples used in the serological screening, confirmation and genotype differentiation tests and the nested real-time PCR are visualized in a graphical heatmap format in Table 1. The colors reflect the intensity of each serological reaction, with values ranging from dark to light blue to white (negative) and from light to dark red (positive). In general, positive serological reactions were seen in all flocks investigated, indicating the presence of SRLV infections. However, overall, remarkable discordance between the results obtained by different serological tests was evident(*), avoiding the recognition of clear serological reactivity patterns within flocks. The highest agreement among the screening and confirmatory test results was seen in the samples of flocks 2, 3 and 5, whereas flock 10 exhibited discordance among the results of the screening tests as well as among screening and confirmation (Table 1).
In order to evaluate the performance of each test in the absence of a generally accepted gold standard, the SRLV true positive status was identified as a composite reference standard as recommended by others [13,16,26,27,28]. For that purpose, the composite concordance of the serological results across the different screening tests was analyzed, as shown in the Venn diagram (Figure 1). A total of 137 samples were positive with all three commercial tests, and therefore were considered as serologically true positives. The IDEXX-, VMRD- and ERADIKIT ELISA tests detected 178, 177 and 170 positive samples, respectively. The results of the Immunoblot (IB) formerly used as a serological and real-time PCR as an additional truth standard were included for comparative purposes (Figure 2). In the non-overlapping areas, one of four single-positive IDEXX ELISA samples showed a positive IB result, and three of them showed a positive real-time PCR result. None out of seven single-positive VMRD ELISA samples were positive in IB; however, three were positive by PCR. Finally, none out of nine single-positive ERADIKIT ELISA samples were positive in IB, and only one was positive by PCR. The proportion of real-time PCR positive samples was highest in single-positive IDEXX ELISA samples. In the overlapping regions between the IDEXX ELISA with either the ERADIKIT ELISA or the VMRD ELISA, more IB and real-time PCR positive results were observed than in the overlapping region between the ERADIKIT- and the VMRD ELISA. Samples’ ELISA reactivity tended to be higher in the overlapping areas of the Venn diagram than that of samples in the non-overlapping regions (data not shown). Based on these results, we arbitrarily defined the condition of a positive reaction with at least two screening ELISAs as a composite standard rule for serological truth, complemented with real-time PCR as an additional, independent truth standard.

2.2. Performance of the Serological Tests Based on the Composite Truth Standard vs. Real-Time PCR

Sensitivity and specificity were calculated by crossing the results of each test with the composite truth standard (Table 2). The IDEXX ELISA exhibited a sensitivity of 92.2%, a specificity of 98.9%, with a positive (PPV) and a negative (NPV) predictive value of 99,4% and 85.9%, respectively, whereas the VMRD ELISA’s sensitivity, specificity, PPV and NPV were 90.1%, 95.7%, 97.7% and 82.2%, respectively. The lowest performance was observed with the ERADIKIT ELISA with a sensitivity of 84.4%, a specificity of 91.3%, a PPV of 95.3% and an NPV of 73.7%. The distribution of the results of the commercial ELISA tests is shown as dot plots in Figure 2, revealing the IDEXX ELISA as the clearest discriminant between the expected negative and positive samples, followed by VMRD and the ERADIKIT ELISA tests. The immunoblot, which was formerly used as a confirmatory test, showed a low sensitivity of 59.2%, a specificity of 92.4%, a PPV of 94.2% and an NPV of 52.2%. The performance of the nested real-time PCR as the new, independent truth standard exceeded that of the immunoblot, with a sensitivity of 75.5%, a specificity and PPV of 100%, and an NPV of 66.2% (Table 3).
Comparing the results regarding the animal host or viral species (Table 4 and Table 5), the immunoblot exhibited more false negative samples in sheep (50%) than in goats (5.9%, Table 4. Still, from the view of the viral species, the results of the immunoblot show a clear tendency toward more false negative results in samples identified as MVV than in samples identified as CAEV by PCR (Table 5). No significance tests were applied due to the small sample size of goats.

2.3. Serological and Molecular Differentiation between MVV and CAEV Infections

The SU5 ELISA had been used to date to differentiate between MVV and CAEV infections in seropositive samples. According to the composite truth standard, 24 samples were categorized as false positive. Due to its use as a differentiation test, we restricted the evaluation of SU5 ELISA to the serological differentiation between MVV and CAEV. In total, 151 out of 184 serologically positive samples could be characterized as MVV- or CAEV-positive. Samples without positive SU5 test results were classified as “SRLV-positive” (Table 1). Using the nested real-time PCR for differentiation, a total of 147 samples were classified either as MVV or CAEV, whereas in six samples, coinfection with both MVV and CAEV was detected. In a total of 57 real-time PCR positive samples, the classification was confirmed by sequencing (Figure 3). The differentiation between MVV and CAEV by real-time PCR was 100% concordant with the phylogenetic analysis of the sequences using both NCBI BLAST (basic local alignment search tool [29]) and phylogenetic analysis. To evaluate the agreement between the SU5 ELISA and the real-time PCR, the results of 116 samples available for both methods were compared. The real-time PCR results were considered as true on the basis of the sequence confirmation. Fifty-nine and 51 samples were differentiated as CAEV and MVV, respectively, by real-time PCR (Figure 2d). Good concordance between the real-time PCR and SU5 ELISA results (48 out of 51 samples) was observed in the samples classified as MVV by real-time PCR (Figure 2d). However, 19 out of 59 samples classified as CAEV were misclassified as MVV by SU5-ELISA. Furthermore, the real-time PCR was able to detect coinfection with both virus types in six samples (Table 1).

2.4. Phylogenetic Analysis of Sequences

The genetic relatedness among the SRLV was analyzed using a 200 bp-long LTR-gag sequence fragment located within the real-time PCR target sequence. A total of 57 sequences supplemented with 14 closely related sequences retrieved from GenBank were analyzed phylogenetically (Figure 3). Two main clusters with clear separation between genotype A and genotype B according to the reference sequences were observed in the phylogenetic tree. Thus, the clusters were denominated as genotype A and genotype B (Figure 3). Moreover, as mentioned above, the classification as MVV or CAEV by real-time PCR was in complete agreement with the genotype classification in the phylogenetic tree.
The genetic relatedness of the sequences characterized was higher within flocks or within given geographic regions. The sequences from Italian samples in the genotype B cluster, split into two different clades closely related to Italian sequences previously characterized as subtype B2 (MG554402) and B3 (JF502417) strains [30], respectively. Additionally, four Italian samples were placed as separate groups in the genotype A cluster. These sequences were closely related to representative sequences of the subtypes A19 (MH374287) and A8 (MH374284), respectively, which similarly originated from Italy. The sequences of Croatian samples were also present in both the genotype A and B clusters of the tree. Two sequences located in the genotype B were found to be related to the subtype B1 reference sequence of the strain CAEV-Co (M33677.1) and three to the representative subtype B1 sequence MG554410. Sequences of genotype A were located in three different clades and were closely related to the representative sequences of the subtypes A18 (MG554409) and A4 (AY445885). Interestingly, due to sequence divergence, two distant clades were related to the same subtype A18 (MG554409) sequence. This divergence was also reflected in the percentages of identity among the samples of the individual clades (data not shown) and additionally in the different ranges of identity observed between the two clades and the subtype A18 (MG554409) sequence (Figure 4). Given the most commonly accepted SRLV subtyping method, it would be interesting to sequence the gag gene of these samples, which was beyond the focus of this work.
The 17 German samples were sent for diagnostic and epidemiological purposes. The only two positive goat samples were serologically positive repetitively in a German SRLV surveillance program. Our serological and genotyping results were in complete concordance with those of the Friedrich-Loeffler Institut (FLI, personal communication). The sequence of the German sample classified as genotype A was closely related to the subtype A2 sequence (MT993908), and that classified as genotype B was linked to the sequence of the subtype B1 CAEV-Co strain.
The Swiss samples were almost exclusively from sheep with only two samples from goats, which tested negative with all the protocols used. Sequence analysis revealed a balanced distribution of the Swiss sheep samples within the genotype A and B clusters. Samples with genotype B sequences were exclusively found in the clade with the subtype B1 CAEV-Co reference strain, whereas the genotype A samples were located in four different clades. The closest relatives of these four clades were sequences of the subtypes A18 (MG554409), A4 (AY445885) and A2 (MT993908), respectively (Figure 3).

3. Discussion

The diagnosis of SRLV infections is based on the serological detection of infected animals [3,12,31,32,33,34,35,36,37]. Due to low viral load and genomic heterogeneity, direct detection of the virus in blood is considered less efficient [3,12,16,32,33,38,39,40,41,42,43,44,45], though not negligible in view of delayed seroconversion [18,46,47,48,49,50,51,52,53]. In this work, using a panel of 290 sera of animals originating from different European countries, we aimed to improve and simplify the quite complex diagnostic protocol established in our diagnostic laboratory consisting of several serological tests run in parallel. Heat-mapping of the results from three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation exhibited a quite complex and challenging pattern of reactivity with a number of inconsistent results. Therefore, it was crucial to define, based on the given serological results, an overall truth standard. We based this decision on the well-accepted concept of using a “composite standard” [13,16,26,27,28] defined by the three screening ELISAs used. A useful complementation of this purely serological approach was the addition of a newly developed, highly sensitive PCR consisting of a primary round of a conventional PCR followed by a nested real-time PCR with the ability to differentiate between CAEV and MVV due to reliably discriminating probes with 100% specificity. The nested approach using serial dilutions of plasmids containing the target sequences exhibited at least 10-fold higher sensitivity compared to simple real-time PCR (data not shown).
This composite truth for determining the condition of infection status of a given animal allowed us to re-evaluate the current diagnostic procedure performed in our laboratory. IDEXX ELISA showed the highest sensitivity and specificity out of the three screening tests and the combination of IDEXX ELISA and VMRD ELISA detected the most “true positives” compared to the combinations of IDEXX-/ERADIKIT ELISA and VMRD-/ERADIKIT ELISA. The Immunoblot based on CAEV whole virus used for confirmation exhibited an unsatisfactory sensitivity of 59.2%. This is in accordance with its tendency toward inconclusive results due to an unexplained nonspecific reactivity of most sera with the capsid protein p25 of CAEV seen over the years (data not shown). Furthermore, we show a weaker reactivity of the immunoblot in the case of MVV compared to CAEV infections (Table 5), which does not appear to be due to either the host species or the antigen used (not shown). Given this rather poor performance, the Immunoblot is a quite laborious, demanding and antigen-consuming confirmatory assay, and we would recommend its exclusion from the diagnostic procedure.
PCR exhibited a nearly equal overall sensitivity (75.5%) compared to the SU5-ELISA set (80.7%), combined with 100% specificity and consequent unambiguous differentiation between the infection source (CAEV or MVV). Therefore, it seems appropriate to exclude the labor-intensive and costly SU5 peptide ELISA approach consisting of five ELISAs in favor of the PCR protocol as a differentiation tool. The application of the PCR in the diagnostic procedure has additional advantages regarding the possibility of detecting seronegative animals and sequencing the amplification products for molecular epidemiological analysis. Furthermore, the chance to achieve specific amplification in a seropositive sample might be significantly higher, apparently depending on the sample quality according to our laboratory experience (up to 85%, not shown). This point combined with the determination of the analytical sensitivity of the nested real-time PCR should be elucidated in further applied studies.
As also shown by others [12,43,54,55,56,57,58,59,60,61,62], the resulting LTR sequence fragments of this work allowed quite a robust and plausible phylogentic reconstruction of the taxonomy of lentiviruses detected in the samples originating from a number of Western European countries. Sequencing of the whole genome or other fragments thereof to exclude homologous recombination could be a useful complementation for future work.
In conclusion, given these results, the diagnosis of SRLV infection by a reference laboratory could actually be reduced to the combined application of well-established screening ELISAs complemented with the PCR described here as a differentiation tool regarding the source of infection, i.e., CAEV or MVV, respectively. Finally, with only minor loss of overall performance (not shown), the screening could be reduced to two ELISA kits, e.g., IDEXX ELISA combined with VMRD ELISA and PCR.

4. Materials and Methods

4.1. Samples

EDTA-anticoagulated whole blood samples from 290 animals originating from different countries including Croatia, Germany, Italy and Switzerland were analyzed (Table 1). Animals were sampled during 2019 and 2020 for serological and molecular diagnosis of SRLV infections. All flocks tested positive for SRLV infection previously. Croatian samples consisted of 50 samples from goats of 5 flocks (10 each). The Italian samples consisted of 51 samples from sheep of 3 flocks located in south of Italy (11, 20 and 20, respectively). German samples consisted of 17 samples from goats of one flock. The samples originating from Switzerland were sent to our laboratory for SRLV diagnosis consisting of 170 sheep and 2 goat samples from 18 different flocks with one or more samples per flock (Table 6).

4.2. Serology

Blood samples were analyzed for the presence of SRLV antibodies using three commercial ELISA kits in parallel, namely the IDEXX ELISA (IDEXX CAEV/MVV Total Ab Test (Idexx Laboratories, Liebefeld, Switzerland; whole-virus antigen based indirect ELISA), VMRD ELISA (Small Ruminant Lentivirus Antibody Test Kit-VMRD, Pullman, WA, USA; genotype B gp135 competitive ELISA) and the ERADIKIT ELISA (Eradikit® SRLV Screening Kit-In3Diagnostic, Torino, Italy; gag and env peptide based indirect ELISA), followed by immunoblotting and an in-house SU5 peptide ELISA test [24]. The cut-off values for the commercial ELISA tests were determined according to the manufacturers’ guidelines.
The Immunoblot confirmation was based on the detection of the viral capsid (p25), matrix (p15) and nucleocapsid (p18) proteins [23]. Reactive bands were visually evaluated and scored from 1 to 3 according to the band intensity. Samples showing staining of at least two bands or single bands scored as 2 or more were considered as positive.
The SU5 peptide ELISA was implemented for genotype differentiation. This test consisted of five ELISA tests containing synthetic peptides of the immunodominant SU5 region of the SRLV subgenotypes A1, A3, A4, B1 and B2. Samples with an OD value equal to or over 30% were considered as positive.

4.3. Multiple Sequence Alignments and Design of Primers and Probes

Primers and probes for the nested real-time PCR system are shown in Table 7. Whole genome sequence alignment comprising 52 published SRLV sequences retrieved from the National Centre for Biotechnology Information (NCBI) was used for the selection of conserved genomic regions and the design of the nested real-time PCR system. The Geneious prime software version 2020 (https://www.geneious.com, accessed on 6 December 2021) was used for alignment and the Primer Express® Software Version 3.0.1 (Applied Biosystems) was used for the design of primers and probes.
The forward primers (outer primer–F) of the first amplification step were designed to anneal to the highly conserved lentiviral RNAtlys primer-binding site (PBS) located at the leader region of the lentiviral genome. The similarly conserved annealing region of the reverse primers (outer primer–R) is located at 87 and 108 bp downstream of the predicted ATG gag initiation codon of the genotype A (GenBank Acc. No M60610) and B (GenBank Acc. No. M33677), respectively (outer primer–R). The forward primers for the genotype-specific real-time PCRs are located in a previously described conserved region encompassing a stem-loop structure that contains the dimer initiation site (DIS), just upstream of the major splice donor (MSD) of the small ruminant lentiviruses [63,64]. The reverse primers and probes are located immediately downstream of the predicted gag start codon. The target sequences of the real-time PCR reverse primers and probes and that of the outer reverse primers used were described previously [65,66]. A schematic diagram showing the localization of the nested real-time PCR target sequences is shown in Figure 3.

4.4. DNA Extraction and Two-Step Nested Real-Time PCR System

Seven hundred and fifty microliters of EDTA blood were treated with ammonium chloride/Tris buffer (0.14 M NH4Cl, 0.17 M Tris, pH 7.2) to obtain the buffy coats [67]. Buffy coats were stored at −20 °C or used directly for DNA extraction. DNA extraction was performed using the Qiagen DNeasy® Blood & Tissue kit (Qiagen, Hilden, Germany) and finally the DNA was eluted in 100 µL of elution buffer according to the manufacturer’s protocol.
The nested real-time PCR was carried out in two successive amplification steps. The first step PCR reaction consisting of a conventional qualitative PCR contained 12.5 µL of Hotstar Taq Master Mix (HotStarTaq DNA Polymerase kit, Qiagen GmbH, Hilden, Germany), 300 nM of each primer and 5 µL of the extracted DNA. Amplification started with the activation of the polymerase at 95 °C for 15 min, followed by 40 cycles at 95 °C for 20 s, and at 60 °C for 30 s. All products of the first PCR were then tested in parallel with the genotype-specific real-time PCRs (second step of the nested real-time PCR protocol) for a sensitive detection and discrimination between genotypes A and B.
Real-time PCR reactions contained 12.5 µL TaqMan™ Universal PCR Master Mix (Applied Biosystems, Life Technologies), 900 nM of each primer, 200 nM probes and 5 µL of the PCR product of the first step. Amplification profiles consisted of a hold stage of 20 s at 95 °C and a PCR stage of 40 cycles at 95 °C for 15 s and 60° C for 1 min. Thermal cycling was performed with a 7300 Real-Time PCR System (Applied Biosystems, Life Technologies).

4.5. Sequencing and Sequence Analysis

Samples with positive nested real-time PCR results were submitted for sequencing of the real-time PCR target region (Microsynth AG, Balgach. Switzerland). Sequences obtained were aligned using the clustal omega algorithm implemented in Geneious Prime software (Geneious Prime 2020.2.4). The phylogeny was inferred using the maximum likelihood method and the Tamura–Nei substitution model. Phylogenetic analyses were conducted using MEGA X [68]. The sequences from PCR products were deposited in GenBank under the following accession numbers: OL456240, OL456241 and OL449029 to OL449084.

4.6. Data Analysis

Sensitivity and specificity of serological tests and PCR were calculated using Single Sample Binary Diagnostic Tests by the software NCSS (NCSS Statistical Software (2019). NCSS, LLC. Kaysville, UT, USA, ncss.com/software/ncss, accessed on 6 December 2021).

Author Contributions

Conceptualization, J.S., Z.C., T.S., S.D., M.G., C.I., F.F., F.P., F.O., R.G.Z. and C.E.A.; Data curation, J.S., R.G.Z. and C.E.A.; Formal analysis, F.O., R.G.Z. and C.E.A.; Investigation, J.S.; Methodology, J.S. and C.E.A.; Project administration, R.G.Z.; Resources, Z.C., T.S., S.D., M.G., C.I., F.F. and F.P.; Supervision, and C.E.A.; Writing—original draft, J.S.; Writing—review and editing, Z.C., T.S., S.D., M.G., C.I., F.F., F.P., F.O., R.G.Z. and C.E.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding. This study was conducted as a part of our core functions as reference laboratory for the small ruminant lentivirus in Switzerland.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequencing data obtained were submitted to NCBI database. Accession numbers are given.

Acknowledgments

We would like to thank Libby Nebel, Sonja Piller, Stephanie Graber and Jasmin Neuenschwander of the diagnostic laboratory at the IVI Berne for their precious technical help.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Narayan, O.; Clements, J.E.; Strandberg, J.D.; Cork, L.C.; Griffin, D.E. Biological characterization of the virus causing leukoencephalitis and arthritis in goats. J. Gen. Virol. 1980, 50, 69–79. [Google Scholar] [CrossRef] [PubMed]
  2. Pasick, J. Maedi-visna virus and caprine arthritis-encephalitis virus: Distinct species or quasispecies and its implications for laboratory diagnosis. Can. J. Vet. Res.-Rev. Can. Rech. Vet. 1998, 62, 241–244. [Google Scholar]
  3. Pepin, M.; Vitu, C.; Russo, P.; Mornex, J.F.; Peterhans, E. Maedi-visna virus infection in sheep: A review. Vet. Res. 1998, 29, 341–367. [Google Scholar] [PubMed]
  4. Shah, C.; Boni, J.; Huder, J.B.; Vogt, H.R.; Muhlherr, J.; Zanoni, R.; Miserez, R.; Lutz, H.; Schupbach, J. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: Evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 2004, 319, 12–26. [Google Scholar] [CrossRef] [Green Version]
  5. Blacklaws, B.A. Small ruminant lentiviruses: Immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus. Comp. Immunol. Microbiol. Infect. Dis. 2012, 35, 259–269. [Google Scholar] [CrossRef] [PubMed]
  6. Blacklaws, B.A.; Berriatua, E.; Torsteinsdottir, S.; Watt, N.J.; de Andres, D.; Klein, D.; Harkiss, G.D. Transmission of small ruminant lentiviruses. Vet. Microbiol. 2004, 101, 199–208. [Google Scholar] [CrossRef] [PubMed]
  7. Ramirez, H.; Reina, R.; Amorena, B.; de Andres, D.; Martinez, H.A. Small Ruminant Lentiviruses: Genetic Variability, Tropism and Diagnosis. Viruses 2013, 5, 1175–1207. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Crawford, T.B.; Adams, D.S. Caprine arthritis-encephalitis: Clinical features and presence of antibody in selected goat populations. J. Am. Vet. Med. Assoc. 1981, 178, 713–719. [Google Scholar]
  9. Krieg, A.; Peterhans, E. Die Caprine Arthritis-Encephalitis in der Schweiz: Epidemiologische und klinische Untersuchungen. SAT 1990, 132, 345–352. [Google Scholar]
  10. Peterhans, E.; Greenland, T.; Badiola, J.; Harkiss, G.; Bertoni, G.; Amorena, B.; Eliaszewicz, M.; Juste, R.A.; Krassnig, R.; Lafont, J.P.; et al. Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes. Vet. Res. 2004, 35, 257–274. [Google Scholar] [CrossRef] [Green Version]
  11. Bulgin, M.S. Ovine progressive pneumonia, caprine arthritis-encephalitis, and related lentiviral diseases of sheep and goats. Vet. Clin. N. Am. Food Anim. Pract. 1990, 6, 691–704. [Google Scholar] [CrossRef]
  12. Brinkhof, J.; van Maanen, K. Detection and control of lentiviral infections in sheep and goats. Vet. Sci. Tommorrow 2010, 26, 1–18. [Google Scholar]
  13. Michiels, R.; Van, M.E.; Quinet, C.; Adjadj, N.R.; Cay, A.B.; De, R.N. Comparative Analysis of Different Serological and Molecular Tests for the Detection of Small Ruminant Lentiviruses (SRLVs) in Belgian Sheep and Goats. Viruses 2018, 10, 696. [Google Scholar] [CrossRef] [Green Version]
  14. Blacklaws, B.A.; Bird, P.; Allen, D.; Roy, D.J.; MacLennan, I.C.M.; Hopkins, J.; Sargan, D.R.; McConnell, I. Initial lentivirus host interactions within lymph nodes: A study of maedi-visna virus infection in sheep. J. Virol. 1995, 69, 1400–1407. [Google Scholar] [CrossRef] [Green Version]
  15. Straub, O.C. Maedi-visna virus infection in sheep. History and present knowledge. Comp. Immunol. Microbiol. Infect. Dis 2004, 27, 1–5. [Google Scholar] [CrossRef]
  16. De Miguel, R.; Arrieta, M.; Rodriguez-Largo, A.; Echeverria, I.; Resendiz, R.; Perez, E.; Ruiz, H.; Perez, M.; de Andres, D.; Reina, R.; et al. Worldwide Prevalence of Small Ruminant Lentiviruses in Sheep: A Systematic Review and Meta-Analysis. Animals 2021, 11, 784. [Google Scholar] [CrossRef]
  17. Reina, R.; Berriatua, E.; Lujan, L.; Juste, R.; Sanchez, A.; de Andres, D.; Amorena, B. Prevention strategies against small ruminant lentiviruses: An update. Vet. J. 2009, 182, 31–37. [Google Scholar] [CrossRef]
  18. De Azevedo, D.A.A.; Pinheiro, R.R.; dos Santos, V.W.S.; Damasceno, E.M.; de Sousa, A.L.M.; Araujo, J.F.; Andrioli, A.; Sider, L.H.; Peixoto, R.M.; Teixeira, M.F.D. Comparison of Serological and Molecular Tests for Diagnosis of Caprine Arthritis Encephalitis and Clinical Evaluation of Mammary Glands of Infected Dairy Goats. Acta Sci. Vet. 2019, 47. [Google Scholar] [CrossRef]
  19. Adjadj, N.R.; Vicca, J.; Michiels, R.; De Regge, N. (Non-)Sense of Milk Testing in Small Ruminant Lentivirus Control Programs in Goats. Comparative Analysis of Antibody Detection and Molecular Diagnosis in Blood and Milk. Viruses 2020, 12, 3. [Google Scholar] [CrossRef] [Green Version]
  20. Thomann, B.; Falzon, L.C.; Bertoni, G.; Vogt, H.R.; Schupbach-Regula, G.; Magouras, I. A census to determine the prevalence and risk factors for caprine arthritis-encephalitis virus and visna/maedi virus in the Swiss goat population. Prev. Vet. Med. 2017, 137, 52–58. [Google Scholar] [CrossRef] [Green Version]
  21. Zanoni, R.G.; Vogt, H.R.; Pohl, B.; B’ttcher, J.; Bommeli, W.; Peterhans, E. An ELISA based on whole virus for the detection of antibodies to small-ruminant lentiviruses. J. Vet. Med. Ser. B 1994, 41, 662–669. [Google Scholar] [CrossRef]
  22. Herrmann, L.M.; Cheevers, W.P.; McGuire, T.; Adams, D.S.; Hutton, M.M.; Gavin, W.G.; Knowles, D.P. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: Diagnostic tool for successful eradication. Clin. Diagn. Lab. Immunol. 2003, 10, 267–271. [Google Scholar] [CrossRef] [Green Version]
  23. Zanoni, R.; Krieg, A.; Peterhans, E. Detection of antibodies to caprine arthritis-encephalitis virus by protein G enzyme-linked immunosorbent assay and immunoblotting. J. Clin. Microbiol. 1989, 27, 580–582. [Google Scholar] [CrossRef] [Green Version]
  24. De Martin, E.; Golomingi, A.; Zahno, M.L.; Cachim, J.; Di Labio, E.; Perler, L.; Abril, C.; Zanoni, R.; Bertoni, G. Diagnostic response to a cross-border challenge for the Swiss caprine arthritis encephalitis virus eradication program. SAT 2019, 161, 93–104. [Google Scholar] [CrossRef]
  25. Chassalevris, T.; Chaintoutis, S.C.; Apostolidi, E.D.; Giadinis, N.D.; Vlemmas, I.; Brellou, G.D.; Dovas, C.I. A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses. Mol. Cell. Probe. 2020, 51, 101528. [Google Scholar] [CrossRef]
  26. Echeverria, I.; de Miguel, R.; de Pablo-Maiso, L.; Glaria, I.; Benito, A.A.; de Blas, I.; de Andres, D.; Lujan, L.; Reina, R. Multi-Platform Detection of Small Ruminant Lentivirus Antibodies and Provirus as Biomarkers of Production Losses. Front. Vet. Sci. 2020, 7, 182. [Google Scholar] [CrossRef]
  27. Potarniche, A.V.; Czopowicz, M.; Szalus-Jordanow, O.; Moroz, A.; Mickiewicz, M.; Witkowski, L.; Markowska-Daniel, I.; Bagnicka, E.; Cerbu, C.; Olah, D.; et al. Diagnostic accuracy of three commercial immunoenzymatic assays for small ruminant lentivirus infection in goats performed on individual milk samples. Prev. Vet. Med. 2021, 191, 9. [Google Scholar] [CrossRef]
  28. Rutjes, A.W.; Reitsma, J.B.; Coomarasamy, A.; Khan, K.S.; Bossuyt, P.M. Evaluation of diagnostic tests when there is no gold standard. A review of methods. Health Technol. Assess. 2007, 11, 1–71. [Google Scholar] [CrossRef]
  29. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  30. Colitti, B.; Coradduzza, E.; Puggioni, G.; Capucchio, M.T.; Reina, R.; Bertolotti, L.; Rosati, S. A new approach for Small Ruminant Lentivirus full genome characterization revealed the circulation of divergent strains. PLoS ONE 2019, 14, e0212585. [Google Scholar] [CrossRef]
  31. Dawson, M. Maedi/visna: A review. Vet. Rec. 1980, 106, 212–216. [Google Scholar] [CrossRef]
  32. Knowles, D.P. Laboratory diagnostic tests for retrovirus infections of small ruminants. Vet. Clin. N. Am. Food Anim. Pract. 1997, 13, 1–11. [Google Scholar] [CrossRef]
  33. De Andres, D.; Klein, D.; Watt, N.J.; Berriatua, E.; Torsteinsdottir, S.; Blacklaws, B.A.; Harkiss, G.D. Diagnostic tests for small ruminant lentiviruses. Vet. Microbiol. 2005, 107, 49–62. [Google Scholar] [CrossRef]
  34. Minguijon, E.; Reina, R.; Perez, M.; Polledo, L.; Villoria, M.; Ramirez, H.; Leginagoikoa, I.; Badiola, J.J.; Garcia-Marin, J.F.; de Andres, D.; et al. Small ruminant lentivirus infections and diseases. Vet. Microbiol. 2015, 181, 75–89. [Google Scholar] [CrossRef]
  35. Ogden, N.; Davies, P.; Lovatt, F. Dealing with maedi visna in UK sheep flocks. In Pract. 2019, 41, 321–328. [Google Scholar] [CrossRef]
  36. Lujan, L.; Perez, M.; de Andres, D.; Reina, R. Pulmonary lentivirus infection in sheep. Small Rumin. Res. 2019, 181, 87–90. [Google Scholar] [CrossRef]
  37. Peixoto, R.M.; de Sousa, A.L.M.; Araujo, J.F.; Pinheiro, R.R. Western Blot in Immunodiagnosis of Small Ruminant Lentivirus. Acta Sci. Vet. 2021, 49, 11. [Google Scholar] [CrossRef]
  38. Zanoni, R.; Pauli, U.; Peterhans, E. Detection of caprine arthritis-encephalitis- and maedi-visna viruses using the polymerase chain reaction. Experientia 1990, 46, 316–319. [Google Scholar] [CrossRef]
  39. Petursson, G.; Andresdottir, V.; Andresson, O.S.; Georgsson, G.; Palsson, P.A.; Rafnar, B.; Torsteinsdottir, S. Lentivirus diseases of sheep and goats - maedi-visna and caprine arthritis-encephalitis. In Progress in Sheep and Goat Research; Wallingford: Oxon, UK, 1992; pp. 107–129. [Google Scholar]
  40. Phelps, S.L.; Smith, M.C. Caprine arthritis-encephalitis virus infection. J. Am. Vet. Med. Assoc. 1993, 203, 1663–1666. [Google Scholar]
  41. Haase, A.T. The role of active and covert infections in lentivirus pathogenesis. In Slow Infections of the Central Nervous System; Bjornsson, J., Carp, R.I., Love, A., Wisniewski, H.M., Eds.; New York Academy of Sciences: New York, NY, USA, 1994; pp. 75–86. [Google Scholar]
  42. McConnell, I.; Blacklaws, B.A.; Bird, P.; Lee, W.C.; Roy, D.J.; Sargan, D. Lentivirus replication in lymphoid tissue: Use of lymphatic cannulation to study the initial stages of infection and immunity. Aids Res. Hum. Retrovir. 1996, 12, 417–420. [Google Scholar] [CrossRef] [PubMed]
  43. Zanoni, R.G. Phylogenetic analysis of small ruminant lentiviruses. J. Gen. Virol. 1998, 79, 1951–1961. [Google Scholar] [CrossRef]
  44. Brodie, S.J.; Delaconcha-Bermejillo, A.; Snowder, G.D.; DeMartini, J.C. Current concepts in the epizootiology, diagnosis, and economic importance of ovine progressive pneumonia in North America. Small Rumin. Res. 1998, 27, 1–17. [Google Scholar] [CrossRef]
  45. Panneum, S.; Rukkwamsuk, T. Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture. Pol. J. Vet. Sci. 2017, 20, 347–353. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Rimstad, E.; East, N.E.; Torten, M.; Higgins, J.; DeRock, E.; Pedersen, N.C. Delayed seroconversion following naturally acquired caprine arthritis-encephalitis virus infection in goats. Am. J. Vet. Res. 1993, 54, 1858–1862. [Google Scholar] [PubMed]
  47. Hanson, J.; Hydbring, E.; Olsson, K. A long term study of goats naturally infected with caprine arthritis-encephalitis virus. Acta Vet. Scand. 1996, 37, 31–39. [Google Scholar] [CrossRef]
  48. Chebloune, Y.; Karr, B.; Sheffer, D.; Leung, K.; Narayan, O. Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep. J. Gen. Virol. 1996, 77, 2037–2051. [Google Scholar] [CrossRef] [PubMed]
  49. Wagter, L.H.; Jansen, A.; BleuminkPluym, N.M.C.; Lenstra, J.A.; Houwers, D.J. PCR detection of lentiviral gag segment DNA in the white blood cells of sheep and goats. Vet. Res. Commun. 1998, 22, 355–362. [Google Scholar] [CrossRef] [PubMed]
  50. Scheer-Czechowski, P.; Vogt, H.R.; Tontis, A.; Peterhans, E.; Zanoni, R. Pilotprojekt zur Sanierung der Maedi-Visna bei Walliser Schwarznasenschafen. SAT 2000, 142, 155–164. [Google Scholar] [PubMed]
  51. Niesalla, H.; McNeilly, T.N.; Ross, M.; Rhind, S.M.; Harkiss, G.D. Experimental infection of sheep with visna/maedi virus via the conjunctival space. J. Gen. Virol. 2008, 89, 1329–1337. [Google Scholar] [CrossRef]
  52. Paula, N.R.D.; Andrioli, A.; Cardoso, J.D.S.; Pinheiro, R.R.; Sousa, F.M.L.; de Souza, K.C.; Alves, F.S.F.; Campello, C.C.; Ricarte, A.R.F.; Teixeira, M.F.D. Profile of the Caprine arthritis-encephalitis virus (CAEV) in blood, semen from bucks naturally and experimentally infected in the semi-arid region of Brazil. Small Rumin. Res. 2009, 85, 27–33. [Google Scholar] [CrossRef]
  53. Kalogianni, A.I.; Bossis, I.; Ekateriniadou, L.V.; Gelasakis, A.I. Etiology, Epizootiology and Control of Maedi-Visna in Dairy Sheep: A Review. Animals 2020, 10, 616. [Google Scholar] [CrossRef] [Green Version]
  54. Brown, A.J.L. Evolutionary relationships of the human immunodeficiency viruses. Trends Ecol. Evol. 1990, 5, 177–181. [Google Scholar] [CrossRef]
  55. Burns, D.P.W.; Desrosiers, R.C. Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys. J. Virol. 1991, 65, 1843–1854. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Turelli, P.; Guiguen, F.; Mornex, J.F.; Vigne, R.; Quérat, G. dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions. J. Virol. 1997, 71, 4522–4530. [Google Scholar] [CrossRef] [Green Version]
  57. Rolland, M.; Mooney, J.; Valas, S.; Perrin, G.; Mamoun, R.Z. Characterisation of an Irish caprine lentivirus strain—SRLV phylogeny revisited. Virus Res. 2002, 85, 29–39. [Google Scholar] [CrossRef]
  58. Germain, K.; Valas, S. Distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected French sheep. Virus Res. 2006, 120, 156–162. [Google Scholar] [CrossRef]
  59. Fras, M.; Leboeuf, A.; Labrie, F.M.; Laurin, M.A.; Sohal, J.S.; L’Homme, Y. Phylogenetic analysis of small ruminant lentiviruses in mixed flocks: Multiple evidence of dual infection and natural transmission of types A2 and B1 between sheep and goats. Infect. Genet. Evol. 2013, 19, 97–104. [Google Scholar] [CrossRef] [PubMed]
  60. L’Homme, Y.; Leboeuf, A.; Arsenault, J.; Fras, M. Identification and characterization of an emerging small ruminant lentivirus circulating recombinant form (CRF). Virology 2015, 475, 159–171. [Google Scholar] [CrossRef] [Green Version]
  61. Olech, M.; Murawski, M.; Kuzmak, J. Molecular analysis of small-ruminant lentiviruses in Polish flocks reveals the existence of a novel subtype in sheep. Arch. Virol. 2019, 164, 1193–1198. [Google Scholar] [CrossRef] [Green Version]
  62. De Azevedo, D.A.A.; Monteiro, J.P.; Pinheiro, R.R.; Mudadu, M.D.; Andrioli, A.; Araujo, J.F.; de Sousa, A.L.M.; Sider, L.H.; Peixoto, R.M.; Teixeira, M.F.D. Molecular characterization of circulating strains of small ruminant lentiviruses in Brazil based on complete gag and pol genes. Small Rumin. Res. 2019, 177, 160–166. [Google Scholar] [CrossRef]
  63. Monie, T.P.; Greatorex, J.S.; Maynard-Smith, L.; Hook, B.D.C.; Bishop, N.; Beales, L.P.; Lever, A.M.L. Identification and visualization of the dimerization initiation site of the prototype lentivirus, maedi visna virus: A potential GACG tetraloop displays structural homology with the alpha- and gamma-retroviruses. Biochemistry 2005, 44, 294–302. [Google Scholar] [CrossRef] [PubMed]
  64. Bjarnadottir, H.; Gudmundsson, B.; Gudnason, J.; Jonsson, J.J. Encapsidation determinants located downstream of the major splice donor in the maedi-visna virus leader region. J. Virol. 2006, 80, 11743–11755. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Kuhar, U.; Barlic-Maganja, D.; Grom, J. Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype A and B small ruminant lentivirus strains. BMC Vet. Res. 2013, 9, 172. [Google Scholar] [CrossRef] [Green Version]
  66. De Regge, N.; Cay, B. Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses. J. Virol. Methods 2013, 194, 250–257. [Google Scholar] [CrossRef] [PubMed]
  67. Sandvik, T.; Krogsrud, J. Evaluation of an antigen-capture ELISA for detection of bovine viral diarrhea virus in cattle blood samples. J. Vet. Diagn. Investig. 1995, 7, 65–71. [Google Scholar] [CrossRef] [PubMed]
  68. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
Pathogens 11 00129 g001 550
Figure 1. Venn diagram showing the concordance between serological results. Number of samples (n), results of the immunoblot test (IB), and real-time PCR results (PCR MVV: positive for Maedi-Visna virus; PCR CAEV: positive for the caprine arthritis-encephalitis virus; and PCR M&C: samples that were positive for both virus types) are indicated in the boxes.
Figure 1. Venn diagram showing the concordance between serological results. Number of samples (n), results of the immunoblot test (IB), and real-time PCR results (PCR MVV: positive for Maedi-Visna virus; PCR CAEV: positive for the caprine arthritis-encephalitis virus; and PCR M&C: samples that were positive for both virus types) are indicated in the boxes.
Pathogens 11 00129 g001
Pathogens 11 00129 g002 550
Figure 2. Jitter plots (a–c) and histogram (d) showing the distribution of results obtained for the commercial ELISAs and the SU5 ELISA. Exp. Positive/Exp. Negative: Expected positive (blue dots) and negative (red dots) results were defined according to the composite reference standard. Sens, Spec, and PPV: sensitivity, specificity and positive predictive value. Pre-specified assay thresholds are shown as red lines for a-c. Agrmt indicates the agreement between the real-time PCR results and the SU5 ELISA.
Figure 2. Jitter plots (a–c) and histogram (d) showing the distribution of results obtained for the commercial ELISAs and the SU5 ELISA. Exp. Positive/Exp. Negative: Expected positive (blue dots) and negative (red dots) results were defined according to the composite reference standard. Sens, Spec, and PPV: sensitivity, specificity and positive predictive value. Pre-specified assay thresholds are shown as red lines for a-c. Agrmt indicates the agreement between the real-time PCR results and the SU5 ELISA.
Pathogens 11 00129 g002
Pathogens 11 00129 g003 550
Figure 3. Phylogenetic tree constructed using a 200-bp LTR-gag region located within the target sequence of the real-time PCR. The tree was constructed using the maximum likelihood method using the Tamura–Nei substitution model. A total of 57 sequences of 2 German, 12 Croatian, 20 Italian and 23 Swiss samples of this study were included in the analysis. Moreover, 14 closely related sequences from Genbank were used as a reference. The sample names contain information regarding the lab ID, species (G = goat and S = sheep), country of origin (HR = Croatia, IT = Italy, DE = Germany, CH = Switzerland) and the continuous numbering of flocks tested. The genotypes groups A and B correspond to MVV and CAEV, respectively, in classical terminology.
Figure 3. Phylogenetic tree constructed using a 200-bp LTR-gag region located within the target sequence of the real-time PCR. The tree was constructed using the maximum likelihood method using the Tamura–Nei substitution model. A total of 57 sequences of 2 German, 12 Croatian, 20 Italian and 23 Swiss samples of this study were included in the analysis. Moreover, 14 closely related sequences from Genbank were used as a reference. The sample names contain information regarding the lab ID, species (G = goat and S = sheep), country of origin (HR = Croatia, IT = Italy, DE = Germany, CH = Switzerland) and the continuous numbering of flocks tested. The genotypes groups A and B correspond to MVV and CAEV, respectively, in classical terminology.
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Pathogens 11 00129 g004 550
Figure 4. Schematic diagram showing the SRLV target regions of primers and probes. Abbreviations: O-PF: outer primer forward; O-PR: outer primer reverse; RT-PF: real-time PCR primer forward; RT-PR: real-time PCR primer reverse; RT-P: real-time PCR fluorescent probes; SL: stem loop structure (according to Bjarnadottir et al. 2006).
Figure 4. Schematic diagram showing the SRLV target regions of primers and probes. Abbreviations: O-PF: outer primer forward; O-PR: outer primer reverse; RT-PF: real-time PCR primer forward; RT-PR: real-time PCR primer reverse; RT-P: real-time PCR fluorescent probes; SL: stem loop structure (according to Bjarnadottir et al. 2006).
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Table
Table 1. Heatmap for the serological and the real-time PCR results of all samples.
Table 1. Heatmap for the serological and the real-time PCR results of all samples.
Serology Serology
IB * 5SU5 * 6Sero. Results * 7PCR-Results * 8 IB * 5SU5 * 6Sero. Results * 7PCR-Results * 8
ID Number * 1IDEXX * 2VMRD * 3Eradikit * 4CAMANCA4A3A1B1B2ID Number * 1IDEXX * 2VMRD * 3Eradikit * 4CAMANCA4A3A1B1B2
716/G/HR_133049.61823130.330.190.180.220.22S-178/S/CH_10−164.430000.010.080.000.110.11--
717/G/HR_1837963000.160.180.260.180.27SM179/S/CH_10−8−1.521010.190.350.420.160.44--
718/G/HR_118139.11473030.460.140.110.140.19MM180/S/CH_10−11−2.431010.060.080.210.030.04--
719/G/HR_133839.41773201.020.340.300.380.64MM181/S/CH_10−15−5.9141010.260.060.00−0.010.05--
720/G/HR_18711.7463010.140.140.150.180.16SM182/S/CH_10−165.4391000.050.090.080.070.06--
721/G/HR_116839.91603010.160.200.150.180.29S-183/S/CH_10−1314.4−11000.010.110.020.220.07--
722/G/HR_1944.6790010.120.130.120.140.13SM184/S/CH_10−18−23100−0.06−0.03−0.040.06−0.01--
723/G/HR_133250.31823130.130.140.140.220.14SM185/S/CH_10−19−4.729100−0.02−0.03−0.040.03−0.01--
724/G/HR_11618.1422000.610.230.170.330.16--186/S/CH_10−17−0.21100−0.07−0.02−0.030.110.02--
725/G/HR_125740.31753130.130.130.120.280.13SM187/S/CH_10−1029.5911000.43−0.03−0.03−0.120.01--
726/G/HR_235787.81763233.152.212.484.002.52CC188/S/CH_10−334.3 1002.680.080.17−0.070.05--
727/G/HR_234492.51813230.520.620.683.110.63CC189/S/CH_10−16−8.733000.030.01−0.03−0.030.05--
728/G/HR_224890.71753230.320.540.753.750.28CC190/S/CH_101935.3 0002.570.120.180.01−0.01--
729/G/HR_233677.11723230.271.803.424.001.15CC191/S/CH_1020917.32161003.262.662.000.090.22M-
730/G/HR_224490.71323230.170.200.191.530.16CC192/G/CH_10−16−2.712100−0.050.00−0.020.080.01--
731/G/HR_235389.71853230.210.350.461.270.25C-193/G/CH_10−17−9.914100−0.050.05−0.010.100.05--
732/G/HR_2302781873230.130.520.794.000.18C-596/S/CH_117962.41131011.432.651.560.330.52MM
733/G/HR_231285.41823230.140.190.161.520.18C-597/S/CH_116057.8810001.192.380.920.080.14MM
734/G/HR_234189.11773230.150.140.144.000.16CC598/S/CH_118049.3790000.390.630.420.300.18MM
735/G/HR_232083.81773230.270.641.221.640.12C-599/S/CH_117343.81270000.921.300.410.040.15MM
736/G/HR_326991.71693120.711.992.084.000.71CC600/S/CH_1135469.51490000.952.321.921.450.06MM
737/G/HR_321190.51493120.180.353.651.040.30C-601/S/CH_1128346.21410000.361.161.400.070.15MM
738/G/HR_320083.31833230.260.290.173.650.14C-602/S/CH_1115546.41690000.360.320.18−0.02−0.04MM
739/G/HR_321789.21283010.200.190.240.360.16CC603/S/CH_1114074.21420003.040.820.320.460.20MM
740/G/HR_335272.11713230.110.480.603.320.11CMC604/S/CH_1110677.71260003.091.800.970.060.04MM
741/G/HR_329292.51623231.811.472.474.002.29CC605/S/CH_11113511350001.670.740.54−0.010.00MM
742/G/HR_320889.61293110.610.501.082.920.56CC606/S/CH_1114955.91430001.920.860.600.040.23MM
743/G/HR_334691.61603230.590.450.634.000.42CC607/S/CH_11−31.3−20000.08−0.010.010.000.05--
744/G/HR_329193.81343230.300.270.413.850.45CC608/S/CH_117931.5500000.020.070.00−0.010.03S-
745/G/HR_335392.71623130.740.850.814.000.75CC609/S/CH_1129367.41550000.870.561.140.080.07MM
746/G/HR_4−911.4−30000.140.130.100.140.12--610/S/CH_1123371.21590000.310.500.140.000.27MM
747/G/HR_4116481483130.190.140.140.160.14S-611/S/CH_1117360.71560000.330.650.120.050.01MM
748/G/HR_48836.8983010.240.350.220.390.41CC612/S/CH_1123245.61270000.892.030.620.020.60MM
749/G/HR_422860.51522130.440.220.120.180.13MM613/S/CH_1114535.71050000.141.170.09−0.010.14MM
750/G/HR_414139.41013010.130.250.110.170.14SM614/S/CH_1124031.3900000.170.310.08−0.010.00SM
751/G/HR_4−95.800000.300.410.120.190.17--615/S/CH_11617.730000.00−0.020.01−0.020.01--
752/G/HR_4−110.400010.220.150.110.140.12--616/S/CH_11937.39000−0.04−0.03−0.010.090.08--
753/G/HR_424379.11193030.160.150.430.140.17M-617/S/CH_1112147.8840000.000.600.050.010.10MM
754/G/HR_4−5−1.351000.140.190.140.220.22--618/S/CH_1110835−32000.06−0.070.020.020.30SM
755/G/HR_424831.61293230.140.140.110.130.14S-619/S/CH_11213.92000−0.010.000.00−0.02−0.03--
756/G/HR_535281.91503230.210.220.214.000.13CC620/S/CH_11516.231000.000.170.030.010.02--
757/G/HR_530692.81283230.270.480.231.240.51CC621/S/CH_11426.890000.040.050.06−0.010.02--
758/G/HR_520087.81573230.951.551.164.000.26CC622/S/CH_112251.1180000.010.310.110.000.05--
759/G/HR_527889.11413230.120.160.132.140.16CC623/S/CH_112725336200−0.010.200.050.000.13SM
760/G/HR_530077.41523230.150.200.241.350.19C-624/S/CH_11224138100−0.04−0.09−0.01−0.030.01--
761/G/HR_523176.91363230.200.200.170.430.28CC625/S/CH_112025.9361000.11−0.060.000.010.07--
762/G/HR_5119501153110.120.150.170.380.12CM626/S/CH_11212265100−0.01−0.040.01−0.020.00--
763/G/HR_518946.3533000.320.120.120.370.11CC627/S/CH_111330520000.260.170.070.000.04--
764/G/HR_515588.81303120.491.191.393.180.63CC628/S/CH_111636.2491000.520.480.200.060.08M-
765/G/HR_521246.41123120.140.130.123.820.15CC629/S/CH_1126856.7 2000.981.081.290.070.07MM
299/S/IT_6305811051010.400.160.180.210.28MM630/S/CH_114123.149100−0.010.130.06−0.020.50MM
300/S/IT_626992.71181011.962.511.652.422.34MC631/S/CH_11107.241000.020.340.030.120.38--
301/S/IT_640091.31221010.812.200.681.820.96MC194/S/CH_12−158.611000.260.060.000.070.05--
302/S/IT_630441.2471001.560.570.331.851.57CC195/S/CH_12−9−3.21000−0.030.03−0.01−0.070.07--
303/S/IT_619294.81221002.672.282.342.912.14CC196/S/CH_12−14−301000.050.070.970.280.35--
304/S/IT_632092.41213233.964.003.274.003.92CC197/S/CH_1221464.42401003.451.483.770.090.73MM
305/S/IT_621990.71051110.150.270.180.331.48CMC198/S/CH_12−121.871000.000.070.040.100.20--
306/S/IT_620572.11171021.661.451.401.250.87MMC199/S/CH_1282.9152000.020.140.250.080.18--
307/S/IT_611776.11251010.121.540.860.050.19MM200/S/CH_12−2110.65100−0.020.040.02−0.010.10--
308/S/IT_629577.41141033.103.673.902.822.60MMC201/S/CH_1216074.31791001.172.960.690.260.48MM
309/S/IT_6−7−17111000.190.250.250.200.15--202/S/CH_12−211.530000.360.110.070.170.20--
310/S/IT_718637.1942000.210.260.380.410.34C-203/S/CH_12−176.434100−0.020.090.000.150.18--
311/S/IT_740083.51212101.790.320.020.071.56MC204/S/CH_128054.42111000.320.280.150.260.32SM
312/S/IT_730986.91191000.330.340.120.220.28S-205/S/CH_126657.11471001.260.660.010.090.11MM
313/S/IT_715850.8541001.120.340.920.040.01MC206/S/CH_12−193.261000.030.230.090.270.31--
314/S/IT_719392.61260000.060.410.110.130.10MC207/S/CH_1221769.32211000.080.040.280.070.05SM
315/S/IT_740084.11253000.310.770.970.500.05MC208/S/CH_12−208.911000.430.292.480.400.32--
316/S/IT_7280801243000.140.350.590.140.14MC209/S/CH_12194682352002.892.571.880.140.25MM
317/S/IT_734087.41182001.602.412.170.451.33MC210/S/CH_12197682501024.004.004.000.140.15MM
318/S/IT_710890.3852000.390.650.660.500.31MC211/S/CH_1248452291000.842.462.490.060.30MM
319/S/IT_717458.7931000.10−0.100.070.040.06S-212/S/CH_1210366.32521002.731.363.270.150.22MM
320/S/IT_710080.41261000.350.570.020.070.10M-213/S/CH_126449.22571000.330.450.890.140.23M-
321/S/IT_740087.81233000.051.261.260.260.08MC214/S/CH_123644.82200000.970.460.690.100.31MM
322/S/IT_730273.31212220.470.820.420.470.68MC215/S/CH_1221267.52261000.691.411.910.050.10MM
323/S/IT_7−5−2471000.210.100.060.250.12--216/S/CH_12828.8350000.140.220.07−0.010.06-M
324/S/IT_732466.7973110.300.450.180.730.22CC217/S/CH_12−341.6810000.151.450.020.030.17M-
325/S/IT_729582.71152110.250.240.180.120.13SC218/S/CH_12146.11451000.040.060.050.10−0.02S-
326/S/IT_7−6−18151000.09−0.060.100.120.08--219/S/CH_12−1620.380000.19−0.010.020.07−0.03--
327/S/IT_7−10−19810000.010.060.020.050.01--220/S/CH_121755.81701000.000.210.070.180.05S-
328/S/IT_734672.21313001.670.190.130.390.48MC221/S/CH_12552.81251000.160.13−0.010.03−0.02S-
329/S/IT_718687.91211000.360.560.360.570.57CC222/S/CH_121625.31941000.470.220.250.210.22--
330/S/IT_8−13−9−11000.290.610.230.070.03--60/S/CH_1315870.11732023.001.873.500.210.20MM
331/S/IT_85749.91151000.820.510.590.210.60M-62/S/CH_1312515.5851003.893.173.252.050.01MM
332/S/IT_8−8−11281000.060.150.040.050.04--64/S/CH_1310930.31452004.163.574.150.030.04MM
333/S/IT_820459.71212010.110.700.270.330.07MM65/S/CH_1310721.8872004.253.904.210.161.23M-
334/S/IT_88649.21131010.230.000.131.730.11CM68/S/CH_137515.4320003.861.901.981.321.28--
335/S/IT_8−8−11−10000.13−0.080.070.220.05--70/S/CH_139256.2241003.112.902.450.250.26MM
336/S/IT_8−12−6.1−40000.070.210.090.070.07--93/S/CH_1437276.1922132.012.252.011.551.78MC
337/S/IT_8−16−11−40000.07−0.070.030.040.01--94/S/CH_1418569.1241000.80−0.13−0.03−0.040.99M-
338/S/IT_8−6−15−10000.080.350.110.070.05--95/S/CH_145681.9281000.04−0.04−0.03−0.08−0.06SC
339/S/IT_813056.21171001.020.171.030.260.99MM99/S/CH_1434961.9651000.380.120.13−0.090.88C-
340/S/IT_8−62480000.970.190.790.150.12-M100/S/CH_14420901131020.860.021.760.140.27MC
341/S/IT_8−15−8.4100000.400.220.050.260.07--101/S/CH_1435966.41011000.05−0.180.07−0.040.46C-
342/S/IT_89380.21111010.971.281.112.640.54CMC102/S/CH_1412870.12100−0.020.37−0.03−0.100.34M-
343/S/IT_816394.41171010.000.060.110.81−0.02CC103/S/CH_1423160.6301000.92−0.18−0.050.000.28MC
344/S/IT_815460.21112011.021.562.172.230.00C-105/S/CH_1519587.7110002.40−0.15−0.05−0.101.58M-
345/S/IT_830987.31243010.660.271.492.620.40CMC106/S/CH_1514286.2361000.34−0.20−0.06−0.100.83C-
346/S/IT_8−22−6.2−10000.020.020.040.030.01--107/S/CH_1545665.3863120.02−0.01−0.040.62−0.10CC
347/S/IT_84030.7942003.040.204.000.400.42M-108/S/CH_152774160000.37−0.17−0.04−0.090.21-C
348/S/IT_8−923.4340000.820.460.220.200.40--109/S/CH_1512387.57100−0.09−0.190.04−0.100.12S-
349/S/IT_8−10−5.700000.350.600.210.260.09--110/S/CH_1518482.545101−0.010.13−0.050.00−0.04S-
255/G/DE_9−17−82300000.03−0.010.12−0.03−0.03--111/S/CH_1516768.5461011.18−0.16−0.04−0.071.04M-
256/G/DE_9−18−80880000.040.00−0.02−0.02−0.01--112/S/CH_151127917100−0.12−0.19−0.08−0.050.14SC
257/G/DE_924649960000.020.39−0.010.00−0.04MM316/S/CH_1618480.3312000.01−0.080.080.350.03CC
258/G/DE_934871.51243000.000.050.010.23−0.05SC318/S/CH_1625779.6443130.060.070.072.170.17CC
259/G/DE_9−17−73−4000−0.020.050.000.07−0.02--319/S/CH_1625771.6753230.220.020.062.210.22CC
260/G/DE_9−18−43−4000−0.010.060.040.150.05--320/S/CH_1623877.8253130.000.540.100.160.03MC
261/G/DE_9−18−54−70000.00−0.020.26−0.01−0.06--321/S/CH_1621580.81743231.212.041.732.342.03C-
262/G/DE_9−13−54−7000−0.01−0.020.140.02−0.03--322/S/CH_1625385.2163200.050.160.140.020.03SC
263/G/DE_9−19−53−70000.000.020.080.040.02--323/S/CH_169376.2830110.02−0.03−0.010.820.23CC
264/G/DE_9−11−47−60000.030.000.01−0.03−0.05--324/S/CH_1622287.2813000.00−0.060.010.040.01SC
265/G/DE_9−15−64−30000.000.04−0.010.01−0.04--325/S/CH_1621066.128313−0.020.020.020.720.68CC
266/G/DE_9−18−38−70000.010.07−0.020.210.06--326/S/CH_1613582.1115200−0.020.000.040.690.03CC
267/G/DE_9−15−31−50000.100.060.130.070.04--327/S/CH_1625085.6563230.290.490.400.120.24MC
268/G/DE_9−17−58−70000.200.29−0.040.02−0.06--328/S/CH_16367.46100−0.020.050.030.030.06--
269/G/DE_9−18−27−60000.010.30−0.01−0.01−0.05--452/S/CH_174535.5401000.200.140.130.080.21SM
270/G/DE_9−15−27−80000.000.01−0.03−0.02−0.08--453/S/CH_175545.9371000.110.190.62−0.010.23MM
271/G/DE_9−18−2920000.000.09−0.030.080.01--455/S/CH_178139161011.170.030.040.120.02M-
151/S/CH_1037411180002.340.010.130.040.14MM456/S/CH_176812.129nd nd nd 1.281.480.520.210.16-M
152/S/CH_10−20−0.413000−0.020.040.000.160.09--457/S/CH_173153.1311000.08−0.010.04−0.020.13-M
153/S/CH_104471.8760002.100.531.120.160.35M-458/S/CH_171337.261000.020.100.030.010.15-M
154/S/CH_103633.5100000.73−0.010.05−0.090.00--459/S/CH_1715430.7512002.362.182.331.522.97CM
155/S/CH_1021401610002.170.060.21−0.010.14MM461/S/CH_1725084.7753230.16−0.050.01−0.104.05C-
156/S/CH_10−131.920000.150.120.01−0.020.07--462/S/CH_1716742.40300−0.02−0.080.020.040.06S-
157/S/CH_106737.5260001.340.610.370.560.36M-463/S/CH_1720582.6493230.100.140.310.000.32SC
158/S/CH_1022970.22340004.000.312.060.080.78MM465/S/CH_1825062603333.77−0.131.53−0.033.69MC
159/S/CH_103245.2470002.890.030.100.070.07M-467/S/CH_1812536.172000.04−0.05−0.010.030.84CC
160/S/CH_1031 0000.090.510.090.870.53--468/S/CH_187926.402000.39−0.100.07−0.080.28-C
161/S/CH_10−13−4.4200000.060.160.050.290.19--469/S/CH_1815759.91512010.830.170.23−0.022.56CC
162/S/CH_1037738.42333004.003.753.460.160.71MM470/S/CH_1821544.2743230.32−0.090.130.030.58CC
163/S/CH_10−174.8601000.010.040.05−0.070.08--471/S/CH_1813687.8702200.650.070.050.740.51CC
164/S/CH_10−143.701000.590.420.050.310.30--472/S/CH_184745.1222010.19−0.09−0.010.070.26SC
165/S/CH_10−14−3.452000.160.140.000.220.12--473/S/CH_1819990.5622230.880.910.550.240.81MC
166/S/CH_105448.62230004.000.490.760.090.07M-123/S/CH_19−1310nd 0000.080.170.120.120.03--
167/S/CH_10−183.850000.060.070.02−0.010.06--124/S/CH_1915326nd2113.574.002.262.901.00-M
168/S/CH_10−123.770000.100.300.330.170.22--21/S/CH_20121251091001.200.610.460.120.14MM
169/S/CH_10−11−3.32000−0.020.130.000.100.14--18/S/CH_214858761001.651.781.330.641.17MM
170/S/CH_10−140.51000−0.040.070.010.090.06--19/S/CH_229953741002.530.262.421.070.71MM
171/S/CH_102210.260001.110.050.08−0.060.06--16/S/CH_2314914231012.710.000.090.010.06-M
172/S/CH_10−165.891000.400.14−0.020.030.12--15/S/CH_236325741002.520.680.990.100.40MM
173/S/CH_10−2−3.314000−0.030.07−0.010.160.06--82/S/CH_249440.81051002.990.930.690.830.66M-
174/S/CH_10−6270000.360.230.390.660.22--78/S/CH_2520364.6442033.994.133.961.440.12M-
175/S/CH_10−18−0.131000.080.020.01−0.020.02--444/S/CH_2618482.971313−0.02−0.10−0.01−0.12−0.01SC
176/S/CH_10−812.201000.070.140.120.250.18--445/S/CH_26−2400000.05−0.050.03−0.060.01--
177/S/CH_10−14−7.640000.010.140.090.100.12--447/S/CH_2725081.7563130.270.530.440.380.54CC
1 ID number consists of lab number, species (G = goat and S = Sheep), country (HR = Croatia, IT = Italy, DE = Germany, CH = Switzerland) and the continuous numbering of flocks tested; 2 results of IDEXX ELISA, corrected OD; 3 results of VMRD ELISA, percent of inhibition; 4 results of ERADIKIT ELISA, corrected OD; 5 Immunoblot, CA (capsid, p25), MA (matrix, p18) and NC (nucleocapisd, p15), 0 = no reaction, 1 = slight reaction, 2 = moderate reaction, 3 = strong reaction; 6 SU5 ELISA, corrected OD. 7 Serological results: S (gray cell background) = SRLV if SU5 is negative but 2 of the commercial ELISAs are positive; M (green cell background) = MVV if SU5 positive at A4, A3, A1; C (orange cell background) = CAEV if SU5 is positive at B1 or B2. 8 PCR results: M for MVV (green cell background) = RT-PCR MVV-positive; C for CAEV (orange cell background) = RT-PCR CAE-positive; MC (yellow cell background) = RT-PCR MVV- and CAE-positive. The colors reflect the intensity of each serological reaction, with values ranging from dark to light blue to white (negative) and from light to dark red (positive).
Table
Table 2. Diagnostic performance of the screening ELISAs, Immunoblot and RT-PCR crossing the results of each test with the composite truth standard.
Table 2. Diagnostic performance of the screening ELISAs, Immunoblot and RT-PCR crossing the results of each test with the composite truth standard.
SensitivitySpecificityPos. Predictive ValueNeg. Predictive Value
Value %95% CI % 1Value %95% CI % 1Value %95% CI % 1Value %95% CI % 1
IDEXX ELISA92.287.4–95.698.994.1–10099.496.2–99.985.977.7–91.9
VMRD ELISA90.185.0–93.995.789.2–98.897.794.3–99.182.273.7–89.0
ERADIKIT ELISA84.478.5–89.291.383.6–96.195.391.2–97.573.764.6–81.5
Immunoblot59.251.8–66.292.485.0–96.994.288.4–97.652.244.2–60.0
Real-time PCR75.568.8–81.410096.1–10010097.5–10066.257.7–74.0
1 95% CI % = 95% Confidence Interval in %.
Table
Table 3. Diagnostic performance of the immunoblot test and the nested real-time PCR.
Table 3. Diagnostic performance of the immunoblot test and the nested real-time PCR.
ImmunoblotNested Real-Time PCR
TruthPosNegTotalPercentTruthPosNegTotalPercent
Pos1137819159.2 (Se)Pos1454719275.5 (Se)
Neg7859292.4 (Sp)Neg09292100 (Sp)
Total120163283100Total145139284100
Percent94.2 (P)52.1 (N)100 Percent100 (P)66.2 (N)100
Se = sensitivity, Sp = specificity, P = positive predictive value; N = negative predictive value.
Table
Table 4. Immunoblot failure by animal host.
Table 4. Immunoblot failure by animal host.
I-Blot pos I-Blot neg
MVVa (%)CAEVa (%)MVVa (%)CAEVa (%)N (% Detected)
Sheep14 (26.9)38 (73.1)43 (82.7)9 (17.3)104 (50.0)
Goats9 (28.1)23 (71.9)2 (100)0 (0)34 (94.1)
Total23 (16.7)45 (32.6)61 (44.2)9 (6.5)138 (49.3)
Viral species assigned by nested real-time PCR.
Table
Table 5. Immunoblot failure by viral species.
Table 5. Immunoblot failure by viral species.
I-Blot posI-Blot neg
SheepGoatsSheepGoatsN (% Detected)
MVVa14 (60.9)9 (39.1)43 (95.6)2 (4.4)68 (33.8)
CAEVa38 (62.3)23 (37.7)9 (100)0 (0)70 (87.1)
Total52 (37.7)32 (23.2)52 (37.7)2 (1.4)138 (60.9)
Viral species assigned by nested real-time PCR.
Table
Table 6. Samples tested in this study listed by country, number of sheep, goats and farms.
Table 6. Samples tested in this study listed by country, number of sheep, goats and farms.
CountryNumber of SheepNumber of GoatsNumber of Farms
Croatia0505
Italy5103
Germany0171
Switzerland170218
Table
Table 7. Sequences of primers and probes for the nested real-time PCR system. IUPAC codes were used to indicate degenerated primers. a F indicates forward primer, R reverse primer and P fluorescent probe, respectively, b GenBank accession number M33677 used as a reference sequence to indicate positions of primers and probes’ binding sites, and c GenBank accession number used as a reference sequence.
Table 7. Sequences of primers and probes for the nested real-time PCR system. IUPAC codes were used to indicate degenerated primers. a F indicates forward primer, R reverse primer and P fluorescent probe, respectively, b GenBank accession number M33677 used as a reference sequence to indicate positions of primers and probes’ binding sites, and c GenBank accession number used as a reference sequence.
Nested Real-Time PCROligonucleotide aPrimer Sequence/Flouorescent DyePosition
First-step PCR:outer primersOuter SRLV-F15′-CGCAGGTGGCGCCCAG-3′158–173 b
Outer SRLV-F25′-CGCAGSTGGCGCCCAA-3′158–173 b
Outer SRLV-R15′-CCTTCTGTCAGGCGCTCCCC-3′622–642 b
Outer SRLV-R25′-CCTTCCGTCAAGGTCTCCTTCC-3′621–642 b
Outer SRLV-R35′-CCTTCTGTCAAGGTCTCCTTCCC-3′620–642 b
Outer SRLV-R45′-CCTTCTGTCAAGTGCTCCCCTCT-3′620–642 b
Outer SRLV-R55′-CCTTCTGTCAGGTGCTCCCCTCT-3′620–642 b
Second-step:Genotype Aspecific real-time PCRRTMVVLTRgag-F 5′-GGGGACGCCTGAAGTRAGGTAA-3′287–308 c
RTMVVLTRgag-R 5′-YTTGAGCTCRGGGTAYCCCTT-3′ 517–537 c
RTMVVLTRgag-P5′-FAM-CTTTGAGCCTTGCKTCGCCATGTCT-TAMRA-3′486–510 c
Genotype Bspecific real-time PCRRTCAELTRgag-F 5′-CTGRAGGAGTAMGGTAAGTRACTCTGC-3′324–350 b
RTCAELTRgag-R 5′-TTGATRCATTTKTCSAKCTCAGGATAA-3′365–591 b
RTCAELTRgag-P 5′-FAM-CCGGAGACTTGCCTCGCCATGTC-TAMRA-3′530–552 b
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Schaer, J.; Cvetnic, Z.; Sukalic, T.; Dörig, S.; Grisiger, M.; Iscaro, C.; Feliziani, F.; Pfeifer, F.; Origgi, F.; Zanoni, R.G.; et al. Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses. Pathogens 2022, 11, 129. https://doi.org/10.3390/pathogens11020129

AMA Style

Schaer J, Cvetnic Z, Sukalic T, Dörig S, Grisiger M, Iscaro C, Feliziani F, Pfeifer F, Origgi F, Zanoni RG, et al. Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses. Pathogens. 2022; 11(2):129. https://doi.org/10.3390/pathogens11020129

Chicago/Turabian Style

Schaer, Jessica, Zeljko Cvetnic, Tomislav Sukalic, Sven Dörig, Martin Grisiger, Carmen Iscaro, Francesco Feliziani, Folke Pfeifer, Francesco Origgi, Reto Giacomo Zanoni, and et al. 2022. "Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses" Pathogens 11, no. 2: 129. https://doi.org/10.3390/pathogens11020129

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