Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter July 4, 2018

Comparison of sensitivity of two primer sets for the detection of Toxoplasma gondii DNA in wildlife

Aleksandra Kornacka, Aleksandra Cybulska and Bożena Moskwa
From the journal Acta Parasitologica

Abstract

Toxoplasma gondii, a coccidian parasite known to infect almost all warm-blooded animals, is the cause of one of the most common zoonotic parasitic diseases. The aim of the study is to determine whether the 529 bp fragment or the TGR1E gene is more useful target for PCR identification of T. gondii, for common use. The brains of 221 carnivores and omnivores collected between 2013 and 2015 from north-eastern Poland were examined for the presence of this parasite. The DNA was extracted and then amplified using specific primers. Positive results were obtained in 24% of brain samples using the TGR1E target and 19% using the 529 bp sequence. The results demonstrate that both TGR1E and 529 bp repeat element are suitable for detecting T. gondii DNA in wildlife animals, and the combination of two methods is necessary to obtain reliable results.

  1. Authors’ contributions: AK and BM were involved in the design and coordination of the study. AC helped performed laboratory tests. All authors contributed in drafting the manuscript. All authors read and approved the final manuscript.

  2. Availability of data and materials: All data generated or analyzed during this study are included in this published article.

  3. Competing interests: The authors declare that they have no competing interests.

  4. Ethics approval: The experiment was approved by the Review of Board of Ethics and conducted in accordance with the guidelines establish by Bioethics Appeals Commission (no 72/2013)

  5. Funding: The authors received no financial support for the research, authorship, and publication of this article.

Acknowledgements

We thank all forest rangers and hunters for their engagement and contribution in this project.

References

Belaz S., Gangneux J.P., Dupretz P., Guiguen C., Gangneuxa F.R. 2015. A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR. Journal of Clinical Microbiology, 53, 1294–1300. 10.1128/JCM.02900-14Search in Google Scholar

Burg J.L., Grover C.M., Pouletty P., Boothroyd J.C. 1989. Direct and sensitive detection of a pathogenic protozoan Toxoplasma gondii, by polymerase chain reaction. Journal of Clinical Microbiology, 27, 1787–179210.1128/jcm.27.8.1787-1792.1989Search in Google Scholar

Burrells A., Bartley P.M., Zimmer I.A., Roy S., Kitchener A.C., Meredith A., et al. 2013. Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK. Parasitology, 140, 1768-1776. 10.1017/S0031182013001169Search in Google Scholar

Cermáková Z., Ryšková O., Plšsková L. 2005. Polymerase chain reaction for detection of Toxoplasma gondii in human biological samples. Folia Microbiologica, 50, 341–34410.1007/BF02931415Search in Google Scholar

Cicchetti D.V., Feinstein A.R. 1990. High agreement but low kappa: II. Resolving the paradoxes. Journal of Clinical Epidemiology, 43, 551–55810.1016/0895-4356(90)90159-MSearch in Google Scholar

De Craeye S., Speybroeck N., Ajzenberg D., Dardé M.L., Collinet F., Tavernier P., et al. 2011. Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae? Veterinary Parasitology, 178, 64–69. 10.1016/j.vetpar.2010.12.016Search in Google Scholar PubMed

De Oliveira Mendonça A., Domingues P.F., Da Silva A.V., Bergamaschi Pezerico S., Langoni H. 2004. Detection of Toxoplasma gondii in swine sausages. Parasitologia Latinoamerica, 59, 42–45. 10.4067/S0717-77122004000100008Search in Google Scholar

Dubey J. P. 1988. Long-term persistence of Toxoplasma gondii in tissues of pigs inoculated with T. gondii oocysts and effect of freezing on viability of tissue cysts in pork. American Journal of Veterinary Research, 49, 910–913Search in Google Scholar

Dubey J. P., Lindsay D. S., Speer C.A. 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clinical Microbiology Reviews, 11, 267–29910.1128/CMR.11.2.267Search in Google Scholar PubMed PubMed Central

Edvinsson B., Lappalainen M., Evengard B. 2006. Real-time PCR targeting a 529 bp repeat element for diagnosis of toxoplasmosis. Clinical Microbiology and Infection, 12, 131–136. 10.1111/j.1469-0691.2005.01332.xSearch in Google Scholar PubMed

Franzen C., Altfeld M., Hegener P., Hartmann P., Arendt G., Jablonowski H., et al. 1997. Limited value of PCR for detection of Toxoplasma gondii in blood from human immunodeficiency virus-infected patients. Journal of Clinical Microbiology, 35, 2639–264110.1128/jcm.35.10.2639-2641.1997Search in Google Scholar PubMed PubMed Central

Herrmann D.C., Maksimov P., Maksimov A., Sutor A., Schwarz S., Jaschke W., et al. 2012. Toxoplasma gondii in foxes and rodents from the German Federal States of Brandenburg and Saxony-Anhalt: seroprevalence and genotypes. Veterinary Parasitology, 185, 78–85. 10.1016/j.vetpar.2011.10.030Search in Google Scholar

Hill D., Dubey J.P. 2002. Toxoplasma gondii: transmission, diagnosis and prevention. Clinical Microbiology and Infection, 8, 634–64010.1046/j.1469-0691.2002.00485.xSearch in Google Scholar

Homan W.L., Vercammen M., De Braekeleer J., Verschueren H. 2000. Identification of a 200-to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. International Journal for Parasitology, 30, 69–7510.1016/S0020-7519(99)00170-8Search in Google Scholar

Hůrková L., Modrý D. 2006. PCR detection of Neospora caninum, Toxoplasma gondii and Encephalitozoon cuniculi in brains of wild carnivores. Veterinary Parasitology, 137, 150–154. 10.1016/j.vetpar.2006.01.005Search in Google Scholar PubMed

Ivovic V., Vujanic M., Zivkovic T., Klun I., Djurkovic-Djakovic O. 2012. Molecular detection and genotyping of Toxoplasma gondii from clinical samples. In: (Eds. Djurkovic-Djakovic O) Toxoplasmosis – Recent Advances. InTech. 10.5772/50830Search in Google Scholar

Juránková J., BassoW., Neumayerová H., Baláž V., Jánová E., Sidler X., et al. 2014. Brain is the predilection site of Toxoplasma gondii in experimentally inoculated pigs as revealed by magnetic capture and real-time PCR. Food Microbiology, 38, 167–170. 10.1016/j.fm.2013.08.011Search in Google Scholar PubMed

Juránková J., Opsteegh M., Neumayerová H., Kovařčik K., Frencová A., Baláž V., et al. 2013. Quantification of Toxoplasma gondii mentally infected goats by magnetic capture and realtime PCR. Veterinary Parasitology, 193, 95–99. 10.1016/j.vetpar.2012.11.016Search in Google Scholar PubMed

Kornacka A., Cybulska A., Bień J., Goździk K., Moskwa B. 2016. The usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals. Veterinary Parasitology, 228, 85–89. 10.1016/j.vetpar.2016.08.010Search in Google Scholar PubMed

Lamoril J., Molina J.M., deGouvello A., Garin Y.J., Deybach J.C., Modai J., Derouin F. 1996. Detection by PCR of Toxoplasma gondii in blood in the diagnosis of cerebral toxoplasmosis in patients with AIDS. Journal of Clinical Pathology, 49, 89–9210.1136/jcp.49.1.89Search in Google Scholar PubMed PubMed Central

Luptakova L., Petrovova E., Mazensky D., Valencakova A., BalentP. 2012. Toxoplasmosis in livestock and pet animals in Slovakia. In: (Eds. Djurkovic-Djakovic O) Toxoplasmosis – Recent Advances, InTech. 2012. 10.5772/47810Search in Google Scholar

Noral M.Y., Dogruman-Al F., Engin E.D., Kustimur S., Babur C., Poyraz A. 2009. The comparison of polymerase chain reaction directed to the 529 bp gene and the B1 gene in the detection of experimental mouse toxoplasmosis. Turkiye Klinikleri Journal of Medical Sciences, 29, 48–56Search in Google Scholar

Opsteegh M., Langelaar M., Sprong H., den Hartog L., De Craeye S., Bokken G., et al. 2010. Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR. International Journal of Food Microbiology, 139, 193–201. 10.1016/j.ijfoodmicro.2010.02.027Search in Google Scholar

Paugam A., Dupouy-Camet J., Sumuyen M.H., Romand S., Lamoril J., Derouin F. 1995. Detection of Toxoplasma gondii parasitemia by polymerase chain reaction in perorally infected mice. Parasitology, 2, 181–184. 10.1051/parasite/1995022181Search in Google Scholar

Piña-Vázquez C., Saavedra R., Hérion P. 2008. A quantitative competitive PCR method to determine the parasite load in the brain of Toxoplasma gondii-infected mice. Parasitology International, 57, 347–353. 10.1016/j.parint.2008.03.001Search in Google Scholar

Reischl U., Bretagne S., Krüger D., Ernault P., Costa J.M. 2003. Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. Bio Med Central Infectious Diseases, 3,7. 10.1186/1471-2334-3-7Search in Google Scholar

Rożej-Bielicka W., Waloch M.E., Goląb E. 2011. Cases of Toxoplasma gondii infection in foetus is and toxoplasmic encephalitis in immunosupressed patients confirmed by PCR method in NIPH-NIH 2009-2010. Przegląd Epidemiologiczny, 65, 593–597Search in Google Scholar

Sedlák K., Bártová E., Literák I., Vodička R., Dubey J.P. 2004. Toxoplasmosis in nilgais (Boselaphus tragocamelus) and a saiga antelope (Saiga tatarica). Journal of Zoo and Wildlife Medicine, 35, 530–533. 10.1638/02-005.1Search in Google Scholar

Spišák F., Turčeková L., Reiterova K., Špilovská S., Dubinský P. 2010. Prevalence estimation and genotypization of Toxoplasma gondii in goats. Biologia, 65, 670–674. 10.2478/s11756-010-0070-2Search in Google Scholar

Şuteu O., MihalcaA.D., Paştiu A.I., Györke A., Matei I.A., Ionică A., et al. 2014. Red Foxes (Vulpes vulpes) in Romania are carriers of Toxoplasma gondii but not Neospora caninum. Journal of Wildlife Diseases, 50, 713–716. 10.7589/2013-07-167Search in Google Scholar

Veronesi F., Santoro A., Milardi G. L., Diaferia M., Branciari R., Miraglia D., et al. 2017. Comparison of PCR assays targeting the multi-copy targets B1 gene and 529 bp repetitive element for detection of Toxoplasma gondii in swine muscle. Food Microbiology, 63, 213–216. 10.1016/j.fm.2016.11.022Search in Google Scholar

Viera A.J., Garrett J.M. 2005. Understanding interobserver agreement: the kappa statistic. Family Medicine, 37, 360–363Search in Google Scholar

Warnekulasuriya M.R., Johnson J.D., Holliman R.E. 1998. Detection of Toxoplasma gondii in cured meats. International Journal of Food Microbiology, 4, 211–215. 10.1016/SO168-1605(98)00158-5Search in Google Scholar

Yang W., Lindquist H.D.A., Cama V., Schaefer F.W., Villegas E., Fayer R., et al. 2009. Detection of Toxoplasma gondii oocysts in water sample concentrates by Real-Time PCR. Applied and Environmental Microbiology, 75, 3477–3483. 10.1128/AEM.00285-09Search in Google Scholar PubMed PubMed Central

Received: 2018-03-13
Revised: 2018-05-18
Accepted: 2018-05-21
Published Online: 2018-07-04
Published in Print: 2018-09-25

© 2018 W. Stefański Institute of Parasitology, PAS