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The mitochondrial genome of Toxocara canis.

Jex AR, Waeschenbach A, Littlewood DT, Hu M, Gasser RB - PLoS Negl Trop Dis (2008)

Bottom Line: In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known.In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy.This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secementean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida).

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia. ajex@unimelb.edu.au

ABSTRACT
Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secementean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts.

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A map of the circular mitochondrial genome (mtDNA) of Toxocara canis.All 12 protein-coding genes and the large and small ribosomal subunits of the rRNA genes are indicated in italics. Each tRNA gene is identified by its anticodon (in brackets). The direction of transcription is indicated by an arrow. The positions of oligonucleotide primers (see table) used for PCR-amplification or sequencing are indicated in the map (drawn to scale).
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pntd-0000273-g001: A map of the circular mitochondrial genome (mtDNA) of Toxocara canis.All 12 protein-coding genes and the large and small ribosomal subunits of the rRNA genes are indicated in italics. Each tRNA gene is identified by its anticodon (in brackets). The direction of transcription is indicated by an arrow. The positions of oligonucleotide primers (see table) used for PCR-amplification or sequencing are indicated in the map (drawn to scale).

Mentions: Using each of the primer pairs MH39F-MH42R and MH5F-MH40R [30],[35],[36], two regions of the entire mitochondrial genome (of ∼5 and 10 kb, respectively) were amplified by the long PCR (Expand 20 kbPLUS kit, Roche) from ∼20 ng of genomic DNA from sample Tcn2. The cycling conditions in a 2400 thermocycler (Perkin Elmer Cetus) were: 92°C, 2 min (initial denaturation); then 92°C, 10 s (denaturation); 50°C, 30 s (annealing); 60°C (∼5 kb region) or 68°C (∼10 kb region), 10 min (extension) for 10 cycles, followed by 92°C, 10 s; 50°C, 30 s; 68°C or 60°C, 10 min for 20 cycles, with an elongation of 10 s for each cycle, and a final extension at 68°C or 60°C for 7 min [30]. Each PCR yielded a single amplicon, detected by agarose gel electrophoresis [30]. Each amplicon was column-purified (PCR-Preps, Promega) and subjected to automated sequencing, either directly or following cloning (TOPO XL PCR cloning kit, Invitrogen, according to instructions provided), employing a “primer-walking” strategy [36] (see Figure 1). Sequencing was performed using BigDye terminator (v.3.1) in a 3730 DNA Analyser (Applied Biosystems). The sequences obtained were assembled manually, aligned with the mitochondrial genome sequence of Ascaris suum [18] using the program Clustal X [37], and the circular map was drawn using the program MacVector v.9.5 (http://www.macvector.com/index.html). Amino acid sequences, translation initiation and termination codons, codon usage, transfer RNA (tRNA or trn) secondary structures, rRNA secondary structures and non-coding regions were predicted using standard approaches [35]. The structure and organization of the mitochondrial genome of T. canis was then compared with those of the nematodes Anisakis simplex (GenBank accession number AY994157; ref. [17]), Ascaris suum (X53453; ref. [18]) (order Ascaridida); Brugia malayi (AF538716; ref. [38]), Dirofilaria immitis (AJ537512; ref. [39]) and Onchocerca volvulus (AF015193; ref. [40]) (order Spirurida); Ancylostoma duodenale (AJ417718; ref. [35]) and Necator americanus (AJ417719; ref. [35]) (order Strongylida).


The mitochondrial genome of Toxocara canis.

Jex AR, Waeschenbach A, Littlewood DT, Hu M, Gasser RB - PLoS Negl Trop Dis (2008)

A map of the circular mitochondrial genome (mtDNA) of Toxocara canis.All 12 protein-coding genes and the large and small ribosomal subunits of the rRNA genes are indicated in italics. Each tRNA gene is identified by its anticodon (in brackets). The direction of transcription is indicated by an arrow. The positions of oligonucleotide primers (see table) used for PCR-amplification or sequencing are indicated in the map (drawn to scale).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483351&req=5

pntd-0000273-g001: A map of the circular mitochondrial genome (mtDNA) of Toxocara canis.All 12 protein-coding genes and the large and small ribosomal subunits of the rRNA genes are indicated in italics. Each tRNA gene is identified by its anticodon (in brackets). The direction of transcription is indicated by an arrow. The positions of oligonucleotide primers (see table) used for PCR-amplification or sequencing are indicated in the map (drawn to scale).
Mentions: Using each of the primer pairs MH39F-MH42R and MH5F-MH40R [30],[35],[36], two regions of the entire mitochondrial genome (of ∼5 and 10 kb, respectively) were amplified by the long PCR (Expand 20 kbPLUS kit, Roche) from ∼20 ng of genomic DNA from sample Tcn2. The cycling conditions in a 2400 thermocycler (Perkin Elmer Cetus) were: 92°C, 2 min (initial denaturation); then 92°C, 10 s (denaturation); 50°C, 30 s (annealing); 60°C (∼5 kb region) or 68°C (∼10 kb region), 10 min (extension) for 10 cycles, followed by 92°C, 10 s; 50°C, 30 s; 68°C or 60°C, 10 min for 20 cycles, with an elongation of 10 s for each cycle, and a final extension at 68°C or 60°C for 7 min [30]. Each PCR yielded a single amplicon, detected by agarose gel electrophoresis [30]. Each amplicon was column-purified (PCR-Preps, Promega) and subjected to automated sequencing, either directly or following cloning (TOPO XL PCR cloning kit, Invitrogen, according to instructions provided), employing a “primer-walking” strategy [36] (see Figure 1). Sequencing was performed using BigDye terminator (v.3.1) in a 3730 DNA Analyser (Applied Biosystems). The sequences obtained were assembled manually, aligned with the mitochondrial genome sequence of Ascaris suum [18] using the program Clustal X [37], and the circular map was drawn using the program MacVector v.9.5 (http://www.macvector.com/index.html). Amino acid sequences, translation initiation and termination codons, codon usage, transfer RNA (tRNA or trn) secondary structures, rRNA secondary structures and non-coding regions were predicted using standard approaches [35]. The structure and organization of the mitochondrial genome of T. canis was then compared with those of the nematodes Anisakis simplex (GenBank accession number AY994157; ref. [17]), Ascaris suum (X53453; ref. [18]) (order Ascaridida); Brugia malayi (AF538716; ref. [38]), Dirofilaria immitis (AJ537512; ref. [39]) and Onchocerca volvulus (AF015193; ref. [40]) (order Spirurida); Ancylostoma duodenale (AJ417718; ref. [35]) and Necator americanus (AJ417719; ref. [35]) (order Strongylida).

Bottom Line: In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known.In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy.This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secementean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida).

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia. ajex@unimelb.edu.au

ABSTRACT
Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secementean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts.

Show MeSH
Related in: MedlinePlus