Accurate diagnosis of small ruminant lentivirus infection is needed for selection of resistant sheep through tmem154 e35k genotyping

pathogens Article Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping Hugo Ramírez 1,† , Irache Echeverría2,†, Alfredo A. Benito 3, Idoia Glaria 2, Julio Benavides 4, Valentín Pérez 5, Damián de Andrés2and Ramsés Reina 2,*   Citation: Ramírez, H.; Echeverría, I.; Benito, A.A.; Glaria, I.; Benavides, J.; Pérez, V.; de Andrés, D.; Reina, R. Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping. Pathogens 2021,10, 83. https://doi.org/10.3390/ pathogens10010083 Received: 30 November 2020 Accepted: 13 January 2021 Published: 19 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Virology, Genetics and Molecular Biology Laboratory, Faculty of Higher Education, Cuautitlan, Veterinary Medicine, Campus 4, National Autonomous University of Mexico, Km. 2.5 Carretera Cuautitlán-Teoloyucan, San Sebastián Xhala, Cuautitlán Izcalli Estado de México C.P. 54714, Mexico; [email protected] 2Animal Health Department, Institute of Agrobiotechnology (IdAB), CSIC-Government of Navarra, 31192 Navarra, Spain; [email protected] (I.E.); [email protected] (I.G.); [email protected] (D.d.A.) 3Molecular and Cell Biology Department, EXOPOL SL, 50840 Zaragoza, Spain; [email protected] 4Mountain Livestock Institute (IGM), CSIC-University of León, 24346 León, Spain; [email protected] 5Department of Animal Health, University of León, 24071 León, Spain; [email protected] *Correspondence: ramses.r[email protected]; Tel.: +34-948-168022 † These authors contributed equally (priority order was decided according to seniority). Abstract: Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation. Keywords: small ruminant lentiviruses; TMEM154; ELISA; PCR 1. Introduction Small ruminant lentivirus (SRLV) infection widely affects animal health and production by causing a wasting disease characterized by chronic inflammation of carpal joints, udder, central nervous system, and/or lungs [ 1 ]. Infection takes place through colostrum/milk consumption from infected ewes, and/or by direct contact with respiratory secretions from infected animals [ 2 , 3 ]. Once infection occurs, immune responses result in production of antibodies that generally remain detectable, for the whole animal’s life, which is at the basis of the current control programs [ 4 , 5 ]. Strategies to control SRLV are based on the identification of seropositive animals since no vaccine is available, despite Pathogens 2021,10, 83. https://doi.org/10.3390/pathogens10010083 https://www.mdpi.com/journal/pathogens Pathogens 2021,10, 83 2 of 16 profuse trials [ 6 ]. However, antigenic heterogeneity of circulating strains may be wider than the covered by available ELISA tests [ 7 – 9 ] making serological response not always detectable. Accordingly, the description of new infection outbreaks in ELISA-controlled flocks [ 9 – 11 ] have practically questioned current control strategies. Molecular diagnosis by PCR may add diagnostic value to serodiagnosis since seronegative animals may show PCR positive results due to low antibody production [ 12 , 13 ]. New molecular methods are being described focused on the design of universal primers, thereby increasing sensitivity to enable the identification and removal of animals with low viral load in vivo [14–17]. In addition to their use in control programs, the detection of antibodies through ELISA tests along with molecular tools has been used to identify association between breeds and susceptibility patterns to SRLV infection. Genome wide association studies (GWAS) have opened the possibility to apply genetic selection programs by describing a number of candidate genes associated to SRLV seroreaction [ 18 – 21 ] or proviral load [ 22 ]. Among them, different studies suggest that TMEM154 haplotypes 1, 2, and 3, the most common haplotypes found in sheep, have an effect on SRLV susceptibility. Sheep with a copy of either haplotype 2 or 3, both of which encode a glutamate amino acid residue at position 35 (E35) of the extracellular portion of TMEM154, have an increased risk of SRLV infection. Conversely, sheep homozygous for haplotype 1, which encodes a lysine residue at position 35 (K35), have a decreased risk of infection in sheep breeds from North America and Germany [ 18 , 23 , 24 ], but also in Asian sheep breeds [ 25 , 26 ]. With the exception of the mentioned countries, there is little information about the TMEM154 haplotype composition in productive breeds and its association with SRLV susceptibility. In this study, we analyzed TMEM154 E/K genotype association with SRLV infectious status in approximately 500 sheep belonging to different flocks, production systems, and breeds according to three different ELISAs and a PCR-based molecular test. 2. Results 2.1. Serodiagnosis SRLV diagnosis through ELISA was carried out using three different commercial tests based on different strains and antigens. An animal was considered infected in the Total ELISA classification when tested positive to at least one of the ELISAs. At the population level, ELISA testing indicated that all the flocks were infected with a seroprevalence ranging from 3.5% to 100%. Individually considered, the different ELISAs showed important differences when examining intraflock seroprevalence. Interestingly, two flocks of the churra breed were classified as uninfected taking into account results from ELISA#1. However, Total ELISA analysis indicated a seroprevalence of 60% (Table S1). Considering animals by breed, assaf flocks were the most infected showing a seroprevalence up to 89%, depending on the ELISA tested. When considering the results from the three ELISA tests, seropositive animals reached 97.3% (Table 1), being all flocks above 90% of seroprevalence. Raza and latxa Navarra breeds showed a moderate seroprevalence according to single ELISA tests, however, when applying the three ELISAs seropositive animals reached 50%. Churra sheep showed moderate to high seroprevalence values, reaching a total ELISA rate of 66.3% (Table 1). ELISA efficiencies, calculated as the proportion of seropositive animals detected by a single ELISA, reached 56%, 74%, 92%, and 91% in raza Navarra, latxa Navarra, assaf, and churra animals, respectively. ELISA#3 was clearly more performant in churra animals, whereas infection in latxa Navarra animals was better detected by ELISA#1 (Table 1). Interestingly, the combination of all ELISAs revealed a global seroprevalence higher than 65%, practically doubling the performance offered by kits individually considered. Indeed, efficiency of individual ELISAs varied from 0% to 100% according to flocks ( Table S1 ) and from 38.46% to 91.67% depending on the breed (Table 1). Pathogens 2021,10, 83 3 of 16 Table 1. Small Ruminant Lentivirus (SRLV) seroprevalence and ELISA efficiency in raza Navarra, latxa Navarra, assaf, and churra ovine breeds. Total ELISA reflects reactivity to any of the ELISAs used. TEST Raza Navarra Latxa Navarra Assaf Churra nPositive Efficiency % nPositive Efficiency % nPositive Efficiency % nPositive Efficiency % n%n%n%n% ELISA#1 114 29 25.4 55.8 194 76 39.2 73.8 74 66 89.2 91.7 101 32 31.7 47.8 ELISA#2 114 28 24.6 53.9 194 60 30.9 58.3 74 61 82.4 84.7 101 42 41.6 62.7 ELISA#3 114 20 17.5 38.5 194 60 30.9 58.3 74 46 62.2 63.9 101 61 60.4 91.0 Total ELISA 114 52 45.6 100.0 194 103 53.1 100.0 74 72 97.3 100.0 101 67 66.3 100.0 2.2. Molecular Diagnosis Diagnosis through commercial PCR resulted as sensitive as ELISA, since the overall PCR reactivity was around 44% compared to 46.4%, 44.9%, and 42.7% for ELISAs #1, #2, and #3, respectively (Table 2). As shown for serological analysis, PCR reactivity also depended on the flock considered, since 77% of the animals were detected in assaf flocks and only 22% in the churra animals (Table S2). Table 2. Small Ruminant Lentivirus (SRLV) provirus detection using real time quantitative PCR (qPCR). Total infected refers to samples positive to any of the diagnostic methods used (ELISA and/or PCR). TEST Raza Navarra Latxa Assaf Churra nPositive Efficiency % nPositive Efficiency % nPositive Efficiency % nPositive Efficiency % n%n%n%n% qPCR 111 33 29.7 45.2 191 90 47.1 65.7 74 57 77.0 79.2 82 18 22.0 32.1 Total infected 114 75 65.8 100.0 194 139 71.6 100.0 74 72 97.3 100.0 101 67 66.3 100.0 Among seronegative samples, 54 out of 179 (30%) were identified as qPCR positive, whereas 135 seropositive samples resulted negative in qPCR. When considering each ELISA individually, PCR detected a 22.37%, 34.53%, and 37.94% of seronegative animals to ELISAs #1, #2, and #3, respectively (Figure 1and Tables S4–S6). Total infected animal classification, revealed by ELISA or PCR, allowed the evaluation of PCR efficiency compared to ELISA. qPCR efficiency reached 79% in assaf animals and decreased to 32% in churra flocks. Intraflock efficiency in assaf flocks peaked at 95.8%, whereas highly seropositive churra flocks were not detected by qPCR (Table S2). Animal classification into infected and uninfected after ELISA (Table S3) and qPCR proviral quantification is represented in Figure 1. 2.3. TMEM154 Genotyping Ovine DNA samples (n= 10) from the studied population were employed to amplify a 335bp region of the TMEM154 gene (Table 3), including residue at position 35, that was cloned and sequenced (Figure 2). Table 3. Primer and probe sequences, amplification product size, and purpose of the corresponding PCR method. Probes/Primers Sequences Product Size (Base Pairs) Purpose Fw 5’-CTGCCTTTGTGGGAGATTTA-3’ 335 Amplification and sequencing for verification of genotyping results Rv 5’-TTCTGTGGTCACTGAAGCAA-3’ Fw 5’-TTCGTCTCCATGACAAGTCTCAAT-3’ 121 Determination of nucleotide substitution G/A, resulting in amino acid substitution E35K. Rv 5’-GCTTAGGGCCTCTGACTCTTCA-3’ HEX-AGGACACAGAACTGT-BHQ-1 6-FAM-AGGACACAAAACTGT-BHQ-1 Pathogens 2021,10, 83 4 of 16 Figure 1. Small ruminant lentivirus (SRLV) diagnosis. Scatter plot distribution of ELISA absorbance (X-axis) and proviral load in 250 ng of DNA (Y-axis) data. Samples concordantly positive or negative between ELISA#1 ( A ), ELISA #2 ( B ), or ELISA#3 ( C ) and PCR ( • ) and discordant samples (O) are represented. The Y-axis intercepted the X-axis at the average value of the corresponding ELISA positivity threshold. Pathogens 2021,10, 83 5 of 16 Figure 2. Identification of TMEM154 E35K genotype. Alignment of partial TMEM154 sequences obtained from selected sheep. Numbers refer to the animal sample and clone analyzed. Amino acid substitution at position 35 is highlighted. Identical residues are indicated by dots. Considering Sanger sequencing, seven samples were identified as homozygotes for allele 1, one as homozygote for allele 2, and two as heterozygotes. Specific clones encoding allele 1 or allele 2 were used for real time PCR standardization. Fluorogenic probes were designed within the E35K SNP, with either FAM or HEX (Table 3), to specifically detect plasmids encoding the corresponding genotype. Equimolar mixes of plasmids encoding each of the alleles were automatically classified as heterozygotes, validating their application in biological samples (Figure 3). Allelic discrimination analysis showed different allele frequencies according to the breed considered (Table 4). The protective genotype (K/K) was predominant in all breeds analyzed, followed by heterozygotes and homozygotes (E/E), except for the assaf breed in which heterozygotes and homozygotes (E/E) were prevalent. Pathogens 2021,10, 83 6 of 16 Figure 3. TMEM E35K genotyping using fluorogenic probes. Scatter plot distribution of relative fluorescence of FAM, representative of allele 1 (X-axis) and HEX (allele 2; Y-axis) of TMEM154 clones. Original animal samples are also shown showing a heterozygote pattern. Table 4. Allelic frequency among TMEM154 E35K genotyping in raza Navarra, latxa, assaf, and churra ovine breeds. Genotype Raza Navarra Latxa Assaf Churra n%n%n%n% K/K 92 80.7 134 69.1 15 20.3 75 74.3 E/K 18 15.8 56 28.9 32 43.2 24 23.8 E/E 4 3.5 4 2.1 27 36.5 2 2.0 Total 114 100 194 100 74 100 101 100 2.4. TMEM154 E35K Association with SRLV Infection Status Genotyped sheep were distributed according to ELISA absorbance and PCR proviral load (Figure 4). Considering breeds in which the K/K allele was predominant, the proportion of seropositive and seronegative samples in ELISAs #1 and #3 was similar in resistant (K/K) or susceptible (E/K and E/E) genotypes (Table 5). Similarly, assaf animals were mostly seropositive irrespective of their TMEM154 genotype (Tables S4–S6). However, when considering all breeds as a whole, significant difference was found between resistant and susceptible genotyped samples, mean absorbance being higher in susceptible samples (p< 0.05 Mann–Whitney). Exceptions to this general picture were evident when analyzing data obtained after ELISA#2 testing of latxa and raza Navarra breeds, since differences were found in ELISA absorbance according to TMEM154 genotype (Figure 4B and Table S5). Distribution of genotyped samples according to proviral load values was similar among resistant (K/K) and susceptible (E/K and E/E) samples, suggesting poor association between TMEM154 genotype and SRLV infection (Figure 4D). Relationship between SRLV infection status and TMEM154 genotyping was evaluated using association and relative risk, and regression statistical analyses. Animals from the assaf and churra breeds did not show significant association between TMEM154 genotype and SRLV antibody occurrence, except for ELISA#2 in churra animals (Table 5). Similarly, animal classification by ELISA#2 of the raza Navarra and latxa Navarra breeds allowed a significant association between SRLV seroreactivity and TMEM154 genotype. Additionally, reactivity to ELISAs #1, 2, and 3 was also associated to TMEM154 genotype in latxa Navarra animals. Total ELISA reactivity was associated to genotyping in the case of the aforementioned breeds (raza Navarra and latxa Navarra), but not in assaf or churra sheep. Pathogens 2021,10, 83 7 of 16 Figure 4. Cont. Pathogens 2021,10, 83 8 of 16 Pathogens 2021, 10, x FOR PEER REVIEW 8 of 17 Figure 4. Distribution of ELISA#1 ( A ), ELISA#2 ( B ), ELISA#3 ( C ) absorbance and qPCR proviral load ( D ) according to TMEM154 genotyped latxa, raza Navarra, assaf, and churra sheep. Animal samples were classified according to the E35K TMEM154 polymorphism into K/K ( ) or E/K and E/E ( ) and analyzed by ELISA and qPCR. Samples were grouped by individual breeds and combined (All breeds). Average cut-off values of individual ELISA are represented as a horizontal dotted line (* Mann–Whitney, p< 0.05). Pathogens 2021,10, 83 9 of 16 Table 5. Small Ruminant Lentivirus (SRLV) infection status and TMEM154 genotyping association. Samples classified into positive or negative according to different methods (ELISAs and qPCR) were re-classified according TMEM154 E35K polymorphism. Statistical probability associated to Fisher’s exact test (p) and to relative risk (RR; p’) are shown. Significant values are in bold. TEST SRLV Raza Navarra Latxa Navarra Assaf Churra TMEM154 Genotype (%) TMEM154 Genotype (%) TMEM154 Genotype (%) TMEM154 Genotype (%) pRR P’ pRR P’ p RR P’ pRR P’ KK EK/EE (95%CI) KK EK/EE (95%CI) KK EK/EE (95%CI) KK EK/EE (95%CI) ELISA#1 Negative 69 16 0.792 1.09 (0.51–2.35) 0.824 95 23 <0.0001 2.12 (1.52–2.95) <0.0001 2 6 0.66 1.04 (0.83–1.29) 0.745 53 16 0.338 1.35 (0.74–2.48) 0.326 Positive 23 6 39 37 13 53 21 10 ELISA#2 Negative 74 12 0.024 2.32 (1.25–4.31) 0.008 111 23 <0.0001 3.59 (2.36–5.48) <0.0001 4 9 0.446 1.16 (0.84–1.60) 0.381 50 9 0.005 2.02 (1.31–3.10) 0.002 Positive 18 10 23 37 11 50 24 17 ELISA#3 Negative 78 16 0.214 1.79 (0.78–4.13) 0.171 101 33 0.007 1.83 (1.22–2.75) 0.0037 5 23 0.772 0.91 (0.60–1.38) 0.674 31 9 0.644 1.11 (0.79–1.56) 0.530 Positive 14 6 33 27 10 36 44 17 TOTAL ELISA Negative 52 10 0.475 1.25 (0.80–1.96) 0.003 73 18 0.002 1.54 (1.20–1.98) 0.0007 0 2 10.97 (0.92–1.01) 0.157 28 6 0.232 1.23 (0.93–1.61) 0.142 Positive 40 12 61 42 15 57 47 20 qPCR Negative 62 16 10.90 (0.42–1.91) 0.781 72 29 0.437 1.15 (0.84–1.56) 0.384 3 14 10.95 (0.71–1.27) 0.747 48 16 0.770 1.12 (0.45–2.76) 0.810 Positive 27 6 59 31 12 45 13 5 Pathogens 2021,10, 83 16 of 16 38. 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