VIOLIN: Vaccine Investigation and Online Information Network

Eimeria tenella

Table of Contents

General Information
Vaccine Related Pathogen Genes
1. 3-1E (Protective antigen, Protective antigen)
2. Chain A, Eimeria Tenella Microneme Protein 3 Mar_b Domain (Protective antigen)
3. GX3262 (Protective antigen, Protective antigen)
4. MIC2 (Protective antigen, Protective antigen)
5. Rho (Protective antigen, Protective antigen)
6. TA4 (Protective antigen, Protective antigen)
Vaccine Information
References

I. General Information

1. NCBI Taxonomy ID:

2. Disease:

Hemorrhagic cecal coccidiosis

3. Introduction

Eimeria tenella is a species of Eimeria that causes hemorrhagic cecal coccidiosis in young poultry. Monoxeous life cycle with definitive (only) host as chickens. Extremely host-specific. Acquired via fecal contamination of food and water (oral fecal route). Goes thru endogenous merogony in crypts of lieberkuhr (intestinal ceca of chicken) and gametogony in epithelial cells of small intestines. Fusion of microgamete and macrogamete forms unsporulated zygote which is released with feces of chicken. Zygote sporulates after 1-5 days and becomes infective. Diagnosis is based on finding oocysts in feces. No effective treatment but can reduce rate of infection via prophylates, anti-coccidial drugs and vaccination of baby chicks (Wiki: Eimeria tenella).

4. Host Ranges and Animal Models

Chickens the only host of Eimeria tenella (Wiki: Eimeria tenella).

5. Host Protective Immunity

Protective immunity against Eimeria infections strongly depends on the cellular immune responses involving T cells, natural killer cells (NK) and macrophages (Wang et al., 2009).

II. Vaccine Related Pathogen Genes

1. 3-1E

Gene Name : 3-1E
Sequence Strain (Species/Organism) : Eimeria tenella
VO ID : VO_0011066
NCBI Protein GI : 126362934
Other Database IDs : CDD:320770
CDD:238085
Taxonomy ID : 5802
Gene Strand (Orientation) : ?
Protein Name : sporozoite antigen 3-1E
Protein pI : 3.96
Protein Weight : 17761.05
Protein Length : 237
Protein Note : Profilin binds actin monomers, membrane polyphosphoinositides such as PI(4,5)P2, and poly-L-proline. Profilin can inhibit actin polymerization into F-actin by binding to monomeric actin (G-actin) and terminal F-actin subunits, but - as a regulator of the...; cl00123

Protein Sequence : Show Sequence

>ABO10495.1 sporozoite antigen 3-1E, partial [Eimeria tenella]
MGEEADTQAWDTSVKEWLVDTGKVYAGGIASIADGCRLFGAAIDNGEDAWSQLVKTGYQIEVLQEDGSST
QEDCDEAETLRQAIVDGRAPNGVYIGGIKYKLAEVKRDFTYNDQNYDVAILGKNKGGGFLIKTPNDNVVI
ALYDEEKEQNKADALTTALAFAEYLYQGGF

Molecule Role : Protective antigen
Molecule Role Annotation : The Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-gamma) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and prol. After challenge the proIE-vaccinated chickens showed protective immunity as demonstrated by significantly reduced oocyst shedding (Xu et al., 2006).
Additional Molecule Role : Protective antigen
Related Vaccine(s): E. tenella DNA vaccine proIE

2. Chain A, Eimeria Tenella Microneme Protein 3 Mar_b Domain

Gene Name : Chain A, Eimeria Tenella Microneme Protein 3 Mar_b Domain
Sequence Strain (Species/Organism) : Eimeria tenella
NCBI Protein GI : 2LBO_A
Other Database IDs : CDD:287526
Taxonomy ID : 5802
Protein Name : Chain A, Eimeria Tenella Microneme Protein 3 Mar_b Domain
Protein pI : 4.68
Protein Weight : 13475.68
Protein Length : 167
Protein Note : helix 1

Protein Sequence : Show Sequence

>pdb|2LBO|A Chain A, Microneme protein 3
APTLQEALDRKCAQFGKESCNSGLASYCGAAIYARYDKGNSSQQTKEWRCYIEKELDFSKSGDGCVDDCG
SMTSCRGSVSGPSTSHLTRPSELQAVIDSDKSNYCTDKSEQQPSQATPPPEIP

Molecule Role : Protective antigen
Molecule Role Annotation : (Lai et al., 2011)

3. GX3262

Gene Name : GX3262
Sequence Strain (Species/Organism) : Eimeria tenella
VO ID : VO_0011067
NCBI Protein GI : 102304
Taxonomy ID : 5802
Gene Strand (Orientation) : ?
Protein Name : sporozoite antigen
Protein pI : 9.06
Protein Weight : 11721.58
Protein Length : 176

Protein Sequence : Show Sequence

>pir||A60111 sporozoite antigen - Eimeria tenella (fragment)
LLLLLLLLLQGAECLLRSSKLALEALLEGARVAATRGLLLVESSKDTVLRSIPHTQEKLAQAYSSFLRGY
QGAAAGRSLGYGAPAAAYGQQQQPSSYGAPPASSQQPSGFFW

Molecule Role : Protective antigen
Molecule Role Annotation : A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites. After challenge with an experimental E. tenella infection, the greatest degree of protection in immunized broiler chickens was seen after only a single immunization of 2-day-old birds with a live recombinant E. coli preparation (Miller et al., 1989).
Additional Molecule Role : Protective antigen
Related Vaccine(s): E. tenella GX3262 protein vaccine

4. MIC2

Gene Name : MIC2
Sequence Strain (Species/Organism) : Eimeria tenella
VO ID : VO_0011063
NCBI Protein GI : 225579627
Other Database IDs : CDD:115333
Taxonomy ID : 5802
Gene Strand (Orientation) : ?
Protein Name : microneme 2
Protein pI : 4.13
Protein Weight : 32481.77
Protein Length : 390
Protein Note : Microneme protein Etmic-2; pfam06670

Protein Sequence : Show Sequence

>ACN93990.1 microneme 2 [Eimeria tenella]
MARALSLVALGLLFSLPPSSAVRTRVPGEDSFSPESGVLSGTDAPERRPIVPGLVEGNCGRLTVRNGPSV
DETIKVTSAGWTKSERDFIVSLVADETRKVVQLRESEGASGASGPGPAPAEKPPSGQGSAEEAPKGEGGQ
EKPSVPLIAVRIHGSGGDKGESAPQSAVLLYGNDESEPTEVPLETAAGPTTPLMVLITQQNPKEVEVRVL
AWISTDATTGKGSWKENSVVVGSSLSGRDLTVNLGDCGPSSLRVYGSASADLVTVKEGMCEADDPELIAL
TRPHTSAASPLPAEEGDVAQDAQQSAGAQQEAEAQEVGEPQQEAAAAEQGSSAAESDTQQSS

Molecule Role : Protective antigen
Molecule Role Annotation : Birds vaccinated with Eimeria tenella microneme recombinant gene (EtMIC2) and encoded protein developed protective immunity against infection by E. tenella as assessed by significantly increased body weight gain and decreased fecal oocyst shedding compared with non-vaccinated controls. Vaccination with the EtMIC2 gene also led to protective immunity against infection by E. acervulina, but not E. maxima (Ding et al., 2005).
Additional Molecule Role : Protective antigen
Related Vaccine(s): E. tenella DNA vaccine EtMIC2

5. Rho

Gene Name : Rho
Sequence Strain (Species/Organism) : Eimeria tenella
VO ID : VO_0011064
NCBI Protein GI : 84043169
Other Database IDs : CDD:304416
Taxonomy ID : 5802
Gene Strand (Orientation) : ?
Protein Name : rhomboid-like protein
Protein pI : 8.07
Protein Weight : 26568.3
Protein Length : 314
Protein Note : Rhomboid family; cl21536

Protein Sequence : Show Sequence

>ABC50099.1 rhomboid-like protein [Eimeria tenella]
MSDIESQRVLGAGIPRTGRLIDVAFPNISLDKSIVVITLAQIAVYIMSCAMSSAFEPTERVLYQLGATYG
PGIRHLQAWRLIMPVFLHVGIVHLLFNVLFILHMGLDKEIKYGRTNFLILYFSSALIGNMFTVLMRPCSL
AVGASTAGFGLVGSILAEILIVWHKLDERTRNMYTLDMTVFGALMILLSYGQTVDIWGHLGGFVCGFAVT
CEFNKNIRDLPHWFDLAKNGTRTICFGVVALTLARVFLGLPLPMGCA

Molecule Role : Protective antigen
Molecule Role Annotation : Two recombinant Mycobacterium bovis BCG (rBCG) strains carrying the Eimeria tenella rhomboid gene (Rho) delivered by extrachromosomal vector pMV261 and integrative vector pMV361 were evaluated for their ability to protect chickens against E. tenella challenge. Chickens were immunized intranasal with BCG, rBCG pMV261-Rho, or rBCG pMV361-Rho twice at a 2-week interval. All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4(+) and CD8(+) cells compared to the control (P<0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge (Wang et al., 2009).
Additional Molecule Role : Protective antigen
Related Vaccine(s): E. tenella vaccine rBCG pMV261-Rho , E. tenella vaccine rBCG pMV361-Rho

6. TA4

Gene Name : TA4
Sequence Strain (Species/Organism) : Eimeria tenella
VO ID : VO_0011065
NCBI Protein GI : 84783094
Other Database IDs : CDD:287966
Taxonomy ID : 5802
Gene Strand (Orientation) : ?
Protein Name : TA4 antigen protein
Protein pI : 4.47
Protein Weight : 22659.37
Protein Length : 293
Protein Note : Sporozoite TA4 surface antigen; pfam11054

Protein Sequence : Show Sequence

>ABC61818.1 TA4 antigen protein, partial [Eimeria tenella]
DYPTAVTLDCKEAMNKMRKAAGLPAFEDAVGDTFVLPAYSHEESRAAPVAETLWKTEICPKVLGGGRSRN
VTEAVRLTGNFAYYPVTDGKKECSDAVEYWKGGLSQFNDTIPPTFQALNDPVVYNDRAVSFVALYNPKTS
PVVSCVLLQCPNAGVGGRRLAAGTTDAVICLTNPAPLEARSQPFDDEQWKKIVDSLSLSEEEEEKGGVSP
VVPSVALISAAVISAFALF

Molecule Role : Protective antigen
Molecule Role Annotation : The immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella, including route, dose, time of immunization and age of primary immunization of chicken, was optimized. results illustrated that 25 microg was the optimal dose and intramuscular injection was the most effective route to induce protective immunity in Chinese Yellow chickens (Song et al., 2009).
Additional Molecule Role : Protective antigen
Related Vaccine(s): E. tenella DNA vaccine pcDNA-TA4-IL-2

III. Vaccine Information

1. E. tenella DNA vaccine EtMIC2

a. Vaccine Ontology ID:

b. Type:

DNA vaccine

c. Status:

Research

d. Antigen

E. tenella microneme 2 (MIC2)

e. Gene Engineering of MIC2

Type: DNA vaccine construction
Description: The EtMIC2 coding sequence was subcloned from EtMIC2-pGEX into the BamHI/HindIII sites of the pMal4c vector with a NH2-terminal maltose-binding protein tag, expressed in E. coli in TY broth (20 g/l tryptone, 10 g/l yeast extract, 10 g/l NaCl) containing 100 μg/ml ampicillin, the bacteria grown to OD600 = 0.5, induced with 1.0 mM isopropyl-β-d-thiogalactopyranoside for 3 h at 37 °C, collected by centrifugation, and disrupted by sonication on ice (Misonix, Farmingdale, NY). The EtMIC2 protein was isolated on an amylose affinity column (New England Biolabs, Beverly, MA) according to the manufacturer's instructions, digested with Factor Xa to release EtMIC2, and re-passed through the amylose column to remove maltose-binding protein (Ding et al., 2005).
Detailed Gene Information: Click here.

f. Vector:

pcDNA vector

g. Immunization Route

in ovo

h. Chicken Response

Vaccination Protocol: To assess anti-EtMIC2 antibody titers and protective immunity to coccidiosis following in ovo vaccination with the EtMIC2 gene, 75 fertile eggs were distributed into five groups (15/group) and non-injected or injected with 100 μl of sterile PBS, 50 μg/egg of the pcDNA empty vector, or 25 or 50 μg/egg of EtMIC2-pcDNA. To determine the effects of post-vaccination boosting with the EtMIC2 gene, 150 fertile eggs were distributed into 10 groups (15/group) and non-injected or injected with 100 μl of sterile PBS, 50 μg/egg of the pcDNA empty vector, or 25 or 50 μg/egg of EtMIC2-pcDNA (Ding et al., 2005).
Challenge Protocol: At day 11 post-hatching, chickens were infected with 10,000 sporulated oocysts of E. tenella. Serum samples were collected at days 1, 10, and 17 post-infection (days 10, 21, and 28 post-hatching) and anti-EtMIC2 antibody titers determined by ELISA (Ding et al., 2005).
Efficacy: Birds vaccinated with Eimeria tenella microneme recombinant gene (EtMIC2) and encoded protein developed protective immunity against infection by E. tenella as assessed by significantly increased body weight gain and decreased fecal oocyst shedding compared with non-vaccinated controls. Vaccination with the EtMIC2 gene also led to protective immunity against infection by E. acervulina, but not E. maxima (Ding et al., 2005).

2. E. tenella DNA vaccine pcDNA-TA4-IL-2

a. Vaccine Ontology ID:

b. Type:

DNA vaccine

c. Status:

Research

d. Antigen

E. tenella sporozoite TA4 surface antigen

e. Gene Engineering of TA4

Type: DNA vaccine construction
Description: PCR amplification of TA4 (without stop codon) was performed using the gene-specific primers with pET28a-TA4 as template. After purification, the amplified TA4 (without stop codon) was digested with BamHI and EcoRI and cloned into the BamHI/EcoRI sites of pcDNA3.1b. The recombinant plasmid pcDNA3.1b-TA4 (without stop codon) was confirmed by PCR amplification and endonuclease cleavage. Using the same method above, amplified chIL-2 product was ligated into pcDNA3.1b-TA4 (without stop codon) and pcDNA4.0c (Invitrogen) to construct pcDNA3.1b-TA4-IL2 and pcDNA4.0c-IL-2 (Song et al., 2009).
Detailed Gene Information: Click here.

f. Vector:

pcDNA3.1 (Song et al., 2009)

g. Immunization Route

Intramuscular injection (i.m.)

h. Chicken Response

Vaccination Protocol: At 14 days of age, chickens were randomly distributed into seven groups (30/group), group 1 negative control, group 2 positive control, groups 3, 4, 5 and 6 immunized with 25 μg, 50 μg, 100 μg and 200 μg of DNA vaccine, respectively and group 7 vector control. Experimental groups were immunized with corresponding doses of DNA vaccine pcDNA3.1b–TA4–IL-2 by leg intramuscular injection. Control group chickens were injected with sterile TE or 100 μg pcDNA3.1b at the same injection site to the experimental groups. A booster immunization was given by the same method as the first immunization 7 days later (Song et al., 2009).
Challenge Protocol: At 28 days of age, the chickens were weighed one-by-one and challenged with 5 × 10⁴ sporulated oocysts of E. tenella JS except the negative control group. The chickens were weighed one-by-one and slaughtered 7 days post challenge (Song et al., 2009).
Efficacy: The immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella, including route, dose, time of immunization and age of primary immunization of chicken, was optimized. results illustrated that 25 microg was the optimal dose and intramuscular injection was the most effective route to induce protective immunity in Chinese Yellow chickens (Song et al., 2009).

3. E. tenella DNA vaccine proIE

a. Vaccine Ontology ID:

b. Type:

DNA vaccine

c. Status:

Research

d. Antigen

Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-gamma)

e. Gene Engineering of 3-1E

Type: DNA vaccine construction
Description: The Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-gamma) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and prol, and then linked by splicing overlap extension by polymerase chain reaction to construct the chimeric plasmid prolE; the chimeric protein (rlE) was expressed in Escherichia coli harboring the constructed plasmid pGEX/IE (Xu et al., 2006).
Detailed Gene Information: Click here.

f. Vector:

proVAX

g. Immunization Route

Intramuscular injection (i.m.)

h. Chicken Response

Host Strain: Broiler
Vaccination Protocol: Broilers were administered two intramuscular injections with the constructed DNA vaccines (50 microg); in the protein booster groups 100 microg of the rlE were given following the proIE prime (Xu et al., 2006).
Challenge Protocol: One week following the booster immunization, all chickens except for the unchallenged control group were orally challenged with 5 x 10⁴ sporulated E. tenella GS oocysts (Xu et al., 2006).
Efficacy: The Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-gamma) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and prol. After challenge the proIE-vaccinated chickens showed protective immunity as demonstrated by significantly reduced oocyst shedding (Xu et al., 2006).

4. E. tenella GX3262 protein vaccine

a. Vaccine Ontology ID:

b. Type:

Subunit vaccine

c. Status:

Research

d. Antigen

E. tenella sporozoite antigen GX3262

e. Gene Engineering of GX3262

Type: Recombinant protein preparation
Description: To produce the 3-galactosidase-GX3262 fusion protein (Pgal-GX3262), recombinant E. coli cells. grown overnight in Luria-Bertani broth with ampicillin and induced with IPTG, were lysed by sonication and centrifuged, and the insoluble cell pellet was washed extensively with PBS (Miller et al., 1989).
Detailed Gene Information: Click here.

f. Adjuvant: complete Freunds adjuvant

VO ID: VO_0000139

g. Immunization Route

Subcutaneous injection

h. Chicken Response

Host Strain: Hubbard broiler
Vaccination Protocol: The basic experimental design involved immunizing birds subcutaneously or orally with partially purified recombinant antigen preparations, heatkilled bacterins. live E. coli cells, or control materials (Miller et al., 1989).
Challenge Protocol: Broilers were challenged with E. tendell/i oocysts; and then evaluated for cecal lesions (Miller et al., 1989).
Efficacy: A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites. After challenge with an experimental E. tenella infection, the greatest degree of protection in immunized broiler chickens was seen after only a single immunization of 2-day-old birds with a live recombinant E. coli preparation (Miller et al., 1989).

5. E. tenella vaccine rBCG pMV261-Rho

a. Vaccine Ontology ID:

b. Type:

Recombinant vector vaccine

c. Status:

Research

d. Antigen

Eimeria tenella rhomboid gene (Rho)

e. Gene Engineering of Rho

Type: Recombinant vector construction
Description: The rhomboid gene segment (GenBank accession no. DQ323509) was generated from the cDNA of E. tenella strains by PCR with primers QF (5’-CTGACTGCAGATGTCGGACATCGAATCCCAGAG-3′) containing PstI restriction site (underlined) and QR (5’-GACTATCGATTTATGCGCATCCCATGGGCAAAGG-3’) containing ClaI restriction site (underlined), and cloned into the pMD18-T vector. The 774-bp rhomboid gene fragment was subcloned into the PstI/ClaI sites of pMV261. The plasmid pMV261-Rho was electrotransfected into BCG and selected by kanamycin (Sigma) (Wang et al., 2009).
Detailed Gene Information: Click here.

f. Vector:

BCG

g. Immunization Route

Intravenous injection (i.v.)

h. Chicken Response

Vaccination Protocol: Chickens were randomly assigned to 4 groups (40 birds in each group) and immunized intranasal (i.n.) with 10⁶ CFU/200 μl of BCG or rBCG strains expressing rhomboid protein, respectivelly. Negative control was immunized i.n. with 200 μl PBS only. 2 weeks after the first vaccination, a second immunization was carried out with the same amounts of each sample (Wang et al., 2009).
Challenge Protocol: All groups were challenged with 3 × 10⁴ sporulated oocysts of E. tenella per bird 2 weeks after the final immunization (Wang et al., 2009).
Efficacy: All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4(+) and CD8(+) cells compared to the control (P<0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge (Wang et al., 2009).

6. E. tenella vaccine rBCG pMV361-Rho

a. Vaccine Ontology ID:

b. Type:

Recombinant vector vaccine

c. Status:

Research

d. Antigen

E. tenella rhomboid gene (Rho)

e. Gene Engineering of Rho

Type: Recombinant vector construction
Description: The rhomboid gene segment (GenBank accession no. DQ323509) was generated from the cDNA of E. tenella strains by PCR with primers QF (5’-CTGACTGCAGATGTCGGACATCGAATCCCAGAG-3′) containing PstI restriction site (underlined) and QR (5’-GACTATCGATTTATGCGCATCCCATGGGCAAAGG-3’) containing ClaI restriction site (underlined), and cloned into the pMD18-T vector. The 774-bp rhomboid gene fragment was subcloned into the PvuII/ClaI site of pMV361. The plasmid pMV361-Rho was electrotransfected into BCG and selected by kanamycin (Sigma) (Wang et al., 2009).
Detailed Gene Information: Click here.

f. Vector:

BCG

g. Immunization Route

Intranasal

h. Chicken Response

Vaccination Protocol: Chickens were randomly assigned to 4 groups (40 birds in each group) and immunized intranasal (i.n.) with 10⁶ CFU/200 μl of BCG or rBCG strains expressing rhomboid protein, respectivelly. Negative control was immunized i.n. with 200 μl PBS only. 2 weeks after the first vaccination, a second immunization was carried out with the same amounts of each sample (Wang et al., 2009).
Challenge Protocol: All groups were challenged with 3 × 10⁴ sporulated oocysts of E. tenella per bird 2 weeks after the final immunization (Wang et al., 2009).
Efficacy: All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4(+) and CD8(+) cells compared to the control (P<0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge (Wang et al., 2009).

7. Livacox

a. Tradename:

Livacox

b. Manufacturer:

BIOPHARM

c. Vaccine Ontology ID:

d. Type:

Live, attenuated vaccine

e. Status:

Clinical trial

f. Host Species for Licensed Use:

Pig

g. Immunization Route

NA

h. Description

Precocious and egg-passaged lines(Williams, 2002)

IV. References

1. Ding et al., 2005: Ding X, Lillehoj HS, Dalloul RA, Min W, Sato T, Yasuda A, Lillehoj EP. In ovo vaccination with the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis. Vaccine. 2005; 23(28); 3733-3740. [PubMed: 15882535].

2. Lai et al., 2011: Lai L, Bumstead J, Liu Y, Garnett J, Campanero-Rhodes MA, Blake DP, Palma AS, Chai W, Ferguson DJ, Simpson P, Feizi T, Tomley FM, Matthews S. The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella. PLoS pathogens. 2011; 7(10); e1002296. [PubMed: 22022267].

3. Miller et al., 1989: Miller GA, Bhogal BS, McCandliss R, Strausberg RL, Jessee EJ, Anderson AC, Fuchs CK, Nagle J, Likel MH, Strasser JM. Characterization and vaccine potential of a novel recombinant coccidial antigen. Infection and immunity. 1989; 57(7); 2014-2020. [PubMed: 2659532].

4. Song et al., 2009: Song X, Xu L, Yan R, Huang X, Shah MA, Li X. The optimal immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella and its cross-immunity to Eimeria necatrix and Eimeria acervulina. Veterinary parasitology. 2009; 159(1); 30-36. [PubMed: 19019543].

5. Wang et al., 2009: Wang Q, Li J, Zhang X, Liu Q, Liu C, Ma G, Cao L, Gong P, Cai Y, Zhang G. Protective immunity of recombinant Mycobacterium bovis BCG expressing rhomboid gene against Eimeria tenella challenge. Veterinary parasitology. 2009; 160(3-4); 198-203. [PubMed: 19117681].

6. Wiki: Eimeria tenella: Eimeria tenella [http://en.wikipedia.org/wiki/Eimeria_tenella]

7. Williams, 2002: Williams RB. Fifty years of anticoccidial vaccines for poultry (1952-2002). Avian diseases. 2002; 46(4); 775-802. [PubMed: 12495038].

8. Xu et al., 2006: Xu SZ, Chen T, Wang M. Protective immunity enhanced by chimeric DNA prime-protein booster strategy against Eimeria tenella challenge. Avian diseases. 2006; 50(4); 579-585. [PubMed: 17274297].