Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA

Abstract

A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers for Echinococcus multilocularis (amplicon size 395 bp) were species-specific as assessed by in silico analysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA from E. granulosus or Taenia spp. was not possible. The primers designed for E. granulosus also amplified DNA (117 bp) from E. vogeli, and those designed for Taenia spp. amplified products (267 bp) from species of Mesocestoides, Dipylidium and Diphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the described E. granulosus genotypes. Taenia spp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.

Echinococcosis in pigs and intestinal infection with Echinococcus spp. in dogs in southwestern Lithuania

“…However, for a better understanding of the epidemiology of E. granulosus in the other parts of the country, further studies should be initiated in Lithuania. c PCR with samples containing taeniid eggs (Trachsel et al, 2007). Three samples with coinfections E. granulosus/Taenia spp.…”

“…DNA extraction from taeniid eggs isolated from faeces was performed as described (Stefanić et al, 2004), and multiplex PCR according to Trachsel et al (2007) for the simultaneous detection of E. granulosus (all strains), E. multilocularis and Taenia spp. was performed.…”

Retrospective and spatial analysis tools for integrated surveillance of cystic echinococcosis and bovine cysticercosis in hypo-endemic areas

“…The presence of taeniid eggs was detected by means of an inverted microscope in a 10 ml closed tube with flattened side, from which the liquid obtained at the end of the isolation procedure was carefully poured into a 15-ml conical Falcon ® centrifuge tube. Positive samples were investigated by polymerase chain reaction (PCR) (Trachsel et al, 2007) after further sedimentation for 30 min, and aspiration of excess fluid from the tube, in order to ensure a final volume of about 0.5 ml. The PCR results were confirmed by the National Reference Centre for Echinococcus granulosus (CeNRE, IZS Sardinia).…”

Fatal Alveolar Echinococcosis of the Lumbar Spine

“…A 395-bp fragment specific to E. multilocularis was detected and led to the final diagnosis. This PCR targets mitochondrial genes for NADPH dehydrogenase subunit 1, cytochrome oxidase subunit 1, and the small subunit of rRNA of taeniids, as described by Trachsel et al (1). The distinction between E. granulosus, E. multilocularis, and other Taenia spp.…”

“…If E. multilocularis lesions are suspected on imaging results, the diagnosis should include a species-specific serology such as ELISA or immunodiffusion combined with Western blotting using Em2 and/or Em18 antigens (1,19), PCR of biopsy specimens, and, if feasible or if PCR could not be done, immunochemistry (12,20,21). The serological tests using purified and/or recombinant Em2 antigens have a high sensitivity of 90 to 100% and a specificity of 95 to 100% (22).…”