Sample collection
Between August and December 2023 blood samples were taken by veterinarians using 9 ml EDTA monovettes from the jugular vein or vena cava cranialis, the latter method is especially used in unshorn sheep [40]. The study involved 54 flocks from 9 German states, which were divided into 3 different study areas: North-western Germany (Schleswig Holstein, North Rhine-Westphalia, Lower Saxony), Eastern Germany (Saxony, Saxony-Anhalt, Brandenburg, Mecklenburg-Western Pomerania) and Southern Germany (Hesse, Baden-Württemberg) (Figure 1). In each flock, all breeding rams over 1 year of age and 1-10 unrelated females per ram line were sampled, in total 530 samples for genotyping of TMEM154 E35K. Additionally, 35 of these flocks participated in the voluntary, but reportable MV monitoring. For this purpose, all RPL sheep over 1 year of age (plus 4 sheep aged 10 months) were sampled per flock, resulting in a total of 849 samples for MVV serology. The average flock size of the MVV-tested flocks was 52 RPL sheep (range: 7-295) (Table 1).
EDTA-blood samples were stored at <8°C after collection for up to 5 days. Samples for MVV serology were centrifuged and plasmas were separated before freezing at -20°C until use.
Questionnaire
A questionnaire was developed and sent to each RPL breeder of the 35 flocks to be completed before, during, or after the farm visit for blood sampling. The aim of the questionnaire was to obtain flock-level information including flock size, management, type of housing, sheep purchases, presence of other sheep breeds or goats on the farm, health, and potentially known MV status.
MVV serology
All plasma samples were tested by using a commercial competitive ELISA test kit (VMRD Inc., Pullman, Washington, USA), according to the manufacturer's instructions. Antibodies in the plasma sample inhibit the binding of horseradish peroxidase (HRP)-labeled monoclonal antibody to CAEV envelope glycoprotein gp135, the viral antigen. The color development after adding an enzyme substrate that binds to the conjugate of the HRP-labeled monoclonal antibody was measured using a photometer. Based on the optical density (OD) results, the percent inhibition (PI) was calculated as follows:
PI = 100 [1-(Sample OD ÷ Negative Control OD)]
According to the manufacturer’s recommendations, a sample was classified as seropositive when it produced ≥ 35% inhibition.
Although based on the CAEV envelope glycoprotein, the sensitivity and specificity of the assay were 98.6% and 96.9%, respectively, when used with sheep sera [41].
SRLV serotyping
Twenty-nine of the 30 seropositive plasmas were serotyped with the indirect Eradikit ELISA for SRLV genotyping (Eradikit ™ SRLV Genotyping IN3 Diagnostic, Torino, Italy). This genotyping ELISA uses plates coated with specific recombinant subunits of genotypes A, B, and E on three separate strips. Genotype-specific antibodies bind to the corresponding antigen and lead to color development. To determine a positive result for one genotype, the measured OD value must be > 0.4, and 40% above the other OD values. The results were classified as indeterminate if the OD values of all genotypes were < 0.4, or if the difference between the two most reactive antigens was too low (< 40%). In the latter case, a double infection could be possible, according to the manufacturer´s instructions.
TMEM154 genotyping
A total of 530 blood samples were analyzed for the nucleotide substitution G>A at position 35 in the coding region of the TMEM154 gene, resulting in amino acids E or K in the protein. DNA extraction was performed using the NucleoMag® Blood 200 μl kit (Machery-Nagel GmbH & Co. KG, Dueren, Germany), which is based on magnetic separation by reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions. The extracted DNA was measured with the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the concentrations were standardized to equal values. Afterwards, TMEM154 E35K was genotyped by an allele-specific PCR method as described by Molaee et al. [20].
Statistical analysis
The data were stored and organized in Microsoft Excel 2016 (Microsoft Corporation, Redmond, USA). The associations between the potential risk factors and the individual and flock-level seropositivity were assessed by using a chi-square or Fisher´s exact test and by calculating the odds ratio (OR). Due to the small number of positive samples, only a univariable analysis was performed for each independent variable. The SAS Studio Version 9.4 program (SAS Institute Inc., Cary, NC, USA) was used for the statistical analyses.