CA1261285A - Antigens and monoclonal antibodies reactive against sporozoites of eimeria spp - Google Patents

Abstract

ANTIGENS AND MONOCLONAL ANTIBODIES REACTIVE
AGAINST SPOROZOITES OF Eimeria spp.

ABSTRACT OF THE DISCLOSURE
Monoclonal antibodies against sporozoites of the Eimeria spp. are obtained by use of hybridoma technology.
Specific sporozoite antigens for use as vaccines in the prevention and treatment of coccidiosis and hybridoma cultures producing monoclonal antibodies are described.

Description

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This application is a divisional of application No.
461,242 filed on August 17th, 1984.
The invention herein described relates to monoclonal antibodies which react specifically against sporozoites of the parasite Eimeria tenella. Hybridoma cultures producing antibodies against E. tenella are described. Such antibodies are obtained by means of hybridoma technology. Sporozoites antigens are identi-fied and characterized. These antigens, along with certain monoclonal antibodies are effective for the prevention and treat-ment of coccidiosis. The antigens of the invention are useful as vaccines against coccidio~is.
By way of background, coccidiosis is a disease of animals caused by a variety of protozoan parasites. Avian coccidiosis is a devastating disease o~ poultry caused by a variety of species of the genus Eimeria. This disease has a complicated lie cycle consisting of both asexual and sexual stages. Chickens are initially infected with the dlsease after ingestion of free-living oocysts which are generally associated with fecal material.
Oocysts develop into invasive asexual sporozoites in the chicken's digestive tract. The sporozoite~ infect epithelial cells and develop into multinucleate structures known as schizonts. Each schizont matures and eventually liberates multiple invasive asexual structures known as merozoites. These merozoites leave the infected cell and reinvade other ~126;~Z8~i epithelial cells. The multiple invasive asexual stages involving sporozoites and merozoites account for much of the pathology of coccidiosis. The sexual cycle of coccidiosis is initiated when merozoites differentiate into gametoc~tes. Fertilization occurs and the fertilization products known as oocysts are released in the feces. Thus the parasite's life cycle is completed.
In chickens, the life cycle of Eimeria tenella, a repre-sentative species, is completed in about 7 to 9 days.
Due to the tremendous economic losses in~licted on the poultry industry by Eimeria species, a vaccine against the parasite is highly desirable. However, due to the complexity of the life cycle of the parasite and the variability of the quantity of antigens present in each stage, it has been observed that deactivated or lS killed parasites have not generated consistent immunity in the past. One solution to this problem is to isolate and characterize particular antigens from the parasite and administer them in a sufficient amount to serve as an immunizing agent. Preferably such antigens will offer protection against infection by all important species.
It is known that various species of Eimeria, as well as different stages in the life cycle of the same species, have both common and specific antigens [Cerna, Z., Folia Parasitologica ~Prague) 17:135-140 ~1970); Davis et al., Immunol 34:879-888 ~1978); Rose, M.E., Immunol.

2:112-122 (1959); Rose et al., Immunol. 5:79-92 (1962);
and Tanielian et al., Acta Parasitol. Yugosl. 7:79-84 ~1976)]. It is also known that development of immunity to Eimeria is species specific and in some species of domestic fowl there is significant strain-specific immunity [Jef~ers, T.K.; In Long, P.L. et al. (eds.), Avian Coccidi~sis, pp. 57-125, Proc. 13th Poultry Sci.
Symp. (1978); Joyner, L.P., Parasitol. 59:725-732 (1969);

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Long, P.L., Parasitol. 69:337-347 ~1974); and Long et al., Parasitol. 79:451-457 (1979)]. Currently immunogens of Eimeria species capable of stimulating protective immunity in avian or mammalian hosts have not yet been isolated or identified. Such Eimeria immunogens,will likely provide successful immunization against coccidiosis.
, The development of lymphocyte hybridoma technology provides a tool for producing relatively large amounts of specific antibodies against various antigens of Elmeria. By fusing specific antibody-producing cells (spleen cells) with cells of a myeloma tumor, it is possible to produce hybridoma cells that secrete monoclonal antibodies directed specifically against the original sensitizing antigen. [Kohler & Milstein, Nature (London) 256:495-497 (1975)]. If monoclonal antibodies against the parasite are obtained, it may be possible to provide such anitbodies to infected or susceptible fowl and to thus provide the host organism with a measure of passive immunity. Once such hybridoma cultures producing monoclonal antibodies are obtained, it is possible by vario~s procedures to utilize such anti-bodies to isolate and identify specific antigens which could in turn be utilized as a vaccine to provide host organisms with a system of active immunity. Various patents concerning hybridoma cultures and monoclanal antibodies are knswn (i.e., U.S. Pat. Nos. 4,172,124;
4,196,265; 4,271,145; 4,361,549; 4,631,550; 4,364,932;
4,364,g33; 4,364,934; 4,364,935; 4,364,936; 4,381,292;
and 4,381,295).
In light of the- foregoing discussion of the economic effects of coccidiosis in the area of animal husbandry and more specifically in the poultry industry ~26128S

control of the protozoan parasite Eimeria is highly desirable.
Accordingly, the invention of the divisional application seeks to provide new and useful monoclonal antibodies obtained against sporozoites of the genus Eimeria. The invention of the parent application seeks to isolate and identify specific antigens of E.
tenella useful as a vaccine for the control of avian coccidiosis.
A preparation of E. tenella sporozoites is used to immunize mice in order to eventually generate monoclonal antibodies ~ollowing the method of K~hler and Milstein as described below.
The monoclonal antibodies are used to identify antigens of the parasite. The antigens which elicit monoclonal antibodies that react with sporozoites, and show neutralization of parasite growth, are considered protective antigens. The protective antigens that occur in various species of Eimeria are regarded as potential candidates for the development of a vaccine against avian coccid-iosis.
Soluble antigens are obtained from sporozoites of E.
tenella. These soluble antigens are separated electrophoretic-ally by molecular weight and those which specifically react with monoclonal antibodies of the present invention are identified.
U~ing appropriate standards, the reactive antigens are then characterized on the basis o molecular weight.
In order to evaluate the ability of the monoclonal anti-bodies to effectively neutralize the infective capability of coccidial sporozoites, chickens are exposed to sporozoites of E.
tenella previously treated with various monoclonal antibodies of the invention. This in vivo experimental system demonstrates the protectiVe ¢apabilitie~ of selected monoclonal antibodies.

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Chickens receiving injections of solubilized sporozoite antigens including those identified by the corresponding monoclonal antibodies were found to be protected against oral challenge. This immunization procecure demonstrates the vaccine potential of spor-ozoite antigens which can be recognized by monoclonal antibodies.
According to the invention of the parent application there is provded a method for preparing an antigenic, immunogenic, proteinaceous vaccine, which is soluble in detergent-containing buffer, containing at least one antigen of Eimeria tenella sporoz-oites, specifically reactive with anti-sporozoite monoclonal anti-bodies seareted by hybridoma clones s5E5 (ATCC No. HB8~03) and having molecular weights of 110 ~ 16 kd and 130 + 20 kd; specific-ally reactive with anti-sporozoite monoclonal antibodies ~ecreted by hybridoma clone s4E2 and having molecular weights of 110 ~ 16 kd and liO ~ 30 kd; specifically reactive with anti-sporozoite monoclonal antibodies secreted by hybridoma clone slC4 (ATCC No.
HB8333), and having molecular weights of 66 i 9 kd; 55 i 8 kd, 20 - 30 kd, 18 ~ 3 kd, 15 ~ 2 kd; ~pecifically reactive with anti-sporozo$te monoclonal antibodies secreted by hybridoma clone s2G8 ~ATCC No. HB8405) and having molecular weight of 55 ' 8 kd;
specifically reactive with anti-sporozoite monoclonal antibodies ~ecreted by hybridoma clone s5B9 (ATCC No. HB8402) and having molecular weight of 55 ~ 8 kd; speciically reactive with anti-sporozoite monoclonal antibodie~ secreted by hybridoma clone SlA
~ATCC No. HB8404) and having molecular weight of 54 - 8 kd;
specifically reactive with anti-sporozoite monoclonal antibodie~
secreted by hybridoma clone s3Cll and having molecular weight of 50 ~ 7 kd; specifically reactive with anti-sporozoite monoclonal ' ", . ' - ;

~26~Z85 antibodi.es secreted by hybridoma clone ~3D3 ~TCC No,HB8331), and haviny a molecular wei~ht of 29 i 4 kd; or specifically reactive with anti-sporozoites monoclonal antibodies secreted by hybridoma clone slE4 (ATCC No. Hs8332)~ and having molecular.weights of 58 * 9 kd and 130 ~ 20 kd; which method comprises the steps of:
(a) extracting, and solubilizing sporozoite antigens of E. tenella;
(b) separating the solubilized material by suitable isolation and purification methods to obtain purified antigen.
According to another aspect of the invention of the parent application there i8 provided an antigenic, immunogenic, proteinaceous vaccine containing at leaæt one antigen of Eimeria tenella sporozoite~ when prepared by the above~referenced process or an obvious chemical equivalent thereof.
According to the invention of the divisional application there is provided a method of producing monoclonal antibodies which, comprises forming by fusion hybridoma cells from a mouse myeloma line and spleen cells from a mouse previously immunized with _imeria~tenella sporozoites and recovering the produced antibodies which ~ a) reacts ~pecifically with antigens o~ Eimeria ~e~. sporoz-oite~ and (b) reacts specifically with antigens of Bi~er-ia tenella having a molecular weight of approximately 13 to 150 kd.
In a pre~erred embodiment the hybridomas are formed by the fuæion of P.3 x 63. Ag 8. 653 my~loma cellæ and spleen cells from BALB/c mice previously immunized with E. ten'ella sporozoites. In a part-icularly pre~erred embodiment the hybridoma used is a clone chosen ', : - 5a -~Z612~

from the group consisting of,: the hybridoma designated clone number sSE5 and de~o~ited as ATCC No. HB8403; the hybridoma designated clone number s4E2; the hybridoma designated clone No. slC4 and deposited as ATCC No. HB8333; the hybridoma designat-ed clone No. s2G8 and deposited as ATCC No. HB8405; the hybridoma designated clone No. s5~9 and deposited as ATCC No. HB8402; the hybridoma designated clone No. sl~ and deposited as ATCC No.
~B8404; the hybridoma desiynated clone No. s3Cll; the hybridoma designated clone No. s3D3 and deposited as ATCC No. HB8331; and the hybridoma designated clone No. slE4 and deposited as ATCC
No. HB8332.
A preferred method of the invention of the divisional application comprises preparing a monoclonal antibody, which reacts with antigens of Eimeria tenella sporozoites, which comprises the steps of:
(a) immunizing mice with ~.'tenel'la sporozoites;
(b) removing the spleen0 from said mice and making a suspen-sion of the spleen cells;
(c) fusing said spleen cells with mouse myeloma cells in the presence of a fusion promoter;
(d) 'diluting and culturing the fu8ed cells ln separate wells in a medium which will not support the growth of unfused myeloma cells;
(,e) evaluating the spuernatant of each well containing a hybridoma for the presence of antibody reactive with E. tenella 0porozoites ~

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(f) selecting and cloning a hybridoma producing antibody reactive with _. tenella sporozoites; and (g) recovering the antibody from the supernatant or ascitic product of said clones.
Another preferred method of the invention of the division-al app]ication comprises preparing monoclonal antibodies which react with antigens of Eimeria spp. sporozoites which comprises either:
(a) culturing clone No. s5E5 (ATCC No. HB8403), s4E2, slC4 (~TCC No. HB8333), s2G8(ATCC No. HB8405), s5B9 (ATCC No. HB8403), slA (ATCC No. HB8404), s3Cll, s3D3 (ATCC No. HB8331), or slE4 (ATCC No. HB8332), in a suitable medium and recovering the anti-body ~xom the ~upernatant of an above said hybridoma culture; or ~b) in;ecting into a mouse a hybridoma culture designated clone No. s5E5 (ATCC No. HB8403), ~4E2, slC4 (ATCC No. HB8333), s2~8 (ATCC No. HB8405), s5B9 (ATCC No. HB8402), slA (ATCC No.
HB840~), s3Cll, s3D3 ~ATCC No. HB8331), or slE4 (ATCC No. HB8332) and recovering said antibody from the ascites or serum of said mouse.
The following non-limiting Examples further serve to illustrate the inventions of the parent and divisional applications.

Construction of Hybridoma Lines Sporozoites of the organism Eimeria ~nella are obtained by excysting sporulated oocysts using established procedures [Doran et al., Proc. Helmintol. Soc. Wash. 34:59-65 (1967)]. A
preparation of E. tenella sporozoites thus obtained is used to - 5c -immunize eighteen-week-old female BALB/c mice by intraperitoneal in]ection. After determining that an immunized mouse is producing anti-sporozoite antibodies using an indirect immunofluorescence assay (IFA) technique known to the art, spleen cells are obtained from the mouse and fused with mouse myeloma cell line P3X63.
Ag8.653. The fusion process is carried out in the presence of 30 to 35% polyethylene glycol (950-1050). The method of generating hybridomas has been previously described (c.f., Kennett et al., Monoclonal Antibodies - Plenum Press: 365-371, 1980). Hybridoma fu~ion products are cultured in HAT medium [Littlefield, J.W., Science, 1~5:709-710 (1964)] containing Iscove's modified Dulbecco's medium (IMDM) with 20% fetal calf serum supplement.
Culture media are monitored for anti-sporozoite antibody product-ion by indirect immunofluorescence assay (IFA) using glutarald-ehyde fixed sporozoites of E. tenella as the antigen source. Of all the cultures tested, 33 wells were found positive by IFA.

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In order to ensure monoclonicity of the hybri-doma cultures, a limiting dilution procedure was em-ployed. Following exposure of E. tenella sporozoites to various monoclonal antibodies of the invention, three major IFA reactivity patterns on treated sporozoites are observed: (1) reaction on the entire surface of sporo-zoites; (2) surface reaction as patches on the sporozoites;

(3) internal reaction around the nuclear membranes of sporozoites. These reaction patterns are confirmed by ferritin labelling and transmission election microscopy (Speer et ?.1., J. of Protozoology in press). As assessed by IFA, hybridoma cultures of the invention generate identical antibodies after cloning. Clones are grown either 1n vitro or in BA~/c mice as peritoneal tumors and the ascites fluid contains antibodies in a concen-tration of up to approximately 10 mg/ml.

Preparation of Antigens Associated with E. tenella sporozoites .
Freshly excysted sporozoites of E. tenella are used for the antigen preparation. The outer membrane components o~ the sporozoites are extracted using deter-gents (i.e., 0.5% Nonidet P40, 0.5 to 2~ CHAPS, or 0.5%
to 1% Triton X-100) in 5 mM sodium phosphate buffer having a pH o 7.8. The buffer contains the following protease inhibitors: aprotinin (2 trypsin units/ml);
antipain ~25 ~g/ml); leupeptin (25 ~g/ml), phenyl methyl sulfonyl fluoride (4 mM) [Yoshida et al., J. Exp. Med., 1 :1225- 1236 (1981)1. The detergent solubilized material is centrifuged at 100,000 x g for 10 minutes to remove particulate matter. The clear supernatant contains soluble antigens associated with E. tenella sporozoites.

.
Antigen Characterization Soluble antigens of E. tenella sporozoites are separated by molecular weight using SDS polyacrylamide gel electrophoresis (PAGE) [Laemmli, U.K., Nature 227:
680-685 (1970)]. The SDS PAGE separated proteins are transferred electrophoretically onto nitrocellulose mem-branes using the Western blotting technique [Towbin et al., Proc., Natl. Acad. Sci. (USA) 76:4350-4354 (1979)].
The nitrocellulose filter is then reacted with either diluted ascites fluid or spent hybridoma culture fluid containing antibodies. Bound monoclonal antibodies are then detected by using a radioimmuno detection method involving 125I labelled anti-mouse IgG antibody (~ew England ~uclear). The unbound second antibody is removed by washing, and the nitrocellulose filters are then exposed with Kodak* X-ray film XAR-5.
Alternatively, specific antigen-monoclonal antibody complexes are identified by an ELISA technique using horseradish peroxidase coupled rabbit IgG antibody (Cappel Lab) against mouse immunoglobulin [Burnette et al., Anal. Biochem. 112:195-203 (1981)]. The Bio-Rad*
Immuno Blot Assay Kit is employed.
The apparent molecular weights of the reactive sporozoite antigens are determined by comparing the electrophoretic Rf values of the antigens with Rf values of known molecular weight compounds run as standards along with the antigens in the same system. The exper-imental molecular weight data of various antigens are presented in Table I.
* Trade Mark TABLE I
MOI~Ct~AR ~lEI~HT D~TI~J OF
V~RIOUS E. ~Fr.rA SPOROZOII~E ANTIOENS

Hybridoma l~noclonal Approximate ~lecular Culture Antikody Weiqht of Antiqen sSE5 s~ 110 + 16, 130 + 20 kd s4E2 s2 ilO + 16, 130 ~ 20 kd slC4 s3 66 + 9 kd, 55 + 8 kd, 20 - 30 kd, 18 + 3 kd, 15 + 2 kd s2G8 s4 55 + 8 kd s5B9 s5 55 + 8 kd slA s6 54 + 8 kd s3C11 s7 50 + 7 kd s3D3 . s8 29 + 4 kd slE4 s9 58 + 9 kd, 130 + 20 kd :~85 Neutralization of Sporozoites of E. tPn~
With Monoclonal Antibodies Usinq In ~i~Q Chicken Assay -An ln vlvo system is employed to evaluate the capability of monoclonal antibodies produced from hybri-doma lines of the invention to neutralize sporozoites of E. tenella. The caeca of the fowl are the sites of infec-tion by E. tenella and are accessable by surgery [Burns et al., Exp. Parasitol 8:515-526 (1959); I,awn et al., J.
Parasitol. 68:1117-1123 ~1982)]. The caeca of chickens are surgically exposed and infused with preparations of E. tenella sporozoites which have been previously treated ~ith monoclonal antibodies of the invention.
Freshly excysted sporozoites are incubated under sterile conditions with heat inactivated ascites fluid ontaining monoclonal antibodies derived from hybridoma lines of the invention. The incubation period is for 30 to 60 minutes at 25 to 37C. An incubation period of 37C for 60 minutes is preferred. Treated sporozoites are then introduced into 'he caeca of three-~eek-old chickens by surgical procedures. At the end of a five-day incubation period, the caeca of the infected chickens are observed for lesions. The five-day incuba tion period represents the most destructive stage of coccidiosis. Results of this experiment are presented in Table II. It is noted that monoclonal antibodies s3 and s8 both provided 60% total protectlon against infection by sporozoites of E. tenella.

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~:~ E t~ 0 I ~ ~ _ ~ I ~ ~ _ _ :~~ o I u, u~ a a I u~ a a a g gl ~1 12~12~5 Immunization with E. tenella s~orrzoite antig~ns Chickens at one week of age w~re immunized intraperitoneally with solubilized E. tenell~ sporoziOte antigens. Initial injections employed proteinaceOus material derived from 1.5 x 107 sporoziotes in Freund's complete adjuvant. Two boosters Eollo~ed at ten-day intervals each employing one-half the initial immunizing dose of material in Freund's incomplete adjuvant. Ten --days after the last booster, the chick~ns were challenged with 50,000 oocysts orally. Five days post-challen~e, caecal lesions were observed. Results on this experiment are presented in Table III. It is noc~d that soluble antigens render significant protection against oral oocyst challenge whereas normal chickens are not protected.

TABLE III

20 Treatment Percent protection (~/O o~ birds tested) .
None Partiai ComDlete Control 100 0 0 Immunized 0 33 66 The new-monoclonal antibodies, No. slC4, No.
s3D3, No. slE4, No. s5B9, No. s5E5, No. slA and No.
s2G8, isolated as described hereinabove, have been deposited with the American Type Culture Collection (ATCC) located in Rockville, Maryland and have been added to its permanent collection. No. slC4 has been assigned the number HB8333; No. s3D3 has the number HB8331, No. slE4 has been designated the number HB8332, No. s5B9 has been designated number HB8402, No. s5E5 has been designated number HB8403, No. slA has been desig-nated number HB8404 and No. s2G8 has been designated number HB8405. Access to the antibodies are available during ~he pendency of the present application to one de~ermined by ~he Commissioner o~ Patents and Trademarks 1261;~5 to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C..122, and all restrictions on the availability to the public of HB8331, HB8332, HB8333, HB8402, HB8403, HB8404 and HB8405 will be irrevocably removed upon the granting of a patent on 5 the present application.
No. slC4, HB8333, No. s3D3, HB8331 and No. slE4, HB8332 were deposited with the ATCC on August 11, 1983. The new monoclonal antibodies No. s5B9, HB8402, No. s5E5, EIB8403, No.
slA, HB8404 and No. s2G8, HB8405 were deposited with the ATCC
on November 1, 1983. -

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of producing monoclonal antibodies which comprises forming by fusion hybridoma cells derived from a mouse myeloma line and spleen cells from a mouse previously immunized with Eimeria tenella sporozoites and recovering the produced antibodies which (a) react specifically with antigens of Eimeria spp. sporozoites; and (b) react specifically with antigens of Eimeria tenella having a molecular weight of approximately 13 to 150 kd.

2. A method according to claim 1 wherein the hybridomas are formed by fusion of P3x63.Ag8.653 myeloma cells and spleen cells from BALB/c mice previously immunized with E. tenella sporozoites.

3. A method according to claim 2 wherein the hybridoma is a clone chosen from the group consisting of: the hybridoma designated clone number s5E5 and deposited as ATCC No. HB8403;
the hybridoma designated clone number s4E2; the hybridoma designated clone No. s1C4 and deposited as ATCC No. HB 8333; the hybridoma designated clone No. s2G8 and deposited as ATCC No.
HB8405; the hybridoma designated clone No. s5B9 and deposited as ATCC No. HB8402; the hybridoma designated clone No. s1A and deposited as ATCC No. HB8404; the hybridoma designated clone No.
s3C11; the hybridoma designated clone No. s3D3 and deposited as ATCC No. HB8331; and the hybridoma designated clone No. s1E4 and deposited as ATCC No. HB8332.

4. A method for preparing a monoclonal antibody, which reacts with antigens of Eimeria tenella sporozoites, which comprises the steps of:
(a) immunizing mice with E. tenella sporozoites;
(b) removing the spleens from said mice and making a suspension of the spleen cells;
(c) fusing said spleen cells with mouse myeloma cells in the presence of a fusion promoter;
(d) diluting and culturing the fused cells in separate wells in a medium which will not support the growth of unfused myeloma cells;
(e) evaluating the supernatant of each well containing a hybridoma for the presence of antibody reactive with E. tenella sporozoites;
(f) selecting and cloning a hybridoma producing anti-body reactive with E. tenella sporozoites; and (g) recovering the antibody from the supernatant or ascitic product of said clones.

5. A method of preparing monoclonal antibodies which react with antigens of Eimeria spp. sporozoites which comprises either:
(a) culturing clone No. s5E5 (ATCC No. HB8403), s4E2, s1C4 (ATCC No. HB8333), s2G8 (ATCC No. HB8405), s5B9 (ATCC No.
HB8403), s1A (ATCC No. HB8404), s3C11, s3D3 (ATCC No. HB8331), or s1E4 (ATCC No. HB8332), in a suitable medium and recovering the antibody from the supernatant of an above said hybridoma culture; or (b) injecting into a mouse a hybridoma culture designated clone No. s5E5 (ATCC No. HB8403), s4E2, s1C4 (ATCC
No. HB8333), s2G8 (ATCC No. HB8405), s5B9 (ATCC No. HB8402), s1A (ATCC No. HB8404), s3C11, s3D3 (ATCC No. HB8331), or s1E4 (ATCC No. HB8332) and recovering said antibody from the ascites or serum of said mouse.

6. A monoclonal antibody which (a) react specifically with antigens of Eimeria spp. sporozoites; and (b) react specifically with antigens of Eimeria tenella having a molecular weight of approximately 13 to 150 kd.

7. A monoclonal antibody according to claim 6 wherein said antibody is derived from a hybridoma formed by fusion of P3x63.Ag8.653 myeloma cells and spleen cells from BALB/c mice previously immunized with E. tenella sporozoites.

8. A monoclonal antibody according to claim 7 wherein the hybridoma is a clone chosen from the group consisting of: the hybridoma designated clone number s5E5 and deposited as ATCC No.
HB8403; the hybridoma designated clone number s4E2; the hybridoma designated clone No. s1C4 and deposited as ATCC No.
HB8333; the hybridoma designated clone No. s2G8 and deposited as ATCC No. HB8405; the hybridoma designated clone No. s5B9 and deposited as ATCC No. HB8402; the hybridoma designated clone No.
s1A and deposited as ATCC No. HB8404; the hybridoma designated clone No. s3C11; the hybridoma designated clone No. s3D3 and deposited as ATCC No. HB8331; and the hybridoma designated clone No. s1E4 and deposited as ATCC No. HB8332.

9. A monoclonal antibody when prepared by a process accord-ing to claim 4 or an obvious chemical equivalent thereof.

10. A monoclonal antibody which reacts with antigens of Eimeria spp. sporozoites, said monoclonal antibody being prepared by either:
(a) culturing clone No. s5E5 (ATCC No. HB8403), s4E2, s1C4 (ATCC No. HB8333), s2G8 (ATCC No. HB8405), s5B9 (ATCC No.
HB8403), s1A (ATCC No. HB8404), s3C11, s3D3 (ATCC No. HB8331), or s1E4 (ATCC No. HB8332), in a suitable medium and recovering the antibody from the supernatant of an above said hybridoma culture; or (b) injecting into a mouse a hybridoma culture designated clone No. s5E5 (ATCC No. HB8403), s4E2, s1C4 (ATCC
No. HB8333), s2G8 (ATCC No. HB8405), s5B9 (ATCC No. HB8402), s1A
(ATCC No. HB8404), s3C11, s3D3 (ATCC No. HB8331), or s1E4 (ATCC
No. HB8332) and recovering said antibdoy from the ascites or serum of said mouse or an obvious chemical equivalent thereof.