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Perinatal priming of calves born to Schistosoma mattheei-infected dams
Veterinary Parasitology 144 (2007) 61–67 www.elsevier.com/locate/vetpar
Perinatal priming of calves born to Schistosoma mattheei-infected dams S. Gabrie¨l a,d, C. Ververken b, J. Vercruysse c, L. Duchateau d, I.K. Phiri a, B.M. Goddeeris b,c,* a
Department of Clinical Studies, School of Veterinary Medicine, University of Zambia, P.O. Box 32379, Lusaka, Zambia b Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium c Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium d Department of Physiology, Biochemistry and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 18 November 2005; received in revised form 5 May 2006; accepted 8 May 2006
Abstract The objective of this study was to elucidate whether calves born to infected dams had been primed against Schistosoma mattheei antigens. Infection-confirmed, pregnant cows were randomly selected for monitoring their offspring. Pre-colostral serum was collected from the neonates for the detection of specific antibodies at birth, as they indicate a transplacental transfer of schistosomespecific antibodies and antigen. At the age of approximately 2 months, peripheral blood mononuclear cells (PBMC) of calves were analysed for specific memory by antigen-specific stimulation in vitro. Twenty-six of the 30 calves demonstrated S. mattheei-specific proliferation. All 12 seropositive-born, as well as 14 of the 18 seronegative-born (before colostrum uptake) calves displayed mattheei-specific proliferation. The results indicate that the calves were primed against S. mattheei and might explain why seropositive-born calves from infected dams are better protected against S. mattheei, and query the impermeability of the damaged ruminant placenta with consequences for antigen transfer. # 2006 Elsevier B.V. All rights reserved. Keywords: Schistosoma mattheei; Ruminants; Perinatal priming
1. Introduction Ruminants have an epithelio-chorial placenta – as opposed to the haemochorial placenta of human – which does not allow transfer of antibodies and/or antigens.
* Corresponding author at: Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium. Tel.: +32 16 321 427; fax: +32 16 321 994. E-mail address: [email protected] (B.M. Goddeeris). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.05.035
However, in recent studies from Gabrie¨l et al. (2002, 2004) antibodies against soluble adult worm antigen preparation (SWAP) mattheei and circulating anodic schistosome antigen (antigens derived from the gut of the worm) were detected in the serum of colostrum-free calves born to Schistosoma mattheei-infected mothers. As congenital S. mattheei infections are not very likely (Gabrie¨l et al., 2002), a transplacental transfer of circulating antigen was assumed. This transfer would only be possible if lesions would have disrupted the placental barrier (Kruse, 1983; Poitras et al., 1986). Gabrie¨l et al. (2005a) detected schistosome eggs on the
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foetal side of the placenta and ascribed them to be the likely cause of placental lesions enabling passive transfer of antibodies and antigens. Gabrie¨l et al. (2002, 2004) also observed the intake of circulating schistosome antigens by newborn calves after the uptake of colostrum originating from infected cows. The amount of antigen detected in the serum from the newborns varied. Early contact of the neonatal with parasite antigens (pre- and/or post-natal) could modulate its future immune response, leading either to sensitization or tolerance development (Rasheed, 1994). The cattle foetus is known to be capable of building immune reactions after contact with antigen from about 120 days of gestation onwards (Osburn et al., 1982; Hein et al., 1988; Jensen et al., 1988). This modulation of the immune response could be beneficial, but may also lead to immunopathological consequences (Carlier and Truyens, 1995). In ruminants, no studies have been performed to analyse whether a priming occurs, either intra-uterine or after birth via the intake of colostrum. The objective of this study was to elucidate whether calves born to infected mothers have been primed against S. mattheei antigens. Therefore, PBMC of such calves were analysed for specific memory by antigenspecific stimulation in vitro.
Around the age of 2 months (since birth all calves were kept in transmission-free areas), PBMC were collected from 30 calves in order to determine their T cell driven memory for S. mattheei in antigen-specific proliferation assays. 2.3. Parasitological techniques To determine faecal egg counts (EPG) a modification of the concentration technique of Lawrence (1970) modified by De Bont et al. (1995) (1 egg = 5 EPG) was used. 2.4. Serological techniques All serum samples were analysed for IgG levels against SWAP mattheei using an enzyme-linked immunosorbent assay (ELISA) as described by Gabrie¨l et al. (2002). Serum samples from the calves on day 0 were also analysed for IgG levels against E. coli using an ELISA as described by Gabrie¨l et al. (2005a). Ig levels were expressed as the difference between the optical densities of the test samples and a known negative sample resulting in DOD = ODx ODneg, with ODx being the individual OD value of the test serum, and ODneg the OD of a known negative sample.
2. Materials and methods 2.5. T cell priming 2.1. Study site and animals The study was carried out on an extensive beef farm in Chisamba, in the Central Province of Zambia. A total of 44 pregnant cows were randomly selected out of a group of 350 cows. Faecal egg counts were performed twice during the selection of the cows for the confirmation of their S. mattheei infection status. All cows were older than 4 years, multiparous, and had been present on the endemic farm from birth. 2.2. Experimental design At partus, serum and colostrum samples were collected from the dams, as well as serum samples from the newborn calves before (day 0) and after intake of colostrum (day 1) for the determination of specific antibodies against SWAP mattheei (cows and calves) and E. coli (calves day 0). Determination of antibodies against E. coli was performed to demonstrate placental transfer. Foetal antibodies against E. coli are probably from maternal origin, transferred via lesions in the placenta, as foetal origin would imply septicaemia in the foetus during gestation.
2.5.1. Experimental setup Thirty calves (around the age of 2 months) were tested for Schistosoma-specific T-cell responses by stimulating PBMC with cleared SWAP mattheei. 2.5.2. Preparation of cleared SWAP mattheei antigen A soluble adult worm antigen preparation (SWAP) of S. mattheei was cleared by sonication and centrifugation (4000 g, 200 , 4 8C). The resulting soluble fraction was filter sterilized (20 mm), dialyzed twice against RPMI1640 (Invitrogen, Carlsbad, USA) and filter sterilized again. The protein concentration was determined using the BCA protein assay method (Pierce, Rockford, USA). The antigens were kept at 80 8C until they were shipped on dry ice. In order to check whether adequate T-cell antigens were present in the antigen preparation, two infectionconfirmed (by faecal egg counts and specific antibody levels) dams were tested for SWAP mattheei-specific Tcell responses as described below. Also, the proliferation of six known negative calves from a Belgian farm in response to SWAP mattheei was assayed to
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exclude the presence of non-specific T-cell mitogens (e.g. lectin). 2.5.3. Lymphocyte proliferation assay Proliferation assays were performed on 3 consecutive days, handling 10 animals/day. PBMC were prepared according to Goddeeris and Morrison (1988). This procedure typically yielded about 4–5 106 cells/ml of blood. The PBMC (5 105 cells/well) were stimulated in duplicate in flat-bottom 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) with 90 mg of cleared SWAP mattheei at 38 8C in a humidified incubator with 5% CO2. Proliferation was measured by the incorporation of 3 H-thymidine (MP Biomedicals, ICN, Irvine, USA) during the last 24 h of culture (1 mCi/well). Separate wells for background proliferation (plain culture medium) and for proliferative capacity of the isolated PMBC (stimulation with 10 mg concanavalin A; Sigma– Aldrich, Steinheim, Germany) were included. ConAstimulated PBMC were harvested after 3 days (maximum proliferation for ConA stimulation, Goddeeris et al., 1986), whereas the antigen-induced proliferation and negative controls were cultured for 5 days. Cultures were harvested on glass fibre filters with a cell harvester (Skatron, Lier, Norway). Filters were placed in 2 ml Lumasave LSC cocktail (Lumac, Groningen, The Netherlands) and DNA-incorporated 3H-thymidine was measured in a b-scintillation counter (Tricarb 2550 TR, Packard Instrument Company, Meriden, USA). As PBMC from young calves quite often show high spontaneous proliferation in plain medium, antigenspecific proliferation of PBMC was determined in relation to their proliferative capacity to the mitogen ConA instead of the more conventional stimulation index (cpm sample/cpm medium): 100 (cpmsample cpmmedium)/cpmConA cpmmedium) with cpm being counts per minute.
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2.6. Statistical analyses Proliferation levels were compared between seropositive- and seronegative-born calves by the nonparametric Mann–Whitney test. The correlation between antibody titers and proliferation levels was determined using the Spearman Rank correlation coefficient. 3. Results 3.1. Dams All 44 cows were confirmed infected with S. mattheei on either one or both of the faecal examinations, with egg counts varying between 0 and 30 EPG with a mean value of 4. All dams had S. mattheeispecific antibodies at birth, with a mean DOD of 0.31 in serum, and 0.96 in colostrum. 3.2. Antibody responses of calves at birth and after intake of colostrum Twelve of the 30 calves were born with S. mattheeispecific serum antibodies (=seropositive-born). All seropositive-born calves were also seropositive for antibodies against E. coli. Mean DOD levels of serum IgG against SWAP mattheei for the seropositive-born calves on day 0 equalled 0.73. Mean DOD levels for E. coli equalled 0.68. After colostrum uptake, IgG levels against SWAP mattheei were detected in all calves (seropositive- as well as seronegative-born), with a mean DOD level of 0.66. 3.3. Antigen preparation and control Antigen-specific and non-specific T-cell stimulatory capacity of the antigen preparations were analysed by in vitro stimulation of PBMC from known immune
Table 1 Specific T-cell proliferation of two positive control cows and six negative control calves Control animals Positive controls
Negative controls
Cow no.
cpm A
cpm C
cpm m
Calf no.
cpm A
cpm C
cpm m
1 2
125,343 181,052
143,702 123,468
2965 1803
1 2 3 4 5 6
18,837 3,379 14,640 3,880 2,794 6,255
167,060 115,954 184,222 156,102 191,596 195,271
51,916 40,088 54,392 423 297 1,118
cpm A: cpm obtained after stimulation with 90 mg antigen; cpm C: stimulation with Con A; cpm m: stimulation with medium.
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Fig. 1. Specific T-cell proliferation of calves born to Schistosoma mattheei-infected cows. The proliferation induced by 90 mg of antigen is shown as the percentage of the proliferation induced with ConA. (A) Proliferation induced in PBMC from known immune and naı¨ve animals. (B) Proliferative response of PBMC from seropositive- (seropos) and seronegative-born (seroneg) calves from S. mattheei-infected cows.
and known naı¨ve animals, respectively. The infected control dams showed a strong proliferative response whereas the negative control calves did not (Table 1 and Fig. 1A), indicating that the necessary T-cell antigens were present in the antigen preparation and that no aspecific mitogenic responses were induced. Therefore, SWAP mattheei could be used for assaying S. mattheei-specific T-cell responses in the calves. The positive proliferative response of the infected dams was in accordance with their high serum antibody levels for S. mattheei. As expected for young calves, autologous background proliferation in the negative control calves was high (range of 297– 54392 cpm). 3.4. S. mattheei-specific proliferation The results of the proliferation tests of the calves from infected dams are presented in Table 2 and Fig. 1B. Twenty-six of the 30 calves (87%) showed antigenspecific proliferation. All 12 seropositive-born calves and 14 of the 18 seronegative-born animals (71%) displayed S. mattheei-specific proliferative responses. There were no significant differences in proliferation levels between the seropositive- and seronegative-born calves (P = 0.53). Compared to the infected control dams, proliferation of the calves was in most cases equally strong. There was no correlation between serum
antibody titers and proliferation levels (correlation coefficient of 0.213, P = 0.53). 4. Discussion In previous studies Gabrie¨l et al. (2002) indicated a transplacental transfer of circulating schistosome anodic antigen through the epithelio-chorial placenta of cows, most probably through lesions in the placenta, as well as a transfer of circulating schistosome antigen via the colostrum. These transfers would imply neonatal contact with schistosome antigens. In this study, we were able to demonstrate that 87% of the calves born to S. mattheei-infected mothers, but kept under S. mattheei-free conditions, were able to respond in vitro to schistosome antigens as strongly as infected dams, and consequently must have been primed as foeti during gestation or as neonates after colostrum uptake of mattheei antigens. The latter possibility is unlikely however, as Gabrie¨l et al. (2005b) could not observe protective differences between seonegative-born – but seropositive after colostrum uptake from their infected dams – calves and calves from non-infected animals. This is the first time that a priming of neonatal calves for schistosome antigens has been demonstrated. In mice and man, intra-uterine priming to schistosome antigen and subsequent tolerance or sensitization has been described (Hang et al., 1974; Camus et al., 1976;
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Table 2 Specific T-cell proliferation of calves born to S. mattheei-infected mothers Proliferation positive
Proliferation negative
Calf no.
cpm A
cpm C
cpmm
Seropositive-born 1 2 3 4 5 6 7 8 9 10 11 12
32,541 104,994 41,539 109,213 68,519 93,815 38,302 118,202 113,239 51,718 49,907 4,705
98,399 98,399 81,036 246,359 162,969 285,745 77,847 195,662 133,376 8,481 101,702 86,805
12,787 2,421 22,459 26,979 26,720 5,455 20,396 9,205 5,551 9,050 1,367 991
Calf no.
cpm A
cpm C
cpm m
131,801 166,809 116,559 134,224
35,498 74,458 921 171,586
No. of calves 12 Seronegative-born 13 14 15 16 17 18 19 20 21 22 23 24 25 26
142,474 52,435 60,178 39,293 148,410 70,458 18,107 102,073 144,527 134,972 15,304 89,067 39,844 60,671
0 128,685 63,480 41,639 90,490 163,457 213,689 99,088 94,346 150,044 320,096 159,190 323,103 242,243 26,722
19,421 3,761 5,181 11,517 27,883 10,582 726 39,924 62,104 4,387 849 65,651 18,849 10,693
27 28 29 30
14,078 38,708 976 128,900
No. of calves 14
4
cpm A: cpm obtained after stimulation with 90 mg antigen; cpm C: stimulation with Con A; cpm m: stimulation with medium.
Tachon and Borojevic, 1978; Novato-Silva et al., 1992; Malhotra et al., 1997). However, man and mouse have haemochorial placentas (three maternal layers disappeared) as opposed to cattle, swine and horse where the epithelio-chorial placenta (three maternal layers present) constitutes a strong barrier between the blood circulations of mother and foetus. This prenatal priming has important consequences for subsequent immunity development and protection of the newborn in endemic areas. Indeed, Gabrie¨l et al. (2004) observed that seropositive-born calves from infected dams, suspected of intra-uterine priming, had lower faecal egg counts and lower schistosome circulating antigen levels in their serum after natural S. mattheei challenge around the age of 5 months, indicating a protective memory. As already mentioned above, this protective effect appeared to be induced
prenatally and not postnatally as seronegative-born calves becoming seropositive after colostrum (antigen and antibody) uptake were not protected (no significant differences in faecal or tissue egg counts, nor in worm counts between calves born to infected and non-infected mothers) when challenged at the age of 7 months (Gabrie¨l et al., 2005b). S. mattheei-specific antibodies in the newborn colostrum-free calves are most probably from maternal origin through placental lesions, although minor foetal origin might not be excluded as has been addressed before (Gabrie¨l et al., 2005a). Western blots on E. coli demonstrated similar recognition patterns of the sera of dams and their newborn seropositive calves. As foetal origin of anti-E. coli antibodies would imply the occurrence of a septicemia in the foetus with resulting abortion, it was assumed that the detected antibodies
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were not of foetal but maternal origin. Moreover, if these antibodies are of maternal origin, they seem not to block priming of the foetus to S. mattheei antigen, as all seropositive-born calves demonstrated antigen-specific proliferation in vitro. This is in agreement with Siegrist et al. (1998) who described that introduction of antigens into a host with high levels of maternal antibodies leads to a failure of antibody induction, without however reducing the antigen-specific T-cell proliferative response. Lesions in the placenta which allow a transfer of antigens can be caused by schistosome eggs, as demonstrated by Gabrie¨l et al. (2005a), but might also be inflicted by other placenta-migrating pathogens such as Brucella (Ghirotti et al., 1991), Leptospira (Jerrett et al., 1984) or Neospora (Bergeron et al., 2001). These pathogens are primarily associated with abortions but can, through damage of the placenta, cause a transfer of antigens leading to foetal contact and in utero priming. One can presume that antigens from several other pathogens might also cross the damaged placenta and consequently prime the neonatal against significantly more pathogens (antigens) than was originally thought. In conclusion, this study demonstrates for the first time that the majority of calves born to S. mattheeiinfected dams are primed against S. mattheei as evidenced by in vitro proliferation of their lymphocytes upon antigen-specific stimulation. These results query the impermeability of the ruminant epithelio-chorial placenta and might explain previous data where seropositive-born calves showed evidence of protection against natural S. mattheei challenge. Acknowledgements This work was supported by the Flemish Inter University Council (VLIR-UNZA IUC Programme Zambia-Belgium). We would like to thank Mr. V. Mkanjo and the workers from the farm for their cooperation in the study, the excellent technical assistance of the technicians of the Department of Clinical Studies, School of Veterinary Medicine, Zambia, and the School of Veterinary Medicine, University of Zambia. References Bergeron, N., Girard, C., Pare, J., Fecteau, G., Robinson, J., Baillargeon, P., 2001. Rare detection of Neospora caninum in placentas from seropositive dams giving birth to full-term calves. J. Vet. Diagn. Invest. 13, 173–175. Camus, D., Carlier, Y., Bina, J.C., Borojevic, R., Prata, A., Capron, A., 1976. Sensitization to schistosoma mansoni antigen in uninfected children born to infected mothers. J. Infect. Dis. 134, 405–408.
Carlier, Y., Truyens, C., 1995. Review: influence of maternal infection on offspring resistance towards parasites. Parasitol. Today 11, 94–99. De Bont, J., Vercruysse, J., Sabbe, F., Southgate, V.R., Rollinson, D., 1995. Schistosoma mattheei infections in cattle: changes associated with season and age. Vet. Parasitol. 57, 299–307. Gabrie¨l, S., De Bont, J., Phiri, I.K., Masuku, M., Riveau, G., Schacht, A.M., Deelder, A.M., Van Dam, G.J., Vercruysse, J., 2002. Transplacental transfer of schistosomal circulating anodic antigens in cows. Parasite Immunol. 24, 521–525. Gabrie¨l, S., Phiri, I.K., Van Dam, G.J., Deelder, A.M., Duchateau, L., Vercruysse, J., 2004. Variations in immune response to natural Schistosoma mattheei infections in calves born to infected mothers. Vet. Parasitol. 119, 177–185. Gabrie¨l, S., Geldhof, P., Phiri, I.K., Cornillie, P., Goddeeris, B.M., Vercruysse, J., 2005a. Placental transfer of immunoglobulins in cattle infected with Schistosoma mattheei. Vet. Immunol. Immunopathol. 104, 265–272. Gabrie¨l, S., Dorny, P., Duchateau, L., Phiri, I.K., Chembensofu, M., Vercruysse, J., 2005b. The influence of colostrum on infection of calves at 7 months of age with Schistosoma mattheei. Vet. Parasitol. 129, 55–60. Ghirotti, M., Semproni, G., De Meneghi, D., Mungaba, F.N., Nannini, D., Calzetta, G., Paganico, G., 1991. Sero-prevalences of selected cattle diseases in the Kafue flats of Zambia. Vet. Res. Commun. 15, 25–36. Goddeeris, B.M., Baldwin, C.L., ole-MoiYoi, O., Morrison, W.I., 1986. Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. Functional evaluation of monocytes in response to lectins. J. Immunol. Methods. 89, 165–173. Goddeeris, B.M., Morrison, W.I., 1988. Techniques for the generation, cloning and characterization of bovine cytotoxic T cells specific for the protozoan Theileria parva. J. Tissue Cult. Methods 11, 101–110. Hang, L.M., Boros, D.L., Warren, K.S., 1974. Induction of immunological hyporesponsiveness to granulomatous hypersensitivity in Schistosoma mansoni infection. J. Infect. Dis. 130, 515–522. Hein, W.R., Shelton, J.N., Simpson-Morgan, M.W., Morris, B., 1988. Traffic and proliferative responses of recirculating lymphocytes in foetal calves. Immunology 64, 621–626. Jensen, J., Rubino, M., Yang, W.C., Cooley, A.J., Schultz, R.D., 1988. Ontogeny of mitogen responsive lymphocytes in the bovine foetus. Dev. Comp. Immunol. 12, 685–692. Jerrett, I.V., McOrist, S., Waddington, J., Browning, J.W., Malecki, J.C., McCausland, I.P., 1984. Diagnostic studies of the fetus, placenta and maternal blood from 265 bovine abortions. Cornell Vet. 74, 8–20. Kruse, P.E., 1983. The importance of colostral immunoglobulins and their absorption from the intestine of the newborn animals. Ann. Rech. Vet. 14, 349–353. Lawrence, J.A., 1970. Examination of ruminant faeces for schistosome eggs. Rhod. Vet. J. 1, 49–52. Malhotra, I., Ouma, J., Wamachi, A., Kioko, J., Mungai, P., Omollo, A., Elson, L., Koech, D., Kazura, J., King, C.L., 1997. In utero exposure to helminth and mycobacterial antigens generates cytokine responses similar to that observed in adults. J. Clin. Invest. 99, 1759–1766. Novato-Silva, E., Gazzinelli, G., Colley, D.G., 1992. Immune responses during human schistosomiasis mansoni. XVIII. Immunologic status of pregnant women and their neonates. Scand. J. Immunol. 35, 429–437. Osburn, B.I., MacLachlan, N.J., Terrell, T.G., 1982. Ontogeny of the immune system. JAVMA 181, 1049–1052.
S. Gabrie¨l et al. / Veterinary Parasitology 144 (2007) 61–67 Poitras, B.J., Miller, R.B., Wilkie, B.N., Bosu, W.T., 1986. The maternal to foetal transfer of immunoglobulins associated with placental lesions in sheep. Can. J. Vet. Res. 50, 68–73. Rasheed, F.N., 1994. Maternal infections influence infection susceptibility in childhood. Med. Hypotheses 42, 76–80. Siegrist, C.-A., Barrios, C., Martinez, X., Brandt, C., Berney, M., Cordova, M., Kovarik, J., Lambert, P.-H., 1998. Influence of
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maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime-boost strategies in mice. Eur. J. Immunol. 28, 4138–4148. Tachon, P., Borojevic, R., 1978. Mother–child relation in human schistosomiasis mansoni: skin test and cord blood reactivity to schistosomal antigens. Trans. R. Soc. Trop. Med. Hyg. 72, 605–609.
Perinatal priming of calves born to Schistosoma mattheei-infected dams S. Gabrie¨l a,d, C. Ververken b, J. Vercruysse c, L. Duchateau d, I.K. Phiri a, B.M. Goddeeris b,c,* a
Department of Clinical Studies, School of Veterinary Medicine, University of Zambia, P.O. Box 32379, Lusaka, Zambia b Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium c Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium d Department of Physiology, Biochemistry and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 18 November 2005; received in revised form 5 May 2006; accepted 8 May 2006
Abstract The objective of this study was to elucidate whether calves born to infected dams had been primed against Schistosoma mattheei antigens. Infection-confirmed, pregnant cows were randomly selected for monitoring their offspring. Pre-colostral serum was collected from the neonates for the detection of specific antibodies at birth, as they indicate a transplacental transfer of schistosomespecific antibodies and antigen. At the age of approximately 2 months, peripheral blood mononuclear cells (PBMC) of calves were analysed for specific memory by antigen-specific stimulation in vitro. Twenty-six of the 30 calves demonstrated S. mattheei-specific proliferation. All 12 seropositive-born, as well as 14 of the 18 seronegative-born (before colostrum uptake) calves displayed mattheei-specific proliferation. The results indicate that the calves were primed against S. mattheei and might explain why seropositive-born calves from infected dams are better protected against S. mattheei, and query the impermeability of the damaged ruminant placenta with consequences for antigen transfer. # 2006 Elsevier B.V. All rights reserved. Keywords: Schistosoma mattheei; Ruminants; Perinatal priming
1. Introduction Ruminants have an epithelio-chorial placenta – as opposed to the haemochorial placenta of human – which does not allow transfer of antibodies and/or antigens.
* Corresponding author at: Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium. Tel.: +32 16 321 427; fax: +32 16 321 994. E-mail address: [email protected] (B.M. Goddeeris). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.05.035
However, in recent studies from Gabrie¨l et al. (2002, 2004) antibodies against soluble adult worm antigen preparation (SWAP) mattheei and circulating anodic schistosome antigen (antigens derived from the gut of the worm) were detected in the serum of colostrum-free calves born to Schistosoma mattheei-infected mothers. As congenital S. mattheei infections are not very likely (Gabrie¨l et al., 2002), a transplacental transfer of circulating antigen was assumed. This transfer would only be possible if lesions would have disrupted the placental barrier (Kruse, 1983; Poitras et al., 1986). Gabrie¨l et al. (2005a) detected schistosome eggs on the
62
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foetal side of the placenta and ascribed them to be the likely cause of placental lesions enabling passive transfer of antibodies and antigens. Gabrie¨l et al. (2002, 2004) also observed the intake of circulating schistosome antigens by newborn calves after the uptake of colostrum originating from infected cows. The amount of antigen detected in the serum from the newborns varied. Early contact of the neonatal with parasite antigens (pre- and/or post-natal) could modulate its future immune response, leading either to sensitization or tolerance development (Rasheed, 1994). The cattle foetus is known to be capable of building immune reactions after contact with antigen from about 120 days of gestation onwards (Osburn et al., 1982; Hein et al., 1988; Jensen et al., 1988). This modulation of the immune response could be beneficial, but may also lead to immunopathological consequences (Carlier and Truyens, 1995). In ruminants, no studies have been performed to analyse whether a priming occurs, either intra-uterine or after birth via the intake of colostrum. The objective of this study was to elucidate whether calves born to infected mothers have been primed against S. mattheei antigens. Therefore, PBMC of such calves were analysed for specific memory by antigenspecific stimulation in vitro.
Around the age of 2 months (since birth all calves were kept in transmission-free areas), PBMC were collected from 30 calves in order to determine their T cell driven memory for S. mattheei in antigen-specific proliferation assays. 2.3. Parasitological techniques To determine faecal egg counts (EPG) a modification of the concentration technique of Lawrence (1970) modified by De Bont et al. (1995) (1 egg = 5 EPG) was used. 2.4. Serological techniques All serum samples were analysed for IgG levels against SWAP mattheei using an enzyme-linked immunosorbent assay (ELISA) as described by Gabrie¨l et al. (2002). Serum samples from the calves on day 0 were also analysed for IgG levels against E. coli using an ELISA as described by Gabrie¨l et al. (2005a). Ig levels were expressed as the difference between the optical densities of the test samples and a known negative sample resulting in DOD = ODx ODneg, with ODx being the individual OD value of the test serum, and ODneg the OD of a known negative sample.
2. Materials and methods 2.5. T cell priming 2.1. Study site and animals The study was carried out on an extensive beef farm in Chisamba, in the Central Province of Zambia. A total of 44 pregnant cows were randomly selected out of a group of 350 cows. Faecal egg counts were performed twice during the selection of the cows for the confirmation of their S. mattheei infection status. All cows were older than 4 years, multiparous, and had been present on the endemic farm from birth. 2.2. Experimental design At partus, serum and colostrum samples were collected from the dams, as well as serum samples from the newborn calves before (day 0) and after intake of colostrum (day 1) for the determination of specific antibodies against SWAP mattheei (cows and calves) and E. coli (calves day 0). Determination of antibodies against E. coli was performed to demonstrate placental transfer. Foetal antibodies against E. coli are probably from maternal origin, transferred via lesions in the placenta, as foetal origin would imply septicaemia in the foetus during gestation.
2.5.1. Experimental setup Thirty calves (around the age of 2 months) were tested for Schistosoma-specific T-cell responses by stimulating PBMC with cleared SWAP mattheei. 2.5.2. Preparation of cleared SWAP mattheei antigen A soluble adult worm antigen preparation (SWAP) of S. mattheei was cleared by sonication and centrifugation (4000 g, 200 , 4 8C). The resulting soluble fraction was filter sterilized (20 mm), dialyzed twice against RPMI1640 (Invitrogen, Carlsbad, USA) and filter sterilized again. The protein concentration was determined using the BCA protein assay method (Pierce, Rockford, USA). The antigens were kept at 80 8C until they were shipped on dry ice. In order to check whether adequate T-cell antigens were present in the antigen preparation, two infectionconfirmed (by faecal egg counts and specific antibody levels) dams were tested for SWAP mattheei-specific Tcell responses as described below. Also, the proliferation of six known negative calves from a Belgian farm in response to SWAP mattheei was assayed to
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exclude the presence of non-specific T-cell mitogens (e.g. lectin). 2.5.3. Lymphocyte proliferation assay Proliferation assays were performed on 3 consecutive days, handling 10 animals/day. PBMC were prepared according to Goddeeris and Morrison (1988). This procedure typically yielded about 4–5 106 cells/ml of blood. The PBMC (5 105 cells/well) were stimulated in duplicate in flat-bottom 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) with 90 mg of cleared SWAP mattheei at 38 8C in a humidified incubator with 5% CO2. Proliferation was measured by the incorporation of 3 H-thymidine (MP Biomedicals, ICN, Irvine, USA) during the last 24 h of culture (1 mCi/well). Separate wells for background proliferation (plain culture medium) and for proliferative capacity of the isolated PMBC (stimulation with 10 mg concanavalin A; Sigma– Aldrich, Steinheim, Germany) were included. ConAstimulated PBMC were harvested after 3 days (maximum proliferation for ConA stimulation, Goddeeris et al., 1986), whereas the antigen-induced proliferation and negative controls were cultured for 5 days. Cultures were harvested on glass fibre filters with a cell harvester (Skatron, Lier, Norway). Filters were placed in 2 ml Lumasave LSC cocktail (Lumac, Groningen, The Netherlands) and DNA-incorporated 3H-thymidine was measured in a b-scintillation counter (Tricarb 2550 TR, Packard Instrument Company, Meriden, USA). As PBMC from young calves quite often show high spontaneous proliferation in plain medium, antigenspecific proliferation of PBMC was determined in relation to their proliferative capacity to the mitogen ConA instead of the more conventional stimulation index (cpm sample/cpm medium): 100 (cpmsample cpmmedium)/cpmConA cpmmedium) with cpm being counts per minute.
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2.6. Statistical analyses Proliferation levels were compared between seropositive- and seronegative-born calves by the nonparametric Mann–Whitney test. The correlation between antibody titers and proliferation levels was determined using the Spearman Rank correlation coefficient. 3. Results 3.1. Dams All 44 cows were confirmed infected with S. mattheei on either one or both of the faecal examinations, with egg counts varying between 0 and 30 EPG with a mean value of 4. All dams had S. mattheeispecific antibodies at birth, with a mean DOD of 0.31 in serum, and 0.96 in colostrum. 3.2. Antibody responses of calves at birth and after intake of colostrum Twelve of the 30 calves were born with S. mattheeispecific serum antibodies (=seropositive-born). All seropositive-born calves were also seropositive for antibodies against E. coli. Mean DOD levels of serum IgG against SWAP mattheei for the seropositive-born calves on day 0 equalled 0.73. Mean DOD levels for E. coli equalled 0.68. After colostrum uptake, IgG levels against SWAP mattheei were detected in all calves (seropositive- as well as seronegative-born), with a mean DOD level of 0.66. 3.3. Antigen preparation and control Antigen-specific and non-specific T-cell stimulatory capacity of the antigen preparations were analysed by in vitro stimulation of PBMC from known immune
Table 1 Specific T-cell proliferation of two positive control cows and six negative control calves Control animals Positive controls
Negative controls
Cow no.
cpm A
cpm C
cpm m
Calf no.
cpm A
cpm C
cpm m
1 2
125,343 181,052
143,702 123,468
2965 1803
1 2 3 4 5 6
18,837 3,379 14,640 3,880 2,794 6,255
167,060 115,954 184,222 156,102 191,596 195,271
51,916 40,088 54,392 423 297 1,118
cpm A: cpm obtained after stimulation with 90 mg antigen; cpm C: stimulation with Con A; cpm m: stimulation with medium.
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Fig. 1. Specific T-cell proliferation of calves born to Schistosoma mattheei-infected cows. The proliferation induced by 90 mg of antigen is shown as the percentage of the proliferation induced with ConA. (A) Proliferation induced in PBMC from known immune and naı¨ve animals. (B) Proliferative response of PBMC from seropositive- (seropos) and seronegative-born (seroneg) calves from S. mattheei-infected cows.
and known naı¨ve animals, respectively. The infected control dams showed a strong proliferative response whereas the negative control calves did not (Table 1 and Fig. 1A), indicating that the necessary T-cell antigens were present in the antigen preparation and that no aspecific mitogenic responses were induced. Therefore, SWAP mattheei could be used for assaying S. mattheei-specific T-cell responses in the calves. The positive proliferative response of the infected dams was in accordance with their high serum antibody levels for S. mattheei. As expected for young calves, autologous background proliferation in the negative control calves was high (range of 297– 54392 cpm). 3.4. S. mattheei-specific proliferation The results of the proliferation tests of the calves from infected dams are presented in Table 2 and Fig. 1B. Twenty-six of the 30 calves (87%) showed antigenspecific proliferation. All 12 seropositive-born calves and 14 of the 18 seronegative-born animals (71%) displayed S. mattheei-specific proliferative responses. There were no significant differences in proliferation levels between the seropositive- and seronegative-born calves (P = 0.53). Compared to the infected control dams, proliferation of the calves was in most cases equally strong. There was no correlation between serum
antibody titers and proliferation levels (correlation coefficient of 0.213, P = 0.53). 4. Discussion In previous studies Gabrie¨l et al. (2002) indicated a transplacental transfer of circulating schistosome anodic antigen through the epithelio-chorial placenta of cows, most probably through lesions in the placenta, as well as a transfer of circulating schistosome antigen via the colostrum. These transfers would imply neonatal contact with schistosome antigens. In this study, we were able to demonstrate that 87% of the calves born to S. mattheei-infected mothers, but kept under S. mattheei-free conditions, were able to respond in vitro to schistosome antigens as strongly as infected dams, and consequently must have been primed as foeti during gestation or as neonates after colostrum uptake of mattheei antigens. The latter possibility is unlikely however, as Gabrie¨l et al. (2005b) could not observe protective differences between seonegative-born – but seropositive after colostrum uptake from their infected dams – calves and calves from non-infected animals. This is the first time that a priming of neonatal calves for schistosome antigens has been demonstrated. In mice and man, intra-uterine priming to schistosome antigen and subsequent tolerance or sensitization has been described (Hang et al., 1974; Camus et al., 1976;
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Table 2 Specific T-cell proliferation of calves born to S. mattheei-infected mothers Proliferation positive
Proliferation negative
Calf no.
cpm A
cpm C
cpmm
Seropositive-born 1 2 3 4 5 6 7 8 9 10 11 12
32,541 104,994 41,539 109,213 68,519 93,815 38,302 118,202 113,239 51,718 49,907 4,705
98,399 98,399 81,036 246,359 162,969 285,745 77,847 195,662 133,376 8,481 101,702 86,805
12,787 2,421 22,459 26,979 26,720 5,455 20,396 9,205 5,551 9,050 1,367 991
Calf no.
cpm A
cpm C
cpm m
131,801 166,809 116,559 134,224
35,498 74,458 921 171,586
No. of calves 12 Seronegative-born 13 14 15 16 17 18 19 20 21 22 23 24 25 26
142,474 52,435 60,178 39,293 148,410 70,458 18,107 102,073 144,527 134,972 15,304 89,067 39,844 60,671
0 128,685 63,480 41,639 90,490 163,457 213,689 99,088 94,346 150,044 320,096 159,190 323,103 242,243 26,722
19,421 3,761 5,181 11,517 27,883 10,582 726 39,924 62,104 4,387 849 65,651 18,849 10,693
27 28 29 30
14,078 38,708 976 128,900
No. of calves 14
4
cpm A: cpm obtained after stimulation with 90 mg antigen; cpm C: stimulation with Con A; cpm m: stimulation with medium.
Tachon and Borojevic, 1978; Novato-Silva et al., 1992; Malhotra et al., 1997). However, man and mouse have haemochorial placentas (three maternal layers disappeared) as opposed to cattle, swine and horse where the epithelio-chorial placenta (three maternal layers present) constitutes a strong barrier between the blood circulations of mother and foetus. This prenatal priming has important consequences for subsequent immunity development and protection of the newborn in endemic areas. Indeed, Gabrie¨l et al. (2004) observed that seropositive-born calves from infected dams, suspected of intra-uterine priming, had lower faecal egg counts and lower schistosome circulating antigen levels in their serum after natural S. mattheei challenge around the age of 5 months, indicating a protective memory. As already mentioned above, this protective effect appeared to be induced
prenatally and not postnatally as seronegative-born calves becoming seropositive after colostrum (antigen and antibody) uptake were not protected (no significant differences in faecal or tissue egg counts, nor in worm counts between calves born to infected and non-infected mothers) when challenged at the age of 7 months (Gabrie¨l et al., 2005b). S. mattheei-specific antibodies in the newborn colostrum-free calves are most probably from maternal origin through placental lesions, although minor foetal origin might not be excluded as has been addressed before (Gabrie¨l et al., 2005a). Western blots on E. coli demonstrated similar recognition patterns of the sera of dams and their newborn seropositive calves. As foetal origin of anti-E. coli antibodies would imply the occurrence of a septicemia in the foetus with resulting abortion, it was assumed that the detected antibodies
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were not of foetal but maternal origin. Moreover, if these antibodies are of maternal origin, they seem not to block priming of the foetus to S. mattheei antigen, as all seropositive-born calves demonstrated antigen-specific proliferation in vitro. This is in agreement with Siegrist et al. (1998) who described that introduction of antigens into a host with high levels of maternal antibodies leads to a failure of antibody induction, without however reducing the antigen-specific T-cell proliferative response. Lesions in the placenta which allow a transfer of antigens can be caused by schistosome eggs, as demonstrated by Gabrie¨l et al. (2005a), but might also be inflicted by other placenta-migrating pathogens such as Brucella (Ghirotti et al., 1991), Leptospira (Jerrett et al., 1984) or Neospora (Bergeron et al., 2001). These pathogens are primarily associated with abortions but can, through damage of the placenta, cause a transfer of antigens leading to foetal contact and in utero priming. One can presume that antigens from several other pathogens might also cross the damaged placenta and consequently prime the neonatal against significantly more pathogens (antigens) than was originally thought. In conclusion, this study demonstrates for the first time that the majority of calves born to S. mattheeiinfected dams are primed against S. mattheei as evidenced by in vitro proliferation of their lymphocytes upon antigen-specific stimulation. These results query the impermeability of the ruminant epithelio-chorial placenta and might explain previous data where seropositive-born calves showed evidence of protection against natural S. mattheei challenge. Acknowledgements This work was supported by the Flemish Inter University Council (VLIR-UNZA IUC Programme Zambia-Belgium). We would like to thank Mr. V. Mkanjo and the workers from the farm for their cooperation in the study, the excellent technical assistance of the technicians of the Department of Clinical Studies, School of Veterinary Medicine, Zambia, and the School of Veterinary Medicine, University of Zambia. References Bergeron, N., Girard, C., Pare, J., Fecteau, G., Robinson, J., Baillargeon, P., 2001. Rare detection of Neospora caninum in placentas from seropositive dams giving birth to full-term calves. J. Vet. Diagn. Invest. 13, 173–175. Camus, D., Carlier, Y., Bina, J.C., Borojevic, R., Prata, A., Capron, A., 1976. Sensitization to schistosoma mansoni antigen in uninfected children born to infected mothers. J. Infect. Dis. 134, 405–408.
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