Praphathip Eamsobhana

Immunological diagnosis of human angiostrongyliasis due to Angiostrongylus cantonensis (Nematoda: Angiostrongylidae)

Angiostrongylus cantonensis-associated eosinophilic meningitis in humans has been commonly reported worldwide. However parasitologically confirmed cases are not common, as the parasite has been recovered only infrequently from the cerebrospinal fluid of patients. The potential value of immunodiagnosis is therefore self-evident. Immunological tests can also help in the differential diagnosis of parasitic (particularly helminths) infections that cause eosinophilic meningitis. This paper summarizes the state of and advances in the immunological diagnosis of human angiostrongyliasis due to Angiostrongylus (= Parastrongylus) cantonensis. A specific antigen is available for the definitive diagnosis and unequivocal differentiation of eosinophilic meningitis due to helminth infections. Rapid diagnostic kits based on dot-blot ELISA have been developed and have proved to be simple, effective, and economical for field use.

Molecular differentiation of Angiostrongylus taxa (Nematoda: Angiostrongylidae) by cytochrome c oxidase subunit I (COI) gene sequences.

Nematodes of the genus Angiostrongylus are parasites of rodents and carnivores. They reside in the pulmonary or mesenteric arteries of their hosts. Two species are pathogenic in humans -Angiostrongylus cantonensis causes eosinophilic meningitis or meningoencephalitis, and Angiostrongylus costaricensis produces abdominal angiostrongyliasis. In addition Angiostrongylus malaysiensis may have the potential of being pathogenic in humans. The mitochondrial gene cytochrome c oxidase subunit I (COI) of these Angiostrongylus species and three geographical isolates (China, Hawaii and Thailand) of A. cantonensis were studied by polymerase chain reaction amplification and DNA sequencing. COI sequences of A. cantonensis, A. costaricensis and Angiostrongylus vasorum in the GenBank were included for comparison. Phylogenetic analysis by maximum-likelihood (ML), maximum-parsimony (MP), neighbour-joining (NJ) and Bayesian inference (BI) produced similar tree topology except variation in the bootstrap support values. There were two major clades - (1) A. cantonensis and A. malaysiensis, and (2) A. costaricensis and A. vasorum. The three geographical isolates of A. cantonensis formed a clade with low to high bootstrap values, and consisted of two subclades: (a) China and Hawaii isolates, and (b) monophyletic Thailand isolate. The individuals of each isolate formed a distinct cluster. In the second major clade, the Europe isolates of A. vasorum were distinctly different from the Brazil isolates. For A. costaricensis, the Costa Rica isolate was distinct from the Brazil isolate with an uncorrected (p) distance of 11.39%, indicating the possible occurrence of cryptic species. The present results indicate that COI sequences might be a useful marker for differentiating geographical isolates of A. cantonensis and in uncovering cryptic species. Efforts are being made to carry out an extensive collaborative study to cover a wide range of Angiostrongylus species and geographical isolates.

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)

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 66kDa 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%.

A dot-blot ELISA comparable to immunoblot for the specific diagnosis of human parastrongyliasis

A dot-blot enzyme-linked immunosorbent assay (dot-blot ELISA) using an electroeluted 31-kDa glycoprotein from adult worms of Parastrongylus cantonensis as the specific antigen was evaluated for the immunological diagnosis of patients infected with P. cantonensis. The sensitivity and specificity for the detection of serum antibody to P. cantonensis in dot-blot ELISA were both 100%, as determined with serum samples of ten P. cantonensis-infected patients, 60 patients with other related parasitic infections, and 20 uninfected controls. The test was as sensitive and specific as the immunoblot test which revealed a reactive band of 31 kDa. Both the dot-blot ELISA and immunoblot detected all sera from ten P. cantonensis-infected individuals, but not with those of other heterologous parasitoses (gnathostomiasis, toxocariasis, filariasis, paragonimiasis, cysticercosis and malaria) or sera from healthy controls. The dot-blot ELISA is much simpler to perform than the immunoblot technique, and the test can be applied under field conditions where sophisticated facilities are lacking.

Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

Molecular phylogeography of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) and genetic relationships with congeners using cytochrome b gene marker.

Angiostrongylus cantonensis is an important emerging zoonotic parasite causing human eosinophilic meningitis (or meningoencephalitis) in many parts of the world. To-date there is only a single study using mitochondrial cytochrome b (CYTB) gene to determine its genetic structure in eight geographical localities in Thailand. The present study examined the molecular phylogeography of this rat lungworm and its phylogenetic relationship with congeners using CYTB gene marker. A total of 15 CYTB haplotypes was found in 37 sequences from 14 geographical localities (covering north, west, east, central and south regions) in Thailand. These CYTB haplotypes were distinct from those of A. cantonensis for China and Hawaii. In Thailand, some CYTB haplotypes appeared to be confined to specific geographical localities. The partial CYTB DNA nucleotide sequences separated unequivocally the A. cantonensis isolates of Thailand, China and Hawaii as well as the congeners Angiostrongylus malaysiensis, A. costaricensis and Angiostrongylus vasorum, with A. malaysiensis grouped with A. cantonensis and A. costaricensis grouped with A. vasorum. Likewise the congeners of Metastrongylus and Onchocerca genera could also be clearly differentiated. The present study added two new definitive hosts (Bandicota savilei and Rattus losea) and three new localities (Mae Hong Son in the north, Tak in the west, and Phang Nga in the south) for A. malaysiensis in Thailand, indicating its wide occurrence in the country. Three CYTB haplotypes were found in the Thailand samples of A. malaysiensis. In addition to differentiation of congeners, CYTB gene marker could be used for determining the genetic diversity of a given population/taxon.

Complete mitochondrial genome of Bactrocera arecae (Insecta: Tephritidae) by next-generation sequencing and molecular phylogeny of Dacini tribe

The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Draft genome of neurotropic nematode parasite Angiostrongylus cantonensis, causative agent of human eosinophilic meningitis

Angiostrongylus cantonensis is a bursate nematode parasite that causes eosinophilic meningitis (or meningoencephalitis) in humans in many parts of the world. The genomic data from A. cantonensis will form a useful resource for comparative genomic and chemogenomic studies to aid the development of diagnostics and therapeutics. We have sequenced, assembled and annotated the genome of A. cantonensis. The genome size is estimated to be ∼260 Mb, with 17,280 genomic scaffolds, 91X coverage, 81.45% for complete and 93.95% for partial score based on CEGMA analysis of genome completeness. The number of predicted genes of ≥300 bp was 17,482. A total of 7737 predicted protein-coding genes of ≥50 amino acids were identified in the assembled genome. Among the proteins of known function, kinases are the most abundant followed by transferases. The draft genome contains 34 excretory-secretory proteins (ES), a minimum of 44 Nematode Astacin (NAS) metalloproteases, 12 Homeobox (HOX) genes, and 30 neurotransmitters. The assembled genome size (260 Mb) is larger than those of Pristionchus pacificus, Caenorhabditis elegans, Necator americanus, Caenorhabditis briggsae, Trichinella spiralis, Brugia malayi and Loa loa, but smaller than Haemonchus contortus and Ascaris suum. The repeat content (25%) is similar to H. contortus. The GC content (41.17%) is lower compared to P. pacificus (42.7%) and H. contortus (43.1%) but higher compared to C. briggsae (37.69%), A. suum (37.9%) and N. americanus (40.2%) while the scaffold N50 is 42,191. This draft genome will facilitate the understanding of many unresolved issues on the parasite and the disorder it causes.

Workshop on research priorities for management and treatment of angiostrongyliasis(1).

In a concluding session of the workshop, the participants developed a list of 115 research and outreach needs, outlining the top 5-7 needs in each of 8 areas (Table). For complete information, including presenter details and abstracts, visit the workshop website at www.hawaii.edu/cowielab/Angio%20website%20home.htm.