Vol. 60, No. 8

INFECTION AND IMMUNITY, Aug. 1992, P. 3442-3445

0019-9567/92/083442-04$02.00/0 Copyright ©) 1992, American Society for Microbiology

Cross-Reactivity of Polyclonal Serum Antibodies Generated against Cryptosporidium parvum Oocysts L. M. ORTEGA

MORA,t* J. M. TRONCOSO, F.

A.

ROJO-VAZQUEZ,t AND M. GOMEZ-BAUTISTA

Departamento de Patologia Animal I (Sanidad Animal), Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain Received 13 November 1991/Accepted 30 April 1992

Polyclonal antibodies raised against Cryptosporidium parvum oocysts were found to cross-react with Eimeria oocyst antigens in an indirect immunofluorescence assay, and sera from Eimeria spp.-infected lambs reacted with some antigens from sonicated C. partum oocysts (between 29 to 30 and 66 to 69 kDa) by Western blot (immunoblot). No cross-reaction was observed with cystozoites of Toxoplasma and Sarcocystis spp. These results show the existence of epitopes common to C. parvum and various Eimeria spp. spp.

Coccidia of the

genus

Cryptosponidium

are

widespread

protozoan parasites that infect many species of vertebrates. In neonatal ruminants and immunocompromised persons,

Cryptosporidium parvum causes a severe, sometimes lifethreatening diarrhea. The diagnosis of cryptosporidiosis in these species is based on detection of oocysts in the feces, most recently by utilizing enzyme-linked immunosorbent assays (ELISAs) designed to detect C. parvum antigens (1, 19, 28) or serum antibodies against the parasite by indirect immunofluorescence assay or ELISA with C. parvum oocysts and sporozoites as the antigen (6, 14, 17, 29). The presence of Cryptosporidium species in the environment is determined by analysis of concentrated samples by using antioocyst monoclonal antibody-based techniques and then epifluorescence microscopic examination (20). Recently, several researchers have reported the presence of similar antigens among stages, species (7, 23), and genera (16) of the suborder Eimeriina. However, little information is available concerning stage-specific and species-specific C. parvum antigens (2, 3, 25, 26), and the existence of common epitopes in C. parvum and other Eimeriina has not been previously reported. The present study describes the development of polyclonal antibodies to C. parvum oocysts and examines the cross-reactivity of these antibodies to different stages of sheep Eimeriina, specifically oocysts of some Eimeria spp. (E. crandallis, E. faurei, E. intricata, E. ovina, and E. parva) and cystozoites of Toxoplasma gondii and Sarcocystis gigantea. C. parvum antigens recognized by sera from lambs naturally infected by Eimeria spp. are also described. Parasites. C. parvum oocysts were obtained from feces of experimentally infected lambs (VPN-1 isolate) and purified as previously described (18). Eimeria spp. oocysts (E. crandallis, E. faurei, E. ovina, E. parva, and E. inticata) were collected from naturally infected lambs and cleaned by sieving and flotation of fecal samples in saturated NaCl solution. Cystozoites of S. gigantea were mechanically isolated (22) from macroscopically visible cysts in the esophagus of a naturally infected sheep. Zoites of T. gondii were obtained from bioMerieux (Toxo-Spot IF). Anti-C. parvum polyclonal antibodies. Polyclonal antibodCorresponding author. t Present address: Departamento de Patologia Animal (Sanidad Animal), Facultad de Veterinaria, Universidad de Le6n, 24007 *

Le6n, Spain.

ies against C. parvum were raised in rabbits and lambs. Two 3-month-old female New Zealand x California rabbits were immunized against C. parvum oocysts utilizing a modification of the Vaitukaitis method (30). Each animal was intradermally inoculated in several parts of the shoulder and back with 2 x 107 oocysts resuspended in 1 ml of Freund's complete adjuvant. Blood samples were taken before and at 6 and 12 weeks after inoculation. Coprological examination of samples concentrated by flotation in saturated NaCl solution were conducted weekly and showed that the animals were free of Eimeria infection. Two female Castellana x Manchega lambs were subcutaneously inoculated with 2 107 C. parvum oocysts resuspended in Freund's complete adjuvant at 4 weeks, and at 8 weeks, each animal was injected intramuscularly with a similar quantity of oocysts resuspended in Freund's incomplete adjuvant. The lambs were bled before the first and second inoculations and 4 and 8 weeks postimmunization. Serum samples containing antibodies to Eimeria spp. were obtained from two naturally infected lambs. Coprological analyses by the Heine (9) and modified Ziehl-Neelsen techniques in either concentrated or unconcentrated samples (8, 10) were done daily from birth to the end of the first month of life and weekly thereafter, which showed these animals to be C. parvum free. However, analysis of fecal samples concentrated by flotation in saturated NaCl solution showed that these animals suffered a natural infection with Eimeria spp. (E. crandallis, E. faurei, and E. ovina) starting at 4 weeks of age. All sera were stored at -20°C. Indirect immunofluorescence assay. The indirect immunofluorescence assay utilized was based on the method described by Johnson (13). Air-dried, acetone-fixed parasites were exposed to either rabbit (1/200) or lamb (1/100) sera and subsequently incubated with either fluorescein isothiocyanate-conjugated rabbit anti-sheep immunoglobulin G (IgG) (Nordic) or goat anti-rabbit IgG (Nordic) at 1/60 and 1/100 dilutions, respectively. Fluorescence reactivity patterns exhibited by the rabbit and lamb immune sera exposed to the C. parvum oocysts consisted of surface and occasionally internal structure fluorescence (Fig. la). Surprisingly, similar patterns and intensities were observed when these sera were tested with the different oocysts of Eimena spp. (Fig. lb). However, neither fluorescence was observed when lamb and rabbit preimmune sera were tested with C. parvum or Eimeria x

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VOL. 60, 1992

FIG. 1. lmmunolluores4 weeks postimmunization).

'.

3443

parvum oocysts (a) and Eimeria spp. oocysts (b) incubated with anti-C. parvum rabbit sera (10

oocysts or when the immune sera were tested with Sarcocystis and Toxoplasma zoites. ELISA and Western blot (immunoblot) assays. Approximately 109 purified oocysts were pelleted and resuspended in 1 ml of 10 mM Tris hydrochloride containing 2 mM of the enzyme inhibitor phenylmethylsulfonyl fluoride. The oocysts were disrupted by ultrasonic treatment (Branson Sonifier 450) in an ice bath, and the material was centrifuged at 10,000 x g for 20 min at 4°C. Finally, the protein content was determined (21), and the supernatant was aliquoted and cryopreserved at -80°C. Optimum ELISA conditions were determined in a checkerboard manner as recommended by Catty and Raykundalia (5). C. parvum antigens were used at a protein concentration of 0.5 ,ug per well. Sera were diluted at 1/100 (rabbit) or 1/40 (lamb), and peroxidase conjugates of either rabbit anti-sheep IgG (1/600) (Bio-Rad) or goat antirabbit IgG (1/3,000) (Bio-Rad) were used. The reaction was developed with ABTS [2,2'-azino-di-(3-ethylbenzothiazatine sulfonic acid)] as the substrate, and after 30 min, optical densities (OD) were read at 405 nm in an EAR 400 ELISA reader. For the Western blot studies, C. parvum soluble proteins (=2 mg/ml) were resuspended in an equal volume of sample buffer containing 2% sodium dodecyl sulfate and 4% mercaptoethanol, boiled for 5 min, and electrophoresed in discontinuous 12.5% polyacrylamide gels with Laemmli buffers (15). Low-molecular-weight markers (Pharmacia) were coelectrophoresed for comparative purposes. Proteins were subsequently transferred electrophoretically to nitrocellulose (27), which in turn was blocked (12), and individual strips were exposed to the samples. Antibody binding was detected utilizing a peroxidase conjugate of either goat anti-rabbit IgG (Bio-Rad) diluted to 1/300 or rabbit antisheep IgG (Bio-Rad) diluted to 1/60 and developed in a solution containing 4-chloro-1-naphthol as the substrate. ELISA specific antibody levels in the rabbit sera rose from 0.264 + 0.027 OD unit before immunization to 1.037 + 0.025 OD units at 6 weeks and 1.174 + 0.045 OD units at 12 weeks postimmunization. A similar increase was observed in the IgG levels of 12-week-old immunized lambs (1.119 + 0.032 OD units) compared with preimmunization levels (0.276 ± 0.004 OD unit). Figure 2 (lanes B and D) shows the antigens from sonicated C. parvum oocysts recognized by rabbit and lamb immune sera. No proteins were recognized before immunization (Fig. 2, lanes A and C), but a strong reaction

was observed in sera from the immunized animals, showing seroconversion specificity. Rabbit anti-oocyst and -sporozoite antibodies recognized at least six antigens with molecular masses of approximately 16 to 20, 44, 52, and 66 to 69 kDa and some which surpassed 94 kDa. Lamb immune sera recognized proteins of approximately 15, 17, 20, 23, 29 to 30, 33, 34, 36, 37, 40 to 42, 44, 48, 52, 58, 67 to 69, 77, and 82 kDa as well as others with molecular masses higher than 94 kDa. Figure 2, lane E, shows the antigens from sonicated C.

A B

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C D

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43-

i.

94-

6 7-WI

a ..'

43-

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3030-

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2014-

FIG. 2. Western blot of C. parvum antigens probed with sera from preimmunized rabbits (lane A), immunized rabbits (10 weeks postimmunization) (lane B), preimmunized lambs (lane C), immunized lambs (4 weeks after the second inoculation) (lane D), and lambs infected with Eimena spp. (lane E). Molecular size markers are represented in kilodaltons.

3444

NOTES

parvum oocysts recognized by serum antibodies from lambs infected with Eimeria spp. Cross-reactivity found by the indirect immunofluorescence assay was confirmed by Western blot assays, and some C. parvum proteins were recognized by sera containing antibodies from lambs suffering natural infections from Eimeria spp. The C. parvum antigens recognized by both anti-C. parvum and anti-Eimena spp. antibodies had approximate molecular masses of 29 to 30, 34, 37, 40, 48, 52, 58, and 66 to 69 kDa. Anti-Eimenia spp. antibodies were not able to recognize proteins of low (less than 25 kDa) or high (more than 94 kDa) molecular mass. Our studies suggest that C. parvum oocysts share common antigens with several ovine Eimena spp. These results also confirm the lack of significant cross-reactivity of C. parvum with Toxoplasma or Sarcocystis species. Similar results have been obtained by Campbell and Current (4). Diagnosis of C. parvum infection in humans and animals has been made by using sonicated, detergent-treated, or whole oocysts or sporozoites used as antigens to detect anti-C. parvum antibodies and by using polyclonal anti-C. parvum oocyst serum used in an ELISA designed to detect C. parvum oocysts in feces (28). The cross-reactivity detected in our experiments implies that care should be taken when using these methods of diagnosis because of the high prevalence of infections caused by Eimeria spp. in domestic ruminants. Although our results are preliminary, the immunoblot data with sera from C. parvum- and Eimena-infected lambs shows the existence of both specific and shared C. parvum oocyst and sporozoite antigens. Peptides with molecular sizes similar to those reported here have proved to be important targets in the immune response to cryptosporidium in humans and animals (11, 24, 26). The 15-kDa protein reacted strongly with both fecal IgA and serum antibodies in the experimentally infected lambs (11) and was among the antigens recognized by hyperimmune bovine colostrum (24), which has been shown to be a possible target for immunotherapy in cryptosporidiosis (26). In summary, the results of the present study support the existence of common antigens among different genera of Eimenina. This research was supported by a grant from the Spanish Ministry of Education and Science, CYCIT (GAN-89/0332). REFERENCES 1. Anusz, K. Z., P. H. Mason, M. W. Riggs, and L. E. Perryman. 1990. Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immuno-

sorbent assay. J. Clin. Microbiol. 28:2770-2774. 2. Arrowood, M. J., J. R. Mead, J. L. Mahrt, and C. R. Sterling. 1989. Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosponidiumparvum infections in neonatal mice. Infect. Immun. 57:2283-2288. 3. Bjorneby, J. M., M. W. Riggs, and L. E. Perryman. 1990. Cryptosporidium parvum merozoites share neutralization-sensitive epitopes with sporozoites. J. Immunol. 145:298-304. 4. Campbell, P. N., and W. L. Current. 1983. Demonstration of serum antibodies to Cryptosponidium sp. in normal and immunodeficient humans with confirmed infections. J. Clin. Microbiol. 18:165-169. 5. Catty, D., and C. Raykundalia. 1989. ELISA and related enzyme immunoassays, p. 97-152. In D. Catty (ed.), Antibodies, a

INFECT. IMMUN.

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VOL. 60, 1992 27. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 28. Ungar, B. L. P. 1990. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens. J. Clin. Microbiol. 28:2491-2495.

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29. Ungar, B. L. P., R. Soave, R. Fayer, and T. E. Nash. 1986. Enzyme immunoassay detection of immunoglobulin M and G antibodies to Cryptosporidium in immunocompetent and immunocompromised persons. J. Infect. Dis. 153:570-578. 30. Vaitukaitis, J. L. 1981. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods Enzymol. 73:46-52.

Cross-reactivity of polyclonal serum antibodies generated against Cryptosporidium parvum oocysts.

Polyclonal antibodies raised against Cryptosporidium parvum oocysts were found to cross-react with Eimeria spp. oocyst antigens in an indirect immunof...
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