The usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals

doi:10.1016/j.vetpar.2016.08.010

Veterinary Parasitology

Volume 228, 15 September 2016, Pages 85-89

https://doi.org/10.1016/j.vetpar.2016.08.010 Get rights and content

Highlights

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We compared the usefulness of two antibody-based methods: DAT and ELISA, with that of PCR for detecting Toxoplasma gondii in samples from naturally-infected wild animals.
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We confirmed that both ELISA and DAT are capable methods for quick screening of meat juice samples.
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PCR should be used to confirm the presence of T. gondii DNA.

Abstract

The aim of the study was to compare the usefulness of two antibody-based methods, the direct agglutination test (DAT) and enzyme linked immuosorbent assay (ELISA), with that of the polymerase chain reaction (PCR) for detecting anti-Toxoplasma gondii in samples derived from naturally-infected wild animals.

Antibodies against T. gondii were detected in meat juice samples collected from 129 free- living carnivores and omnivores. T. gondii seroprevalence was confirmed in 73,6% of examined samples when DAT and ELISA were used separately, but in only 88,4% samples when both immunological tests were used in parallel.

PCR results confirmed the presence of DNA of the parasite in 24 of all the 129 samples. Sixteen samples were classified as positive when all three tests were used.

A moderate degree of agreement was found between DAT and ELISA (κ = 0.55). However, no agreement was found between the molecular and serological tests: κ = − 1.75 for DAT versus PCR; κ = − 1.67 ELISA versus PCR.

By using both serological tests, antibodies against T. gondii were found in 77.5% of red foxes, 12.5% of badgers, 40% of martens and 8.3% of raccoon dogs. Antibodies against the parasite were detected also in one mink, but not in the sample derived from a polecat. T.gondii DNA was found in the brain tissue of 20 red foxes, three badgers and one raccoon dog.

Our studies confirm that ELISA and DAT are suitable and reliable techniques for T. gondii antibody detection in meat juice from wild animals when serum samples are unavailable. Positive results obtained by immunological tests do not always reflect that the host was infected by T. gondii. They indicate only a contact with parasite. PCR should be used to confirm te presence of DNA from T. gondii.

Introduction

Toxoplasma gondii is a parasite found worldwide, with the ability to infect most warm-blooded animals, including humans (Dubey, 2008, Wallander et al., 2015). The parasite has a complex life cycle with felines as a definitive host (Herrmann et al., 2012), although many other animal species can act as potential intermediate hosts. Infection may occur by the ingestion of oocysts excreted to the environment with the feces of the definitive hosts, or by consuming meat containing tissue cysts. Additionally, transplacental transmision of T. gondii from mother to fetus has been reported. Most animals infected by T. gondii do not show any clinical sympthoms of parasitosis (Śmielewska-Łoś and Turniak, 2004). The parasite affects several organs; however, the predilection sites are the lungs, central nervous system and eyes (Dubey, 2008).

The most commonly-used, and the fastest, way to confirm that an examined animal has been in contact with the parasite is by the detection of antibodies in serum samples. Several different immunological tests for the detection of T. gondii-specific immunoglobulin (IgG and IgM) are commercially available, with the most frequently-used methods in seroprevalence studies being DAT and ELISA (Mainar-Jaime and Barberán, 2007, Wallander et al., 2015). ELISA is a serological test used to detect anti-Toxoplasma antibodies in serum and meat juice in different animal species. It is easy to perform and large number of samples can be tested in a short time. However, studies on the seroprevalence of T. gondii antibodies in wild animals suggest that DAT is equally useful. Using this method, Jokelainen et al. (2015) found the seroprevalence of T. gondii to be 23.99% in meat juice samples from free-roaming wild boars. Infection and disease can be detected with greater specificity by molecular methods of diagnosis such as the polymerase chain reaction (PCR). This method has been proven to be a valuable method of detecting T. gondii DNA in both domestic and wild animals, such as sheep, goats, pigs and mink (Hůrková and Modrý, 2006, Shaapan et al., 2008, Turčeková et al., 2014). Of the numerous DNA sequences which could be used for PCR analysis, the

TGR1E gene has been suggested as the most suitable for detecting toxoplasmosis in wildlife animals (Hill et al., 2005, Luptakova et al., 2010, Turčeková et al., 2014).

The present study compares the usefulness of three methods of detecting T. gondii in samples taken from wild animals: commercial DAT and ELISA tests to detect antibodies against T. gondii, and PCR to confirm the presence of DNA.

Section snippets

Sample collection

A total of 129 animals were examined, including 102 red foxes (Vulpes vulpes), 12 raccoon dogs (Nyctereutes procyonoides), eight badgers (Meles meles), five martens (Martes martes), one mink (Neovison vison) and one polecat (Mustela putorius). The animals were taken from the Głeboki Bród Forest District (Fig. 1). Samples of tongue, diaphragm and brain were collected individually and delivered frozen for further examination.

The meat juice samples were collected from tongue and diaphragm, and

DNA isolation

Brain samples were prepared as described by Dubey (1998), with some modification. Briefly, tissue was digested for one hour with HCl/pepsin/H₂O solution (2.10 ml/3 g/300 ml), homogenate was centrifuged and the pellet was used for further DNA isolation. Three samples (300 μl each) were taken from each pellet for genomic DNA extraction by a commercially available NucleoSpin Tissue kit (Machery Nagel, Germany) according to the manufacturer’s instructions.

PCR

Amplification of the isolated DNA was carried

Statistical analysis

Statistical analyses were performed using Microsoft Excel 2008 (Microsoft Corporation, Redmond, USA). The positive and negative results obtained by DAT, ELISA and PCR were classified into two-by-two contingency tables. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using ELISA as a reference method. Additionally, the results were analyzed based on binominal analysis, by calculating the proportion of positive (P_A), negative (N_A) and overal (p_o)

Results

T. gondii antibodies were detected in 95 of the 129 (73.6%) examinated samples in DAT and ELISA separately. However, only 84 samples gave a positive result for both immunological tests. Antibodies against T. gondii were found in 77. 5% red foxes, 12.5% badgers, 40% martens and 8.3% raccoon dogs. Antibodies against the parasite were found also in one mink but not in the polecat sample. The detailed results for three tests are presented in Table 1. Of the 129 brain samples, the presence of T.

Discussion

Serological assays, such as ELISA and DAT, are important tools for the diagnosis of toxoplasmosis in animals and humans, as well as for performing epidemiological studies. Many commercially available tests are available for the detection of specific antibodies against T. gondii in various types of samples. For example, a variety of ELISA systems have been developed to analyse sera, plasma or meat juice obtained from meat-producing animals such as pigs and ruminants (Witkowski et al., 2015).

Conclusion

Most of the serological tests have not been validated for wild carnivores and omnivores samples. Due to the specificity, sensitivity and cut-off value which have not been evaluated, more than one serological test is advisable in the analysis of the prevalence of T. gondii in wildlife. Results described in this study indicated that both ELISA and DAT are simple and rapid tools for screening meat juice samples collected from wild animals for Toxoplasma infection. PCR could be used as confirmatory

Acknowledgments

The paper is a result of reaserch work done within the frames of internal project for young scientists state-funded by the Ministry of National Education of Poland. The authors would like to express their gratitude to the Forest District Manager of the Forest District Głęboki Bród, Mr. Tadeusz Wilczyński MSc, for his help in collecting the materials.

Animals were collected as a part of the project LIFE11 NAT/PL428 “Active protection of lowland population of capercaillie (Tetrao urogallus L.) in

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Research paperThe usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals

Highlights

Abstract

Introduction

Section snippets

Sample collection

DNA isolation

PCR

Statistical analysis

Results

Discussion

Conclusion

Acknowledgments

Vet. Parasitol.

J. Clin. Eidemiol.

Vet. Parasitol.

Vet. Parasitol.

Int. J. Parasitol. Parasites. Wildl.

Vet. Parasitol.

Vet. Parasitol.

Clin. Mic. Inf.

Vet. Parasitol.

Vet. Parasitol.

Vet. Parasitol.

Vet. Parasitol.

Risk factors for Toxoplasma gondii exposure in semiaquatic mammals in a freshwater ecosystem

J. Wildl. Dis.

Antibodies to Toxoplasma gondii in Eurasian badgers

J. Wildl. Dis.

Research paper
The usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals