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Molecular analysis of Baylisascaris columnaris revealed mitochondrial and nuclear polymorphisms.

Franssen F, Xie K, Sprong H, van der Giessen J - Parasit Vectors (2013)

Bottom Line: Four different multi-locus genotypes were found in B. columnaris, based on 14 single nucleotide polymorphisms (SNPs) and two insertions / deletions in CO1, CO2, ITS1-5.8S-ITS2 and 28S.These polymorphisms could be used as a tool to differentiate B. columnaris from B. procyonis in molecular diagnostic assays, and to identify B. columnaris by PCR, in addition to or replacing morphometric analysis.This might lead to more insight into the zoonotic relevance of B. columnaris in humans.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute for Public Health and Environment, RIVM, Center for Zoonoses and Environmental Microbiology, cZ&O, Antonie van Leeuwenhoeklaan 9, PO Box 1, Bilthoven 3720 BA, The Netherlands. frits.franssen@rivm.nl

ABSTRACT

Background: Baylisascaris species are intestinal nematodes of skunks, raccoons, badgers, and bears belonging to the genus Ascarididae. Oral uptake of embryonated Baylisascaris sp. eggs by a wide variety of mammals and birds can lead to visceral, ocular and neurological larva migrans. B. procyonis, the raccoon roundworm, is known to cause severe illness in intermediate hosts and in humans, whereas the skunk roundworm B. columnaris is probably less pathogenic. Skunks and raccoons are kept as pets in Europe, sometimes together with cats and dogs, living in close contact with humans. B. procyonis and B. columnaris are difficult to differentiate based on morphological criteria and molecular and phylogenetic information concerning B. columnaris is missing. This is the first study on the genetic characterisation of B. columnaris, based on mitochondrial and nuclear molecular markers.

Methods: B. columnaris worms were isolated from pet skunks, and used for molecular analysis. PCR primers targeted at mitochondrial cytochrome c oxidase 1 and 2 (CO1 and CO2), ribosomal ITS1-5.8S-ITS2 and ribosomal 28S genes were used. DNA sequences from B. columnaris, B. procyonis and B. transfuga from bears were analysed by cluster analysis.

Results: Four different multi-locus genotypes were found in B. columnaris, based on 14 single nucleotide polymorphisms (SNPs) and two insertions / deletions in CO1, CO2, ITS1-5.8S-ITS2 and 28S.

Conclusions: The genetic characteristics of B. columnaris show close resemblance to those of B. procyonis, but in contrast to B. procyonis, show several polymorphisms in both mitochondrial and nuclear markers. These polymorphisms could be used as a tool to differentiate B. columnaris from B. procyonis in molecular diagnostic assays, and to identify B. columnaris by PCR, in addition to or replacing morphometric analysis. This might lead to more insight into the zoonotic relevance of B. columnaris in humans.

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G-A tandem repeats with different length on the ITS2 gene.B. procyonis has 9 G-A tandem repeats on the ITS2 gene, where B. columnaris has two tandem repeats of different length, six or seven G-A repeats. B. transfuga has a much shorter G-A tandem repeat sequence, which is one nucleotide shorter and different from the tandem repeat in B. schroederi.
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Figure 2: G-A tandem repeats with different length on the ITS2 gene.B. procyonis has 9 G-A tandem repeats on the ITS2 gene, where B. columnaris has two tandem repeats of different length, six or seven G-A repeats. B. transfuga has a much shorter G-A tandem repeat sequence, which is one nucleotide shorter and different from the tandem repeat in B. schroederi.

Mentions: Ribosomal ITS1-5.8S-ITS2 amplicons (644 or 647 bp) were obtained from fifteen B. columnaris isolates, twelve B. procyonis isolates (650 bp) and two B. transfuga isolates (630 bp). Sequence alignment revealed an insertion as compared to B. procyonis in the ITS1 gene (nt 1–228) at nucleotide position 152 and a SNP at nt 201. The sequence of 5.8S rDNA (nt 229–385) was homologous between B. columnaris and B. procyonis isolates, but differed from B. transfuga (nt 230, 337 and 346) (Table 4). In the ITS2 gene region (nt 386 and further), a tandem repeat was found commencing at nucleotide position 526 and containing a number of G-A inserts varying with Baylisascaris species. Nine G-A tandem repeats were identified for B. procyonis, which was corroborated by comparison with Genbank accession number AB051231. Two genotypes were found in B. columnaris: one with six G-A tandem repeats and another with seven. B. transfuga had only two G-A tandem repeats, which was corroborated by Genbank accession number EU642819, and was different from the tandem repeat in B. schroederi [JN210911 and JN210912] (Figure 2).


Molecular analysis of Baylisascaris columnaris revealed mitochondrial and nuclear polymorphisms.

Franssen F, Xie K, Sprong H, van der Giessen J - Parasit Vectors (2013)

G-A tandem repeats with different length on the ITS2 gene.B. procyonis has 9 G-A tandem repeats on the ITS2 gene, where B. columnaris has two tandem repeats of different length, six or seven G-A repeats. B. transfuga has a much shorter G-A tandem repeat sequence, which is one nucleotide shorter and different from the tandem repeat in B. schroederi.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643864&req=5

Figure 2: G-A tandem repeats with different length on the ITS2 gene.B. procyonis has 9 G-A tandem repeats on the ITS2 gene, where B. columnaris has two tandem repeats of different length, six or seven G-A repeats. B. transfuga has a much shorter G-A tandem repeat sequence, which is one nucleotide shorter and different from the tandem repeat in B. schroederi.
Mentions: Ribosomal ITS1-5.8S-ITS2 amplicons (644 or 647 bp) were obtained from fifteen B. columnaris isolates, twelve B. procyonis isolates (650 bp) and two B. transfuga isolates (630 bp). Sequence alignment revealed an insertion as compared to B. procyonis in the ITS1 gene (nt 1–228) at nucleotide position 152 and a SNP at nt 201. The sequence of 5.8S rDNA (nt 229–385) was homologous between B. columnaris and B. procyonis isolates, but differed from B. transfuga (nt 230, 337 and 346) (Table 4). In the ITS2 gene region (nt 386 and further), a tandem repeat was found commencing at nucleotide position 526 and containing a number of G-A inserts varying with Baylisascaris species. Nine G-A tandem repeats were identified for B. procyonis, which was corroborated by comparison with Genbank accession number AB051231. Two genotypes were found in B. columnaris: one with six G-A tandem repeats and another with seven. B. transfuga had only two G-A tandem repeats, which was corroborated by Genbank accession number EU642819, and was different from the tandem repeat in B. schroederi [JN210911 and JN210912] (Figure 2).

Bottom Line: Four different multi-locus genotypes were found in B. columnaris, based on 14 single nucleotide polymorphisms (SNPs) and two insertions / deletions in CO1, CO2, ITS1-5.8S-ITS2 and 28S.These polymorphisms could be used as a tool to differentiate B. columnaris from B. procyonis in molecular diagnostic assays, and to identify B. columnaris by PCR, in addition to or replacing morphometric analysis.This might lead to more insight into the zoonotic relevance of B. columnaris in humans.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute for Public Health and Environment, RIVM, Center for Zoonoses and Environmental Microbiology, cZ&O, Antonie van Leeuwenhoeklaan 9, PO Box 1, Bilthoven 3720 BA, The Netherlands. frits.franssen@rivm.nl

ABSTRACT

Background: Baylisascaris species are intestinal nematodes of skunks, raccoons, badgers, and bears belonging to the genus Ascarididae. Oral uptake of embryonated Baylisascaris sp. eggs by a wide variety of mammals and birds can lead to visceral, ocular and neurological larva migrans. B. procyonis, the raccoon roundworm, is known to cause severe illness in intermediate hosts and in humans, whereas the skunk roundworm B. columnaris is probably less pathogenic. Skunks and raccoons are kept as pets in Europe, sometimes together with cats and dogs, living in close contact with humans. B. procyonis and B. columnaris are difficult to differentiate based on morphological criteria and molecular and phylogenetic information concerning B. columnaris is missing. This is the first study on the genetic characterisation of B. columnaris, based on mitochondrial and nuclear molecular markers.

Methods: B. columnaris worms were isolated from pet skunks, and used for molecular analysis. PCR primers targeted at mitochondrial cytochrome c oxidase 1 and 2 (CO1 and CO2), ribosomal ITS1-5.8S-ITS2 and ribosomal 28S genes were used. DNA sequences from B. columnaris, B. procyonis and B. transfuga from bears were analysed by cluster analysis.

Results: Four different multi-locus genotypes were found in B. columnaris, based on 14 single nucleotide polymorphisms (SNPs) and two insertions / deletions in CO1, CO2, ITS1-5.8S-ITS2 and 28S.

Conclusions: The genetic characteristics of B. columnaris show close resemblance to those of B. procyonis, but in contrast to B. procyonis, show several polymorphisms in both mitochondrial and nuclear markers. These polymorphisms could be used as a tool to differentiate B. columnaris from B. procyonis in molecular diagnostic assays, and to identify B. columnaris by PCR, in addition to or replacing morphometric analysis. This might lead to more insight into the zoonotic relevance of B. columnaris in humans.

Show MeSH
Related in: MedlinePlus