- Email: [email protected]
Presence of caprine arthritis–encephalitis virus (CAEV) infected cells in flushing media following oviductal-stage embryo collection
Theriogenology 57 (2002) 931±940
Presence of caprine arthritis±encephalitis virus (CAEV) infected cells in ¯ushing media following oviductal-stage embryo collection F. Fienia,*, J. Rowea, K. Van Hooseara, C. Burucoab, S. Oppenheimc, G. Andersonc, J. Murraya, R. BonDuranta 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 2 November 1999; accepted 18 July 2001
Abstract To improve the knowledge on the risk of transmission of the caprine arthritis±encephalitis virus (CAEV) during embryo manipulations, we conducted a double-nested polymerase chain reaction (PCR) for CAEV proviral-DNA on ¯ushing media recovered from the oviducts 48 h after the beginning of estrus and on blood from 89 donor does. Sixty-four does had negative blood and ¯ushing media by PCR. Among the 25 CAEV infected goats (blood PCR positive), 11 were PCR ¯ushing media positive (P < 0:01). Cell lysate from ¯ushing media samples that were PCR positive were serially diluted 10 times at 1:100. Starting with the second 1:100 dilution all the cell lysate samples were PCR negative. The mean number of embryos recovered was not signi®cantly different between goats with ¯ushing media PCR positive and goats with ¯ushing media PCR negative (6:0 5:4 versus 7:8 4:4, respectively: mean S:D:) nor between goats with blood PCR positive and goats with blood PCR negative (7:0 5:0 versus 5:9 5:3: mean S:D:). The presence of CAEV infected cells in oviductal ¯ushing media from infected donor does was indicated for the ®rst time during this study. The absence of ¯ushing media PCR positive for goat blood PCR negative seemed to allow the use of the blood PCR test to con®dently predict the absence of CAEV provirus in the oviductal ¯uid. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Caprine arthritis±encephalitis virus; Flushing media; Embryo; Goat; CAEV
* Corresponding author. Present address: Department of Biotechnology and Pathology of Reproduction, National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France. Tel.: 33-2-40-68-77-10; fax: 33-240-68-77-48. 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 1 ) 0 0 6 9 8 - 7
932
F. Fieni et al. / Theriogenology 57 (2002) 931±940
1. Introduction Caprine arthritis±encephalitis virus (CAEV) is a very common disease in dairy goats. In adult does, CAEV infection causes chronic arthritis, interstitial pneumonia and indurative mastitis with decreased milk production [1]. Less frequently, CAEV causes leukoencephlomyelitis in young goats [2]. CAEV is an enveloped, single-stranded RNA virus. It has been classi®ed as a Lentivirus, subfamily of Retroviridae [3]. This group of cell-associated viruses is characterized by the presence of an RNA-dependent DNA polymerase, and the CAEV is functional only when the viral RNA is transformed into viral DNA and is integrated into the host cell DNA. Thus, the viral genome becomes a part of the cellular DNA and replicates ef®ciently in nondividing, terminally differentiated cells [4]. The virus has a tropism for monocytes/macrophages and causes chronic in¯ammatory disease [5]. CAEV infection has been detected worldwide, but is most prevalent in countries where goats are intensively dairied [6]. Approximately 30±80% of dairy goats in the USA, Canada and Europe are infected, compared to 0±10% in other countries in Africa and South America [6±10]. In dairy herds, mammary tropism has economic consequences on milk production and mammary pathology [11,12]. The major route of CAEV transmission is the ingestion of virus-laden colostrum or milk from infected does [13±15]. Prevention of CAEV transmission is based on the removal of kids from their mothers at birth and the feeding on heat-treated goat colostrum [16,17]. But in large herds using this pasteurized method, up to 10% incidence of unexplained seroconversion has been observed [13,18]. Less ef®cient routes of transmission must be clari®ed in order to improve control and eradication methods [11,13]. Embryo manipulation is a common technique used for fundamental research such as gene transfer or cloning and for commercial exchanges. For both uses, embryo biotechnologies have to present a complete sanitary guarantee against infectious disease transmission. Risk of disease transmission through animal embryos collected and processed in accordance with the standards and procedures prescribed by the International Embryo Transfer Society (IETS) tends to be extremely small [19,20], but has to be studied for each species, each infectious agent and each biological structure transmitted from donor to recipient. Two biological products coming from embryo collection can be infectious: ®rst, embryos and second, ¯ushing medias, which are permanently in contact with embryos during embryo manipulations. In goats, only one in vivo study has been done to examine the consequence of embryo transfer, and this study included only a small number of animals. Using seropositive embryo donors and bucks, Wolfe et al. [21] could not isolate, by inoculation of cellular culture, CAEV from uterine ¯ushings, from recipients' colostrum or placentas, or from the resulting fetuses and live kids. No seroconversion appeared in recipients after transfer. Currently, the use of the polymerase chain reaction (PCR) technique has allowed us to directly examine various samples for the presence of CAEV proviralDNA. The goal of our study was to determine whether CAEV infected cells might be found in ¯ushing media recovered during early embryo collection from slaughtered donors.
F. Fieni et al. / Theriogenology 57 (2002) 931±940
933
2. Materials and methods 2.1. Animals Eighty-nine nonlactating dairy goats of various breeds (Alpine, Saanen and Nubian) were used as embryo donors in groups of 12 or 13 from September to December 1998. Estrous cycles of donors were synchronized via intravaginal pessaries containing 40 mg progestin (Redopharm, Halfway House, Republic of South Africa). The pessaries were left in place for 10 days. The embryo donors received injections of a follicle-stimulating hormone (FSH-P) (Sioux Biochemical, Sioux Center, IA) twice daily in decreasing doses (5, 4, 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, PharmaciaUpjohn Co., Kalamazoo, MI) intramuscularly. Twenty-four hours after pessary removal, the does were given 50 mg of a gonadotropin-releasing hormone (Cystorelin1, Rhone Merieux, Athens, GA) intravenously. Donors 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 On the day of embryo collection, blood samples (8 ml in acid citrate dextrose (ACD)) were obtained by jugular venipuncture from each goat. 2.2.2. Flushing media and embryos 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 exanguination. Immediately afterwards, at the laboratory, ovulations were counted and embryos ¯ushed from the oviducts. The mesosalpinx were trimmed to straighten out the uterine tubes. A blunt, 20-gauge needle was inserted through the utero-tubal junction and 20 ml of phosphate-buffered saline (PBS) containing 1% heattreated calf serum and 1% antibiotic/antimycotic (penicillin, streptomycin, polymixin B, Gibco, Grand Island, NE) 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. 2.3. Preparation of blood and flushing media 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, 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 hydrochlorideÐpH 8.3, 0.45% NP-40, 0.45% Tween-20, 50 mg proteinase
934
F. Fieni et al. / Theriogenology 57 (2002) 931±940
K per 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. Manipulations were made in a class II laminar ¯ow hood. Blood samples were stored at 208C until the PCR reactions were performed. 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. Manipulations were made in a class II laminar ¯ow hood. Flushing media samples were stored at 20 8C until the PCR reaction was performed. In a second step, cell lysate from double-nested PCR (dn-PCR) positive ¯ushing media samples were serially diluted at 1:100, 10 times. After each dilution, the presence or absence of CAEV proviral-DNA was determined by dn-PCR. 2.4. Procedure for double-nested PCR CAEV proviral-DNA was tested by a dn-PCR technique as described by Barlough et al. [22]. 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 ), 953±975 bp and P2 (50 -TCCTACCCCCATAATTTGATCCAC-30 ), 1249±1226 bp. The inner primer were P3 (50 -GTTCCAGCAACTGCAAACAGTAGCAATG-30 ), 997±1024 bp and P4 (50 -ACCTTTCTGCTTCTTCATTTAATTTCCC-30 ), 1181±1154 bp. The primers were designed using the published sequence of CAEV strain CO Saltarelli et al. [23] 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 with 47 ml of a mixture containing 10 mM Tris±HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 0.001% w/v gelatin, 400 mM each dNTP, 5 mM tetramethylammonium chloride (TMAC), 20 pmol (in 1 ml) outer primers 1 and 2, 2 UI Taq DNA polymerase (AmpliTaq, PerkinElmer, 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 of (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 round 2 was added to 2 ml dye buffer per well inside 2% agarose gels 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. A Hae III Digest of fX174 yielding 11 suitable fragments was used as the molecular weight standard for the agarose gel electrophoresis (New UK Biolabs, Beverly, MA). To prevent DNA contamination, all mixture preparations were performed in a class II laminar ¯ow hood. Nondisposable materials were decontaminated before use in a UV
F. Fieni et al. / Theriogenology 57 (2002) 931±940
935
crosslinker (FB UVXL-1000, Fisher Scienti®c, Pittsburg, 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 [22]. 2.5. Statistical analyses Quantitative values are presented as means (M) and standard deviation (S.D.). Differences with associated P-values of P < 0:05 were considered to be signi®cant. A Chi-square test with Yates correction was used to compare the frequency of CAEV proviral-DNA in ¯ushing media between blood-positive and blood-negative donor does. The same statistical test was used to compare the frequency of CAEV proviral-DNA in serially diluted samples. The mean ova and embryos recovery rates for ¯ushing media positive and negative goats and for blood positive and negative goats were compared using the Student±Fisher test for independent samples [24]. 3. Results Ampli®cation of CAEV proviral-DNA by the gag primer resulted in a predictable 184 bp band included between 194 and 118 bp bands of the molecular weight standards for agarose gel electrophoresis use (Fig. 1). Of the 89 goats examined, 25 (28.1%) were blood PCR positive and 64 (71.9%) blood PCR negative for CAEV proviral-DNA. All blood PCR negative was also ¯ushing media
Fig. 1. Representative double-nested PCR products from blood, flushing media, tissue samples and controls. Lanes 1 and 14: fX174RFDNA Hae III Digest used as molecular weight standards. Lanes 2 and 5: blood samples (lanes 2 and 5, positive; lanes 3 and 4, negative). Lanes 6±10: flushing media samples (lanes 6, 8, 10, negative; lanes 7 and 9, positive). Lane 11: positive control. Lane 12: negative control. Lane 13: distilled water.
936
F. Fieni et al. / Theriogenology 57 (2002) 931±940
Table 1 Cumulative number of blood PCR positive goats with PCR positive and PCR negative cell lysate from flushing media Cell lysate from flushing media
dn-PCR positive dn-PCR negative
Without dilution
First dilution
Second±tenth dilution
11 14
1 24
0 25
PCR negative. The presence of CAEV proviral-DNA in the ¯ushing media was signi®cantly different between the blood PCR positive does and the blood PCR negative does (P < 0:01). During this experiment, a total of 253 ova and 256 embryos were recovered (mean 8:75 6:00 per goat: mean S:D:). The mean number of embryos recovered was not signi®cantly different between goats with ¯ushing media PCR positive and goats with ¯ushing media PCR negative (6:0 5:4 versus 7:8 4:4, respectively; mean S:D:) nor between goats with blood PCR positive and goats with blood PCR negative (7:0 5:0 versus 5:9 5:3: mean S:D:). Among the 25 infected goats (blood PCR positive) the cumulative number of goats that were dn-PCR positive and dn-PCR negative in ¯ushing media are shown in Table 1. The frequency of the presence of CAEV proviral-DNA in the undiluted ¯ushing media was 44%. This frequency decreased signi®cantly from the ®rst 1:100 dilution (P < 0:01). All the ¯ushing media samples after the second dilution were dn-PCR negative. 4. Discussion Using a double nested PCR, this study indicates, for the ®rst time, the presence of CAEV infected cells in ¯ushing media from infected donor goats. We chose dn-PCR for its high sensitivity [22,25±27]. Pathogenicity studies have shown that while other tissues could be infected, monocytes are the major target cells in CAEV infection. Only a small population of monocytes are infected, and expression of infectious virus occurs only when these monocytes mature into macrophages in the target tissues [4,27,28]. Previous reports have described infection rates of 1±8% of monocytes/macrophages [4,22,29]. The dn-PCR used in this study required at least 3000 monocytes/macrophages, containing between 30 and 240 infected cells to produce a signal visible in agarose gels [22]. In this study, we did not characterize the cells present in ¯ushing ¯uid. But detection of monocytes/macrophages in embryo ¯ushing media would be surprising. Usually, the presence of mononuclear cells suggests the development of a sub-acute to chronic in¯ammatory process. On the uterine tube lumen, in¯ammation can modify gland secretions such that the substrate becomes incompatible with fertilization and embryo survival. That was not the case in our experiment in which 5:9 5:3 (mean S:D:) embryos per goat were recovered without any signi®cant difference between CAEV infected and CAEV noninfected does. This embryo recovery rate was similar to those published in previous studies [30,31].
F. Fieni et al. / Theriogenology 57 (2002) 931±940
937
At least three explanations can be given for the presence of CAEV proviral-DNA in embryo ¯ushing media. First, monocytes/macrophages could be present in the uterine tube lumen at a suf®cient concentration to be detected, but not enough to modify the uterine tube secretions. Second, proviral-DNA could come from other cellular types like epithelial cells, which are very commonly found in embryo ¯ushing media. Recent in vitro research has demonstrated that caprine oviduct epithelial cells are susceptible to infection by CAEV in vitro with a high viral replication rate [32]. Third, the presence of mononuclear cells in ¯ushing media can also be the result of experimental conditions. On one hand, the stress due to the slaughter, which occurs before genital tract recovery, can be responsible for a self-defense process with mononuclear cell transfers from blood or tissues into the uterine lumen and uterine tube lumen. On the other hand, immediately after being euthanized, the does were hung upside down in order to allow exanguination. This position might allow the entry of cervical mucus (and any cells it might contain) into the uterine cavity [33]. In our experiment, embryo recovery carried out for gene transfer happened very early after the beginning of estrus (48 h). Because estrous cervical mucus contains substantial numbers of mononuclear cells, and because CAEV proviral-DNA has already been found in cervical mucus from infected does (Rowe, unpublished data), it is possible that the uterine lumen was contaminated postmortem by hanging the goats upside down. Whatever the origin of CAEV-infected cells, detection of CAEV-proviral DNA in ¯ushings following oviductal stage embryo collection would suggest potential risk for CAEV transmission during embryo manipulation for research studies or commercial uses. However, we were not able to examine for the presence of infective virus, so the absolute risk of CAEV transmission remains unknown. The commercial use of goat embryo transfer is common, safe and useful to exchange genetic material between countries. For this purpose, embryos are recovered by a ¯ush of the uterine cavity 7 days after the beginning of heat. Nevertheless, fundamental studies in genetics are up to date, and goats are an interesting species to use as a model. Genetic experiments such as gene transfers use early developmental stage embryos recovered by a ¯ush of the oviduct. That is the stage of embryo development that we used in this experiment. After embryo collection from infected donor goats, embryos and ¯ushing media can be contaminated by the CAE virus. Flushing media were particularly interesting because they gave indications about infectious agents that can exist on the oviduct or uterine lumen of the donor. Infectious agents present in ¯ushing media could be eliminated by appropriate techniques [20]. The International Embryo Transfer Society (IETS) recommends ¯ushing 10 times in 100-fold dilutions with medium [34]. In this experiment, as early as the second dilution, CAEV proviral-DNA could not be detected from cell lysate derived from ¯ushing media. Due to experimental conditions these serial dilutions could not be made directly on ¯ushing media, but were made on ¯ushing media prepared for PCR which included cellular lysates and DNA degradation products. These dilution conditions, involving smaller viral structures, should have increased the likelihood that CAEV proviral-DNA would be detected in serial dilutions. So these experimental conditions seemed to be more conclusive than the clinical practice of diluting ¯ushing media. These observations con®rm the effectiveness of IETS's recommendations. But other experiments in clinical practice with serial dilution of the embryo ¯ushing media have to be done to determine the amount of dilution needed to avoid any risk of infection.
938
F. Fieni et al. / Theriogenology 57 (2002) 931±940
One other way to prevent contamination is to use noninfected embryo donor does. In this experiment, the blood PCR test seems to give good results that predict which does will be ¯ushing media negative, but these results have to be con®rmed using a larger number of animals. Until this con®rmation, the best way to choose donor does could be based on repeated serological negative tests at appropriate intervals. These serological negative results would be complemented by a blood PCR test just before the embryo recovery due to the possibility of delayed seroconversion [35]. Actually, previous work using virus isolation or single-round PCR, together with epidemiological studies, has revealed that approximately 10±12% of antibody negative goats may be infected with CAEV [13,22,36,37]. Although the mean time for seroconversion is 3±12 weeks after oral, intravenous or intra-articular exposure to CAEV, in some animals the time from infection to seroconversion or from blood PCR detection to seroconversion may be 3±8 months or more [6]. Our ®ndings indicate that early recovered embryos from donor CAEV-infected does could induce a risk of ¯ushing media contamination with CAEV-infected cells. Prospective studies are to be done ®rst to verify if this contamination depends on the embryo recovery method (euthanatized versus surgery) or on the time of embryo recovery (2 versus 7 days) and second to explore the contamination risk from cells ®xing themselves onto the embryo pellucid. Until the results from such studies are available, screening uninfected healthy donors by sensitive techniques such as CAEV-PCR of blood is indicated. 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 US Department of Agriculture (CALV-AH-161).
References [1] Cheevers WP, Knowles DP, McGuire TC, Cunningham DR, Adams DS, Gorham JR. Chronic disease in goats orally infected with two isolates of the caprine arthritis±encephalitis lentivirus. Lab Invest 1988;58(5):510±7. [2] Cork LC, Hadlow WJ, Crawford TB, Gorham JR, Piper RC. Infectious leukoencephalomyelitis of young goats. J Infe Dis 1974;129:134±41. [3] Dawson M. Lentivirus diseases of domesticated animals. J Comp Pathol 1988;99:401±19. [4] Narayan O, Kennedy-Stoskopf S, Sheffer D, Grif®n DE, Clements JE. Activation of caprine arthritis± encephalitis virus expression during maturation of monocytes to macrophages. Inf Immun 1983;41(1):67± 73. [5] Narayan O, Zink M, Gorrell S, Crane D, Huso P, Jolly P, et al. The lentiviruses of sheep and goats. In: Levy JA (Ed.), The Retroviridae. New York, NY: Plenum Press, 1993. [6] Rowe JD, East NE. Risk factors for transmission and methods for control of caprine arthritis±encephalitis virus infection. Vet Clin North Am-Food Anim Practice 1997;13(1):35±53. [7] 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(19):493±5.
F. Fieni et al. / Theriogenology 57 (2002) 931±940
939
[8] Crawford TB, Adams DS. Caprine arthritis±encephalitis: clinical features and presence of antibody in selected goat populations. J Am Vet Med Assoc 1981;178(7):713±9. [9] 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(2):182±6. [10] Phelps SL, Smith MC. Caprine arthritis±encephalitis virus infection. J Am Vet Med Assoc 1993;203(12):1663±6. [11] Greenwood PL. Effects of caprine arthritis±encephalitis virus on productivity and health of dairy goats in New-South-Wales, Australia. Prevent Vet Med 1995;22(1±2):71±87. [12] Smith MC, Cutlip RC. Effects of infection with caprine arthritis±encephalitis virus on milk production in goats. J Am Vet Med Assoc 1987;193:63. [13] East NE, Rowe JD, Dahlberg JE, Theilen GH, Pedersen NC. Modes of transmission of caprine arthritis± encephalitis virus infection. Sm Ruminant Res 1993;10(3):251±62. [14] 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(4):850±5. [15] 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. New Zealand Vet J 1987;35:184± 6. [16] 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(9):1670±5. [17] Peretz G, Bugnard F, Calavas D. Study of a prevention programme for caprine arthritis±encephalitis. Vet Res 1994;25(2/3):322±6. [18] 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(12):2386±95. [19] Evans B, Faul A, Bielanski A, Renwick S, Van Derlinden I. Risk analysis and international trade principles applied to the importation into Canada of caprine embryos from South Africa. Rev Sci Tech Int Epiz 1997;16(1):265±70. [20] Wrathall AE. Embryo transfer and disease transmission in livestock: a review of recent research. Theriogenology 1995;43:81±8. [21] Wolfe DE, Nusbaum KE, Lauerman PW, Mysinger MG, Riddell MR, Putnam LS, et al. Embryo transfer from goats seropositive for caprine arthritis±encephalitis virus. Theriogenology 1987;28:307±16. [22] Barlough J, East N, Rowe JD, Vanhoosear K, Derock E, Bigornia L, et al. Double-nested polymerase chain reaction for detection of caprine arthritis±encephalitis virus proviral DNA in blood, milk, and tissues of infected goats. J Virol Meth 1994;50(1±3):101±13. [23] Saltarelli M, Querat G, Konings DAM, Vigne R, Clements JE. Nucleotide sequence and transcriptional analysis of molecular clones of CAEV which generate infectious virus. Virology 1990;179:347±64. [24] Snedecor GW, Cochran WG. Statitical methods. Iowa: Iowa State University Press, 1989. [25] Reddy PG, Sapp WJ, Heneine W. Detection of caprine arthritis±encephalitis virus by polymerase chain reaction. J Clin Microbiol 1993;31(11):3042±53. [26] Zanoni RG, Cordano P, Nauta IM, Peterhans E. PCR for the detection of lentiviruses from small ruminants. Schw Archiv Tierheilkunde 1996;138(2):93±8. [27] Zanoni R, Pauli U, Peterhans E. Detection of caprine arthritis±encephalitis and maedi-visna viruses using the polymerase chain reaction. Experientia 1990;46(3):316±9. [28] Gorrell MD, Brandon MR, Sheffer D, Adams RJ, Narayan O. Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J Virol 1992;66(5):2679±88. [29] Jolly PE, Narayan O. Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses. J Virol 1989;63(11):4682±8. [30] Fieni F, Beckers JF, Buggin M, Bruyas JF, Perrin J, Daubie M, Tainturier D. Evaluation of cryopreservation techniques for goat embryos. Reprod Nutr Dev 1995;35:367±73. [31] Li R, Cameron AWN, Batt PA, Trounson AO. Maximum survival of frozen goat embryos is attained at the expanded, hatching and hatched blastocyst stages of development. Reprod Fertil Dev 1990;2:345±50. [32] Lamara A, Fieni F, Chebloume Y, Bruyas JF, Tainturier D. Susceptibility of caprine oviduct epithelial cells to caprine arthritis±encephalitis virus (CAEV)Ðpreliminary results. In: Proceedings of the World Veterinary Congress. Lyon, France, 23±26 September 1999 [Abstract].
940
F. Fieni et al. / Theriogenology 57 (2002) 931±940
[33] Nakamura R, Walt M, Bennett R. Studies of bovine genital tracts: mycoplasma isolations from dairy cows. Theriogenology 1977;7(6):351±5. [34] IETS, (International, Embryo, Transfer, Society). Manual of the International Embryo Transfer Society: a procedural guideline and general information for the use of embryo transfer technology emphasizing sanitary procedures. Champlain (IL): Internat Embryo Tranfer Society, 1987. p. 31±32. [35] Hanson J, Hydbring E, Olsson K. A long term study of goats naturally infected with caprine arthritis± encephalitis virus. Acta Vet Scand 1996;37(1):31±9. [36] Rimstad E, East NE, Torten M, Higgins J, DeRock E, Pedersen NC. Delayed seroconversion following naturally acquired caprine arthritis±encephalitis virus infection in goats. Am J Vet Res 1993;54(11):1858± 62. [37] Rowe JD, East NE, Franti CE, Thurmond MC, Pedersen NC. Theilen GH. Risk factors associated with the incidence of seroconversion to caprine arthritis±encephalitis virus in goats on California dairies. Am J Vet Res 1992;53(12):2396±403.
Presence of caprine arthritis±encephalitis virus (CAEV) infected cells in ¯ushing media following oviductal-stage embryo collection F. Fienia,*, J. Rowea, K. Van Hooseara, C. Burucoab, S. Oppenheimc, G. Andersonc, J. Murraya, R. BonDuranta 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 2 November 1999; accepted 18 July 2001
Abstract To improve the knowledge on the risk of transmission of the caprine arthritis±encephalitis virus (CAEV) during embryo manipulations, we conducted a double-nested polymerase chain reaction (PCR) for CAEV proviral-DNA on ¯ushing media recovered from the oviducts 48 h after the beginning of estrus and on blood from 89 donor does. Sixty-four does had negative blood and ¯ushing media by PCR. Among the 25 CAEV infected goats (blood PCR positive), 11 were PCR ¯ushing media positive (P < 0:01). Cell lysate from ¯ushing media samples that were PCR positive were serially diluted 10 times at 1:100. Starting with the second 1:100 dilution all the cell lysate samples were PCR negative. The mean number of embryos recovered was not signi®cantly different between goats with ¯ushing media PCR positive and goats with ¯ushing media PCR negative (6:0 5:4 versus 7:8 4:4, respectively: mean S:D:) nor between goats with blood PCR positive and goats with blood PCR negative (7:0 5:0 versus 5:9 5:3: mean S:D:). The presence of CAEV infected cells in oviductal ¯ushing media from infected donor does was indicated for the ®rst time during this study. The absence of ¯ushing media PCR positive for goat blood PCR negative seemed to allow the use of the blood PCR test to con®dently predict the absence of CAEV provirus in the oviductal ¯uid. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Caprine arthritis±encephalitis virus; Flushing media; Embryo; Goat; CAEV
* Corresponding author. Present address: Department of Biotechnology and Pathology of Reproduction, National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France. Tel.: 33-2-40-68-77-10; fax: 33-240-68-77-48. 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 1 ) 0 0 6 9 8 - 7
932
F. Fieni et al. / Theriogenology 57 (2002) 931±940
1. Introduction Caprine arthritis±encephalitis virus (CAEV) is a very common disease in dairy goats. In adult does, CAEV infection causes chronic arthritis, interstitial pneumonia and indurative mastitis with decreased milk production [1]. Less frequently, CAEV causes leukoencephlomyelitis in young goats [2]. CAEV is an enveloped, single-stranded RNA virus. It has been classi®ed as a Lentivirus, subfamily of Retroviridae [3]. This group of cell-associated viruses is characterized by the presence of an RNA-dependent DNA polymerase, and the CAEV is functional only when the viral RNA is transformed into viral DNA and is integrated into the host cell DNA. Thus, the viral genome becomes a part of the cellular DNA and replicates ef®ciently in nondividing, terminally differentiated cells [4]. The virus has a tropism for monocytes/macrophages and causes chronic in¯ammatory disease [5]. CAEV infection has been detected worldwide, but is most prevalent in countries where goats are intensively dairied [6]. Approximately 30±80% of dairy goats in the USA, Canada and Europe are infected, compared to 0±10% in other countries in Africa and South America [6±10]. In dairy herds, mammary tropism has economic consequences on milk production and mammary pathology [11,12]. The major route of CAEV transmission is the ingestion of virus-laden colostrum or milk from infected does [13±15]. Prevention of CAEV transmission is based on the removal of kids from their mothers at birth and the feeding on heat-treated goat colostrum [16,17]. But in large herds using this pasteurized method, up to 10% incidence of unexplained seroconversion has been observed [13,18]. Less ef®cient routes of transmission must be clari®ed in order to improve control and eradication methods [11,13]. Embryo manipulation is a common technique used for fundamental research such as gene transfer or cloning and for commercial exchanges. For both uses, embryo biotechnologies have to present a complete sanitary guarantee against infectious disease transmission. Risk of disease transmission through animal embryos collected and processed in accordance with the standards and procedures prescribed by the International Embryo Transfer Society (IETS) tends to be extremely small [19,20], but has to be studied for each species, each infectious agent and each biological structure transmitted from donor to recipient. Two biological products coming from embryo collection can be infectious: ®rst, embryos and second, ¯ushing medias, which are permanently in contact with embryos during embryo manipulations. In goats, only one in vivo study has been done to examine the consequence of embryo transfer, and this study included only a small number of animals. Using seropositive embryo donors and bucks, Wolfe et al. [21] could not isolate, by inoculation of cellular culture, CAEV from uterine ¯ushings, from recipients' colostrum or placentas, or from the resulting fetuses and live kids. No seroconversion appeared in recipients after transfer. Currently, the use of the polymerase chain reaction (PCR) technique has allowed us to directly examine various samples for the presence of CAEV proviralDNA. The goal of our study was to determine whether CAEV infected cells might be found in ¯ushing media recovered during early embryo collection from slaughtered donors.
F. Fieni et al. / Theriogenology 57 (2002) 931±940
933
2. Materials and methods 2.1. Animals Eighty-nine nonlactating dairy goats of various breeds (Alpine, Saanen and Nubian) were used as embryo donors in groups of 12 or 13 from September to December 1998. Estrous cycles of donors were synchronized via intravaginal pessaries containing 40 mg progestin (Redopharm, Halfway House, Republic of South Africa). The pessaries were left in place for 10 days. The embryo donors received injections of a follicle-stimulating hormone (FSH-P) (Sioux Biochemical, Sioux Center, IA) twice daily in decreasing doses (5, 4, 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, PharmaciaUpjohn Co., Kalamazoo, MI) intramuscularly. Twenty-four hours after pessary removal, the does were given 50 mg of a gonadotropin-releasing hormone (Cystorelin1, Rhone Merieux, Athens, GA) intravenously. Donors 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 On the day of embryo collection, blood samples (8 ml in acid citrate dextrose (ACD)) were obtained by jugular venipuncture from each goat. 2.2.2. Flushing media and embryos 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 exanguination. Immediately afterwards, at the laboratory, ovulations were counted and embryos ¯ushed from the oviducts. The mesosalpinx were trimmed to straighten out the uterine tubes. A blunt, 20-gauge needle was inserted through the utero-tubal junction and 20 ml of phosphate-buffered saline (PBS) containing 1% heattreated calf serum and 1% antibiotic/antimycotic (penicillin, streptomycin, polymixin B, Gibco, Grand Island, NE) 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. 2.3. Preparation of blood and flushing media 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, 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 hydrochlorideÐpH 8.3, 0.45% NP-40, 0.45% Tween-20, 50 mg proteinase
934
F. Fieni et al. / Theriogenology 57 (2002) 931±940
K per 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. Manipulations were made in a class II laminar ¯ow hood. Blood samples were stored at 208C until the PCR reactions were performed. 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. Manipulations were made in a class II laminar ¯ow hood. Flushing media samples were stored at 20 8C until the PCR reaction was performed. In a second step, cell lysate from double-nested PCR (dn-PCR) positive ¯ushing media samples were serially diluted at 1:100, 10 times. After each dilution, the presence or absence of CAEV proviral-DNA was determined by dn-PCR. 2.4. Procedure for double-nested PCR CAEV proviral-DNA was tested by a dn-PCR technique as described by Barlough et al. [22]. 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 ), 953±975 bp and P2 (50 -TCCTACCCCCATAATTTGATCCAC-30 ), 1249±1226 bp. The inner primer were P3 (50 -GTTCCAGCAACTGCAAACAGTAGCAATG-30 ), 997±1024 bp and P4 (50 -ACCTTTCTGCTTCTTCATTTAATTTCCC-30 ), 1181±1154 bp. The primers were designed using the published sequence of CAEV strain CO Saltarelli et al. [23] 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 with 47 ml of a mixture containing 10 mM Tris±HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 0.001% w/v gelatin, 400 mM each dNTP, 5 mM tetramethylammonium chloride (TMAC), 20 pmol (in 1 ml) outer primers 1 and 2, 2 UI Taq DNA polymerase (AmpliTaq, PerkinElmer, 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 of (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 round 2 was added to 2 ml dye buffer per well inside 2% agarose gels 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. A Hae III Digest of fX174 yielding 11 suitable fragments was used as the molecular weight standard for the agarose gel electrophoresis (New UK Biolabs, Beverly, MA). To prevent DNA contamination, all mixture preparations were performed in a class II laminar ¯ow hood. Nondisposable materials were decontaminated before use in a UV
F. Fieni et al. / Theriogenology 57 (2002) 931±940
935
crosslinker (FB UVXL-1000, Fisher Scienti®c, Pittsburg, 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 [22]. 2.5. Statistical analyses Quantitative values are presented as means (M) and standard deviation (S.D.). Differences with associated P-values of P < 0:05 were considered to be signi®cant. A Chi-square test with Yates correction was used to compare the frequency of CAEV proviral-DNA in ¯ushing media between blood-positive and blood-negative donor does. The same statistical test was used to compare the frequency of CAEV proviral-DNA in serially diluted samples. The mean ova and embryos recovery rates for ¯ushing media positive and negative goats and for blood positive and negative goats were compared using the Student±Fisher test for independent samples [24]. 3. Results Ampli®cation of CAEV proviral-DNA by the gag primer resulted in a predictable 184 bp band included between 194 and 118 bp bands of the molecular weight standards for agarose gel electrophoresis use (Fig. 1). Of the 89 goats examined, 25 (28.1%) were blood PCR positive and 64 (71.9%) blood PCR negative for CAEV proviral-DNA. All blood PCR negative was also ¯ushing media
Fig. 1. Representative double-nested PCR products from blood, flushing media, tissue samples and controls. Lanes 1 and 14: fX174RFDNA Hae III Digest used as molecular weight standards. Lanes 2 and 5: blood samples (lanes 2 and 5, positive; lanes 3 and 4, negative). Lanes 6±10: flushing media samples (lanes 6, 8, 10, negative; lanes 7 and 9, positive). Lane 11: positive control. Lane 12: negative control. Lane 13: distilled water.
936
F. Fieni et al. / Theriogenology 57 (2002) 931±940
Table 1 Cumulative number of blood PCR positive goats with PCR positive and PCR negative cell lysate from flushing media Cell lysate from flushing media
dn-PCR positive dn-PCR negative
Without dilution
First dilution
Second±tenth dilution
11 14
1 24
0 25
PCR negative. The presence of CAEV proviral-DNA in the ¯ushing media was signi®cantly different between the blood PCR positive does and the blood PCR negative does (P < 0:01). During this experiment, a total of 253 ova and 256 embryos were recovered (mean 8:75 6:00 per goat: mean S:D:). The mean number of embryos recovered was not signi®cantly different between goats with ¯ushing media PCR positive and goats with ¯ushing media PCR negative (6:0 5:4 versus 7:8 4:4, respectively; mean S:D:) nor between goats with blood PCR positive and goats with blood PCR negative (7:0 5:0 versus 5:9 5:3: mean S:D:). Among the 25 infected goats (blood PCR positive) the cumulative number of goats that were dn-PCR positive and dn-PCR negative in ¯ushing media are shown in Table 1. The frequency of the presence of CAEV proviral-DNA in the undiluted ¯ushing media was 44%. This frequency decreased signi®cantly from the ®rst 1:100 dilution (P < 0:01). All the ¯ushing media samples after the second dilution were dn-PCR negative. 4. Discussion Using a double nested PCR, this study indicates, for the ®rst time, the presence of CAEV infected cells in ¯ushing media from infected donor goats. We chose dn-PCR for its high sensitivity [22,25±27]. Pathogenicity studies have shown that while other tissues could be infected, monocytes are the major target cells in CAEV infection. Only a small population of monocytes are infected, and expression of infectious virus occurs only when these monocytes mature into macrophages in the target tissues [4,27,28]. Previous reports have described infection rates of 1±8% of monocytes/macrophages [4,22,29]. The dn-PCR used in this study required at least 3000 monocytes/macrophages, containing between 30 and 240 infected cells to produce a signal visible in agarose gels [22]. In this study, we did not characterize the cells present in ¯ushing ¯uid. But detection of monocytes/macrophages in embryo ¯ushing media would be surprising. Usually, the presence of mononuclear cells suggests the development of a sub-acute to chronic in¯ammatory process. On the uterine tube lumen, in¯ammation can modify gland secretions such that the substrate becomes incompatible with fertilization and embryo survival. That was not the case in our experiment in which 5:9 5:3 (mean S:D:) embryos per goat were recovered without any signi®cant difference between CAEV infected and CAEV noninfected does. This embryo recovery rate was similar to those published in previous studies [30,31].
F. Fieni et al. / Theriogenology 57 (2002) 931±940
937
At least three explanations can be given for the presence of CAEV proviral-DNA in embryo ¯ushing media. First, monocytes/macrophages could be present in the uterine tube lumen at a suf®cient concentration to be detected, but not enough to modify the uterine tube secretions. Second, proviral-DNA could come from other cellular types like epithelial cells, which are very commonly found in embryo ¯ushing media. Recent in vitro research has demonstrated that caprine oviduct epithelial cells are susceptible to infection by CAEV in vitro with a high viral replication rate [32]. Third, the presence of mononuclear cells in ¯ushing media can also be the result of experimental conditions. On one hand, the stress due to the slaughter, which occurs before genital tract recovery, can be responsible for a self-defense process with mononuclear cell transfers from blood or tissues into the uterine lumen and uterine tube lumen. On the other hand, immediately after being euthanized, the does were hung upside down in order to allow exanguination. This position might allow the entry of cervical mucus (and any cells it might contain) into the uterine cavity [33]. In our experiment, embryo recovery carried out for gene transfer happened very early after the beginning of estrus (48 h). Because estrous cervical mucus contains substantial numbers of mononuclear cells, and because CAEV proviral-DNA has already been found in cervical mucus from infected does (Rowe, unpublished data), it is possible that the uterine lumen was contaminated postmortem by hanging the goats upside down. Whatever the origin of CAEV-infected cells, detection of CAEV-proviral DNA in ¯ushings following oviductal stage embryo collection would suggest potential risk for CAEV transmission during embryo manipulation for research studies or commercial uses. However, we were not able to examine for the presence of infective virus, so the absolute risk of CAEV transmission remains unknown. The commercial use of goat embryo transfer is common, safe and useful to exchange genetic material between countries. For this purpose, embryos are recovered by a ¯ush of the uterine cavity 7 days after the beginning of heat. Nevertheless, fundamental studies in genetics are up to date, and goats are an interesting species to use as a model. Genetic experiments such as gene transfers use early developmental stage embryos recovered by a ¯ush of the oviduct. That is the stage of embryo development that we used in this experiment. After embryo collection from infected donor goats, embryos and ¯ushing media can be contaminated by the CAE virus. Flushing media were particularly interesting because they gave indications about infectious agents that can exist on the oviduct or uterine lumen of the donor. Infectious agents present in ¯ushing media could be eliminated by appropriate techniques [20]. The International Embryo Transfer Society (IETS) recommends ¯ushing 10 times in 100-fold dilutions with medium [34]. In this experiment, as early as the second dilution, CAEV proviral-DNA could not be detected from cell lysate derived from ¯ushing media. Due to experimental conditions these serial dilutions could not be made directly on ¯ushing media, but were made on ¯ushing media prepared for PCR which included cellular lysates and DNA degradation products. These dilution conditions, involving smaller viral structures, should have increased the likelihood that CAEV proviral-DNA would be detected in serial dilutions. So these experimental conditions seemed to be more conclusive than the clinical practice of diluting ¯ushing media. These observations con®rm the effectiveness of IETS's recommendations. But other experiments in clinical practice with serial dilution of the embryo ¯ushing media have to be done to determine the amount of dilution needed to avoid any risk of infection.
938
F. Fieni et al. / Theriogenology 57 (2002) 931±940
One other way to prevent contamination is to use noninfected embryo donor does. In this experiment, the blood PCR test seems to give good results that predict which does will be ¯ushing media negative, but these results have to be con®rmed using a larger number of animals. Until this con®rmation, the best way to choose donor does could be based on repeated serological negative tests at appropriate intervals. These serological negative results would be complemented by a blood PCR test just before the embryo recovery due to the possibility of delayed seroconversion [35]. Actually, previous work using virus isolation or single-round PCR, together with epidemiological studies, has revealed that approximately 10±12% of antibody negative goats may be infected with CAEV [13,22,36,37]. Although the mean time for seroconversion is 3±12 weeks after oral, intravenous or intra-articular exposure to CAEV, in some animals the time from infection to seroconversion or from blood PCR detection to seroconversion may be 3±8 months or more [6]. Our ®ndings indicate that early recovered embryos from donor CAEV-infected does could induce a risk of ¯ushing media contamination with CAEV-infected cells. Prospective studies are to be done ®rst to verify if this contamination depends on the embryo recovery method (euthanatized versus surgery) or on the time of embryo recovery (2 versus 7 days) and second to explore the contamination risk from cells ®xing themselves onto the embryo pellucid. Until the results from such studies are available, screening uninfected healthy donors by sensitive techniques such as CAEV-PCR of blood is indicated. 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 US Department of Agriculture (CALV-AH-161).
References [1] Cheevers WP, Knowles DP, McGuire TC, Cunningham DR, Adams DS, Gorham JR. Chronic disease in goats orally infected with two isolates of the caprine arthritis±encephalitis lentivirus. Lab Invest 1988;58(5):510±7. [2] Cork LC, Hadlow WJ, Crawford TB, Gorham JR, Piper RC. Infectious leukoencephalomyelitis of young goats. J Infe Dis 1974;129:134±41. [3] Dawson M. Lentivirus diseases of domesticated animals. J Comp Pathol 1988;99:401±19. [4] Narayan O, Kennedy-Stoskopf S, Sheffer D, Grif®n DE, Clements JE. Activation of caprine arthritis± encephalitis virus expression during maturation of monocytes to macrophages. Inf Immun 1983;41(1):67± 73. [5] Narayan O, Zink M, Gorrell S, Crane D, Huso P, Jolly P, et al. The lentiviruses of sheep and goats. In: Levy JA (Ed.), The Retroviridae. New York, NY: Plenum Press, 1993. [6] Rowe JD, East NE. Risk factors for transmission and methods for control of caprine arthritis±encephalitis virus infection. Vet Clin North Am-Food Anim Practice 1997;13(1):35±53. [7] 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(19):493±5.
F. Fieni et al. / Theriogenology 57 (2002) 931±940
939
[8] Crawford TB, Adams DS. Caprine arthritis±encephalitis: clinical features and presence of antibody in selected goat populations. J Am Vet Med Assoc 1981;178(7):713±9. [9] 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(2):182±6. [10] Phelps SL, Smith MC. Caprine arthritis±encephalitis virus infection. J Am Vet Med Assoc 1993;203(12):1663±6. [11] Greenwood PL. Effects of caprine arthritis±encephalitis virus on productivity and health of dairy goats in New-South-Wales, Australia. Prevent Vet Med 1995;22(1±2):71±87. [12] Smith MC, Cutlip RC. Effects of infection with caprine arthritis±encephalitis virus on milk production in goats. J Am Vet Med Assoc 1987;193:63. [13] East NE, Rowe JD, Dahlberg JE, Theilen GH, Pedersen NC. Modes of transmission of caprine arthritis± encephalitis virus infection. Sm Ruminant Res 1993;10(3):251±62. [14] 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(4):850±5. [15] 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. New Zealand Vet J 1987;35:184± 6. [16] 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(9):1670±5. [17] Peretz G, Bugnard F, Calavas D. Study of a prevention programme for caprine arthritis±encephalitis. Vet Res 1994;25(2/3):322±6. [18] 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(12):2386±95. [19] Evans B, Faul A, Bielanski A, Renwick S, Van Derlinden I. Risk analysis and international trade principles applied to the importation into Canada of caprine embryos from South Africa. Rev Sci Tech Int Epiz 1997;16(1):265±70. [20] Wrathall AE. Embryo transfer and disease transmission in livestock: a review of recent research. Theriogenology 1995;43:81±8. [21] Wolfe DE, Nusbaum KE, Lauerman PW, Mysinger MG, Riddell MR, Putnam LS, et al. Embryo transfer from goats seropositive for caprine arthritis±encephalitis virus. Theriogenology 1987;28:307±16. [22] Barlough J, East N, Rowe JD, Vanhoosear K, Derock E, Bigornia L, et al. Double-nested polymerase chain reaction for detection of caprine arthritis±encephalitis virus proviral DNA in blood, milk, and tissues of infected goats. J Virol Meth 1994;50(1±3):101±13. [23] Saltarelli M, Querat G, Konings DAM, Vigne R, Clements JE. Nucleotide sequence and transcriptional analysis of molecular clones of CAEV which generate infectious virus. Virology 1990;179:347±64. [24] Snedecor GW, Cochran WG. Statitical methods. Iowa: Iowa State University Press, 1989. [25] Reddy PG, Sapp WJ, Heneine W. Detection of caprine arthritis±encephalitis virus by polymerase chain reaction. J Clin Microbiol 1993;31(11):3042±53. [26] Zanoni RG, Cordano P, Nauta IM, Peterhans E. PCR for the detection of lentiviruses from small ruminants. Schw Archiv Tierheilkunde 1996;138(2):93±8. [27] Zanoni R, Pauli U, Peterhans E. Detection of caprine arthritis±encephalitis and maedi-visna viruses using the polymerase chain reaction. Experientia 1990;46(3):316±9. [28] Gorrell MD, Brandon MR, Sheffer D, Adams RJ, Narayan O. Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J Virol 1992;66(5):2679±88. [29] Jolly PE, Narayan O. Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses. J Virol 1989;63(11):4682±8. [30] Fieni F, Beckers JF, Buggin M, Bruyas JF, Perrin J, Daubie M, Tainturier D. Evaluation of cryopreservation techniques for goat embryos. Reprod Nutr Dev 1995;35:367±73. [31] Li R, Cameron AWN, Batt PA, Trounson AO. Maximum survival of frozen goat embryos is attained at the expanded, hatching and hatched blastocyst stages of development. Reprod Fertil Dev 1990;2:345±50. [32] Lamara A, Fieni F, Chebloume Y, Bruyas JF, Tainturier D. Susceptibility of caprine oviduct epithelial cells to caprine arthritis±encephalitis virus (CAEV)Ðpreliminary results. In: Proceedings of the World Veterinary Congress. Lyon, France, 23±26 September 1999 [Abstract].
940
F. Fieni et al. / Theriogenology 57 (2002) 931±940
[33] Nakamura R, Walt M, Bennett R. Studies of bovine genital tracts: mycoplasma isolations from dairy cows. Theriogenology 1977;7(6):351±5. [34] IETS, (International, Embryo, Transfer, Society). Manual of the International Embryo Transfer Society: a procedural guideline and general information for the use of embryo transfer technology emphasizing sanitary procedures. Champlain (IL): Internat Embryo Tranfer Society, 1987. p. 31±32. [35] Hanson J, Hydbring E, Olsson K. A long term study of goats naturally infected with caprine arthritis± encephalitis virus. Acta Vet Scand 1996;37(1):31±9. [36] Rimstad E, East NE, Torten M, Higgins J, DeRock E, Pedersen NC. Delayed seroconversion following naturally acquired caprine arthritis±encephalitis virus infection in goats. Am J Vet Res 1993;54(11):1858± 62. [37] Rowe JD, East NE, Franti CE, Thurmond MC, Pedersen NC. Theilen GH. Risk factors associated with the incidence of seroconversion to caprine arthritis±encephalitis virus in goats on California dairies. Am J Vet Res 1992;53(12):2396±403.