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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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Morphological differentiation of Baylisascaris procyonis (A, D) from B. columnaris (B, E and C, F). A - C: Unstained cross section of lateral cuticle through anterior end of adult female worms (about 10 centimetres in length), 6 millimetres behind mouthparts and rotated 90° in plane. Cross section in lateral view showing outer cuticle lining (ol), inner cuticle lining (il), hypodermis (h) and cervical support in hyaline layer (arrow). Sometimes, cervical supports of the deeper layer are visible, though not in focus (arrowhead). A: Female B. procyonis (isolate Bp9), wide arch-like cervical support B: Female B. columnaris (K19), narrow A-like support C: Female B. columnaris (K10), narrow A-like support. D - F: Posterior end of males showing pre-cloacal papillae (p), pericloacal roughened areas (R) with rounded posterior margin in figure E, bare pre-cloacal rim (b), extruded spicules (S, not in focus in D), post- cloacal papillae (short arrow), terminal part of tail knob (D) or spike (G; E and F out of focus) shaped (long arrow). D: Ventrolateral view of B. procyonis (Bp19), precloacal roughened patch 42 μm and postcloacal patch 72 μm in width. E: Lateral view of B. columnaris (K23), F: Lateral view of B. columnaris (K22). G: Lateral view of posterior end of male B. columnaris (P27), showing spike shaped posterior end of the tail (arrow) and pericloacal roughened patches in close up; precloacal patch (right) 63 μm and postcloacal patch (left) 79 μm in width. Scale bars in μm. See Table 3 for molecular classification of isolate numbers.
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Figure 1: Morphological differentiation of Baylisascaris procyonis (A, D) from B. columnaris (B, E and C, F). A - C: Unstained cross section of lateral cuticle through anterior end of adult female worms (about 10 centimetres in length), 6 millimetres behind mouthparts and rotated 90° in plane. Cross section in lateral view showing outer cuticle lining (ol), inner cuticle lining (il), hypodermis (h) and cervical support in hyaline layer (arrow). Sometimes, cervical supports of the deeper layer are visible, though not in focus (arrowhead). A: Female B. procyonis (isolate Bp9), wide arch-like cervical support B: Female B. columnaris (K19), narrow A-like support C: Female B. columnaris (K10), narrow A-like support. D - F: Posterior end of males showing pre-cloacal papillae (p), pericloacal roughened areas (R) with rounded posterior margin in figure E, bare pre-cloacal rim (b), extruded spicules (S, not in focus in D), post- cloacal papillae (short arrow), terminal part of tail knob (D) or spike (G; E and F out of focus) shaped (long arrow). D: Ventrolateral view of B. procyonis (Bp19), precloacal roughened patch 42 μm and postcloacal patch 72 μm in width. E: Lateral view of B. columnaris (K23), F: Lateral view of B. columnaris (K22). G: Lateral view of posterior end of male B. columnaris (P27), showing spike shaped posterior end of the tail (arrow) and pericloacal roughened patches in close up; precloacal patch (right) 63 μm and postcloacal patch (left) 79 μm in width. Scale bars in μm. See Table 3 for molecular classification of isolate numbers.

Mentions: In total, 119 B. columnaris worms were isolated from seven skunks (Table 1). Among these are five stray animals (D, N, V, C and A). Worm body size varied, ranging from 3–4 centimetres for adult males and young females, to a maximum of 14 centimetres for adult females. All worms were morphologically identified as Baylisascaris sp., based on the absence of cervical alae in adult worms, the morphology and size of intra-uterine eggs (76.7 ± 5.1 by 64.6 ± 5.8 μm, n=10) that were isolated from gravid females, the morphology and size of faecal eggs (72.5 ± 4.1 by 63.2 ± 7.5 μm, n=10) in coprological examination, the typical ascarid mouthparts with three developed lips (one dorsal and two subventral), the presence of precloacal genital papillae, two spicules and perianal roughened patches in adult males. Species identification was carried out using differentiating features as defined by Sprent (1968)[22], which can discriminate between different Baylisascaris species. Figure 1 shows characteristic morphological features of B. procyonis (A and D), and B. columnaris (B, E and C, F) isolates of this present study. Figure 1G zooms in on typical male B. columnaris features, being perianal roughened patches of more or less equal width and the spike-shaped terminal end of the tail.


Molecular analysis of Baylisascaris columnaris revealed mitochondrial and nuclear polymorphisms.

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

Morphological differentiation of Baylisascaris procyonis (A, D) from B. columnaris (B, E and C, F). A - C: Unstained cross section of lateral cuticle through anterior end of adult female worms (about 10 centimetres in length), 6 millimetres behind mouthparts and rotated 90° in plane. Cross section in lateral view showing outer cuticle lining (ol), inner cuticle lining (il), hypodermis (h) and cervical support in hyaline layer (arrow). Sometimes, cervical supports of the deeper layer are visible, though not in focus (arrowhead). A: Female B. procyonis (isolate Bp9), wide arch-like cervical support B: Female B. columnaris (K19), narrow A-like support C: Female B. columnaris (K10), narrow A-like support. D - F: Posterior end of males showing pre-cloacal papillae (p), pericloacal roughened areas (R) with rounded posterior margin in figure E, bare pre-cloacal rim (b), extruded spicules (S, not in focus in D), post- cloacal papillae (short arrow), terminal part of tail knob (D) or spike (G; E and F out of focus) shaped (long arrow). D: Ventrolateral view of B. procyonis (Bp19), precloacal roughened patch 42 μm and postcloacal patch 72 μm in width. E: Lateral view of B. columnaris (K23), F: Lateral view of B. columnaris (K22). G: Lateral view of posterior end of male B. columnaris (P27), showing spike shaped posterior end of the tail (arrow) and pericloacal roughened patches in close up; precloacal patch (right) 63 μm and postcloacal patch (left) 79 μm in width. Scale bars in μm. See Table 3 for molecular classification of isolate numbers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Morphological differentiation of Baylisascaris procyonis (A, D) from B. columnaris (B, E and C, F). A - C: Unstained cross section of lateral cuticle through anterior end of adult female worms (about 10 centimetres in length), 6 millimetres behind mouthparts and rotated 90° in plane. Cross section in lateral view showing outer cuticle lining (ol), inner cuticle lining (il), hypodermis (h) and cervical support in hyaline layer (arrow). Sometimes, cervical supports of the deeper layer are visible, though not in focus (arrowhead). A: Female B. procyonis (isolate Bp9), wide arch-like cervical support B: Female B. columnaris (K19), narrow A-like support C: Female B. columnaris (K10), narrow A-like support. D - F: Posterior end of males showing pre-cloacal papillae (p), pericloacal roughened areas (R) with rounded posterior margin in figure E, bare pre-cloacal rim (b), extruded spicules (S, not in focus in D), post- cloacal papillae (short arrow), terminal part of tail knob (D) or spike (G; E and F out of focus) shaped (long arrow). D: Ventrolateral view of B. procyonis (Bp19), precloacal roughened patch 42 μm and postcloacal patch 72 μm in width. E: Lateral view of B. columnaris (K23), F: Lateral view of B. columnaris (K22). G: Lateral view of posterior end of male B. columnaris (P27), showing spike shaped posterior end of the tail (arrow) and pericloacal roughened patches in close up; precloacal patch (right) 63 μm and postcloacal patch (left) 79 μm in width. Scale bars in μm. See Table 3 for molecular classification of isolate numbers.
Mentions: In total, 119 B. columnaris worms were isolated from seven skunks (Table 1). Among these are five stray animals (D, N, V, C and A). Worm body size varied, ranging from 3–4 centimetres for adult males and young females, to a maximum of 14 centimetres for adult females. All worms were morphologically identified as Baylisascaris sp., based on the absence of cervical alae in adult worms, the morphology and size of intra-uterine eggs (76.7 ± 5.1 by 64.6 ± 5.8 μm, n=10) that were isolated from gravid females, the morphology and size of faecal eggs (72.5 ± 4.1 by 63.2 ± 7.5 μm, n=10) in coprological examination, the typical ascarid mouthparts with three developed lips (one dorsal and two subventral), the presence of precloacal genital papillae, two spicules and perianal roughened patches in adult males. Species identification was carried out using differentiating features as defined by Sprent (1968)[22], which can discriminate between different Baylisascaris species. Figure 1 shows characteristic morphological features of B. procyonis (A and D), and B. columnaris (B, E and C, F) isolates of this present study. Figure 1G zooms in on typical male B. columnaris features, being perianal roughened patches of more or less equal width and the spike-shaped terminal end of the tail.

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