- Email: [email protected]
Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)
Experimental Parasitology 126 (2010) 564–569
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae) Praphathip Eamsobhana a,*, Phaik Eem Lim b,c, Hongman Zhang d, Xiaoxian Gan e, Hoi Sen Yong b a
Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia d Guangxi Zhang Autonomous Region, Center for Disease Control and Prevention, Nanning 530028, Guangxi, PR China e Institute of Parasitic Diseases, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China b c
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 28 May 2010 Accepted 1 June 2010 Available online 8 June 2010 Keywords: Angiostrongylus cantonensis Angiostrongylus costaricensis Angiostrongylus malaysiensis Molecular differentiation Phylogenetic relationships DNA sequences
a b s t r a c t The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades – Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5–400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Angiostrongylus cantonensis, a nematode parasite inhabiting the pulmonary arteries of rats, is a primary cause of human eosinophilic meningitis or eosinophilic meningoencephalitis (cerebral angiostrongyliasis) in Asia and the Pacific Islands (Eamsobhana and Tungtrongchitr, 2005; Eamsobhana, 2006). Angiostrongylus costaricensis which inhabits the mesenteric arteries of rats produces abdominal angiostrongyliasis in humans in Central and South America (Morera, 1985). Angiostrongylus malaysiensis is very similar to A. cantonensis and also inhabits the lung of rats. It has been shown to produce neurologic abnormality in infected rodent host (Cross, 1979), but the potential of being pathogenic to humans need further elucidation.
* Corresponding author. E-mail address: [email protected] (P. Eamsobhana). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.06.001
Biochemical and immunological approaches had been used to discriminate antigenic variability in Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis) (Eamsobhana et al., 1998; Sawabe and Mikiya, 1994). More recently, molecular analysis has been used to differentiate various Angiostrongylus species. Restriction fragment length polymorphisms (RFLPs) have proved valuable in differentiating A. cantonensis, A. costaricensis, and Angiostrongylus vasorum (Caldeira et al., 2003). The small-subunit ribosomal DNA sequences have been used for constructing phylogenetic tree of five species of Angiostrongylus, viz. A. cantonensis, A. costaricensis, Angiostrongylus dujardini, A. malaysiensis and A. vasorum (van Megan et al., 2009; Fontanilla and Wade, 2008). Phylogenetic tree has also been constructed with internal transcribed spacer 2 (ITS-2) for A. cantonensis, A. costaricensis (from Costa Rica and Brazil) and A. vasorum (from Brazil and Europe) (Jefferies et al., 2009). In addition other nucleotide sequences, including the mitochondrial cytochrome-c oxidase subunit I (COI) have been deposited in the GenBank.
565
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
Of the 15–16 species of Angiostrongylus in the world, nucleotide sequences are available in the GenBank for A. cantonensis (209 entries), A. costaricensis (12), A. dujardini (2), A. malaysiensis (1) and A. vasorum (22). There is only a single entry for the adult-specific muscle protein gene, namely for A. cantonensis. It is evident that there had been few studies of the pathogenic A. cantonensis in different endemic localities. In recent years, there have been evidences for wide geographic distribution of A. cantonensis and increasing incidence of human angiostrongyliasis worldwide (Wang et al., 2008). Furthermore, the parasites of different geographical locality show different infectivity, severity and pathogenicity in experimental hosts (Cross, 1979). The purpose of this study was to determine the phylogenetic relationships of three Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis). Different geographical isolates of A. cantonensis from Thailand, Hawaii, Japan and China were also determined to see the level of their molecular variation, and to assess the potential of the mRNA nucleotide sequences of a 66-kDa muscle-associated protein gene of A. cantonensis as a candidate marker for species and isolates differentiation. 2. Materials and methods 2.1. Angiostrongylus worms and other nematodes The Thailand isolate of A. cantonensis was maintained in the Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, with passages through albino rats as definitive rodent host and Biomphalaria glabrata as intermediate snail host. The Hawaii isolate of A. cantonensis was kindly provided by Dr. Akira Ishih of Hamamatsu University, Japan and maintained in the Department of Parasitology, Mahidol University. Both the Thailand and Hawaii adult worm samples have been kept in absolute alcohol since August 2008. The China specimens of A. cantonensis were obtained from rodents caught in Guangxi Province, PR China in September 2009. Individual worm homogenate was applied and dried onto the FTA card before shipment to Bangkok for DNA extractions. The A. cantonensis worms of Japan, collected in May 2006 by Dr. Asato and stored in absolute ethanol, were kindly provided by Professor Ichiro Miyagi and Professor T. Toma, University of the Ryukyus, Okinawa, Japan. A. malaysiensis male and female worms were obtained from the pulmonary arteries of wild caught Rattus tiomanicus on 26 June 2008 in Pahang, Malaysia and stored in absolute ethanol until DNA was extracted. A. costaricensis adult specimens preserved in RNAlater (RNA stabilization solution) were a gift from Dr. Elizabeth Abrahams and Dr. Maria Solano, Department of Parasitology, University of Costa Rica. We were not able to obtain specimens of A. vasorum and other Angiostrongylus species. Adult worms of Ascaris suum from pig, Ascaris lumbricoides from a human patient, Toxocara canis from cat, larvae of Anisakis simplex from marine fish, Trichinella spiralis from experimentally infected mouse and Gnathostoma spinigerum from naturally infected eels, were used as out-groups. All these nematode samples have been kept in absolute ethanol at the Department of Parasitology, Faculty of Medicine Siriraj Hospital, since 2008. 2.2. DNA extraction Genomic DNA extraction from individual adult worms of A. cantonensis (Thailand isolate – 2 males, 2 females; Hawaii isolate – 2 males, 2 females) was tested using a QIAamp tissue kit (QIAGEN, Germany) and the FTA card method (Whatman BioScience). Both procedures yielded similar DNA banding patterns (Fig. 1) after PCR amplification using specific primers AC1 and AC2 for adult A. cantonensis. The FTA card method which is more rapid and conve-
bp
M
1
2
3
4
1000
500 400 300 200
Fig. 1. Electrophoretic patterns of PCR products using the specific primers AC1 and AC2 for adult A. cantonensis on DNA extracted by FTA card method (Whatman BioScience) – Lanes 1 and 2, and QIAamp tissue kit (QIAGEN, Germany) – Lanes 3 and 4. Lane M, molecular weight markers (100 bp ladder).
nient to perform was employed for DNA preparation in the present study. Genomic DNA extraction from individual adult female and male worms of A. cantonensis (Thailand isolate – 6 males, 6 females; Hawaii isolate – 6 males, 6 females; Japan isolate – 2 males, 2 females; China isolate – 4 males, 4 females), A. malaysiensis (2 males, 2 females) and A. costaricensis (3 males, 3 females), was carried out using FTA technology (Whatman BioScience) following the manufacturer’s instruction. In brief, individual worm was homogenized in 100–150 lL of sterilized PBS, pH 7.4. The homogenate was applied and dried onto the FTA card according to the Whatman FTA tissue protocol. A sample disc was taken using a 2.0 mm diameter Harris micro punch, washed with FTA purification reagent, and used in PCR for DNA analysis.
2.3. PCR amplification and DNA sequencing The amplification by polymerase chain reaction was conducted using the primers AC1: 50 CTCGGCTTAATCTTTGCGAC-30 and AC2: 50 AACGAGCGGCAGTAGAAAAA-30 (Silva et al., 2003). The sequence of the primers was designed based on the adult-specific native 66 kDa protein mRNA, clone L5–400 of A. cantonensis (GenBank Accession No. U17585) (Bessarab and Joshua, 1997). The PCR reaction was performed as described by Silva et al. (2003) with slight modification. The amplification was carried out in 50 lL containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris– HCl (pH 8.3), 200 lM each of dATP, dGTP, dCTP and dTTP, 150 pmol of each primer, and 1 U of Taq DNA polymerase, with a 2.0 mm DNA punched disc (FTA card) that contained extracted DNA from individual worm samples in a DNA thermal cycler (Perkin–Elmer Cetus). Amplification of 35 cycles consisted of denaturation at 94 °C for 2 min, annealing at 58 °C for 3 min, and extension at 72 °C for 3 min. Negative and positive controls were run with each round of amplification. The PCR products were electrophoresed in 1.5% agarose gel. After electrophoresis, the agarose gel containing DNA fragments was stained with 0.5 lg/mL of ethidium bromide and visualized by ultraviolet transilluminator. Amplified products were purified using a QIAquick PCR Purification kit (QIAGEN, Germany). Sequencing reactions were performed using an ABI PrismDyeTerminator Cycle Sequencing Core kit (Applied Biosystems, USA).
566
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
2.4. Sequences from GenBank The partial sequences of a 66-kDa protein gene of A. cantonensis clone L5–400 from Taiwan was obtained from the GenBank (Accession No.: ACU17585). In addition, all the available 9 SSU sequences of Angiostrongylus species were retrieved from the GenBank – A. cantonensis (AY295804); A. costaricensis (DQ116748; EF514913); A. dujardini (AY542282; EF514915); A. malaysiensis (EF514914); and A. vasorum (AJ920365; EF514916; EU915247) – to construct a more accurate phylogeny. Didelphostrongylus hayesi (GenBank Accession No. AY295806) was used as outgroup for phylogenetic analysis. 2.5. Sequence alignment and phylogenetic analysis Sequences from this study were preliminarily aligned using the CLUSTAL X program (Thompson et al., 1994) and subsequently manually aligned. The aligned sequences were subjected to maximum-parsimony (MP) and maximum-likelihood (ML) analyses using PAUP* 4.0b10 (Swofford, 2002). The MP tree was constructed using the heuristic search option, 100 random sequences additions, tree bisection reconnection (TBR) branch swapping, and unordered and unweighted characters. Bootstrap percentage (BP) was computed with 1000 replications. To find the least-rejected model of sequences by an Akaike Information Criterion (AIC), the program ModelTest v.3.7 (Posada and Crandall, 1998) was used. The AIC indicated that the least-rejected model for the present study data set was HKY85+G; with the number of substitution types = 2 (HKY85 variant); Transition/ transversion ratio = 1.5049 (j = 2.9340413) and nucleotide frequencies A = 0.3318, C = 0.1936, G = 0.2425, T = 0.2320. The maxi-
mum-likelihood (ML) was performed using TBR swapping with 10 random sequence additions in PAUP using the estimated parameters from the ModelTest. BP was computed with 100 replications. 3. Results The aligned sequences consisted of 304 sites, of which 207 characters were constant, 23 characters were parsimony informative and 74 characters parsimony uninformative. MP analysis resulted in 3071 equally most parsimonious trees, of which only the strict consensus tree is shown (Fig. 2); the strict consensus MP tree was slightly different from the ML tree (Fig. 3) but gave very similar results in the overall topologies and branching order of the major clades. The MP consensus tree (Fig. 2) showed that the Angiostrongylus taxa could be divided into two major clades – Clade 1 (A. costaricensis) and Clade 2 (comprising A. cantonensis and A. malaysiensis) with a full support bootstrap value. Clade 2 was divided into two subclades: Clade 2a (A. cantonensis) and Clade 2b (A. malaysiensis) with 100% bootstrap support value. Clade 2a was further subdivided into Clade 2ai (BP = 64); Clade 2aii (BP = 57%); Clade 2aiii (BP = 74%) and Clade 2aiv (BP = 50%). Clade 1 samples (A. costaricensis) with a full bootstrap support were the most basal group among the Angiostrongylus taxa. The lnL likelihood for ML tree was 881.16. The ML tree (Fig. 3) contained two major clades – Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. Clade 2 was divided into two subclades: Clade 2a (A. cantonensis) with BP = 100% and 2b (A. malaysiensis) with 83% bootstrap support value. Clade 2a was further subdivided into Clade 2ai
Fig. 2. Maximum-parsimony (MP) tree of Angiostrongylus costaricensis (ACOS), A. malaysiensis (AM), A. cantonensis (AC) of different geographical origin, and ACU17585 (A. cantonensis clone L5–400 from Taiwan) from the GenBank – based on partial sequences of a 66-kDa protein gene. F, female; M, male; J, Japan; T, Thailand; H, Hawaii; C, China. The bootstrap values are shown above the branches.
567
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
Fig. 3. Maximum-likelihood (ML) tree of Angiostrongylus costaricensis (ACOS), A. malaysiensis (AM), A. cantonensis (AC) of different geographical origin, and ACU17585 (A. cantonensis clone L5–400 from Taiwan) from the GenBank – based on partial sequences of a 66-kDa protein gene. F, female; M, male; J, Japan; T, Thailand; H, Hawaii; C, China. The bootstrap values are shown above the branches.
Table 1 Percentage of uncorrected ‘‘p” distance matrix between the selected samples of A. costaricensis (ACOS), A. malaysiensis (AM), A. cantonensis (AC) from various locaties, and ACU17585 (A. cantonensis clone L5–400 from Taiwan) from the GenBank. F, female; M, male; J, Japan; T, Thailand; H, Hawaii; C, China. Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
1. ACOS_F2 2. ACOS_M2 3. AC_J_F1 4. AC_J_M1 5. AC_T_F1 6. AC_T_M1 7. AC_H_F1 8. AC_H_M1 9. AC_C_F1 10. AC_C_M2 11. AM_F2 12. AM_M2 13. AC_U17585
0.00 4.08 4.42 5.44 5.77 4.08 4.77 3.74 3.76 5.10 5.10 29.92
4.08 4.42 5.44 5.77 4.08 4.77 3.74 3.76 5.10 5.10 29.92
0.34 1.36 1.69 0.68 0.68 0.34 0.34 2.38 2.38 26.87
1.02 1.36 0.34 0.34 0.68 0.68 2.72 2.72 27.22
1.69 1.36 1.36 1.70 1.70 3.74 3.74 27.56
1.69 1.02 2.03 2.04 4.08 4.08 27.92
0.68 0.34 0.34 2.38 2.38 27.57
1.02 1.03 3.07 3.07 27.31
0.00 2.04 2.04 27.22
1.70 1.70 27.36
0.00 28.93
28.93
(BP = 58%) and Clade 2aiii (no bootstrap support) which were similar as shown in MP tree (Fig. 2). However, the samples that were grouped in Clade 2aii and Clade 2aiv did not group as in the MP tree. In the ML tree (Fig. 3), the samples of Clade 2aii in the MP tree (AC_J_F2; U17585; both of A. cantonensis) were grouped with AC_C_M3 (Clade 2 aiv in MP tree; also of A. cantonensis) and AC_C_F2 (A. cantonensis) as another clade, Clade 2av with no bootstrap support value. Clade 1 samples were the most basal group among the Angiostrongylus taxa. From both the MP and ML analyses, A. costaricensis (ACOS) was the most distant taxon, and possibly the earliest divergence group in evolutionary history. A. cantonensis (AC samples) is a sister group to A. malaysiensis (AM samples). These two taxa (species)
are clearly separated. There is no clear distinction between the A. cantonensis (AC) samples from different geographical localities; only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values as shown for the AC samples in Clades 2ai, 2aii, 2aiii, 2aiv and 2av (Figs. 2 and 3). The uncorrected p-distance values between ACU17585 and representative samples of A. costaricensis, A. cantonensis and A. malaysiensis are given in Table 1. Of the A. cantonensis from different geographical localities, AC_T_M1 from Thailand was exceptionally different from the others. The 50% majority-rule consensus tree resulting from maximumlikelihood analysis of all the available Angiostrongylus SSU DNA se-
568
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
Fig. 4. The 50% majority-rule consensus tree resulting from maximum-likelihood analysis of available Angiostrongylus SSU DNA sequences from the GenBank with an outgroup of Didelphostronglyus hayesi. Ln likelihood was 2833.45904. The bootstrap values (ML/MP/NJ/Bayesian Inference) are shown above the branches. Bootstrap values below 50% are indicated as ‘‘–’’.
quences from the GenBank, with D. hayesi as an outgroup, resulted in A. cantonensis and A. malaysiensis forming a distinct clade from the other species (Fig. 4). In the other clade, A. costaricensis was closer related to A. dujardini than A. vasorum. 4. Discussion Prior to 1971, the rat lungworm occurring in Malaysia was referred to as A. cantonensis. It was subsequently found to be a distinct species and named as A. malaysiensis (Bhaibulaya and Cross, 1971; Cross and Bhaibulaya, 1974). Recently, A. vasorum from South America and Europe have been shown to represent distinct lineages on the basis of the mitochondrial COI gene and the second ribosomal internal transcribed spacer (Jefferies et al., 2009). The partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes of A. cantonensis were among those used for phylogenetic analysis of the Metastrongyloidea (Carreno and Nadler, 2003). The cytochrome-c oxidase subunit I and 12S rRNA yielded poorly resolved phylogenetic hypotheses for a sample of Metastrongyloidea (Carreno and Nadler, 2003). A phylogeny based on analysis of 1628 nucleotides of the SSU rRNA gene, with one gene sequence for each taxon, indicated that A. cantonensis clustered more distantly (P = 0.98 BI, 63% ML bootstraps, 70% NJ bootstraps) to A. costaricensis and A. malaysiensis (P = 0.84 BI, 53% ML bootstraps, 51% NJ bootstraps) (Fontanilla and Wade, 2008). Another analysis based on about 1200 full-length small-subunit ribosomal DNA sequences, but including two gene sequences for each of these taxa, indicated that A. cantonensis and A. malaysiensis are closer related (same cluster) than A. costaricensis (van Megan et al., 2009). Our analysis based on all available SSU rRNA sequences for Angiostrongylus species from the GenBank indicates that A. cantonensis and A. malaysiensis form a cluster
(clade) distinct from A. costaricensis, A. dujardini and A. vasorum (Fig. 4). This is perhaps a more accurate phylogeny as more than one gene sequence where available, was used for each taxon. The present study which included more gene sequences for each of these taxa also shows A. cantonensis and A. malaysiensis forming a clade distinctly separated from A. costaricensis (Figs. 2 and 3). Taking into consideration their morphology, biology and geographical distribution, it is more probable that A. malaysiensis is closer related to A. cantonensis than A. costaricensis. As far as we are aware there is only a single report (GenBank Accession No. U17585, A. cantonensis) on the partial sequences of the 66-kDa protein gene. The present results show that the AC primers [GenBank Accession No. U17585] could be used successfully to amplify the genomic DNA of three Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis). No amplification was detected with the genomic DNA of six other species of nematodes (A. suum, A. lumbricoides, T. canis, A. simplex, G. spinigerum and T. spiralis). The absence of amplification had been reported for A. suum and T. canis, in addition to Ancylostoma caninum and Strongyloides ratti (Bessarab and Joshua, 1997). Further studies on other species of angiostrongylids and other nematodes are needed to ascertain if the primers are genus-specific or family-specific. In the present study, the samples of A. cantonensis did not cluster unequivocally according to their geographical origin (Thailand, China, Japan and Hawaii). A possible reason could be the sequences used in this study are too short. In order to have a better resolution tree and relationship, longer sequences and region with higher resolution should be explored. This will form part of our continuing studies on the Angiostrongylus parasites. Two genes, cytochromec oxidase subunit I (COI) and internal transcribed spacer 2 (ITS-2) are being investigated. It is noteworthy that the nucleotide sequences of A. cantonensis from Taiwan (GenBank Accession No. U17585) appear to be very distant from the rest of the A. cantonensis samples (Fig. 3), with the uncorrected p-distance values ranging from 26.87% to 29.92% (Table 1). This apparent discrepancy will be investigated. More detailed studies to determine the extent of association between specific genotype of A. cantonensis and its intermediate snail host in different geographical localities will provide a foundation to better understand the origin, occurrence, pattern, and spreading of the disease in humans. In sum, the present study has added knowledge to the relatively not well understood 66-kDa protein gene of Angiostrongylus nematodes. The partial nucleotide sequences of this gene could unequivocally differentiate A. cantonensis, A. costaricensis and A. malaysiensis. On the basis of these nucleotide sequences, A. cantonensis is closer related to A. malaysiensis than A. costaricensis. Acknowledgments The authors would like to thank Mr Paibulaya Punthuprapasa, Mr Adisak Yoolek and Ms Jeeranan Pinnak for their excellent technical assistance. We also thank the Mahidol University and University of Malaya for support and facilities. This work received financial support from a grant for research (to P. Eamsobhana) on infectious diseases from the Department of Disease Control, Ministry of Public Health, Thailand; and a special research fund (to H.S. Yong) from the University of Malaya. The comments and suggestions by the reviewers and editor have greatly improved the paper and are gratefully acknowledged. References Bessarab, I.N., Joshua, G.W.P., 1997. Stage-specific gene expression in Angiostrongylus cantonesis: characterization and expression of an adultspecific gene. Mol. Biochem. Parasitol. 88, 73–84.
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569 Bhaibulaya, M., Cross, J.H., 1971. Angiostrongylus malaysiensis (Nematoda: Metastrongylidae), a new species of rat lungworm from Malaysia. Southeast Asian J. Trop. Med. Public Health 2, 527–533. Caldeira, R.L., Carvalho, O.S., Mendonca, C.L.F.G., Graeff-Teixeira, C., Silva, M.C.F., Ben, R., Maurer, R., Lima, W.S., Lenzi, H.L., 2003. Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis and A. vasorum by polymerase chain reaction-restriction fragment length polymorphism. Mem. Inst. Oswaldo Cruz. 98, 1039–1043. Carreno, R.A., Nadler, S.A., 2003. Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences. J. Parasitol. 89, 965–973. Cross, J.H., 1979. Experimental studies on Angiostrongylus species and strains in monkeys and laboratory animals. In: Cross, J.H. (Ed.), Studies On Angiostrongyliasis in Eastern Asia and Australia. NAMRU-2-SP-44. U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, pp. 118–137. Cross, J.H., Bhaibulaya, M., 1974. Validity of Angiostrongylus malaysiensis, Bhaibulaya and Cross, 1971. Southeast Asian J Trop Med Public Health 5, 374–378. Eamsobhana, P., 2006. The Rat Lungworm Parastrongylus cantonensis: parasitology, immunology, eosinophilic meningitis, epidemiology and laboratory diagnosis. Wankaew (IQ) Book Center Co., Ltd., Bangkok. P. 156. Eamsobhana, P., Tungtrongchitr, A., 2005. Angiostrongyliasis in Thailand. In: Arizono, N., Chai, J.Y., Nawa, Y., Takahashi, T. (Eds.), Food-Borne Helminthiasis in Asia. The Federation of Asian parasitologist, Chiba, Japan, pp. 32–47. Eamsobhana, P., Yong, H.S., Mak, J.W., Tungtrongchitr, A., 1998. Use of a monoclonal antibody to identify antigenic differences among species and isolates of Parastrongylus (Nematoda: Metastrongylidae). Trop. Biomed. 15, 51–56.
569
Fontanilla, I.K.C., Wade, C.M., 2008. The small subunit (SSU) ribosomal (r) RNA as a genetic marker for identifying infective 3rd juvenile stage Angiostrongylus cantonensis. Acta Trop. 105, 181–186. Jefferies, R., Shaw, S.E., Viney, M.E., Morgan, E.R., 2009. Angiostrongylus vasorum from South America and Europe represent distinct lineages. Parasitology 136, 107–115. Morera, P., 1985. Abdominal angiostrongyliasis: a problem of public health. Parsitol. Today 6, 173–175. Posada, D., Crandall, K.A., 1998. ModelTest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Sawabe, K., Mikiya, K., 1994. Genetic variability in isozymes of Angiostrongylus malaysiensis. Southeast Asian J. Trop. Med. Public. Health 25, 728–734. Silva, A.C.A., Graeff-Teixeira, C., Zaha, A., 2003. Diagnosis of abdominal angiostrongyliasis by PCR from sera of patients. Rev Inst Med Trop S Paulo 45, 295–297. Swofford, D.L., 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 425–430. van Megan, H., van den Elsen, S., Holterman, M., et al., 2009. A phylogenetic tree of nematodes based on about 1200 full-lengh small subunit ribosomal DNA sequences. Nematology 11, 927–950. Wang, Q.P., Lai, D.H., Zhu, X.Q., Chen, X.G., Lun, Z.R., 2008. Human angiostrongyliasis. Lancet Infect. Dis. 8, 621–630.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae) Praphathip Eamsobhana a,*, Phaik Eem Lim b,c, Hongman Zhang d, Xiaoxian Gan e, Hoi Sen Yong b a
Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia d Guangxi Zhang Autonomous Region, Center for Disease Control and Prevention, Nanning 530028, Guangxi, PR China e Institute of Parasitic Diseases, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China b c
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 28 May 2010 Accepted 1 June 2010 Available online 8 June 2010 Keywords: Angiostrongylus cantonensis Angiostrongylus costaricensis Angiostrongylus malaysiensis Molecular differentiation Phylogenetic relationships DNA sequences
a b s t r a c t The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades – Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5–400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Angiostrongylus cantonensis, a nematode parasite inhabiting the pulmonary arteries of rats, is a primary cause of human eosinophilic meningitis or eosinophilic meningoencephalitis (cerebral angiostrongyliasis) in Asia and the Pacific Islands (Eamsobhana and Tungtrongchitr, 2005; Eamsobhana, 2006). Angiostrongylus costaricensis which inhabits the mesenteric arteries of rats produces abdominal angiostrongyliasis in humans in Central and South America (Morera, 1985). Angiostrongylus malaysiensis is very similar to A. cantonensis and also inhabits the lung of rats. It has been shown to produce neurologic abnormality in infected rodent host (Cross, 1979), but the potential of being pathogenic to humans need further elucidation.
* Corresponding author. E-mail address: [email protected] (P. Eamsobhana). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.06.001
Biochemical and immunological approaches had been used to discriminate antigenic variability in Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis) (Eamsobhana et al., 1998; Sawabe and Mikiya, 1994). More recently, molecular analysis has been used to differentiate various Angiostrongylus species. Restriction fragment length polymorphisms (RFLPs) have proved valuable in differentiating A. cantonensis, A. costaricensis, and Angiostrongylus vasorum (Caldeira et al., 2003). The small-subunit ribosomal DNA sequences have been used for constructing phylogenetic tree of five species of Angiostrongylus, viz. A. cantonensis, A. costaricensis, Angiostrongylus dujardini, A. malaysiensis and A. vasorum (van Megan et al., 2009; Fontanilla and Wade, 2008). Phylogenetic tree has also been constructed with internal transcribed spacer 2 (ITS-2) for A. cantonensis, A. costaricensis (from Costa Rica and Brazil) and A. vasorum (from Brazil and Europe) (Jefferies et al., 2009). In addition other nucleotide sequences, including the mitochondrial cytochrome-c oxidase subunit I (COI) have been deposited in the GenBank.
565
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
Of the 15–16 species of Angiostrongylus in the world, nucleotide sequences are available in the GenBank for A. cantonensis (209 entries), A. costaricensis (12), A. dujardini (2), A. malaysiensis (1) and A. vasorum (22). There is only a single entry for the adult-specific muscle protein gene, namely for A. cantonensis. It is evident that there had been few studies of the pathogenic A. cantonensis in different endemic localities. In recent years, there have been evidences for wide geographic distribution of A. cantonensis and increasing incidence of human angiostrongyliasis worldwide (Wang et al., 2008). Furthermore, the parasites of different geographical locality show different infectivity, severity and pathogenicity in experimental hosts (Cross, 1979). The purpose of this study was to determine the phylogenetic relationships of three Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis). Different geographical isolates of A. cantonensis from Thailand, Hawaii, Japan and China were also determined to see the level of their molecular variation, and to assess the potential of the mRNA nucleotide sequences of a 66-kDa muscle-associated protein gene of A. cantonensis as a candidate marker for species and isolates differentiation. 2. Materials and methods 2.1. Angiostrongylus worms and other nematodes The Thailand isolate of A. cantonensis was maintained in the Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, with passages through albino rats as definitive rodent host and Biomphalaria glabrata as intermediate snail host. The Hawaii isolate of A. cantonensis was kindly provided by Dr. Akira Ishih of Hamamatsu University, Japan and maintained in the Department of Parasitology, Mahidol University. Both the Thailand and Hawaii adult worm samples have been kept in absolute alcohol since August 2008. The China specimens of A. cantonensis were obtained from rodents caught in Guangxi Province, PR China in September 2009. Individual worm homogenate was applied and dried onto the FTA card before shipment to Bangkok for DNA extractions. The A. cantonensis worms of Japan, collected in May 2006 by Dr. Asato and stored in absolute ethanol, were kindly provided by Professor Ichiro Miyagi and Professor T. Toma, University of the Ryukyus, Okinawa, Japan. A. malaysiensis male and female worms were obtained from the pulmonary arteries of wild caught Rattus tiomanicus on 26 June 2008 in Pahang, Malaysia and stored in absolute ethanol until DNA was extracted. A. costaricensis adult specimens preserved in RNAlater (RNA stabilization solution) were a gift from Dr. Elizabeth Abrahams and Dr. Maria Solano, Department of Parasitology, University of Costa Rica. We were not able to obtain specimens of A. vasorum and other Angiostrongylus species. Adult worms of Ascaris suum from pig, Ascaris lumbricoides from a human patient, Toxocara canis from cat, larvae of Anisakis simplex from marine fish, Trichinella spiralis from experimentally infected mouse and Gnathostoma spinigerum from naturally infected eels, were used as out-groups. All these nematode samples have been kept in absolute ethanol at the Department of Parasitology, Faculty of Medicine Siriraj Hospital, since 2008. 2.2. DNA extraction Genomic DNA extraction from individual adult worms of A. cantonensis (Thailand isolate – 2 males, 2 females; Hawaii isolate – 2 males, 2 females) was tested using a QIAamp tissue kit (QIAGEN, Germany) and the FTA card method (Whatman BioScience). Both procedures yielded similar DNA banding patterns (Fig. 1) after PCR amplification using specific primers AC1 and AC2 for adult A. cantonensis. The FTA card method which is more rapid and conve-
bp
M
1
2
3
4
1000
500 400 300 200
Fig. 1. Electrophoretic patterns of PCR products using the specific primers AC1 and AC2 for adult A. cantonensis on DNA extracted by FTA card method (Whatman BioScience) – Lanes 1 and 2, and QIAamp tissue kit (QIAGEN, Germany) – Lanes 3 and 4. Lane M, molecular weight markers (100 bp ladder).
nient to perform was employed for DNA preparation in the present study. Genomic DNA extraction from individual adult female and male worms of A. cantonensis (Thailand isolate – 6 males, 6 females; Hawaii isolate – 6 males, 6 females; Japan isolate – 2 males, 2 females; China isolate – 4 males, 4 females), A. malaysiensis (2 males, 2 females) and A. costaricensis (3 males, 3 females), was carried out using FTA technology (Whatman BioScience) following the manufacturer’s instruction. In brief, individual worm was homogenized in 100–150 lL of sterilized PBS, pH 7.4. The homogenate was applied and dried onto the FTA card according to the Whatman FTA tissue protocol. A sample disc was taken using a 2.0 mm diameter Harris micro punch, washed with FTA purification reagent, and used in PCR for DNA analysis.
2.3. PCR amplification and DNA sequencing The amplification by polymerase chain reaction was conducted using the primers AC1: 50 CTCGGCTTAATCTTTGCGAC-30 and AC2: 50 AACGAGCGGCAGTAGAAAAA-30 (Silva et al., 2003). The sequence of the primers was designed based on the adult-specific native 66 kDa protein mRNA, clone L5–400 of A. cantonensis (GenBank Accession No. U17585) (Bessarab and Joshua, 1997). The PCR reaction was performed as described by Silva et al. (2003) with slight modification. The amplification was carried out in 50 lL containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris– HCl (pH 8.3), 200 lM each of dATP, dGTP, dCTP and dTTP, 150 pmol of each primer, and 1 U of Taq DNA polymerase, with a 2.0 mm DNA punched disc (FTA card) that contained extracted DNA from individual worm samples in a DNA thermal cycler (Perkin–Elmer Cetus). Amplification of 35 cycles consisted of denaturation at 94 °C for 2 min, annealing at 58 °C for 3 min, and extension at 72 °C for 3 min. Negative and positive controls were run with each round of amplification. The PCR products were electrophoresed in 1.5% agarose gel. After electrophoresis, the agarose gel containing DNA fragments was stained with 0.5 lg/mL of ethidium bromide and visualized by ultraviolet transilluminator. Amplified products were purified using a QIAquick PCR Purification kit (QIAGEN, Germany). Sequencing reactions were performed using an ABI PrismDyeTerminator Cycle Sequencing Core kit (Applied Biosystems, USA).
566
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
2.4. Sequences from GenBank The partial sequences of a 66-kDa protein gene of A. cantonensis clone L5–400 from Taiwan was obtained from the GenBank (Accession No.: ACU17585). In addition, all the available 9 SSU sequences of Angiostrongylus species were retrieved from the GenBank – A. cantonensis (AY295804); A. costaricensis (DQ116748; EF514913); A. dujardini (AY542282; EF514915); A. malaysiensis (EF514914); and A. vasorum (AJ920365; EF514916; EU915247) – to construct a more accurate phylogeny. Didelphostrongylus hayesi (GenBank Accession No. AY295806) was used as outgroup for phylogenetic analysis. 2.5. Sequence alignment and phylogenetic analysis Sequences from this study were preliminarily aligned using the CLUSTAL X program (Thompson et al., 1994) and subsequently manually aligned. The aligned sequences were subjected to maximum-parsimony (MP) and maximum-likelihood (ML) analyses using PAUP* 4.0b10 (Swofford, 2002). The MP tree was constructed using the heuristic search option, 100 random sequences additions, tree bisection reconnection (TBR) branch swapping, and unordered and unweighted characters. Bootstrap percentage (BP) was computed with 1000 replications. To find the least-rejected model of sequences by an Akaike Information Criterion (AIC), the program ModelTest v.3.7 (Posada and Crandall, 1998) was used. The AIC indicated that the least-rejected model for the present study data set was HKY85+G; with the number of substitution types = 2 (HKY85 variant); Transition/ transversion ratio = 1.5049 (j = 2.9340413) and nucleotide frequencies A = 0.3318, C = 0.1936, G = 0.2425, T = 0.2320. The maxi-
mum-likelihood (ML) was performed using TBR swapping with 10 random sequence additions in PAUP using the estimated parameters from the ModelTest. BP was computed with 100 replications. 3. Results The aligned sequences consisted of 304 sites, of which 207 characters were constant, 23 characters were parsimony informative and 74 characters parsimony uninformative. MP analysis resulted in 3071 equally most parsimonious trees, of which only the strict consensus tree is shown (Fig. 2); the strict consensus MP tree was slightly different from the ML tree (Fig. 3) but gave very similar results in the overall topologies and branching order of the major clades. The MP consensus tree (Fig. 2) showed that the Angiostrongylus taxa could be divided into two major clades – Clade 1 (A. costaricensis) and Clade 2 (comprising A. cantonensis and A. malaysiensis) with a full support bootstrap value. Clade 2 was divided into two subclades: Clade 2a (A. cantonensis) and Clade 2b (A. malaysiensis) with 100% bootstrap support value. Clade 2a was further subdivided into Clade 2ai (BP = 64); Clade 2aii (BP = 57%); Clade 2aiii (BP = 74%) and Clade 2aiv (BP = 50%). Clade 1 samples (A. costaricensis) with a full bootstrap support were the most basal group among the Angiostrongylus taxa. The lnL likelihood for ML tree was 881.16. The ML tree (Fig. 3) contained two major clades – Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. Clade 2 was divided into two subclades: Clade 2a (A. cantonensis) with BP = 100% and 2b (A. malaysiensis) with 83% bootstrap support value. Clade 2a was further subdivided into Clade 2ai
Fig. 2. Maximum-parsimony (MP) tree of Angiostrongylus costaricensis (ACOS), A. malaysiensis (AM), A. cantonensis (AC) of different geographical origin, and ACU17585 (A. cantonensis clone L5–400 from Taiwan) from the GenBank – based on partial sequences of a 66-kDa protein gene. F, female; M, male; J, Japan; T, Thailand; H, Hawaii; C, China. The bootstrap values are shown above the branches.
567
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
Fig. 3. Maximum-likelihood (ML) tree of Angiostrongylus costaricensis (ACOS), A. malaysiensis (AM), A. cantonensis (AC) of different geographical origin, and ACU17585 (A. cantonensis clone L5–400 from Taiwan) from the GenBank – based on partial sequences of a 66-kDa protein gene. F, female; M, male; J, Japan; T, Thailand; H, Hawaii; C, China. The bootstrap values are shown above the branches.
Table 1 Percentage of uncorrected ‘‘p” distance matrix between the selected samples of A. costaricensis (ACOS), A. malaysiensis (AM), A. cantonensis (AC) from various locaties, and ACU17585 (A. cantonensis clone L5–400 from Taiwan) from the GenBank. F, female; M, male; J, Japan; T, Thailand; H, Hawaii; C, China. Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
1. ACOS_F2 2. ACOS_M2 3. AC_J_F1 4. AC_J_M1 5. AC_T_F1 6. AC_T_M1 7. AC_H_F1 8. AC_H_M1 9. AC_C_F1 10. AC_C_M2 11. AM_F2 12. AM_M2 13. AC_U17585
0.00 4.08 4.42 5.44 5.77 4.08 4.77 3.74 3.76 5.10 5.10 29.92
4.08 4.42 5.44 5.77 4.08 4.77 3.74 3.76 5.10 5.10 29.92
0.34 1.36 1.69 0.68 0.68 0.34 0.34 2.38 2.38 26.87
1.02 1.36 0.34 0.34 0.68 0.68 2.72 2.72 27.22
1.69 1.36 1.36 1.70 1.70 3.74 3.74 27.56
1.69 1.02 2.03 2.04 4.08 4.08 27.92
0.68 0.34 0.34 2.38 2.38 27.57
1.02 1.03 3.07 3.07 27.31
0.00 2.04 2.04 27.22
1.70 1.70 27.36
0.00 28.93
28.93
(BP = 58%) and Clade 2aiii (no bootstrap support) which were similar as shown in MP tree (Fig. 2). However, the samples that were grouped in Clade 2aii and Clade 2aiv did not group as in the MP tree. In the ML tree (Fig. 3), the samples of Clade 2aii in the MP tree (AC_J_F2; U17585; both of A. cantonensis) were grouped with AC_C_M3 (Clade 2 aiv in MP tree; also of A. cantonensis) and AC_C_F2 (A. cantonensis) as another clade, Clade 2av with no bootstrap support value. Clade 1 samples were the most basal group among the Angiostrongylus taxa. From both the MP and ML analyses, A. costaricensis (ACOS) was the most distant taxon, and possibly the earliest divergence group in evolutionary history. A. cantonensis (AC samples) is a sister group to A. malaysiensis (AM samples). These two taxa (species)
are clearly separated. There is no clear distinction between the A. cantonensis (AC) samples from different geographical localities; only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values as shown for the AC samples in Clades 2ai, 2aii, 2aiii, 2aiv and 2av (Figs. 2 and 3). The uncorrected p-distance values between ACU17585 and representative samples of A. costaricensis, A. cantonensis and A. malaysiensis are given in Table 1. Of the A. cantonensis from different geographical localities, AC_T_M1 from Thailand was exceptionally different from the others. The 50% majority-rule consensus tree resulting from maximumlikelihood analysis of all the available Angiostrongylus SSU DNA se-
568
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569
Fig. 4. The 50% majority-rule consensus tree resulting from maximum-likelihood analysis of available Angiostrongylus SSU DNA sequences from the GenBank with an outgroup of Didelphostronglyus hayesi. Ln likelihood was 2833.45904. The bootstrap values (ML/MP/NJ/Bayesian Inference) are shown above the branches. Bootstrap values below 50% are indicated as ‘‘–’’.
quences from the GenBank, with D. hayesi as an outgroup, resulted in A. cantonensis and A. malaysiensis forming a distinct clade from the other species (Fig. 4). In the other clade, A. costaricensis was closer related to A. dujardini than A. vasorum. 4. Discussion Prior to 1971, the rat lungworm occurring in Malaysia was referred to as A. cantonensis. It was subsequently found to be a distinct species and named as A. malaysiensis (Bhaibulaya and Cross, 1971; Cross and Bhaibulaya, 1974). Recently, A. vasorum from South America and Europe have been shown to represent distinct lineages on the basis of the mitochondrial COI gene and the second ribosomal internal transcribed spacer (Jefferies et al., 2009). The partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes of A. cantonensis were among those used for phylogenetic analysis of the Metastrongyloidea (Carreno and Nadler, 2003). The cytochrome-c oxidase subunit I and 12S rRNA yielded poorly resolved phylogenetic hypotheses for a sample of Metastrongyloidea (Carreno and Nadler, 2003). A phylogeny based on analysis of 1628 nucleotides of the SSU rRNA gene, with one gene sequence for each taxon, indicated that A. cantonensis clustered more distantly (P = 0.98 BI, 63% ML bootstraps, 70% NJ bootstraps) to A. costaricensis and A. malaysiensis (P = 0.84 BI, 53% ML bootstraps, 51% NJ bootstraps) (Fontanilla and Wade, 2008). Another analysis based on about 1200 full-length small-subunit ribosomal DNA sequences, but including two gene sequences for each of these taxa, indicated that A. cantonensis and A. malaysiensis are closer related (same cluster) than A. costaricensis (van Megan et al., 2009). Our analysis based on all available SSU rRNA sequences for Angiostrongylus species from the GenBank indicates that A. cantonensis and A. malaysiensis form a cluster
(clade) distinct from A. costaricensis, A. dujardini and A. vasorum (Fig. 4). This is perhaps a more accurate phylogeny as more than one gene sequence where available, was used for each taxon. The present study which included more gene sequences for each of these taxa also shows A. cantonensis and A. malaysiensis forming a clade distinctly separated from A. costaricensis (Figs. 2 and 3). Taking into consideration their morphology, biology and geographical distribution, it is more probable that A. malaysiensis is closer related to A. cantonensis than A. costaricensis. As far as we are aware there is only a single report (GenBank Accession No. U17585, A. cantonensis) on the partial sequences of the 66-kDa protein gene. The present results show that the AC primers [GenBank Accession No. U17585] could be used successfully to amplify the genomic DNA of three Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis). No amplification was detected with the genomic DNA of six other species of nematodes (A. suum, A. lumbricoides, T. canis, A. simplex, G. spinigerum and T. spiralis). The absence of amplification had been reported for A. suum and T. canis, in addition to Ancylostoma caninum and Strongyloides ratti (Bessarab and Joshua, 1997). Further studies on other species of angiostrongylids and other nematodes are needed to ascertain if the primers are genus-specific or family-specific. In the present study, the samples of A. cantonensis did not cluster unequivocally according to their geographical origin (Thailand, China, Japan and Hawaii). A possible reason could be the sequences used in this study are too short. In order to have a better resolution tree and relationship, longer sequences and region with higher resolution should be explored. This will form part of our continuing studies on the Angiostrongylus parasites. Two genes, cytochromec oxidase subunit I (COI) and internal transcribed spacer 2 (ITS-2) are being investigated. It is noteworthy that the nucleotide sequences of A. cantonensis from Taiwan (GenBank Accession No. U17585) appear to be very distant from the rest of the A. cantonensis samples (Fig. 3), with the uncorrected p-distance values ranging from 26.87% to 29.92% (Table 1). This apparent discrepancy will be investigated. More detailed studies to determine the extent of association between specific genotype of A. cantonensis and its intermediate snail host in different geographical localities will provide a foundation to better understand the origin, occurrence, pattern, and spreading of the disease in humans. In sum, the present study has added knowledge to the relatively not well understood 66-kDa protein gene of Angiostrongylus nematodes. The partial nucleotide sequences of this gene could unequivocally differentiate A. cantonensis, A. costaricensis and A. malaysiensis. On the basis of these nucleotide sequences, A. cantonensis is closer related to A. malaysiensis than A. costaricensis. Acknowledgments The authors would like to thank Mr Paibulaya Punthuprapasa, Mr Adisak Yoolek and Ms Jeeranan Pinnak for their excellent technical assistance. We also thank the Mahidol University and University of Malaya for support and facilities. This work received financial support from a grant for research (to P. Eamsobhana) on infectious diseases from the Department of Disease Control, Ministry of Public Health, Thailand; and a special research fund (to H.S. Yong) from the University of Malaya. The comments and suggestions by the reviewers and editor have greatly improved the paper and are gratefully acknowledged. References Bessarab, I.N., Joshua, G.W.P., 1997. Stage-specific gene expression in Angiostrongylus cantonesis: characterization and expression of an adultspecific gene. Mol. Biochem. Parasitol. 88, 73–84.
P. Eamsobhana et al. / Experimental Parasitology 126 (2010) 564–569 Bhaibulaya, M., Cross, J.H., 1971. Angiostrongylus malaysiensis (Nematoda: Metastrongylidae), a new species of rat lungworm from Malaysia. Southeast Asian J. Trop. Med. Public Health 2, 527–533. Caldeira, R.L., Carvalho, O.S., Mendonca, C.L.F.G., Graeff-Teixeira, C., Silva, M.C.F., Ben, R., Maurer, R., Lima, W.S., Lenzi, H.L., 2003. Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis and A. vasorum by polymerase chain reaction-restriction fragment length polymorphism. Mem. Inst. Oswaldo Cruz. 98, 1039–1043. Carreno, R.A., Nadler, S.A., 2003. Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences. J. Parasitol. 89, 965–973. Cross, J.H., 1979. Experimental studies on Angiostrongylus species and strains in monkeys and laboratory animals. In: Cross, J.H. (Ed.), Studies On Angiostrongyliasis in Eastern Asia and Australia. NAMRU-2-SP-44. U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, pp. 118–137. Cross, J.H., Bhaibulaya, M., 1974. Validity of Angiostrongylus malaysiensis, Bhaibulaya and Cross, 1971. Southeast Asian J Trop Med Public Health 5, 374–378. Eamsobhana, P., 2006. The Rat Lungworm Parastrongylus cantonensis: parasitology, immunology, eosinophilic meningitis, epidemiology and laboratory diagnosis. Wankaew (IQ) Book Center Co., Ltd., Bangkok. P. 156. Eamsobhana, P., Tungtrongchitr, A., 2005. Angiostrongyliasis in Thailand. In: Arizono, N., Chai, J.Y., Nawa, Y., Takahashi, T. (Eds.), Food-Borne Helminthiasis in Asia. The Federation of Asian parasitologist, Chiba, Japan, pp. 32–47. Eamsobhana, P., Yong, H.S., Mak, J.W., Tungtrongchitr, A., 1998. Use of a monoclonal antibody to identify antigenic differences among species and isolates of Parastrongylus (Nematoda: Metastrongylidae). Trop. Biomed. 15, 51–56.
569
Fontanilla, I.K.C., Wade, C.M., 2008. The small subunit (SSU) ribosomal (r) RNA as a genetic marker for identifying infective 3rd juvenile stage Angiostrongylus cantonensis. Acta Trop. 105, 181–186. Jefferies, R., Shaw, S.E., Viney, M.E., Morgan, E.R., 2009. Angiostrongylus vasorum from South America and Europe represent distinct lineages. Parasitology 136, 107–115. Morera, P., 1985. Abdominal angiostrongyliasis: a problem of public health. Parsitol. Today 6, 173–175. Posada, D., Crandall, K.A., 1998. ModelTest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Sawabe, K., Mikiya, K., 1994. Genetic variability in isozymes of Angiostrongylus malaysiensis. Southeast Asian J. Trop. Med. Public. Health 25, 728–734. Silva, A.C.A., Graeff-Teixeira, C., Zaha, A., 2003. Diagnosis of abdominal angiostrongyliasis by PCR from sera of patients. Rev Inst Med Trop S Paulo 45, 295–297. Swofford, D.L., 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 425–430. van Megan, H., van den Elsen, S., Holterman, M., et al., 2009. A phylogenetic tree of nematodes based on about 1200 full-lengh small subunit ribosomal DNA sequences. Nematology 11, 927–950. Wang, Q.P., Lai, D.H., Zhu, X.Q., Chen, X.G., Lun, Z.R., 2008. Human angiostrongyliasis. Lancet Infect. Dis. 8, 621–630.