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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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Maximum likelihood trees inferred from Baylisascaris sp. mitochondrial and ribosomal gene sequences. A: ML of concatenated mitochondrial markers CO1 and CO2 shows a clear separation between B. procyonis and B. columnaris, which in turn is divided into two different groups. B: ML of concatenated ribosomal markers ITS1-5.8S-ITS2 and 28S again shows two B. columnaris groups, which separate from B. procyonis, although to a lesser extent, due to the number of G-A repeats (9, 7 and 6) as most important difference (see also Table 4).
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Figure 4: Maximum likelihood trees inferred from Baylisascaris sp. mitochondrial and ribosomal gene sequences. A: ML of concatenated mitochondrial markers CO1 and CO2 shows a clear separation between B. procyonis and B. columnaris, which in turn is divided into two different groups. B: ML of concatenated ribosomal markers ITS1-5.8S-ITS2 and 28S again shows two B. columnaris groups, which separate from B. procyonis, although to a lesser extent, due to the number of G-A repeats (9, 7 and 6) as most important difference (see also Table 4).

Mentions: The inferred ML tree of concatenated CO1 and CO2 sequences shows two B. columnaris clusters (type 1 and 2, 15 isolates, and type 3 and 4, 23 isolates), which are separated from B. procyonis, supported by high bootstrap values (Figure 4A). The B. columnaris intra-species difference based on concatenated CO1 and CO2 sequences was 0.68%, whereas the inter-species differences with B. procyonis were 0.71% and 0.91% respectively. An inferred ML tree from concatenated ribosomal markers (ITS1-5.8S-ITS2 and 28S) again revealed two B. columnaris groups which diverge from B. procyonis (Figure 4B), but are not identical to the ML shown in Figure 4A. However, this is based on only three SNPs and one nucleotide insert, which is too few to obtain a reliable differentiation.


Molecular analysis of Baylisascaris columnaris revealed mitochondrial and nuclear polymorphisms.

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

Maximum likelihood trees inferred from Baylisascaris sp. mitochondrial and ribosomal gene sequences. A: ML of concatenated mitochondrial markers CO1 and CO2 shows a clear separation between B. procyonis and B. columnaris, which in turn is divided into two different groups. B: ML of concatenated ribosomal markers ITS1-5.8S-ITS2 and 28S again shows two B. columnaris groups, which separate from B. procyonis, although to a lesser extent, due to the number of G-A repeats (9, 7 and 6) as most important difference (see also Table 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Maximum likelihood trees inferred from Baylisascaris sp. mitochondrial and ribosomal gene sequences. A: ML of concatenated mitochondrial markers CO1 and CO2 shows a clear separation between B. procyonis and B. columnaris, which in turn is divided into two different groups. B: ML of concatenated ribosomal markers ITS1-5.8S-ITS2 and 28S again shows two B. columnaris groups, which separate from B. procyonis, although to a lesser extent, due to the number of G-A repeats (9, 7 and 6) as most important difference (see also Table 4).
Mentions: The inferred ML tree of concatenated CO1 and CO2 sequences shows two B. columnaris clusters (type 1 and 2, 15 isolates, and type 3 and 4, 23 isolates), which are separated from B. procyonis, supported by high bootstrap values (Figure 4A). The B. columnaris intra-species difference based on concatenated CO1 and CO2 sequences was 0.68%, whereas the inter-species differences with B. procyonis were 0.71% and 0.91% respectively. An inferred ML tree from concatenated ribosomal markers (ITS1-5.8S-ITS2 and 28S) again revealed two B. columnaris groups which diverge from B. procyonis (Figure 4B), but are not identical to the ML shown in Figure 4A. However, this is based on only three SNPs and one nucleotide insert, which is too few to obtain a reliable differentiation.

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