Presence of caprine arthritis–encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does

Theriogenology 59 (2003) 1515±1523

Presence of caprine arthritis±encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does F. Fienia,*, J. Rowea, K. Van Hooseara, C. Burucoab, S. Oppenheimc, G. Andersonc, J. Murrayb, R. BonDurantb a

Department of Population Health and Reproduction, University of California, Davis, CA 95616, USA b Department of Medical Microbiology, University of California, Davis, CA 95616, USA c Embryo Transfer Laboratory, University of California, Davis, CA 95616, USA Received 31 December 2001; accepted 14 June 2002

Abstract Transmission of caprine arthritis±encephalitis virus (CAEV) is not completely understood and the vertical route of infection from the goat to the embryo or to the fetus needs to be investigated. This route of infection involves the presence of CAEV in the genital tract tissues. Prior studies have detected CAEV-infected cells in genital secretions and in ¯ushing media recovered during embryo collection from infected goats. To specify the origin of these cells, we conducted a double-nested polymerase chain reaction (PCR) test on embryo ¯ushing media and on mammary gland, mammary lymph node, synovial membrane, pelvic lymph node, uterus and oviduct tissues from 25 CAEVinfected (blood PCR positive) embryo donor goats for the presence of CAEV proviral DNA. The presence of proviral DNA was found in 22 of 25 mammary gland samples, 14 of 25 uterus samples, and in 16 of 25 oviduct samples. Nineteen of 25 goats had at least one positive genital tract sample. Flushing media from 11 goats were PCR positive. All goats with positive-¯ushing media were oviduct positive. Of this group of does, except for 1 of the 11, infection of ¯ushing media correlated with infection of almost all the other tissues examined. The frequency of positive tissues for ¯ushing media-positive goats (61/66; 92%) was signi®cantly higher than that for ¯ushing medianegative goats (50/84; 60%) (P < 0:01). This study demonstrated the presence of CAEV-infected cells in the goat genital tract. The presence of CAEV-infected cells in the uterus and oviducts suggests potential for vertical transmission of CAEV from doe to embryo or fetus. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Caprine arthritis±encephalitis virus; Flushing media; Uterus; Oviduct; Goat; CAEV * Corresponding author. Present address: Department of Biotechnology and Pathology of Reproduction, National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France. Tel.: ‡33-2-40687710; fax: ‡33-2-40687748. E-mail address: [email protected] (F. Fieni).

0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 9 4 - 9

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1. Introduction Caprine arthritis±encephalitis infection has been detected worldwide, but is most prevalent in countries where dairy goats are intensively raised [1]. Approximately 30±80% of dairy goats in the U.S.A, Canada, and Europe are infected, compared to 0±10% in Africa and South America [1±5]. This very common disease in dairy goats is induced by an enveloped, single-stranded RNA virus which has been classi®ed in the Lentivirus subfamily of Retroviridae [6]. Caprine arthritis±encephalitis virus (CAEV) has a tropism for monocytes and macrophages and causes chronic in¯ammatory disease [7]. Consequently, all body secretions and excretions could be a source of infection. This tropism accounts for the fact that a major route of CAEV transmission is the ingestion of virus-laden colostrum or milk from infected does [8±10]. Prevention of CAEV transmission is based on the removal of kids from their mothers at birth and feeding of kids with heat-treated goat colostrum [11,12]. However, in large herds using this pasteurized feeding method, an unexplained seroconversion incidence of up to 10% has been observed [8,13]. Less ef®cient routes of transmission, such as the vertical route, must be investigated in order to improve control and eradication methods [14,15]. Suspicion of the presence of CAEV virus in the genital tract of infected goats and the possibility of a vertical or a horizontal route of transmission was established thanks to two ®ndings: ®rst, the detection of CAEV-infected cells in postpartum genital secretion of dairy does [16] and second, the detection of CAEV-infected cells in oviductal ¯ushing media recovered during early embryo collection from infected goats [17]. To determine the origin of the infected cells found in ¯ushing media and to determine if the CAEV virus was able to infect genital tract tissues, the aim of this study was to examine uterine and oviductal tissues from CAEV-infected embryo donor goats for the presence of CAEV proviral DNA and to compare this with the incidence of infection in tissues outside the reproductive tract. 2. Materials and methods 2.1. Animals Twenty ®ve CAEV-infected (blood PCR positive) nonlactating dairy goats of various breeds (Alpine, Saanen, and Nubian) were used as embryo donors for this study, from September to December 1998. Estrous cycles were synchronized via intravaginal pessaries containing 40 mg of progestin (Redopharm, Halfway House, Republic of South Africa). The pessaries were left in place for 10 days. The does received treatments with follicle-stimulating hormone (FSH-P) (Sioux Biochemical, Sioux Center, IA) twice daily, by subcutaneous route, in decreasing doses (5, 4, and 3 IU) the day before, the day of, and the day after pessary removal. Twelve hours prior to pessary removal, the does received 15 mg of prostaglandin F2a (Lutalyse1, Pharmacia-Upjohn Co., Kalamazoo, MI) intramuscularly. Twenty-four hours after pessary removal, the does were given 50 mg of a gonadotropin-releasing hormone (Cystorelin1, Rhone Merieux,

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Athens, GA) intravenously. Superovulated goats were hand-mated twice daily to fertile bucks (i.e. by natural service), allowing the doe to mate twice in the morning and twice in the evening. 2.2. Sample collection 2.2.1. Blood Blood samples (8 ml in acid citrate dextrose (ACD)) were obtained by jugular venipuncture from each goat just before the recovery of tissues and ¯ushing media. 2.2.2. Flushing media Approximately 60±65 h after pessary removal, (i.e. 36±48 h after the beginning of estrus), the reproductive tracts were collected from donor goats at slaughter immediately following exsanguination. A new scalpel blade was used for each goat. Immediately afterward, at the laboratory, ovulations were counted and embryos ¯ushed from the oviducts. The mesosalpinx was trimmed to straighten out the oviducts. A sterile, blunt, 20-gauge needle was inserted through the utero-tubal junction and 20 ml of phosphate-buffered saline (PBS) containing 1% heat-treated calf serum and 1% antibiotic/antimycotic (penicillin, streptomycin, polymixin B, Gibco, Grand Island, NY) was ¯ushed out the infundibulum into a scored petri dish. After recovery of embryos, ¯ushing media from each goat was transferred into a sterile glass bottle and maintained at 4 8C. 2.2.3. Tissues At the slaughterhouse, samples of mammary gland, mammary lymph node, synovial membrane, and pelvic lymph node were collected and were frozen at 70 8C. At the laboratory immediately after ¯ushing, samples of uterus and oviducts were collected and were frozen at 70 8C. At the slaughterhouse and at the laboratory, we used a different scalpel blade per goat. When collecting different samples recovered from the same goat, the scalpel blade was ®rst washed in RBS Viro (Fluka Chemical Corp, New York, NY), then wiped on a sheet of absorbent paper and disinfected with ethanol (70 8C), and ®nally, dried with a new sheet of absorbent paper. 2.3. Preparation of blood, ¯ushing media, and tissues for PCR 2.3.1. Blood Mononuclear cells were recovered from 8 ml of ACD blood by density-gradient centrifugation (1925  g for 30 min) through a cushion of Ficoll (Histopaque-1077, Sigma Chemical Co., St. Louis, MO). The cells were washed three times in sterile PBS and frozen at 20 8C. After thawing, blood mononuclear cells were resuspended in 100 ml of lysis buffer (10 mM Tris hydrochloride, pH 8.3, 0.45% NP-40, 0.45% Tween-20, 50 mg proteinase K/ml), and incubated in a water bath at 56 8C for 3 h. The proteinase K was subsequently inactivated by incubating the samples at 100 8C for 15 min. Blood samples were then stored at 20 8C until the PCR reactions were performed. Manipulations were made in a class II laminar ¯ow hood.

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2.3.2. Flushing media Cells were recovered from ¯ushing media by centrifugation (1900  g for 5 min). The supernatant was carefully poured off. The cells were washed one time in 1 ml sterile PBS and then frozen at 20 8C. After thawing, the cells were lysed as described for the blood samples. Cell lysates were then stored at 20 8C until the PCR reaction was performed. Manipulations were made in a class II laminar ¯ow hood. 2.3.3. Tissues Total DNA was puri®ed by using a QIAamp Tissue kit (QUIAGEN Inc., Calencia, CA) following to the manufacturer's protocol. Brie¯y, after thawing, the samples (0.25 mg) were diluted in 200 ml tissue lysis buffer containing proteinase K, and incubated overnight on a rotator at 55 8C. When the tissues were completely lysed, the proteinase K was subsequently inactivated by incubating the samples at 70 8C for 10 min. The DNA was precipitated with 210 ml ethanol. Puri®cation of DNA was obtained by applying the samples to a QIAamp spin column. The DNA was adsorbed onto the QIAamp silica membrane during a brief centrifugation step. The QIAamp spin columns were washed twice. The DNA was eluted twice with 200 ml of distilled water preheated at 70 8C. Samples were processed in groups of 18. One negative tissue control and one positive tissue control were used for each group of 18 tissue samples tested. After DNA puri®cation, the samples were stored at 20 8C until the PCR reactions were performed. 2.4. Procedure for double-nested PCR The CAEV proviral DNA was tested by a dn-PCR technique as described by Barlough et al. [18]. The DNA fragments of 184 bp corresponding to gag sequences of CAEV were ampli®ed with two pairs of oligononucleotide primers. The outer primers used were P1 (50 -CAAGCAGCAGGAGGGAGAAGCTG-30 ), bp 953±975, and P2 (50 -TCCTACCCCCATAATTTGATCCAC-30 ), bp 1249±1226. The inner primer was P3 (50 -GTTCCAGCAACTGCAAACAGTAGCAATG-30 ), bp 997±1024, and P4 (50 -ACCTTTCTGCTTCTTCATTTAATTTCCC-30 ), bp 1181±1154. The primers were designed using the published sequence of CAEV strain CO by Saltarelli et al. [19] and a commercially available software package (Amplify, University of Wisconsin, Madison, WI), and were synthesized on a Gene Assembler Plus (Life Technologies, Grand Island, NY). Ampli®cation was carried out in a thermal cycler (PTC 100TM Programmable Thermal Controller, MJ Research Inc., Watertown, MA). For the ®rst round reaction, 3 ml of extracted DNA was added to 47 ml of a mixture containing 10 mM Tris±HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 0.001% wt/v gelatin, 400 mM each dNTP, 5 mM tetramethylammonium chloride (TMAC), 20 pmol (in 1 ml) outer primers 1 and 2, 2 IU Taq DNA polymerase (AmpliTaq, Perkin±Elmer, Branchburg, NJ). For the second round reaction, 1 ml aliquots from the ®rst round were again ampli®ed in the same reaction mixture except that 20 pmol (in 1 ml) inner primers 3 and 4 were used in place of outer primers 1 and 2. For each round, after denaturation at 94 8C for 5 min, the samples were submitted to 34 cycles with denaturation at 94 8C for 30 s, annealing at 55 8C for 30 s, and extension at 72 8C for 90 s. Each round was followed by a ®nal extension at 72 8C for 5 min. Products were visualized by gel electrophoresis: 18 ml PCR (second round) was added to 2 ml dye buffer per well inside 2% agarose gel

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containing ethidium bromide in 0.5 TBE buffer. After 40 min of electrophoreses at 100 V, appropriately sized PCR products were visualized by transillumination with UV light. An HaeIII digest of fX174 yielding 11 suitable fragments was used as the molecular weight standard for the agarose gel electrophoresis (New England Biolabs, Beverly, MA). To reduce the likelihood of DNA contamination, all mixture preparations were performed in a class II laminar ¯ow hood. Nondisposable materials were decontaminated before use in a UV Crosslinker (FB UVXL-1000, Fisher Scienti®c, Pittsburgh, PA) at 1:2  105 mJ/cm3. Samples were processed in groups of 17. For each gel, we used a CAEV positive control, a CAEV negative control, and a distilled water control. Positive and negative CAEV controls were blood samples from well-known serological and dn-PCR positive and negative goats that had been under close surveillance for many years. An internal gag probe was employed to verify the gag product of positive control samples using a Southern blotting procedure [18]. 2.5. Statistical analyses Quantitative values are presented as means (M) and standard deviation (S.D.). Differences with associated P-values of <0.05 were considered to be signi®cant. A Chi-square test with Yates correction was used to study the distribution of CAEV proviral DNA in tissue samples. The mean embryo recovery rate for oviduct positive and negative goats were compared using the Student±Fisher test for independent samples [20]. 3. Results Ampli®cation of CAEV proviral DNA by the nested gag primers resulted in a predictable 184-bp band included between the 194- and 118-bp bands of the molecular weight standards for agarose gel electrophoresis use. Of the 25 blood PCR positive goats examined, 11 were PCR positive-¯ushing media. A total of 150 sample tissues were examined by PCR for the presence of CAEV proviral DNA. Of these, 111 (74%) sample tissues were found to be positive. The presence of proviral DNA was found in 88% (22/25) of mammary gland samples, 84% (21/25) of mammary lymph node samples, 68% (17/25) of synovial membrane samples, 72% (18/25) of pelvic lymph node samples, 68% (17/25) of uterus samples, and in 64% (16/25) of oviduct samples. Seventy-six percent of the goats (19/25) had at least one positive genital tract sample (Tables 1 and 2). The frequency of positive tissues for ¯ushing media-positive goats (61/66; 92%) was signi®cantly higher than that for ¯ushing media-negative goats (50/84; 60%) (P < 0:01). All goats with positive-¯ushing media were oviduct positive (Table 1). The presence of CAEV proviral DNA in oviductal tissues differed signi®cantly between the positive¯ushing media does and the negative ones (P < 0:01). Of the group of positive-¯ushing media does, except for 1 of 11 does, infection of ¯ushing media correlated with infection of almost all the other tissues examined (Table 1). Of the group of negative-¯ushing media does, two does were PCR negative for all tissue samples. For the other does, mammary gland samples were always positive (Table 2). The

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Table 1 Repartition of tissue samples PCR positive for PCR positive-flushing media goats (n ˆ 11) and for PCR negative-flushing media goats (n ˆ 14) Status

Number of tissues PCR positive Goats (n)

Positive-flushing media goats Goats with all tissue samples PCR positive Goats with at least one tissue sample PCR positive Frequency of tissues samples PCR positive (%)

Mammary Mammary gland lymph node

Synovial membrane

Pelvic lymph node

Uterus

Oviduct

9

9

9

9

9

9

9

2

1 0 90.9

1 0 90.9

0 1 90.9

1 0 90.9

1 0 90.9

1 1 100.0a

12

11

7

8

7

5

0

0

0

0

0

0

84.6

50.0

57.1

50.0

37.7c

Negative-flushing media goats Goats with at least one tissue 12 samples PCR positive Goats with no tissue sample 2 PCR positive Frequency of tissues samples PCR positive (%)

85.7b

Values with different superscripts (a, c) (P < 0:01) and (b, c) (P < 0:05) differ.

frequency of tissue infection decreased from mammary glands (87.5%) to oviducts (37.7%). The CAEV proviral DNA was signi®cantly (P < 0:05) more often found in mammary gland samples than in oviduct samples. Except for one doe from the group of positive-¯ushing media, which had only synovial membrane and oviductal tissues as positive samples, all does which had uterus or oviduct positive samples also had mammary gland and mammary lymph node positive samples (Tables 1 and 2). During this experiment a total of 175 embryos were recovered (7:0  5:0 per goat). The mean number of embryos recovered was not signi®cantly different between goats with oviduct PCR positive and goats with oviduct PCR negative (6:6  5:8 versus 7:6  3:2, respectively). Table 2 Detail of relative repartition of tissue samples PCR positive for PCR negative-flushing media goats (n ˆ 14) Tissue samples PCR positive

Repartition of the other tissue samples PCR positive Mammary gland

Mammary lymph node

Synovial membrane

Pelvic lymph node

Uterus

Oviduct

Mammary gland Mammary lymph node Synovial membrane Pelvic lymph node Uterus Oviducts

12 11 7 8 7 5

11 11 6 8 7 5

7 6 7 6 2 3

8 8 6 8 4 4

7 7 2 4 7 4

5 5 3 4 4 5

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4. Discussion This study demonstrated, for the ®rst time, the presence of CAEV-infected cells in goat genital tissues. Cells of the monocyte and macrophage lineage are the principal targets for CAEV infection in vivo, and expression of infectious virus depends upon cellular differentiation [21,22]. Taking into account the clinical expression, tissues in which it is usual to ®nd infected cells are areas of in¯ammation in the brain, spinal cord, lung, synovium, and mammary gland [23]. However, viral transcripts have also been detected in epithelial cells from several organs of infected goats, including small intestine, thyroid, and kidneys [23]. It has also recently been demonstrated that epithelial cells in goat milk are susceptible to CAEV infection both in vitro and in vivo [24], and that granulosa cells derived from goat ovarian and epithelial oviductal cells are fully susceptible to CAEV infection in vitro [25,26]. In this experiment, we did not proceed with histological study to determine the origin (mononuclear or epithelial) of infected cells from the genital tract. Usually, local in¯ammation interferes with fertilization. As in this experiment, the number of embryos recovered was similar to those reported in previous studies [27,28], we assume that there was no, or only a very small, local in¯ammation process. Alternatively, CAEV could infect the resident monocytes and macrophages. The use of in situ hybridization would allow determination of the cellular localization of viral transcripts in the genital tissues. The evidence of CAEV-infected cells on the uterus and oviduct accounts for the ®nding that these cells have been recovered in postpartum genital secretions [16] and in ¯ushing media during embryo collection [17]. In theory, the cotyledonary epithelio-chorial placentation of the goat does not allow any contact between fetal and maternal blood. However, an exchange of cells can occur during a local in¯ammatory process, which could be induced by CAEV infection. Similarly, uterine lesions with leukocyte in®ltration have been observed in some cases of uterine disease [29]. The presence of CAEV-infected cells in the goat genital tract indicates the possible risk of vertical transmission by infection of the embryo or the fetus during oviductal or uterine life or at the parturition period when the fetus passes through the birth canal. This vertical transmission could explain the observation of seroconversion in 2 of 32 colostrum-deprived kids following cesarean delivery and in 1 of 10 kids after vaginal delivery [11], and the observation of seroconversion in 3 of 40 kids 3 months after natural birth and immediate separation from the mother, and 6 other seroconversions 2 months later [8]. In sheep, in utero-ovine lentivirus infection in lambs has been demonstrated [30]. This route of infection can be responsible for 13% of lamb infection [31]. The risk of vertical infection has, also, to be taken into account during embryo biotechnology. After in vitro fertilization, early stage embryos are cultivated on a feeder layer of epithelial cells from goat oviduct [32±34] which provides crucial support for in vitro development before uterine transfer into a recipient goat [35,36]. Caprine epithelial oviduct cells, used for embryo culture, are usually collected at the slaughterhouse from animals of unknown viral status. The use of CAEV-infected epithelial cells for in vitro culture may contribute to CAEV transmission to the cultured embryos and dissemination via embryo transfer. It should be noted that if the safety procedures prescribed by the

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International Embryo Transfer Society [37] are respected, the risk of CAEV transmission by embryo transfer will be extremely small because it has been demonstrated that intact zona pellucida protects early embryos from CAEV infection [26]. Acknowledgements The authors are grateful to Alice Moyer from U.C.D. Animal Science Department Embryo Transfer Laboratory for her technical assistance. This research was supported by the France-Berkeley Fund, ``Conference Nationale des Grandes Ecoles FrancËaises'', and U.S. Department of Agriculture (CALV-AH-161). References [1] Rowe JD, East NE. Risk factors for transmission and methods for control of caprine arthritis±encephalitis virus infection. Vet Clin North Am Food Anim Prac 1997;13(1):35±53. [2] Adams DS, Oliver RE, Ameghino E, DeMartini JC, Verwoerd DW, Houwers DJ, et al. Global survey of serological evidence of caprine arthritis±encephalitis virus infection. Vet Rec 1984;115:493±5. [3] Crawford TB, Adams DS. Caprine arthritis±encephalitis: clinical features and presence of antibody in selected goat populations. J Am Vet Med Assoc 1981;178:713±9. [4] East NE, Rowe JD, Madewell BR, Floyd K. Serologic prevalence of caprine arthritis±encephalitis virus in California goat dairies. J Am Vet Med Assoc 1987;190:182±6. [5] Phelps SL, Smith MC. Caprine arthritis±encephalitis virus infection. J Am Vet Med Assoc 1993;203: 1663±6. [6] Dawson M. Lentivirus diseases of domesticated animals. J Comp Pathol 1988;99:401±19. [7] Narayan O, Zink M, Gorrell S, Crane D, Huso P, Jolly P, et al. The lentiviruses of sheep and goats. In: Levy JA, editor. The Retroviridae. New York, NY: Plenum Press; 1993. [8] East NE, Rowe JD, Dahlberg JE, Theilen GH, Pedersen NC. Modes of transmission of caprine arthritis± encephalitis virus infection. Small Rumin Res 1993;10:251±62. [9] Lerondelle C, Greenland T, Jane M, Mornex JF. Infection of lactating goats by mammary instillation of cell-borne caprine arthritis±encephalitis virus. J Dairy Sci 1995;78:850±5. [10] Mackenzie RW, Olivier RE, Rooney JP. A successful attempt to raise goat kids free of infection with caprine arthritis±encephalitis virus in an endemically infected goat herd. NZ Vet J 1987;35:184±6. [11] Adams DS, Klevjer-Anderson P, Carlson JL, McGuire TC, Gorham JR. Transmission and control of caprine arthritis±encephalitis virus. Am J Vet Res 1983;44:1670±5. [12] Peretz G, Bugnard F, Calavas D. Study of a prevention programme for caprine arthritis±encephalitis. Vet Res 1994;25:322±6. [13] Rowe JD, East NE, Thurmond MC, Franti CE, Pedersen NC. Cohort study of natural transmission and two methods for control of caprine arthritis±encephalitis virus infection in goats on a California dairy. Am J Vet Res 1992;53:2386±95. [14] Greenwood PL. Effects of caprine arthritis±encephalitis virus on productivity and health of dairy goats in New South Wales, Australia. Prev Vet Med 1995;22:71±87. [15] East NE, Rowe JD, Dahlberg JE, Theilen GH, Pedersen NC. Modes of transmission of CAEV infection. Small Rumin Res 1993;10:251±62. [16] Rowe JD, Van Hoosear K, East N, DeRock EJ. The presence of caprine arthritis±encephalitis virus proviral DNA in postpartum genital secretion of dairy goat does. In: Proceedings of the 26th World Veterinary Congress, WVA, 1999 Sept 23±26; Lyon, France. [17] Fieni F, Rowe JD, Van Hoosear K, Burucoa C, Oppenheim S, Anderson G, et al. Presence of caprine arthritis±encephalitis virus (CAEV) infected cells in ¯ushing media following oviductal-stage embryo collection. Theriogenology 2002;57:931±40.

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