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Studies on Neospora caninum DNA detection in the oocytes and embryos collected from infected cows
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Veterinary Parasitology 158 (2008) 370–375 www.elsevier.com/locate/vetpar
Short communication
Studies on Neospora caninum DNA detection in the oocytes and embryos collected from infected cows Boz˙ena Moskwa *, Katarzyna Goz´dzik, Justyna Bien´, Władysław Cabaj Witold Stefanski Institute of Parasitology of the Polish Academy of Sciences, Twarda Street 51/55, 00-818 Warsaw, Poland Received 28 March 2008; received in revised form 21 August 2008; accepted 15 September 2008
Abstract Neosporosis is a major cause of abortion in cattle over the world. One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is embryo transfer (ET) from infected dams into uninfected recipients. Oocytes and embryos taken from seropositive cows were examined for the presence of Neospora caninum DNA. A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams. The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams. Published by Elsevier B.V. Keywords: Neospora caninum; Oocytes; Embryos; DNA; PCR; Vertical transmission
1. Introduction Neospora caninum, an apicomplexan parasite with a worldwide distribution that infects warm-blooded vertebrates, is a causative agent of infertility and abortion (Dubey and Lindsay, 1996; Dubey, 1999). The infection leads to important economical losses in cattle due to reproductive failure associated with abortion and mortality in congenitally infected calves (Anderson et al., 1995; Wouda et al., 1999; Cabaj et al., 2000; Davison et al., 2001; Campero et al., 2003; Moskwa et al., 2005).
* Corresponding author. Tel.: +48 22 620 62 26; fax: +48 22 620 62 27. E-mail address: [email protected] (B. Moskwa). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2008.09.018
The transmission of infection is possible in two ways, vertical transmission from one generation to the next via placental transfer to the fetus or horizontal transmission by ingestion of oocysts shed by definitive hosts (Pare et al., 1996; Anderson et al., 1997; Schares and Conraths, 2001). Recently, modern nomenclature for describing two different modes of transplacental transmission for neosporosis was proposed. Accordingly, endogenous and exogenous transplacental infection modes were introduced to adequately distinguish infection from pre-existing chronic infection of the dam and from an acute infection of the dam (Trees and Williams, 2005; Długon´ska, 2007). Limited data is available on transmission of infection with N. caninum by embryo transfer (ET). The passing of N. caninum by ET was investigated on the basis of the serological status of ET donors and recipients (Baillargeon et al., 2001; Landmann et al., 2002). These
B. Moskwa et al. / Veterinary Parasitology 158 (2008) 370–375
results suggested that the risk of N. caninum transmission by ET is not related to the serological status of the embryo donors, but is rather linked to the seropositivity of the recipients. On the other hand, ET into seronegative recipients, using the procedure proposed by the IETS, is an effective way to prevent vertical transmission of neosporosis (Stringfellow, 1998; Baillargeon et al., 2001). There are no published reports, which show that oocytes or embryos obtained from seropositive donors are attached by the tachyzoites and can transmit the parasite by the uterine route or that tachyzoites adhere firmly to the zona pellicula of preimplantation embryos and can be passed to the uterine environment during embriotransfer (Bielanski, 2002; Bielanski et al., 2002). Additionally, the authors concluded that bovine embryos are protected by the zona pellicula against Neospora invasion. The aim of the study was to examine oocytes and embryos collected from seropositive cows-donors in order to determine the occurrence of parasite DNA. 2. Materials and methods 2.1. Cows and their serological status of neosporosis Eight cows with the history of abortion were used in this study. Animals were from the experimental station belonging to the Polish Academy of Sciences. The animals were naturally infected with N. caninum and infection was assessed by the detection of serum specific antibodies by a commercial ELISA kit (IDEXX Laboratories, Inc., Westbrook, ME, USA) according to the manufacturer’s instructions. Samples where the S/P ratio was greater than or equal to 0.50 were regarded as positive and those where ratio was less than 0.5 were regarded as negative for N. caninum antibodies. To avoid false negative results, cows were tested four times for evidence of N. caninum exposure and they were always seropositive. 2.2. Collection of oocysts and embryos To induce superovulation, the donor dams were injected intramuscularly with follicle stimulating hormone FSH Folltropin (Agtech, Inc., Canada). Oocytes were collected at the non-matured stage (prophase stage of the first division of meiosis). Embryos were obtained through in vivo fertilization and were collected at late morula or a blastocyst stages.
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Thirty oocytes and 30 embryos were used in the study. Oocysts and embryos were unwashed or washed three times using TCM-199 medium (Sigma) (Duszewska et al., 2000; Duszewska and Reklewski, 2001). According to the results obtained by Torres and Ortega (2006) medium was not supplemented with 5% fetal calf serum. Oocysts and embryos were not matured in vitro. No more than two oocytes and embryos were isolated in one session from the superovulated seropositive donors. On account of the fewness of isolated oocytes and embryos obtained, this superovulation was repeated several times. The samples were stored at 20 8C until testing. 2.3. PCR Genomic DNA was isolated from oocytes and embryos and from tachyzoites of the N. caninum NC-1 reference strain, using the Nucleospin Tissue DNA extraction kit (Macherey-Nagel, Germany). The primers, Np21 and Np6, based on the NC-5 region specific for N. caninum were used in this study (Yamage et al., 1996; Moskwa et al., 2003, 2007). To avoid carry-over contamination in the PCR reaction, a uracil N-glycosylase (UNG) protocol was implemented (Rys and Persing, 1993). The standard PCR master mixture contained 1 PCR buffer, 10 mM of each dATP, dCTP, dGTP and dUTP (substituted for dTTP in this protocol), 100 pmol of each primer Np21 and Np6, 1 U Taq DNA polymerase and 0.5 U UNG (uracil DNA glycosylase) and 1 ml of DNA. Water was added to a 50 ml final reaction volume. All reagents used for PCR were purchased from Fermentas (MBI Fermentas, USA). The mixtures were incubated at room temperature for 10 min for deactivation of any dU-containing templates and than were denatured at 95 8C for 10 min (for inactivation of enzyme) prior to 30 cycles of denaturation (95 8C, 30 s), annealing (55 8C, 30 s) and primer extension (72 8C, 1 min). After the last cycle, additional extension was applied for 10 min at 72 8C, all carried out in a Genius (Techne, USA) thermocycler. Amplification products were analyzed by electrophoresis in a 1% agarose gel stained with ethidium bromide and visualised under UV light using the Kodak Electrophoresis Documentation and Analysis System (EDAS) 290. To determine the sensitivity of PCR amplicons of examined samples, DNA was extracted from N. caninum tachyzoites cultured in Vero cells. The DNA
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The number of isolated oocytes and embryos was low (no more than two in one session). The PCR assay did not yield the expected 328 bp products in oocytes and embryos unwashed or washed three times in TCM 199. These results revealed that N. caninum DNA was not present in any of the examined samples. Because all examined samples were negative in relation to the presence of N. caninum DNA, Fig. 2 represents only chosen results. Fig. 1. Agarose gel (1%) electrophoresis of the products obtained in the PCR experiments. DNA isolated from N. caninum tachyzoites was used as a template. A limited dilution series from 9 pg/ml (lane 1), down to 0.017 pg/ml (lane 10). Sample contained 0.070 pg/ml (lane 8) was clearly visible on the original gel. Lane M, molecular size marker (100 bp DNA ladder, Fermentas).
concentration was determined using Spectrophotometr NanoDrop ND1000. A limited dilution series from 10 ng/ml, down to 0.017 pg/ml was used in the study. The amount of DNA in one N. caninum tachyzoite is 0.07 pg/ml (Dubey and Sibley, personal information). 3. Results The S/P ratio reflected IgG level in serum of seropositive cows, achieved high values in the first examination of sera from eight cows (2.12, 2.27, 1.97, 3.42, 2.07, 2.34, 3.11 and 2.51 respectively). The subsequent examinations revealed that the S/P ratios had achieved a stable level and fluctuated around the above values. After 30 cycles of amplification, DNA of one N. caninum tachyzoite could be detected, thus demonstrating the sensitivity of the designed PCR system (Fig. 1).
4. Discussion The majority cases of N. caninum transmission occurs by endogenous and exogenous infection modes (Trees and Williams, 2005), from an infected dam to her foetus in utero. It was reported that more than 80% of calves from infected dams are born with congenital N. caninum infection and that the parasite can cycle within a cow family for many generation (Landmann et al., 2002). One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is ET from infected dams into uninfected recipients. Development of new technology related to in vitro embryo production allowed for the commercial use of this method of reproduction. After insemination of naturally cyclic cows, approximately 85% of the ovulated oocytes will develop into an embryo. To induce superovulation, the donor dams used to be injected intramuscularly with follicle stimulating hormone FSH Folltropin (Sirard et al., 1999; Hendriksen et al., 2000).
Fig. 2. Agarose gel (1%) electrophoresis. Negative PCR from washed and unwashed oocytes and embryos from different N. caninum seropositive cows. M: molecular marker; lane 1: unwashed oocytes; lanes 2–3: washed oocytes; lane 4: unwashed embryos; lane 5: washed embryos; lane 6: positive control (DNA from the reference strain of N. caninum, NC-1); lane 7: negative control.
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The effect of FSH treatment on oocyte yields and developmental competence may depend on the mode of administration of FSH (Goodhand et al., 1999). Although, variability in the number of isolated oocytes and embryos was observed between individuals among examined donors, FSH treatments effectively induce superovulation in cattle (Bousquet et al., 1999; Hendriksen et al., 2000; Kimura et al., 2007). In our experiment, the superovulation effect after FSH treatment was not observed. The number of isolated oocytes and embryos was low (no more than two in one session). The number of oocytes and embryos recovered form N. caninum seropositive donors was comparable or lower than in in vivo conventional procedures (Bousquet et al., 1999; Duszewska et al., 2003). We can speculate that N. caninum infection which has been shown to be the major cause of bovine reproductive disorders may also influence the ovulation process of the seropositive cattle. An infectious agent present during the production of bovine embryos might reduce the number and the quality of embryos generated, resulting in transmission of disease to recipients and offspring (Stringfellow and Givens, 2000). However, the authors concluded that a relatively simple embryo processing procedure will ensure that many infectious agents are effectively diluted, dislodged or inactivated. Contrary to this statement, earlier reports indicated that embryos might function as vectors for transmission of pathogens. Gillespie et al. (1990) reported the adherence of bovine herpesvirus-1, bluetongue virus and bovine viral diarrhea virus to bovine embryos. Bielanski and Surujballi (1996, 1998) revealed that the washing procedure according to IETS standards was not completely effective for removing Leptospira borgpetersenii virus from bovine embryos. There is no documented information on the susceptibility of bovine embryos to N. caninum. The studies undertaken by Baillargeon et al. (2001), Landmann et al. (2002) and Campero et al. (2003) suggest that embryos harvested from seropositive dams and implanted into non-infected recipients, result in the birth of non-infected offspring. Baillargeon et al. (2001) stated that ET from seropositive dams to uninfected recipients led to 63 pregnancies, but 4 of them were aborted (6.3%). ET from seronegative dams to seropositive recipients led to four pregnancies, but one of them was aborted (25%). Landmann et al. (2002) revealed a similar rate of abortion (25%) when embryos were transferred from seropositive dams to seronegative recipients. However, there is no information available to show that, under natural conditions, embryos are exposed to N. caninum in the uterine cavity.
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The adhesion of N. caninum tachyzoites to preimplantation embryos was investigated in in vitro studies only (Bielanski et al., 2002). In vitro fertilized embryos were exposed to tachyzoites of N. caninum in culture with a monolayer Vero cells. Using transmission electron microscope, dividing tachyzoites of the parasite were observed within trophoblastic cells of hatched blastocytes only when the zona pellicula was absent. The results of that study implicated that with an intact zona pellicula, preimplantation embryos are protected by the zona pellicula against N. caninum invasion. However, the author pointed out, that the obtained results may not reflect the natural parasite biology and differences between the properties of the zona pellicula of in vivo and in vitro embryos. Molecular techniques such as PCR offer a highly sensitive and specific alternative to immunological methods for the diagnostics of neosporosis. PCR was suitable to confirm the presence of N. caninum DNA in various tissues such as brain, spinal cord, muscle and blood as well as in the milk and colostrum of infected cows (Guy et al., 2001; Wis´niewski et al., 2002; Moskwa et al., 2003,2007; Ferre et al., 2005; Ortega-Mora et al., 2003, 2005). Using a PCR, protocol the absence of N. caninum DNA in oocytes and embryos obtained from seropositive dams was revealed. In fact, the absence of N. caninum DNA was revealed in both washed and unwashed oocyte and embryo samples. In conclusion, the ET procedure recommended by IETS seems to be an effective way to prevent the vertical transmission of N. caninum (Stringfellow, 1998). But the main pathway of reducing the potential risk of transmission is by ensuring the absence of N. caninum forms. The results of our studies indicate that the oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams. Acknowledgements The research was supported by the grants No. 3 P06K 030 23 of the State Committee for the Scientific Research in Warsaw, Poland and ADIPAR—Advanced Diagnostics for Parasitic Disease Transfer of Knowledge Project in the 6th Framework Programme. References Anderson, M.L., Palmer, C.W., Thurmond, M.C., 1995. Evaluation of abortions in cattle attributable to neosporosis in selected
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dairy herds in California. J. Am. Vet. Med. Assoc. 207, 1206– 1210. Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.V., Packham, A.E., Barr, B.C., Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210, 1169–1172. Baillargeon, P., Fecteau, G., Pare, J., Lomothe, P., Sauve, R., 2001. Evaluation of the embryo transfer procedure proposed by the International Embryo Transfer Society as a method of controlling vertical transmission of Neospora caninum in cattle. J. Am. Vet. Med. Assoc. 218, 1803–1806. Bielanski, A., 2002. Neosporosis and embryo transfer. Embryo Transf. Newslett. 20, 10–14. Bielanski, A., Robinson, J., Philips-Tood, B., 2002. Effect of Neospora caninum on in vitro development of preimplantation stage bovine embryos and adherence to the zona pellucida. Vet. Rec. 150, 316–318. Bielanski, A., Surujballi, O., 1996. Association of Leptospira borgpetersenii serovar hardjo type herdjobovis with bovine ova and embryos produced by in vitro fertilization. Theriogenology 46, 45–55. Bielanski, A., Surujballi, O., 1998. Leptospira borgpetersenii serovar hardjo type herdjobovis in embryos fertilized in vitro. Can. J. Vet. Res. 62, 234–236. Bousquet, D., Twagiramungu, H., Morin, N., Brisson, C., Carboneau, G., Durocher, J., 1999. In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. Theriogenology 51, 59–70. Cabaj, W., Choroman´ski, L., Rodgers, S., Moskwa, B., Malczewski, A., 2000. Neospora caninum infections in aborting dairy cows in Poland. Acta Parasitol. 45, 113–114. Campero, C.M., Moore, D.P., Lagomarsino, H., Odeon, A.C., Castro, M., Visca, H., 2003. Serological status and abortion rate in progeny obtained by natural service or embryo transfer from Neospora caninum-seropositive cows. J. Vet. Med. Sci. B 50, 458–460. Davison, H.C., Guy, C.S., McGarry, J.W., Guy, F., Willams, D.J.L., Kelly, D.F., Trees, A.J., 2001. Experimental studies on the transmission of Neospora caninum between cattle. Res. Vet. Sci. 70, 163–168. Długon´ska, H., 2007. The transplacental transmission of Toxoplasma gondii and Neospora caninum protozoans. Wiad. Parazytol. 53, 9– 13. Dubey, J.P., 1999. Recent advances in Neospora caninum and neosporosis. Vet. Parasitol. 84, 349–367. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Duszewska, A.M., Reklewski, Z., 2001. Isolation of oocytes from pregnant heifers by OPU. Med. Wet. 57, 41–43. Duszewska, A.M., Reklewski, Z., Pien´kowski, M., Karasiewicz, J.A., Modlin´ski, J.A., 2000. Development of bovine embryos on Vero/ BRL cell monolayers (mixes co-culture). Theriogenology 54, 1239–1247. Duszewska, A.M., Relclewski, Z., Woidan, J., Gawrori, W., Korwinkossakowskl, M., Cybuiska, M., 2003. Evaluating heifers used for embryo production by OPU-IVM-JVF-TVC. Mcd Wet. 59, 58–60. Ferre, I., Aduriz, G., del-Pozo, I., Regidor-Cerrillo, J., Atxaerandio, R., Collantes-Fernandez, E., Hurtado, A., Ugarte-Garagalza, C., Ortega-Mora, L.M., 2005. Detection of Neospora caninum in semen and blood of naturally infected bulls. Theriogenology 1504–1518.
Gillespie, J.H., Schlafer, D.H., Foote, R.H., Quick, S., Dougherty, E., Schiff, E., Allen, S., 1990. Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure. Dtsch. Tierarztl. Wschr. 97, 65–68. Goodhand, K.L., Watt, R.G., Staines, M.E., Hutchinson, J.S.M., Broadbent, P.J., 1999. In vitro oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment. Theriogenology 51, 851–961. Guy, C.S., Williams, D.J.L., Kelly, D.F., McGarry, J.W., Guy, F., Bjorkman, C., Smith, R.F., Trees, A.J., 2001. Neospora caninum in persistently infected, pregnant cows: spontaneous transplacental infection is associated with an acute increase in maternal antibody. Vet. Rec. 149, 443–453. Hendriksen, P.J.M., Vos, P.L.A.M., Steenweg, W.N.M., Bevers, M.M., Dieleman, S.J., 2000. Bovine follicular development and its effect on the in vitro competence of oocytes. Theriogenology 53, 11–20. Kimura, K., Hirako, M., Iwata, H., Aoki, M., Kawaguchi, M., Seki, M., 2007. Successful superovulation of cattle by a single administration of FSH in aluminium hydroxide gel. Theriogenology 68, 633–639. Landmann, J.K., Jillella, D., O’Donoghue, P.J., McGowan, M.R., 2002. Confirmation of the prevention of vertical transmission of Neospora caninum in cattle by the use of embryo transfer. Aust. Vet. J. 80, 502–503. Moskwa, B., Cabaj, W., Pastusiak, K., Bien, J., 2003. The suitability of milk in detection of Neospora caninum infection in cows. Acta Parasitol. 48, 138–141. Moskwa, B., Cabaj, W., Pastusiak, K., Bien, J., 2005. Current studies on neosporosis in Poland. Wiad. Parazytol. 51 (Suppl.), 65–67. Moskwa, B., Pastusiak, K., Bien, J., Cabaj, W., 2007. The first detection of Neospora caninum DNA in the colostrum of infected cows. Parasitol. Res. 3, 633–636. Ortega-Mora, L.M., Ferre, I., del-Pozo, I., Caetano-da-Silva, A., Collantes-Fernandez, E., Regidor-Cerrillo, J., Ugarte-Garagalza, C., Aduriz, G., 2003. Detection of Neospora caninum in semen of bulls. Vet. Parasitol. 117, 301–308. Ortega-Mora, L.M., Gomez-Bautista, M., Fernandez-Garcia, A., 2005. Neospora and neosporosis: achievements and perspectives in diagnosis. Wiad. Parazytol. 51 (Suppl.), 63. Pare, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133–139. Rys, P.N., Persing, D.H., 1993. Preventing false positive: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products. J. Clin. Microb. 31, 2356– 2360. Schares, G., Conraths, F.J., 2001. Placentophagia—an alternative way for horizontal transmission of Neospora caninum in cattle? Trends Parasitol. 17, 574. Sirard, M.A., Picard, L., Dery, M., Coenen, K., Blondin, P., 1999. The time interval between FSH administration and ovarian aspiration influences the development of cattle oocytes. Theriogenology 51, 699–708. Stringfellow, D.A., 1998. Recommendation for the sanitary handling of in vivo derived embryos. In: Stringfellow, D.A., Seidel, S.M. (Eds.), Manual of the International Embryo Transfer Society. International Embryo Transfer Society, Savoy, pp. 79–84. Stringfellow, D.A., Givens, M.D., 2000. Infectious agents in bovine embryo production: hazard and solution. Theriogenology 53, 85– 94. Torres, M.P., Ortega, Y.R., 2006. Neospora caninum antibodies in commercial fetal bovine serum. Vet. Parasitol. 140, 352–355.
B. Moskwa et al. / Veterinary Parasitology 158 (2008) 370–375 Trees, A.J., Williams, D.J., 2005. Endogenous and exogenous transplacental infection in Neospora caninum and Toxoplasma gondii. Trends Parasitol. 21, 558–561. Yamage, M., Flechtner, O., Gottstein, B., 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain ‘‘cyst’’ DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J. Parasitol. 82, 272–279.
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Wis´niewski, M., Cabaj, W., Moskwa, B., We˛drychowicz, H., 2002. The first detection of Neospora caninum DNA in brains of calves in Poland. Acta Vet. (Beograd) 52, 393–400. Wouda, W., Bartels, C.J.M., Moen, A.R., 1999. Characteristics of Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997). Theriogenology 52, 233– 245.
Veterinary Parasitology 158 (2008) 370–375 www.elsevier.com/locate/vetpar
Short communication
Studies on Neospora caninum DNA detection in the oocytes and embryos collected from infected cows Boz˙ena Moskwa *, Katarzyna Goz´dzik, Justyna Bien´, Władysław Cabaj Witold Stefanski Institute of Parasitology of the Polish Academy of Sciences, Twarda Street 51/55, 00-818 Warsaw, Poland Received 28 March 2008; received in revised form 21 August 2008; accepted 15 September 2008
Abstract Neosporosis is a major cause of abortion in cattle over the world. One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is embryo transfer (ET) from infected dams into uninfected recipients. Oocytes and embryos taken from seropositive cows were examined for the presence of Neospora caninum DNA. A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams. The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams. Published by Elsevier B.V. Keywords: Neospora caninum; Oocytes; Embryos; DNA; PCR; Vertical transmission
1. Introduction Neospora caninum, an apicomplexan parasite with a worldwide distribution that infects warm-blooded vertebrates, is a causative agent of infertility and abortion (Dubey and Lindsay, 1996; Dubey, 1999). The infection leads to important economical losses in cattle due to reproductive failure associated with abortion and mortality in congenitally infected calves (Anderson et al., 1995; Wouda et al., 1999; Cabaj et al., 2000; Davison et al., 2001; Campero et al., 2003; Moskwa et al., 2005).
* Corresponding author. Tel.: +48 22 620 62 26; fax: +48 22 620 62 27. E-mail address: [email protected] (B. Moskwa). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2008.09.018
The transmission of infection is possible in two ways, vertical transmission from one generation to the next via placental transfer to the fetus or horizontal transmission by ingestion of oocysts shed by definitive hosts (Pare et al., 1996; Anderson et al., 1997; Schares and Conraths, 2001). Recently, modern nomenclature for describing two different modes of transplacental transmission for neosporosis was proposed. Accordingly, endogenous and exogenous transplacental infection modes were introduced to adequately distinguish infection from pre-existing chronic infection of the dam and from an acute infection of the dam (Trees and Williams, 2005; Długon´ska, 2007). Limited data is available on transmission of infection with N. caninum by embryo transfer (ET). The passing of N. caninum by ET was investigated on the basis of the serological status of ET donors and recipients (Baillargeon et al., 2001; Landmann et al., 2002). These
B. Moskwa et al. / Veterinary Parasitology 158 (2008) 370–375
results suggested that the risk of N. caninum transmission by ET is not related to the serological status of the embryo donors, but is rather linked to the seropositivity of the recipients. On the other hand, ET into seronegative recipients, using the procedure proposed by the IETS, is an effective way to prevent vertical transmission of neosporosis (Stringfellow, 1998; Baillargeon et al., 2001). There are no published reports, which show that oocytes or embryos obtained from seropositive donors are attached by the tachyzoites and can transmit the parasite by the uterine route or that tachyzoites adhere firmly to the zona pellicula of preimplantation embryos and can be passed to the uterine environment during embriotransfer (Bielanski, 2002; Bielanski et al., 2002). Additionally, the authors concluded that bovine embryos are protected by the zona pellicula against Neospora invasion. The aim of the study was to examine oocytes and embryos collected from seropositive cows-donors in order to determine the occurrence of parasite DNA. 2. Materials and methods 2.1. Cows and their serological status of neosporosis Eight cows with the history of abortion were used in this study. Animals were from the experimental station belonging to the Polish Academy of Sciences. The animals were naturally infected with N. caninum and infection was assessed by the detection of serum specific antibodies by a commercial ELISA kit (IDEXX Laboratories, Inc., Westbrook, ME, USA) according to the manufacturer’s instructions. Samples where the S/P ratio was greater than or equal to 0.50 were regarded as positive and those where ratio was less than 0.5 were regarded as negative for N. caninum antibodies. To avoid false negative results, cows were tested four times for evidence of N. caninum exposure and they were always seropositive. 2.2. Collection of oocysts and embryos To induce superovulation, the donor dams were injected intramuscularly with follicle stimulating hormone FSH Folltropin (Agtech, Inc., Canada). Oocytes were collected at the non-matured stage (prophase stage of the first division of meiosis). Embryos were obtained through in vivo fertilization and were collected at late morula or a blastocyst stages.
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Thirty oocytes and 30 embryos were used in the study. Oocysts and embryos were unwashed or washed three times using TCM-199 medium (Sigma) (Duszewska et al., 2000; Duszewska and Reklewski, 2001). According to the results obtained by Torres and Ortega (2006) medium was not supplemented with 5% fetal calf serum. Oocysts and embryos were not matured in vitro. No more than two oocytes and embryos were isolated in one session from the superovulated seropositive donors. On account of the fewness of isolated oocytes and embryos obtained, this superovulation was repeated several times. The samples were stored at 20 8C until testing. 2.3. PCR Genomic DNA was isolated from oocytes and embryos and from tachyzoites of the N. caninum NC-1 reference strain, using the Nucleospin Tissue DNA extraction kit (Macherey-Nagel, Germany). The primers, Np21 and Np6, based on the NC-5 region specific for N. caninum were used in this study (Yamage et al., 1996; Moskwa et al., 2003, 2007). To avoid carry-over contamination in the PCR reaction, a uracil N-glycosylase (UNG) protocol was implemented (Rys and Persing, 1993). The standard PCR master mixture contained 1 PCR buffer, 10 mM of each dATP, dCTP, dGTP and dUTP (substituted for dTTP in this protocol), 100 pmol of each primer Np21 and Np6, 1 U Taq DNA polymerase and 0.5 U UNG (uracil DNA glycosylase) and 1 ml of DNA. Water was added to a 50 ml final reaction volume. All reagents used for PCR were purchased from Fermentas (MBI Fermentas, USA). The mixtures were incubated at room temperature for 10 min for deactivation of any dU-containing templates and than were denatured at 95 8C for 10 min (for inactivation of enzyme) prior to 30 cycles of denaturation (95 8C, 30 s), annealing (55 8C, 30 s) and primer extension (72 8C, 1 min). After the last cycle, additional extension was applied for 10 min at 72 8C, all carried out in a Genius (Techne, USA) thermocycler. Amplification products were analyzed by electrophoresis in a 1% agarose gel stained with ethidium bromide and visualised under UV light using the Kodak Electrophoresis Documentation and Analysis System (EDAS) 290. To determine the sensitivity of PCR amplicons of examined samples, DNA was extracted from N. caninum tachyzoites cultured in Vero cells. The DNA
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The number of isolated oocytes and embryos was low (no more than two in one session). The PCR assay did not yield the expected 328 bp products in oocytes and embryos unwashed or washed three times in TCM 199. These results revealed that N. caninum DNA was not present in any of the examined samples. Because all examined samples were negative in relation to the presence of N. caninum DNA, Fig. 2 represents only chosen results. Fig. 1. Agarose gel (1%) electrophoresis of the products obtained in the PCR experiments. DNA isolated from N. caninum tachyzoites was used as a template. A limited dilution series from 9 pg/ml (lane 1), down to 0.017 pg/ml (lane 10). Sample contained 0.070 pg/ml (lane 8) was clearly visible on the original gel. Lane M, molecular size marker (100 bp DNA ladder, Fermentas).
concentration was determined using Spectrophotometr NanoDrop ND1000. A limited dilution series from 10 ng/ml, down to 0.017 pg/ml was used in the study. The amount of DNA in one N. caninum tachyzoite is 0.07 pg/ml (Dubey and Sibley, personal information). 3. Results The S/P ratio reflected IgG level in serum of seropositive cows, achieved high values in the first examination of sera from eight cows (2.12, 2.27, 1.97, 3.42, 2.07, 2.34, 3.11 and 2.51 respectively). The subsequent examinations revealed that the S/P ratios had achieved a stable level and fluctuated around the above values. After 30 cycles of amplification, DNA of one N. caninum tachyzoite could be detected, thus demonstrating the sensitivity of the designed PCR system (Fig. 1).
4. Discussion The majority cases of N. caninum transmission occurs by endogenous and exogenous infection modes (Trees and Williams, 2005), from an infected dam to her foetus in utero. It was reported that more than 80% of calves from infected dams are born with congenital N. caninum infection and that the parasite can cycle within a cow family for many generation (Landmann et al., 2002). One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is ET from infected dams into uninfected recipients. Development of new technology related to in vitro embryo production allowed for the commercial use of this method of reproduction. After insemination of naturally cyclic cows, approximately 85% of the ovulated oocytes will develop into an embryo. To induce superovulation, the donor dams used to be injected intramuscularly with follicle stimulating hormone FSH Folltropin (Sirard et al., 1999; Hendriksen et al., 2000).
Fig. 2. Agarose gel (1%) electrophoresis. Negative PCR from washed and unwashed oocytes and embryos from different N. caninum seropositive cows. M: molecular marker; lane 1: unwashed oocytes; lanes 2–3: washed oocytes; lane 4: unwashed embryos; lane 5: washed embryos; lane 6: positive control (DNA from the reference strain of N. caninum, NC-1); lane 7: negative control.
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The effect of FSH treatment on oocyte yields and developmental competence may depend on the mode of administration of FSH (Goodhand et al., 1999). Although, variability in the number of isolated oocytes and embryos was observed between individuals among examined donors, FSH treatments effectively induce superovulation in cattle (Bousquet et al., 1999; Hendriksen et al., 2000; Kimura et al., 2007). In our experiment, the superovulation effect after FSH treatment was not observed. The number of isolated oocytes and embryos was low (no more than two in one session). The number of oocytes and embryos recovered form N. caninum seropositive donors was comparable or lower than in in vivo conventional procedures (Bousquet et al., 1999; Duszewska et al., 2003). We can speculate that N. caninum infection which has been shown to be the major cause of bovine reproductive disorders may also influence the ovulation process of the seropositive cattle. An infectious agent present during the production of bovine embryos might reduce the number and the quality of embryos generated, resulting in transmission of disease to recipients and offspring (Stringfellow and Givens, 2000). However, the authors concluded that a relatively simple embryo processing procedure will ensure that many infectious agents are effectively diluted, dislodged or inactivated. Contrary to this statement, earlier reports indicated that embryos might function as vectors for transmission of pathogens. Gillespie et al. (1990) reported the adherence of bovine herpesvirus-1, bluetongue virus and bovine viral diarrhea virus to bovine embryos. Bielanski and Surujballi (1996, 1998) revealed that the washing procedure according to IETS standards was not completely effective for removing Leptospira borgpetersenii virus from bovine embryos. There is no documented information on the susceptibility of bovine embryos to N. caninum. The studies undertaken by Baillargeon et al. (2001), Landmann et al. (2002) and Campero et al. (2003) suggest that embryos harvested from seropositive dams and implanted into non-infected recipients, result in the birth of non-infected offspring. Baillargeon et al. (2001) stated that ET from seropositive dams to uninfected recipients led to 63 pregnancies, but 4 of them were aborted (6.3%). ET from seronegative dams to seropositive recipients led to four pregnancies, but one of them was aborted (25%). Landmann et al. (2002) revealed a similar rate of abortion (25%) when embryos were transferred from seropositive dams to seronegative recipients. However, there is no information available to show that, under natural conditions, embryos are exposed to N. caninum in the uterine cavity.
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The adhesion of N. caninum tachyzoites to preimplantation embryos was investigated in in vitro studies only (Bielanski et al., 2002). In vitro fertilized embryos were exposed to tachyzoites of N. caninum in culture with a monolayer Vero cells. Using transmission electron microscope, dividing tachyzoites of the parasite were observed within trophoblastic cells of hatched blastocytes only when the zona pellicula was absent. The results of that study implicated that with an intact zona pellicula, preimplantation embryos are protected by the zona pellicula against N. caninum invasion. However, the author pointed out, that the obtained results may not reflect the natural parasite biology and differences between the properties of the zona pellicula of in vivo and in vitro embryos. Molecular techniques such as PCR offer a highly sensitive and specific alternative to immunological methods for the diagnostics of neosporosis. PCR was suitable to confirm the presence of N. caninum DNA in various tissues such as brain, spinal cord, muscle and blood as well as in the milk and colostrum of infected cows (Guy et al., 2001; Wis´niewski et al., 2002; Moskwa et al., 2003,2007; Ferre et al., 2005; Ortega-Mora et al., 2003, 2005). Using a PCR, protocol the absence of N. caninum DNA in oocytes and embryos obtained from seropositive dams was revealed. In fact, the absence of N. caninum DNA was revealed in both washed and unwashed oocyte and embryo samples. In conclusion, the ET procedure recommended by IETS seems to be an effective way to prevent the vertical transmission of N. caninum (Stringfellow, 1998). But the main pathway of reducing the potential risk of transmission is by ensuring the absence of N. caninum forms. The results of our studies indicate that the oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams. Acknowledgements The research was supported by the grants No. 3 P06K 030 23 of the State Committee for the Scientific Research in Warsaw, Poland and ADIPAR—Advanced Diagnostics for Parasitic Disease Transfer of Knowledge Project in the 6th Framework Programme. References Anderson, M.L., Palmer, C.W., Thurmond, M.C., 1995. Evaluation of abortions in cattle attributable to neosporosis in selected
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