The presence of small ruminant lentiviruses in Mexican Pelibuey sheep

Theriogenology xxx (2016) 1–5

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The presence of small ruminant lentiviruses in Mexican Pelibuey sheep José H. Sánchez a, Humberto A. Martínez a, María M. García b, Germán Garrido c, Luis Gómez d, José A. Aguilar b, Damián F. de Andrés e, Ramsés Reina e, Hugo Ramírez a, * a

Laboratorio de Virología, Genética y Biología Molecular, Facultad de Estudios Superiores Cuautitlán (FESC) Campo 4, Universidad Nacional Autónoma de México (UNAM), Estado de México, México b Laboratorio de Inmuno-virología, Unidad de Investigación Médica en Inmunología, UMAE Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México c Laboratorio de apoyo a Histología y Biología, FESC, Campo 4, Universidad Nacional Autónoma de México, Estado de México, México d Laboratorio de Virología CENID Microbiología Animal, Instituto Nacional de Investigación Forestal Agrícola y Pecuaria (INIFAP), Ciudad de México, México e Instituto de Agrobiotecnología, UPNA-CSIC-Gobierno de Navarra, Navarra, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 January 2016 Received in revised form 9 June 2016 Accepted 15 June 2016

The transmission frequency of small ruminant lentiviruses (SRLVs) through the placenta is controversial and may be associated with breed susceptibility. In Mexico, SRLV infections in sheep have been poorly studied. This work explores the presence of antibodies and proviral DNA in Mexican Pelibuey sheep. Enzyme-linked immunosorbent assays (ELISAs; three commercial kits and two on the basis of synthetic peptides) and polymerase chain reaction (PCR; amplifying the long terminal repeat and gag segments) were performed to diagnose SRLV infection in 25 adult Pelibuey ewes with an average age of 2.5 years and 32 fetuses with gestational ages ranging from 40 to 90 days without clinical signs of SRLV. Two of the three commercial ELISAs and the synthetic peptide-based ones were positive for SRLV antibody detection in 28% and 24% of the ewes, respectively, whereas none of the fetuses were positive by any of the ELISAs. By PCR, 31% of the ewes and, interestingly, two fetuses were positive. Characteristic SRLV lesions were not found in the fetal and/or ewe tissues, including those with positive PCR results. These findings demonstrate the susceptibility of Pelibuey sheep to SRLV infection and the low transmission frequency through the placenta. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: ELISA PCR Pelibuey breed Small ruminant lentiviruses Synthetic peptides

1. Introduction Caprine arthritis encephalitis virus (CAEV) and Visna–Maedi virus (VMV) belong to the Lentivirus genus, Retroviridae family; both viruses are currently known as small ruminant lentiviruses (SRLVs). SRLVs are classified into five genotypes (A, B, C, D, and E) and 24 subtypes that are widely distributed among sheep and goats [1]. * Corresponding author. Tel.: þ52 55 5623 1920; fax: þ52 55 5870 5671. E-mail address: [email protected] (H. Ramírez). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.06.017

Breed susceptibility and resistance to SRLV infection have been well described, with selected breeds, such as Assaf [2], Texel, Border Leicester, and Finnish Landrace sheep being highly susceptible [3] and indigenous breeds, such as Roccaverano [4], Columbia, Rambouillet, and Suffolk, being potentially resistant [3]. Mexico has one of the highest sheep populations in Latin America and hosts very diverse breeds in which SRLV infection is considered an exotic event. Therefore, few studies regarding SRLV susceptibility and transmission in Mexican sheep have been conducted. The Pelibuey breed is widely distributed throughout the country, and it is

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considered highly robust and adaptable. However, no information regarding this breed’s susceptibility to SRLV infection is available [5,6]. The main SRLV transmission routes are horizontal, occurring through direct contact between adult animals, or lactogenic, occurring through the consumption of infected colostrum and/or milk. Other routes have been considered as being less important from the epidemiologic perspective and include intrauterine and iatrogenic routes. Both the importance of intrauterine transmission [7,8] and the fetal infection frequency remain controversial, suggesting that viral and host genetics may be involved [6,9]. The virus can infect embryonic cells at the oocyte maturation stage when the zona pellucida is lost, and the intrauterine environment can provide as a source of infectious virus [10,11]. The SRLV susceptibility of epithelial cells from goat oviducts has been demonstrated in vitro [12]. Furthermore, the presence of antibodies against SRLVs has been confirmed in ovine and caprine fetuses extracted before the end of gestation [13], and the presence of antibodies and proviral DNA has been shown in the peripheral blood mononuclear cells (PBMCs) of newborn lambs [14]. The objective of the present work was to determine the presence of SRLV infection (antibodies and proviral DNA) in Pelibuey fetuses and ewes and to evaluate SRLV transmission to fetuses and the presence of SRLV-related histologic lesions in the tissues of fetuses and ewes.

desensitization. After slaughter, fetuses were collected in aseptic conditions, and their gestational ages were determined as described previously [16]. Intracardiac blood collection was performed in fetuses with anticoagulant tubes. Plasma was obtained by centrifugation, and PBMCs were separated by density-gradient centrifugation. Serum samples were kept at 20  C until use. Additionally, the thymus and spleen were collected from the fetuses, and the umbilical cord and cotyledons were collected from the fetuses and ewes; all organs were stored in buffered formalin (pH 7.2) for histopathologic studies and proviral DNA detection by polymerase chain reaction (PCR).

2. Materials and methods

2.4. Peptide ELISAs

2.1. Animals

Synthetic peptides from SRLV genotype A (126M2) or B (126M1) were used in a homemade ELISA test as previously described [17]. The positive and negative controls were evaluated previously by Western blot. Absorbance values were calculated by subtracting the negative control’s optical density at 405 nm (OD405) (diluent only; no peptide) from each sample’s OD405. Samples with absorbance values greater than 0.35 were considered positive on the basis of a threshold value determined previously for sera from uninfected sheep [17].

2.3. Commercial ELISAs Three commercial serologic tests were used: competitive ELISA (cELISA CAEV; VMRD Inc., Pullman, WA, USA) with monoclonal antibodies to a CAEV-type viral envelope protein (ENV-SU, gp135), the AG-CHEKIT (CAEV/Maedi– Visna virus [MVV] kit; IDEXX, Bern, Switzerland) using the whole virus as the antigen, and ELITEST-MVV (Hyphen Biomed, Neuville-sur-Oise, France) using Gag p25 recombinant protein and a transmembrane (TM; gp46) synthetic peptide derived from genotype A, following the manufacturer’s protocols.

Pregnant Pelibuey sheep (n ¼ 25) from three Mexican states (10 from Jalisco, five from Veracruz, and 10 from Chiapas), which were destined for sacrifice, were used. The age range of the female sheep was 2–3 years (Table 1), and clinical signs related to SRLV infection were not present. The study protocol was approved by the Comité Interno para el Cuidado y Uso de los Animales en Experimentación de la Facultad de Estudios Superiores Cuautitlán (Internal Committee for the Care and Use of Experimental Animals from the Faculty of Superior Studies Cuautitlan) under number C12_20. The animals were humanely sacrificed on the basis of the Nom-033, 2015 [15].

2.5. Polymerase chain reaction DNA was obtained from the PBMCs of ewes and fetuses and fetal/ewe tissues using a commercial kit (FavorPrep; DNA Extraction FAVORGEN, Pingtung, Taiwán) and was quantified and stored at ‒20  C until use. The primers used to amplify a region of the gag and long terminal repeat (LTR) genes, which had expected products of 514 and 232 bp, respectively, were as follows:

2.2. Sample collection Blood was collected from the ewes by jugular vein puncture using tubes with anticoagulant (BD Vacutainer Sodium Heparine Franklin Lakes, NJ, USA) before

Table 1 Serological responses against SRLV and proviral DNA in Pelibuey ewes and fetuses. Sheep

\

Age range

F

25 32 Ewes Fetuses

Years

Days

\

F

2‒3

40‒90

Origin

Commercial ELISA kits

Ver

Jal

Chis

ELITEST

CHEKIT

VMRD

5 7

10 12

10 13

(þ) 7 0

(þ) 7 0

(þ) 0 0

(‒) 18 32

(‒) 18 32

Abbreviations: Chis ¼ Chiapas; \ ¼ Ewes; F ¼ Fetuses; Jal ¼ Jalisco; Ver ¼ Veracruz.

(‒) 25 32

Peptide ELISAs

PCR

Genotype A

Genotype B

LTR region

(þ) 11 0

(þ) 11 0

(þ) 7 2

(‒) 14 32

(‒) 14 32

gag region (‒) 18 30

(þ) 9 1

(‒) 16 31

J.H. Sánchez et al. / Theriogenology xxx (2016) 1–5

Fw gag (ATGATGCCTGGRAAYAGAGC), Rv gag (GCTTGTGCTAAYAAYTGCAT), Fw LTR (GAAACTTCGGGGACGCCTG), and Rv LTR (CCTCGCCATGTCTCTATCTAG). The PCR reaction mix consisted of reaction buffer 1 (Thermo Scientific, Waltham, MA, USA), 2-mM MgCl2 (Thermo Scientific), 224-mM each dNTP (Thermo Scientific), 600 nM each primer, 0.04 U/mL Taq DNA polymerase (Thermo Scientific), and 0.5–1 mg of DNA in a final volume of 25 mL. The PCR conditions were as follows: initial denaturation for 5 minutes at 94  C; 45 cycles of 94  C for 30 seconds, annealing at 53  C for 40 seconds or 62  C for 15 seconds for the gag and LTR regions, respectively, and extension at 72  C for 40 seconds; and final extension at 72  C for 10 minutes. The amplified products were separated by electrophoresis in 1.5% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. 2.6. Cohen’s kappa coefficient Kappa coefficient was used to determine the degree of concordance among commercial ELISA tests, ELISA peptides, and PCR, which according to the obtained value is extrapolates and is defined according to the following criteria: slight agreement from 0.01 to 0.20, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 substantial agreement, and 0.81 to 0.99 almost perfect agreement [18]. 2.7. Histopathology Samples of thymus and spleen (fetuses) and umbilical cord and cotyledons (fetuses/ewes) were processed by routine methods: paraffin embedding, cutting to 20 mm/25 mg, and staining with hematoxylin–eosin. The slides were observed under an optical microscope at  40 to inspect the tissue architecture and at  100 to determine the presence of mononuclear cell infiltration (monocytes and lymphocytes) that is characteristic of SRLV infection. 3. Results 3.1. ELISA commercial kits and peptide ELISAs Fifty-seven plasma samples were evaluated (25 ewes and 32 fetuses) by three commercially available ELISA kits. Both ELITEST and CHEKIT identified seven seropositive ewes, but the VMRD commercial kit detected none (Table 1). The seropositive animals originated from Chiapas (n ¼ 1) and Jalisco (n ¼ 6) (Table S1). Peptide ELISA detected 11 seropositive ewes of genotype A and 11 of genotype B (Table 1 and Table S1), corresponding to six seropositive ewes in addition to those identified using commercial ELISAs (one from Chiapas, three from Jalisco, and two from Veracruz; Table S1). Specifically, one ewe from Jalisco was shown to be positive by the commercial kits, only one ewe from Veracruz was identified by the peptide ELISA of genotype A, and one ewe from Jalisco was detected by the genotype B peptide ELISA (Table S1). Among the total seropositive population sampled, 36% (n ¼ 9) were from Jalisco, 8% (n ¼ 2) were from Chiapas, and 8% (n ¼ 2) were

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from Veracruz. All fetuses were negative according to the commercial and peptide ELISAs (Table 1 and Table S1). 3.2. PCR assays Positive PCR amplifications were achieved for PBMC DNA but not for tissue DNA. PCR–gag identified nine positive ewes, whereas PCR–LTR detected seven positive ewes out of 25 (Table 1). Only three ewes were found to be positive by both PCR assays (Table S1). One of the two PCR-positive fetuses was identified by both PCRs and came from a genotype A peptide-seropositive ewe. The other one, which was LTR–PCR positive, was obtained from a seronegative ewe from Chiapas that was identified as positive by both PCRs (Table S1). Proviral DNA was identified in a total of 13 ewes by combining the results from both PCRs, and of these, nine were seropositive (eight from Jalisco and one from Chiapas; Table 1). 3.3. Overall PCR and ELISA results Altogether, serology and proviral DNA detection identified 17 SRLV infection-positive animals, corresponding to 68% of the sampled ewes. The agreement between PCR and ELISA was evaluated by calculating Cohen’s kappa coefficient and was found to be moderate when comparing PCR and commercial ELISAs (K ¼ 0.563) and when comparing both peptide ELISAs (K ¼ 0.403) and fair (K ¼ 0.337) when the genotype A peptide ELISAs were considered individually (Fig. 1). Agreement between the commercial and homemade ELISAs was also moderate (K ¼ 0.564), with peptide ELISA detecting more PCR-positive animals. No evidence of the characteristic lesions of SRLV was found in the histologic preparations of the fetuses/ewes (data not shown), and the presence of proviral DNA was not identified by PCR in the sampled tissues. 4. Discussion Despite the initial reports indicating that VMV infection is unique to sheep and CAEV to goats, genetic studies have demonstrated the presence of VMV in goats and vice versa [9], which has important implications for control programs [19]. Here, we present evidence of SRLV infection in Mexican Pelibuey sheep on the basis of positive reactions in commercial and homemade ELISAs and PCR. Although all the fetuses were seronegative according to the ELISAs used, some were identified as positive by PCR performed on blood, suggesting transmission from mothers to lambs before lambing. In ruminants, maternal antibody transfer occurs through colostrum intake after parturition and not during the gestational period [20]. Therefore, an antibody response in unborn fetuses should be directly linked to the fetus’s immune response. The presence of antibodies against SRLV has been demonstrated in sheep fetuses with gestational ages of 80 and 90 days using an ELISA commercial test [13]; however, in the present study, none of the fetuses were seropositive. This finding may be related to their ages because only 18% (n ¼ 6) were in the 80- to 90-day age group. The maturity of the immune system is influenced by the fetuses’ age. Indeed, fetuses

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A

3

126M2

2.5 2 1.5 1 0.5 0 0

0.5

1

1.5

2

2.5

3

126M1

B

4 3.5

CHEKIT

3 2.5 2 1.5 1 0.5 0 0

0.5

1

1.5

2

2.5

3

3.5

4

ELITEST Fig. 1. Net absorbance dispersion against peptides 126M2 (genotype A; y axis) and 126M1 (genotype B; x axis) (A) and against commercial ELITEST and CHEKIT (B). The solid vertical and horizontal lines represent the corresponding ELISA cutoff values. Sample reactivity in one test (blue squares) or both tests (black squares) is represented. Samples that were close to the cutoff are shown in both colors and negative samples are represented by empty squares. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

have been shown to be able to develop adequate responses against infections by Simian Virus 40 (SV40) at Day 90, bluetongue virus at Day 122, and lymphocytic choriomeningitis virus at Day 140 [13]. The lack of seropositive fetuses may also be related to differences between the antigenic spectrum covered by the ELISAs applied and the antigenic composition of the infected virus or low virus exposure to the nascent immune system because of the low SRLV transmission rate through the placenta. Nonetheless, even in the absence of antibodies indicative of SRLV infection, proviral DNA was detected in the blood of two fetuses that were approximately 40 days old. These fetuses were raised by two different pregnant ewes: one seronegative but positive to gag–PCR and the other seropositive to genotype A peptide and negative to PCR. This finding highlights the low concordance between ELISA and PCR and supports their complementary use in SRLV diagnosis. However, the presence of proviral DNA in tissues from the studied fetuses and ewes could not be detected, likely because of the fixing solution used (buffered formalin). Indeed, cetyltrimethylammonium bromide has been shown to be more effective in obtaining good-quality DNA [21]. Using other preservative methods, satisfactory amplification of proviral DNA in tissues from the reproductive system of infected ewes has been achieved [22].

Some studies have proposed that SRLV infection may originate locally in fetuses through the infected tissues of ewes, such as the ovary [10,23], oviduct [10,11,22,24], and uterus [10,22,23,25] or at the time of birth through vaginal secretions [26] and the vaginal canal [22]. However, embryos that maintain an intact zona pellucida are apparently not susceptible to SRLV infection [8,11,12,27] or have a risk of infection of only 5% [28]. This could explain the low percentage (6%) of infected fetuses found in this study. Similar results have been previously reported in goat fetuses [25] and sheep [22] obtained by caesarean section from seropositive ewes. Few works have addressed SRLV infection in Mexican sheep [1,13]. In this study, the Pelibuey breed was investigated because of its robustness and resistance to infections [29,30]. However, a high percentage of infected ewes was detected, indicating that this breed is susceptible to SRLV infection. In contrast, studies performed in Rambouillet, Columbia, and Polypay sheep reported the absence of proviral DNA and antibodies against SRLV in offspring in close contact with seropositive ewes fed natural infected colostrum [31], suggesting breed resistance to SRLV infection. However, the presence of antibodies in newborns previously separated from their mothers has been demonstrated in susceptible breeds [10,11]. Although the placental and intrauterine transmission frequencies are low, the present work demonstrates that this route could favor the persistence of the virus in herds. This finding highlights the importance of establishing control and detection measures for SRLV infection, even in animals that have been separated from their mothers since birth. 4.1. Conclusions The presence of antibodies against SRLV was detected at a high frequency in Mexican Pelibuey ewes. No lesions suggestive of SRLV infection nor the presence of antibodies or proviral DNA in tissues obtained from fetuses (thymus and spleen) and fetuses/ewes (umbilical cord and cotyledon) was found. However, proviral DNA was detected in the PBMCs of ewes and fetuses, confirming the serologic results. Thus, both the Pelibuey breed’s susceptibility to SRLV infection and a low rate of vertical transmission were demonstrated in this small study group of Mexican sheep. Competing interests The authors have no financial or personal interests that could influence or bias the content of this article. Acknowledgments This study was supported by the PIAPIC27/FESC/UNAM and FIS/IMSS/PROT/G12/1109 programs. The authors extend special thanks to their collaborators (teaching staff and students) in the Laboratory of Virology, Genetics, and Molecular Biology. Ramsés Reina was supported by the Spanish Ministry of Science and Innovation ‘Ramón y Cajal’ contract.

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[15] Secretaria de Agricultura, Ganaderia, Desarrollo Rural, Pesca y Alimentación. Norma Oficial Mexicana. NOM-033-SAG/ZOO-2014, Métodos para dar muerte a los animales domésticos y silvestres. Diario Oficial 26 de agosto del 2015. México. [16] Lyngset O. Studies on reproduction in the goat. VII. Pregnancy and the development of the foetus and the foetal accessories of the goat. Acta Vet Scand 1971;12:185–201. [17] de Andres X, Ramirez H, Bertolotti L, San Roman B, Glaria I, Crespo H, et al. An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections. Vet Immunol Immunopathol 2013;152:277–88. [18] Viera AJ, Garrett JM. Understanding interobserver agreement: the kappa statistic. Fam Med 2005;37:360–3. [19] Reina R, Berriatua E, Lujan L, Juste R, Sanchez A, de Andres D, et al. Prevention strategies against small ruminant lentiviruses: an update. Vet J 2009;182:31–7. [20] Fahey KJ, Morris B. Humoral immune responses in foetal sheep. Immunology 1978;35:651–61. [21] Paireder S, Werner B, Bailer J, Werther W, Schmid E, Patzak B, et al. Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues. Anal Biochem 2013;439:152–60. [22] Fieni F, Rowe J, Van Hoosear K, Burucoa C, Oppenheim S, Anderson G, et al. Presence of caprine arthritis-encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does. Theriogenology 2003;59:1515–23. [23] Cortez Romero C, Fieni F, Roux C, Russo P, Guibert JM, Guiguen F, et al. Detection of ovine lentivirus in the cumulus cells, but not in the oocytes or follicular fluid, of naturally infected sheep. Theriogenology 2006;66:1131–9. [24] Lamara A, Fieni F, Mselli-Lakhal L, Tainturier D, Chebloune Y. Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis-encephalitis virus (CAEV). Virus Res 2002;87:69–77. [25] Ali Al Ahmad MZ, Dubreil L, Chatagnon G, Khayli Z, Theret M, Martignat L, et al. Goat uterine epithelial cells are susceptible to infection with Caprine Arthritis Encephalitis Virus (CAEV) in vivo. Vet Res 2012;43:5. [26] Ali Al Ahmad MZ, Chebloune Y, Bouzar BA, Baril G, Bouvier F, Chatagnon G, et al. Lack of risk of transmission of caprine arthritisencephalitis virus (CAEV) after an appropriate embryo transfer procedure. Theriogenology 2008;69:408–15. [27] Lamara A, Fieni F, Mselli-Lakhal L, Chatagnon G, Bruyas JF, Tainturier D, et al. Early embryonic cells from in vivo-produced goat embryos transmit the caprine arthritis-encephalitis virus (CAEV). Theriogenology 2002;58:1153–63. [28] Al Ahmad MZ, Chebloune Y, Chatagnon G, Pellerin JL, Fieni F. Is caprine arthritis encephalitis virus (CAEV) transmitted vertically to early embryo development stages (morulae or blastocyst) via in vitro infected frozen semen? Theriogenology 2012;77:1673–8. [29] Gonzalez JL, Lopez-Arellano ME, Olazaran-Jenkins S, LiebanoHernandez E, de Gives PM, Vazquez-Prats V, et al. Phenotype characterization of Pelibuey native lambs resistant to Haemonchus contortus. Ann N Y Acad Sci 2008;1149:177–9. [30] Morteo-Gómez R, González-Garduño R, Torres-Hernández G, Nuncio-Ochoa G, Becerril-Pérez CM, Gallegos-Sánchez J, et al. Effect of the phenotipic variation in the resistance of pelibuey lambs to the infestation with gastrointestinal nematodos. Agrociencia 2004;38: 395–404. [31] Broughton-Neiswanger LE, White SN, Knowles DP, Mousel MR, Lewis GS, Herndon DR, et al. Non-maternal transmission is the major mode of ovine lentivirus transmission in a ewe flock: a molecular epidemiology study. Infect Genet Evol 2010;10: 998–1007.

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Table S1 Serological and PCR results for the detection of SRLV infection in 25 pregnant ewes and 32 fetuses. Origin

Identification

Serological tests

PCR

Commercial ELISAs

Chiapas Chiapas

Chiapas

Chiapas Chiapas Chiapas

Chiapas Chiapas Chiapas Chiapas Jalisco Jalisco

Jalisco

Jalisco Jalisco Jalisco Jalisco Jalisco Jalisco Jalisco Veracruz Veracruz

Veracruz

Veracruz Veracruz

E1 F1 E2 F2 F2 E3 F3 F3 E4 F4 E5 F5 E6 F6 F6 E7 F7 E8 F8 E9 F9 E10 F10 E11 F11 E12 F12 F12 E13 F13 F13 E14 F14 E15 F15 E16 F16 E17 F17 E18 F18 E19 F19 E20 F20 E21 F21 E22 F22 F22 E23 F23 F23 E24 F24 E25 F25

Peptide ELISAs

ELITEST

VMRD

CHEKIT

Genotype B

Genotype A

NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS NEG NEG POS NEG NEG NEG POS NEG POS NEG POS NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS NEG NEG POS NEG NEG NEG POS NEG POS NEG POS NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

NEG NEG POS NEG NEG NEG NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS NEG POS NEG NEG POS NEG NEG POS NEG POS NEG POS NEG NEG NEG POS NEG POS NEG NEG NEG NEG NEG NEG NEG NEG POS NEG NEG NEG NEG NEG NEG

NEG NEG POS NEG NEG NEG NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS NEG NEG POS NEG NEG POS NEG POS NEG POS NEG NEG NEG POS NEG POS NEG NEG NEG NEG NEG NEG NEG NEG POS NEG NEG POS NEG NEG NEG

Positive results are indicated in bold. Abbreviations: E ¼ Ewes; F ¼ Fetuses; NEG ¼ negative; POS ¼ positive.

LTR

gag

NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS POS NEG NEG NEG NEG NEG NEG NEG POS NEG NEG NEG NEG POS NEG NEG NEG POS NEG POS NEG POS NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

NEG NEG POS NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG POS NEG POS NEG NEG NEG NEG NEG POS NEG POS POS NEG NEG NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS NEG