Hostname: page-component-cd9895bd7-dk4vv Total loading time: 0 Render date: 2024-12-18T13:55:35.985Z Has data issue: false hasContentIssue false
  • English
  • Français

A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl

Published online by Cambridge University Press:  17 October 2003

S. FERNANDEZ ,
A. H. PAGOTTO ,
M. M. FURTADO ,
Â. M. KATSUYAMA ,
A. M. B. N. MADEIRA  and
A. GRUBER
S. FERNANDEZ
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
A. H. PAGOTTO
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
M. M. FURTADO
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
Â. M. KATSUYAMA
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
A. M. B. N. MADEIRA
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
A. GRUBER
Affiliation:
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia – USP, Av. Prof. Orlando Marques de Paiva 87, São Paulo SP, 05508-000, Brazil
Get access Rights & Permissions [Opens in a new window]

Abstract

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1–5 pg, which corresponds approximately to 2–8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

Keywords

Eimeriadomestic fowlmultiplexpolymerase chain reactionSequence-Characterised Amplified Region (SCAR)molecular diagnosis
Type
Research Article
Information
Parasitology , Volume 127 , Issue 4 , October 2003 , pp. 317 - 325
Copyright
2003 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

Note: Additional figures not included in this paper are available as supplementary material at the web address http://www.lbm.fmvz.usp.br/eimeria/multiplex/

References

REFERENCES

ALLEN, P. C. & FETTERER, R. H. (2002). Recent advances in biology and immunobiology of Eimeria species and diagnosis and control of infection with these coccidian parasites of poultry. Clinical Microbiology Reviews 15, 58–65.CrossRefGoogle Scholar
BRISSE, S., DUJARDIN, J. C. & TIBAYRENC, M. (2000). Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers. Molecular and Biochemical Parasitology 111, 95–105.CrossRefGoogle Scholar
CERE, N., HUMBERT, J. F., LICOIS, D., CORVIONE, M., AFANASSIEFF, M. & CHANTELOUP, N. (1996). A new approach for the identification and the diagnosis of Eimeria media parasite of the rabbit. Experimental Parasitology 82, 132–138.CrossRefGoogle Scholar
CERE, N., LICOIS, D. & HUMBERT, J. F. (1995). Study of the inter- and intraspecific variation of Eimeria spp. from the rabbit using random amplified polymorphic DNA. Parasitology Research 81, 324–328.Google Scholar
CHAPMAN, H. D., CHERRY, T. E., DANFORTH, H. D., RICHARDS, G., SHIRLEY, M. W. & WILLIAMS, R. B. (2002). Sustainable coccidiosis control in poultry production: the role of live vaccines. International Journal for Parasitology 32, 617–629.CrossRefGoogle Scholar
CORNELISSEN, A. W. C. A., OVERDULVE, J. P. & VAN DER PLOEG, M. (1984). Determination of nuclear DNA of five eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella, E. acervulina and Plasmodium berghei, with special reference to gamontogenesis and meiosis in I. (T.) gondii. Parasitology 88, 531–553.Google Scholar
FERNANDEZ, S., COSTA, A. C., KATSUYAMA, Â. M., MADEIRA, A. M. B. N. & GRUBER, A. (2003). A survey of the inter- and intraspecific RAPD markers of Eimeria spp. of the domestic fowl and the development of reliable diagnostic tools. Parasitology Research 89, 437–445.Google Scholar
GASSER, R. B., WOODS, W. G., WOOD, J. M., ASHDOWN, L., RICHARDS, G. & WHITHEAR, K. G. (2001). Automated, fluorescence-based approach for the specific diagnosis of chicken coccidiosis. Electrophoresis 22, 3546–3550.3.0.CO;2-8>CrossRefGoogle Scholar
HAMMOND, D. M. & LONG, P. L. (1973). The Coccidia. University Park Press, Baltimore, USA.
HENEGARIU, O., HEEREMA, N. A., DLOUHY, S. R., VANCE, G. H. & VOGT, P. H. (1997). Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504–511.Google Scholar
JEFFERS, T. K. (1975). Attenuation of Eimeria tenella through selection for precociousness. Journal of Parasitology 61, 1083–1090.CrossRefGoogle Scholar
JOHNSTON, D. A. & FERNANDO, M. A. (1995). Eimeria spp. of the domestic fowl: analysis of genetic variability between species and strains using DNA polymorphisms amplified by arbitrary primers and denaturing gradient-gel electrophoresis. Parasitology Research 81, 91–97.Google Scholar
JOHNSTON, D. A. & FERNANDO, M. A. (1997). Isoenzymes of Eimeria from the domestic fowl: Electrophoretic variants among species, strains and clones. Parasitology Research 83, 464–470.CrossRefGoogle Scholar
JOYNER, L. P. & LONG, P. L. (1974). The specific characters of the Eimeria, with special reference to the coccidia of the fowl. Avian Pathology 3, 145–157.CrossRefGoogle Scholar
LONG, P. L. & JOYNER, L. P. (1984). Problems in the identification of species of Eimeria. Journal of Protozoology 31, 535–541.CrossRefGoogle Scholar
LONG, P. L., MILLARD, B. J., JOYNER, L. P. & NORTON, C. C. (1976). A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Veterinaria Latina 6, 201–217.Google Scholar
MacPHERSON, J. M., ECKSTEIN, P. E., SCOLES, G. J. & GAJADHAR, A. A. (1993). Variability of the random amplified polymorphic DNA assay among thermal cyclers, and effects of primer and DNA concentration. Molecular and Cellular Probes 7, 293–299.CrossRefGoogle Scholar
MacPHERSON, J. M. & GAJADHAR, A. A. (1993). Differentiation of seven Eimeria species by random amplified polymorphic DNA. Veterinary Parasitology 45, 257–266.CrossRefGoogle Scholar
McDOUGALD, L. R. & JEFFERS, T. K. (1976). Eimeria tenella (Sporozoa; Coccidia): gametogony following a single asexual generation. Science 192, 258–259.CrossRefGoogle Scholar
McDOUGALD, L. R. & REID, W. M. (1997). Coccidiosis. In Diseases of Poultry, 10th Edn (ed. Calnek, B. W., Barnes, H. J., Beard, C. W., McDougald, L. R. & Saif, Y. M.), pp. 865–883. Iowa State University Press, Ames, USA.
MORGAN, U. M., O'BRIEN, P. A. & THOMPSON, R. C. A. (1996). The development of diagnostic PCR primers for Cryptosporidium using RAPD PCR. Molecular and Biochemical Parasitolology 77, 103–108.CrossRefGoogle Scholar
ORLANDI, P. A. & LAMPEL, K. A. (2000). Extraction-free, filter-based template preparation for rapid and sensitive PCR detection of pathogenic parasitic protozoa. Journal of Clinical Microbiology 38, 2271–2277.Google Scholar
PARAN, I. & MICHELMORE, R. W. (1993). Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics 85, 985–993.CrossRefGoogle Scholar
PROCUNIER, J. D., FERNANDO, M. A. & BARTA, J. R. (1993). Species and strain differentiation of Eimeria spp. of the domestic fowl using DNA polymorphisms amplified by arbitrary primers. Parasitology Research 79, 98–102.Google Scholar
SCHIERWATER, B. & ENDER, A. (1993). Different thermostable DNA polymerases may amplify different RAPD products. Nucleic Acids Research 21, 4647–4648.CrossRefGoogle Scholar
SCHNITZLER, B. E., THEBO, P. L., MATTSSON, J. G., TOMLEY, F. M. & SHIRLEY, M. W. (1998). Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic Eimeria species of the chicken. Avian Pathology 27, 490–497.CrossRefGoogle Scholar
SCHNITZLER, B. E., THEBO, P. L., TOMLEY, F. M., UGGLA, A. & SHIRLEY, M. W. (1999). PCR identification of chicken Eimeria: a simplified read-out. Avian Pathology 28, 89–93.CrossRefGoogle Scholar
SHIRLEY, M. W. (1975). Enzyme variation in Eimeria species of the chicken. Parasitology 71, 369–376.CrossRefGoogle Scholar
SHIRLEY, M. W. (1994). Coccidial parasites from the chicken: discrimination of different populations of Eimeria tenella by DNA hybridisation. Research in Veterinary Science 57, 10–14.CrossRefGoogle Scholar
SHIRLEY, M. W. (1995). Eimeria species and strains of chickens. In Biotechnology – Guidelines on Techniques in Coccidiosis Research (ed. Eckert, J., Braun, R., Shirley, M. W. & Coudert, P.), pp. 1–24. European Commission COST 89/820, Luxembourg.
SHIRLEY, M. W. & BUMSTEAD, N. (1994). Intra-specific variation within Eimeria tenella detected by the random amplification of polymorphic DNA. Parasitology Research 80, 346–351.CrossRefGoogle Scholar
SHIRLEY, M. W., McDONALD, V., CHAPMAN, H. D. & MILLARD, B. J. (1984). Eimeria praecox: selection and characteristics of precocious lines. Avian Pathology 13, 669–682.CrossRefGoogle Scholar
STUCKI, U., BRAUN, R. & RODITI, I. (1993). Eimeria tenella: characterization of a 5S ribosomal RNA repeat unit and its use as a species-specific probe. Experimental Parasitology 76, 68–75.CrossRefGoogle Scholar
TSUJI, N., KAWAZU, S., OHTA, M., KAMIO, T., ISOBE, T., SHIMURA, K. & FUJISAKI, K. (1997). Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction. Journal of Parasitology 83, 966–970.CrossRefGoogle Scholar
WELSH, J. & McCLELLAND, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213–7218.CrossRefGoogle Scholar
WILLIAMS, J. G. K., KUBELIK, A. R., LIVAK, K. J., RAFALSKI, J. A. & TINGEY, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531–6535.CrossRefGoogle Scholar
WOODS, W. G., WHITHEAR, K. G., RICHARDS, D. G., ANDERSON, G. R., JORGENSEN, W. K. & GASSER, R. B. (2000). Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia. International Journal for Parasitology 30, 1019–1023.CrossRefGoogle Scholar
ZHAO, X., DUSZYNSKI, D. W. & LOKER, E. S. (2001). A simple method of DNA extraction for Eimeria species. Journal of Microbiological Methods 44, 131–137.CrossRefGoogle Scholar