Quantitation of Cytokine MRNA by Real-time RT-PCR During a Vaccination Trial in a Rabbit Model of Fascioliasis

Overview

Journal Vet Parasitol

Publisher Elsevier

Specialties Parasitology
Veterinary Medicine

Date 2010 Jan 9

PMID 20056331

Citations 16

Authors

Ana M Espino

Francheska Rivera

Affiliations

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Abstract

Use of the rabbit as disease model has long been hampered by a lack of immunological assays specific to this species. In the present study we developed a SYBR Green-based, real-time RT-PCR protocol to quantitate cytokine mRNA in freshly harvested rabbit peripheral mononuclear cells. The method was validated in the course of a vaccination trial in which animals vaccinated with the recombinant antigen FhSAP2 were challenged with Fasciola hepatica metacercariae. Changes in the levels of rabbit interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) mRNA were determined. Messenger RNA from the universally expressed housekeeping gene GAPDH was used as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. Rabbits vaccinated with FhSAP2 showed an 83.3% reduction in liver fluke burden after challenge infection when compared to non-vaccinated controls. All cytokine mRNAs were found at detectable levels; however, the levels of IFNgamma, TNFalpha, IL-2 and IL-10 were significantly higher in the vaccinated group compared to the non-vaccinated group. These results suggest that protection conferred by FhSAP2 protein could be associated with a mixed Th1/Th2 immune response in which Th1 cytokines are dominant. The real-time RT-PCR method described herein can be a useful tool for monitoring changes in basic immune functions in the rabbit model of fascioliasis and may also aid in studies of human diseases for which the rabbit is an important experimental model.

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References

Windhagen A, Anderson D, Carrizosa A, Williams R, Hafler D . IL-12 induces human T cells secreting IL-10 with IFN-gamma. J Immunol. 1996; 157(3):1127-31. View

Thompson B, Chilton P, Ward J, Evans J, Mitchell T . The low-toxicity versions of LPS, MPL adjuvant and RC529, are efficient adjuvants for CD4+ T cells. J Leukoc Biol. 2005; 78(6):1273-80. DOI: 10.1189/jlb.0305172. View

Palmer K, Benko A, Doucette S, Cameron T, Foster T, Hanley K . Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes. Vaccine. 2006; 24(26):5516-25. DOI: 10.1016/j.vaccine.2006.04.058. View

Baeza E, Poitou I, Delers F, Boulard C . Influence of anti-inflammatory treatments on experimental infection of rats with Fasciola hepatica: changes in serum levels of inflammatory markers during the early stages of fasciolosis. Res Vet Sci. 1994; 57(2):172-9. DOI: 10.1016/0034-5288(94)90054-x. View

Brewer J, Alexander J . Cytokines and the mechanisms of action of vaccine adjuvants. Cytokines Cell Mol Ther. 1998; 3(4):233-46. View

Silvennoinen O, Nishigaki H, Kitanaka A, Kumagai M, Ito C, Malavasi F . CD38 signal transduction in human B cell precursors. Rapid induction of tyrosine phosphorylation, activation of syk tyrosine kinase, and phosphorylation of phospholipase C-gamma and phosphatidylinositol 3-kinase. J Immunol. 1996; 156(1):100-7. View

Mena A, Ioannou X, Van Kessel A, Popowych Y, Babiuk L, Godson D . Th1/Th2 biasing effects of vaccination in cattle as determined by real-time PCR. J Immunol Methods. 2002; 263(1-2):11-21. DOI: 10.1016/s0022-1759(02)00029-7. View

Hussein A, Khalifa R . Experimental infections with Fasciola in snails, mice and rabbits. Parasitol Res. 2008; 102(6):1165-70. DOI: 10.1007/s00436-008-0888-5. View

Zafra R, Buffoni L, Martinez-Moreno A, Perez-Ecija A, Martinez-Moreno F, Perez J . A study of the liver of goats immunized with a synthetic peptide of the Sm14 antigen and challenged with Fasciola hepatica. J Comp Pathol. 2008; 139(4):169-76. DOI: 10.1016/j.jcpa.2008.06.004. View

10.

Brisbin J, Zhou H, Gong J, Sabour P, Akbari M, Haghighi H . Gene expression profiling of chicken lymphoid cells after treatment with Lactobacillus acidophilus cellular components. Dev Comp Immunol. 2007; 32(5):563-74. DOI: 10.1016/j.dci.2007.09.003. View

11.

Meeusen E, Piedrafita D . Exploiting natural immunity to helminth parasites for the development of veterinary vaccines. Int J Parasitol. 2003; 33(11):1285-90. DOI: 10.1016/s0020-7519(03)00162-0. View

12.

Vilar M, Barrientos F, Almeida M, Thaumaturgo N, Simpson A, Garratt R . An experimental bivalent peptide vaccine against schistosomiasis and fascioliasis. Vaccine. 2003; 22(1):137-44. DOI: 10.1016/s0264-410x(03)00300-1. View

13.

Kindt T . Rabbit immunoglobulin allotypes: structure, immunology, and genetics. Adv Immunol. 1975; 21:35-86. DOI: 10.1016/s0065-2776(08)60218-7. View

14.

Pfister K, Daveau C, Ambroise-Thomas P . Partial purification of somatic and excretory-secretory products of adult Fasciola hepatica and their application for the serodiagnosis of experimental and natural fascioliasis using an ELISA. Res Vet Sci. 1984; 37(1):39-43. View

15.

Yang G, Obiakor H, Sinha R, Newman B, Hood B, Conrads T . Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix. Proc Natl Acad Sci U S A. 2005; 102(47):17083-8. PMC: 1282565. DOI: 10.1073/pnas.0501338102. View

16.

Law R, Smooker P, Irving J, Piedrafita D, Ponting R, Kennedy N . Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection. Infect Immun. 2003; 71(12):6921-32. PMC: 308908. DOI: 10.1128/IAI.71.12.6921-6932.2003. View

17.

Boeuf P, Vigan-Womas I, Jublot D, Loizon S, Barale J, Akanmori B . CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use. BMC Immunol. 2005; 6:5. PMC: 555737. DOI: 10.1186/1471-2172-6-5. View

18.

Ramajo V, Oleaga A, Casanueva P, Hillyer G, Muro A . Vaccination of sheep against Fasciola hepatica with homologous fatty acid binding proteins. Vet Parasitol. 2001; 97(1):35-46. DOI: 10.1016/s0304-4017(01)00388-0. View

19.

Clemons K, Capilla J, Stevens D . Experimental animal models of coccidioidomycosis. Ann N Y Acad Sci. 2007; 1111:208-24. DOI: 10.1196/annals.1406.029. View

20.

Espino A, Dumenigo B, Fernandez R, Finlay C . Immunodiagnosis of human fascioliasis by enzyme-linked immunosorbent assay using excretory-secretory products. Am J Trop Med Hyg. 1987; 37(3):605-8. DOI: 10.4269/ajtmh.1987.37.605. View