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Absence of Wolbachia endobacteria in the non-filariid nematodes Angiostrongylus cantonensis and A. costaricensis.

Foster JM, Kumar S, Ford L, Johnston KL, Ben R, Graeff-Teixeira C, Taylor MJ - Parasit Vectors (2008)

Bottom Line: We were unable to detect Wolbachia in either species using these methodologies.In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes.This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. mark.taylor@liverpool.ac.uk.

ABSTRACT
The majority of filarial nematodes harbour Wolbachia endobacteria, including the major pathogenic species in humans, Onchocerca volvulus, Brugia malayi and Wuchereria bancrofti. These obligate endosymbionts have never been demonstrated unequivocally in any non-filariid nematode. However, a recent report described the detection by PCR of Wolbachia in the metastrongylid nematode, Angiostrongylus cantonensis (rat lungworm), a leading cause of eosinophilic meningitis in humans. To address the intriguing possibility of Wolbachia infection in nematode species distinct from the Family Onchocercidae, we used both PCR and immunohistochemistry to screen samples of A. cantonensis and A. costaricensis for the presence of this endosymbiont. We were unable to detect Wolbachia in either species using these methodologies. In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes. This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples.

No MeSH data available.


Related in: MedlinePlus

Minimum evolution trees based on alignments of A) the Wolbachia16S (770 nucleotides) reported from A. cantonensis[GenBank:AY652762], B) the Wolbachia 16S (639 nucleotides) of D. circumlita c2 [GenBank:AY486072], and C) the Wolbachia ftsZ (431 nucleotides) reported from A. cantonensis [GenBank:DQ159068 ]. Sequences were aligned using ClustalX version 2.0.7 [14] using default parameters for slow/accurate alignment [Gap Opening:10, Gap Extend: 0.1, IUB DNA weight matrix]. After alignment, sequences were manually trimmed to the endpoints of the 16S sequences of the Wolbachia from A. cantonensis (see additional file 1: Wolbachia 16S multiple sequence alignment – A. cantonensis) and D. circumlita (see additional file 2: Wolbachia 16S multiple sequence alignment – D. circumlita), and to the endpoints of the ftsZ sequence of the Wolbachia from A. cantonensis (see additional file 3: Wolbachia ftsZ multiple sequence alignment). Phylogenetic trees were calculated using the Minimum Evolution method in MEGA4 [15]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary distances were computed using the Maximum Composite Likelihood. The Minimum Evolution tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option).
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Figure 1: Minimum evolution trees based on alignments of A) the Wolbachia16S (770 nucleotides) reported from A. cantonensis[GenBank:AY652762], B) the Wolbachia 16S (639 nucleotides) of D. circumlita c2 [GenBank:AY486072], and C) the Wolbachia ftsZ (431 nucleotides) reported from A. cantonensis [GenBank:DQ159068 ]. Sequences were aligned using ClustalX version 2.0.7 [14] using default parameters for slow/accurate alignment [Gap Opening:10, Gap Extend: 0.1, IUB DNA weight matrix]. After alignment, sequences were manually trimmed to the endpoints of the 16S sequences of the Wolbachia from A. cantonensis (see additional file 1: Wolbachia 16S multiple sequence alignment – A. cantonensis) and D. circumlita (see additional file 2: Wolbachia 16S multiple sequence alignment – D. circumlita), and to the endpoints of the ftsZ sequence of the Wolbachia from A. cantonensis (see additional file 3: Wolbachia ftsZ multiple sequence alignment). Phylogenetic trees were calculated using the Minimum Evolution method in MEGA4 [15]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary distances were computed using the Maximum Composite Likelihood. The Minimum Evolution tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option).

Mentions: We performed similar nucleotide comparisons of the 16S [GenBank:AY652762] and ftsZ [GenBank:DQ159068] sequences attributed to Wolbachia from A. cantonensis and constructed phylogenetic trees for each. Wolbachia sequences used for multiple alignments and phylogenetic tree construction are as follows, with the first GenBank sequence for each invertebrate host organism corresponding to 16S and the second to ftsZ: Brugia malayi [GenBank:AJ010275], [GenBank:AJ010269]; B. pahangi [GenBank:AJ012646], [GenBank:AJ010270]; Litomosoides sigmodontis [GenBank:AF069068], [GenBank:AJ010271]; Onchocerca volvulus [GenBank:CU062464], GenBank:AJ276501]; O. gutturosa [GenBank:AJ276498], [GenBank:AJ010266]; Dirofilaria immitis [Genbank:Z49261], [GenBank:AJ010272]; Drosophila melanogaster [GenBank: NC_002978.6; genome coordinates: 1167943–1169389], [GenBank:U28189]; D. simulans wRiverside [GenBank:DQ412085], [GenBank:U28178]; Trichogramma cordubensis [GenBank:L02883], [GenBank:U28200]; Culex pipiens [Genbank:U23709], [GenBank:U28209]; Folsomia candida [GenBank:AF179630], [GenBank:AJ344216]; Kalotermes flavicollis [GenBank:Y11377], [GenBank:AJ292345], Mansonella perstans 16S [GenBank:AY278355]; Mansonella sp. ftsZ [GenBank:AJ628414]. The underlined characters for each host species represent the abbreviations shown in the trees. The 16S sequence attributed to Wolbachia from A. cantonensis had highest nucleotide identity (99%) to Wolbachia 16S from D. immitis [GenBank:Z49261]. This identity was better than that between Wolbachia 16S sequences from sister species within the genus Dirofilaria, namely D. immitis and D. repens (98%). An equal match was detected for Wolbachia apparently from an engorged dog tick, Rhipicephalus sanguineus [GenBank:AF304445], but since this tick lacks Wolbachia [12], we propose that this Wolbachia sequence derived from D. immitis acquired during a blood feed on a heartworm-infected dog. The 16S phylogenetic tree resolved supergroups A to F and confirmed the near identity of the sequence amplified from A. cantonensis to the Wolbachia 16S from D. immitis (Figure 1a; see additional file 1: Wolbachia 16S multiple sequence alignment – A. cantonensis). The available 16S sequences for Wolbachia from A. cantonensis and from D. circumlita are partial gene sequences and have very little overlap with each other. Therefore, we were unable to include D. circumlita Wolbachia 16S in the same alignment and phylogenetic tree as the 16S reported from Wolbachia from A. cantonensis. However, a separate alignment and tree using 16S fragments corresponding to that from Wolbachia of D. circumlita showed that this sequence is quite distinct from the D. immitis Wolbachia 16S and clusters with sequences from Wolbachia of arthropods (Figure 1b; see additional file 2: Wolbachia 16S multiple sequence alignment – D. circumlita), as expected based on recent Wolbachia MLST [1]. Therefore, while the wsp gene reported for Wolbachia from A. cantonensis has high identity to sequences from the endosymbionts of the arthropods, M. genurostris and D. circumlita, the 16S sequence is nearly identical to Wolbachia 16S from filarial nematodes, notably D. immitis (supergroup C). The results of our analyses of ftsZ were very similar to those obtained for 16S. We observed 99% identity to the ftsZ reported from the Wolbachia from D. immitis [GenBank:AJ010272]. The level of identity between these two sequences also exceeded that between the Wolbachia ftsZ sequences from the sister species D. immitis and D. repens (92%). A phylogenetic analysis of ftsZ sequences representing Wolbachia supergroups A to F (Figure 1c; see additional file 3: Wolbachia ftsZ multiple sequence alignment) also resolved these six groups and confirmed the high similarity of the sequence reported for Wolbachia from A. cantonensis and that from D. immitis.


Absence of Wolbachia endobacteria in the non-filariid nematodes Angiostrongylus cantonensis and A. costaricensis.

Foster JM, Kumar S, Ford L, Johnston KL, Ben R, Graeff-Teixeira C, Taylor MJ - Parasit Vectors (2008)

Minimum evolution trees based on alignments of A) the Wolbachia16S (770 nucleotides) reported from A. cantonensis[GenBank:AY652762], B) the Wolbachia 16S (639 nucleotides) of D. circumlita c2 [GenBank:AY486072], and C) the Wolbachia ftsZ (431 nucleotides) reported from A. cantonensis [GenBank:DQ159068 ]. Sequences were aligned using ClustalX version 2.0.7 [14] using default parameters for slow/accurate alignment [Gap Opening:10, Gap Extend: 0.1, IUB DNA weight matrix]. After alignment, sequences were manually trimmed to the endpoints of the 16S sequences of the Wolbachia from A. cantonensis (see additional file 1: Wolbachia 16S multiple sequence alignment – A. cantonensis) and D. circumlita (see additional file 2: Wolbachia 16S multiple sequence alignment – D. circumlita), and to the endpoints of the ftsZ sequence of the Wolbachia from A. cantonensis (see additional file 3: Wolbachia ftsZ multiple sequence alignment). Phylogenetic trees were calculated using the Minimum Evolution method in MEGA4 [15]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary distances were computed using the Maximum Composite Likelihood. The Minimum Evolution tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 1: Minimum evolution trees based on alignments of A) the Wolbachia16S (770 nucleotides) reported from A. cantonensis[GenBank:AY652762], B) the Wolbachia 16S (639 nucleotides) of D. circumlita c2 [GenBank:AY486072], and C) the Wolbachia ftsZ (431 nucleotides) reported from A. cantonensis [GenBank:DQ159068 ]. Sequences were aligned using ClustalX version 2.0.7 [14] using default parameters for slow/accurate alignment [Gap Opening:10, Gap Extend: 0.1, IUB DNA weight matrix]. After alignment, sequences were manually trimmed to the endpoints of the 16S sequences of the Wolbachia from A. cantonensis (see additional file 1: Wolbachia 16S multiple sequence alignment – A. cantonensis) and D. circumlita (see additional file 2: Wolbachia 16S multiple sequence alignment – D. circumlita), and to the endpoints of the ftsZ sequence of the Wolbachia from A. cantonensis (see additional file 3: Wolbachia ftsZ multiple sequence alignment). Phylogenetic trees were calculated using the Minimum Evolution method in MEGA4 [15]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary distances were computed using the Maximum Composite Likelihood. The Minimum Evolution tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option).
Mentions: We performed similar nucleotide comparisons of the 16S [GenBank:AY652762] and ftsZ [GenBank:DQ159068] sequences attributed to Wolbachia from A. cantonensis and constructed phylogenetic trees for each. Wolbachia sequences used for multiple alignments and phylogenetic tree construction are as follows, with the first GenBank sequence for each invertebrate host organism corresponding to 16S and the second to ftsZ: Brugia malayi [GenBank:AJ010275], [GenBank:AJ010269]; B. pahangi [GenBank:AJ012646], [GenBank:AJ010270]; Litomosoides sigmodontis [GenBank:AF069068], [GenBank:AJ010271]; Onchocerca volvulus [GenBank:CU062464], GenBank:AJ276501]; O. gutturosa [GenBank:AJ276498], [GenBank:AJ010266]; Dirofilaria immitis [Genbank:Z49261], [GenBank:AJ010272]; Drosophila melanogaster [GenBank: NC_002978.6; genome coordinates: 1167943–1169389], [GenBank:U28189]; D. simulans wRiverside [GenBank:DQ412085], [GenBank:U28178]; Trichogramma cordubensis [GenBank:L02883], [GenBank:U28200]; Culex pipiens [Genbank:U23709], [GenBank:U28209]; Folsomia candida [GenBank:AF179630], [GenBank:AJ344216]; Kalotermes flavicollis [GenBank:Y11377], [GenBank:AJ292345], Mansonella perstans 16S [GenBank:AY278355]; Mansonella sp. ftsZ [GenBank:AJ628414]. The underlined characters for each host species represent the abbreviations shown in the trees. The 16S sequence attributed to Wolbachia from A. cantonensis had highest nucleotide identity (99%) to Wolbachia 16S from D. immitis [GenBank:Z49261]. This identity was better than that between Wolbachia 16S sequences from sister species within the genus Dirofilaria, namely D. immitis and D. repens (98%). An equal match was detected for Wolbachia apparently from an engorged dog tick, Rhipicephalus sanguineus [GenBank:AF304445], but since this tick lacks Wolbachia [12], we propose that this Wolbachia sequence derived from D. immitis acquired during a blood feed on a heartworm-infected dog. The 16S phylogenetic tree resolved supergroups A to F and confirmed the near identity of the sequence amplified from A. cantonensis to the Wolbachia 16S from D. immitis (Figure 1a; see additional file 1: Wolbachia 16S multiple sequence alignment – A. cantonensis). The available 16S sequences for Wolbachia from A. cantonensis and from D. circumlita are partial gene sequences and have very little overlap with each other. Therefore, we were unable to include D. circumlita Wolbachia 16S in the same alignment and phylogenetic tree as the 16S reported from Wolbachia from A. cantonensis. However, a separate alignment and tree using 16S fragments corresponding to that from Wolbachia of D. circumlita showed that this sequence is quite distinct from the D. immitis Wolbachia 16S and clusters with sequences from Wolbachia of arthropods (Figure 1b; see additional file 2: Wolbachia 16S multiple sequence alignment – D. circumlita), as expected based on recent Wolbachia MLST [1]. Therefore, while the wsp gene reported for Wolbachia from A. cantonensis has high identity to sequences from the endosymbionts of the arthropods, M. genurostris and D. circumlita, the 16S sequence is nearly identical to Wolbachia 16S from filarial nematodes, notably D. immitis (supergroup C). The results of our analyses of ftsZ were very similar to those obtained for 16S. We observed 99% identity to the ftsZ reported from the Wolbachia from D. immitis [GenBank:AJ010272]. The level of identity between these two sequences also exceeded that between the Wolbachia ftsZ sequences from the sister species D. immitis and D. repens (92%). A phylogenetic analysis of ftsZ sequences representing Wolbachia supergroups A to F (Figure 1c; see additional file 3: Wolbachia ftsZ multiple sequence alignment) also resolved these six groups and confirmed the high similarity of the sequence reported for Wolbachia from A. cantonensis and that from D. immitis.

Bottom Line: We were unable to detect Wolbachia in either species using these methodologies.In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes.This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. mark.taylor@liverpool.ac.uk.

ABSTRACT
The majority of filarial nematodes harbour Wolbachia endobacteria, including the major pathogenic species in humans, Onchocerca volvulus, Brugia malayi and Wuchereria bancrofti. These obligate endosymbionts have never been demonstrated unequivocally in any non-filariid nematode. However, a recent report described the detection by PCR of Wolbachia in the metastrongylid nematode, Angiostrongylus cantonensis (rat lungworm), a leading cause of eosinophilic meningitis in humans. To address the intriguing possibility of Wolbachia infection in nematode species distinct from the Family Onchocercidae, we used both PCR and immunohistochemistry to screen samples of A. cantonensis and A. costaricensis for the presence of this endosymbiont. We were unable to detect Wolbachia in either species using these methodologies. In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes. This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples.

No MeSH data available.


Related in: MedlinePlus