Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes.

BACKGROUND: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack. CONCLUSIONS/SIGNIFICANCE: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

Ancient horizontal transfers of retrotransposons between birds and ancestors of human pathogenic nematodes.

Parasite host switches may trigger disease emergence, but prehistoric host ranges are often unknowable. Lymphatic filariasis and loiasis are major human diseases caused by the insect-borne filarial nematodes Brugia, Wuchereria and Loa. Here we show that the genomes of these nematodes and seven tropical bird lineages exclusively share a novel retrotransposon, AviRTE, resulting from horizontal transfer (HT). AviRTE subfamilies exhibit 83-99% nucleotide identity between genomes, and their phylogenetic distribution, paleobiogeography and invasion times suggest that HTs involved filarial nematodes. The HTs between bird and nematode genomes took place in two pantropical waves, >25-22 million years ago (Myr ago) involving the Brugia/Wuchereria lineage and >20-17 Myr ago involving the Loa lineage. Contrary to the expectation from the mammal-dominated host range of filarial nematodes, we hypothesize that these major human pathogens may have independently evolved from bird endoparasites that formerly infected the global breadth of avian biodiversity.

Characterization of a ribosomal DNA clone of Brugia malayi.

The structure of ribosomal DNA (rDNA) clone pBmr7 from microfilariae of the human parasite Brugia malayi has been examined in detail by Southern blot analysis and S-1 mapping techniques. The results demonstrate that this clone contains regions homologous to 28S, 18S and 5.8S rDNAs. A noncoding or 'spacer' region lies between the 3' end of 28S rDNA and the 5' end of 18S rDNA. An AccI-Sau3AI fragment of approximately 900 bp from this spacer region cross-hybridizes to genomic DNA fragments of different sizes from Brugia pahangi and Dirofilaria immitis. The differences observed in hybridization suggest that this rDNA fragment can be used to differentiate between various filariid species.

A DNA probe cloned in Escherichia coli for the identification of Brugia malayi.

This report describes a specific and sensitive DNA probe for the identification of Brugia malayi. A genomic DNA library produced from subperiodic B. malayi microfilariae was screened to detect clones containing DNA sequences which are highly repeated within the parasite genome. Several clones were further analyzed to identify those which hybridize specifically with B. malayi DNA but not with DNA from B. pahangi and Dirofilaria immitis. From these, clone pBm15 was selected because it hybridized with high sensitivity to B. malayi DNA as detected by autoradiography. Clone pBm15 was sensitive enough to detect two infective larvae or five microfilariae or 300 pg of purified B. malayi microfilarial DNA. This study forms the basis for the development of a specific and sensitive DNA probe for the identification of B. malayi in field specimens.

Identification of Brugia malayi in vectors with a species-specific DNA probe.

We evaluated the potential value of a cloned sequence of genomic DNA of Brugia malayi as a species-specific probe. Clone pBm 15 reacted with all stages of 8 different geographic isolates of B. malayi and cross-hybridized with microfilariae of B. timori. It did not hybridize with Wuchereria bancrofti or with B. pahangi, W. kalimantani, Dirofilaria repens, Breinlia booliati or Cardiofilaria species, animal filariids that can be sympatric with B. malayi. P32-labeled clone pBm 15 correctly identified mosquitoes infected even with 1 infective larva of B. malayi. This specific DNA probe should be an invaluable tool to monitor control programs of Brugian filariasis.

Periodicity studies of Brugia malayi in Indonesia: recent findings and a modified classification of the parasite.

We have recently reinvestigated the position of Brugia malayi in Indonesia. Periodicity patterns of microfilariae from several endemic areas were mathematically determined. We have also designed a simple method to quantify microfilaria periodicities in these studies. To determine whether periodicity patterns of microfilariae were stable, repeated studies were performed in the same individual or community. Other biological features of the parasite were also investigated. The parasite from each isolate was taxonomically identified as B. malayi. It could be classified into two distinct biological types, one nocturnally periodic and the other aperiodic, nocturnally subperiodic, or nocturnally periodic. We therefore propose to modify Wilson's classification, using the biological behaviour of the parasite in animals as the discriminating feature, and classify the two types as zoophilic and anthropophilic B. malayi.

The microfilaria of Brugia timori (Partono et al. 1977 = Timor microfilaria, David and Edeson, 1964): morphologic description with comparison to Brugia malayi of Indonesia.

The microfilaria of Brugia timori was compared with microfilariae of Indonesian strains of periodic and subperiodic Brugia malayi using alcohol-fixed (stained) and formalin-fixed (unstained) preparations. As noted by other observers of the Timor microfilaria, the absence of a stained sheath in Giemsa preparations, a long cephalic space with a length-to-width ratio of about 3:1, and a great overall body length are features which most readily distinguish this parasite. Additionally, B. timori has greater numbers of single row nuclei in the terminal column of body cells and a lesser bulge of the cuticle surrounding nuclei in the distal portion of the tail than does B. malayi. About 60% of B. timori microfilariae were exsheathed in haemalum-stained thick blood films. Brugia timori microfilariae were found to be distinct from microfilariae of B. malayi by comparing percentages of total body length included between the cephalic tip and major internal anatomic markers.

Brugia timori sp. n. (nematoda: filarioidea) from Flores Island, Indonesia.

Brugia timori sp. n. from experimentally-infected Mongolian jirds (Meriones unguiculatus) is described. The adult male differs from other Brugia species, except Brugia malayi, in having a spicular ratio of 3 : 1; it differs from B. malayi in having greater numbers of subventral adanal papillae (up to 5 on each side) that are loosely spaced and irregularly positioned about the cloaca, a greater diameter of the capitulum of the left spicule, greater lengths of the proximal- and midsections of the left spicule, and a greater length of the proximal section of the right spicule. The adult female has an ovejector of greater length and width than that of B. malayi. Microfilariae in the blood of persons from whom the parasite lines of this study originated were typical of the Timor type, having a greater total body length than other Brugia spp., a length to width cephalic space ratio of about 3 : 1, and a sheath which did not stain when processed in the usual manner with Giemsa.

Differentiation of species and life cycle stages of Brugia spp. by isoenzyme analysis.

The isoenzyme patterns of glucose phosphate isomerase and phosphoglucomutase of 3 species of Brugia, B. pahangi, subperiodic B. malayi, and B. patei, and 3 life cycle stages, adult, third-stage larva, and microfilaria were compared using the technique of isoelectricfocusing on polyacrylamide gels. The results demonstrated that the adults of all 3 species could be identified from one another and that differences existed between the sexes of any one species. Hybridization between B. pahangi and B. patei could be detected in the progeny of the cross. Both the third-stage larvae and microfilariae of B. malayi and B. pahangi were differentiated and the epidemiological significance and the application of these findings to arthropod-borne filarial infections were discussed.

Brugia lepori sp. n. (Filarioidea: Onchocercidae) from rabbits (Sylvilagus aquaticus, S. floridanus) in Louisiana.

Brugia lepori sp. n., a filarial nematode from the abdominal lymphatics and subcutaneous tissues of rabbits (Sylvilagus aquaticus, S. floridanus), from St. Tammany Parish, Louisiana, is described. Brugia lepori is of moderate size (males 12 to 19 mm, females 39 to 45 mm) and within the genus most closely resembles Brugia beaveri of the raccoon, from which it can be distinguished by its larger size, smaller spicules, and smaller microfilaria which has a shorter cephalic space. Brugia lepori is only the second species of Brugia described from North America and the third species reported from the Western Hemisphere.

The differentiation of brugia malayi, B. pahangi, B. tupaiae and Wuchereria bancrofti.

During studies on filariasis in Thailand, attempts were made to differentiate Wuchereria bancrofti and Brugia spp. by morphological characteristics, as well as by vector species.

Molecular phylogenetic studies on Brugia filariae using Hha I repeat sequences.

This paper is the first molecular phylogenetic study on Brugia parasites (family Onchocercidae) which includes 6 of the 10 species of this genus: B. beaveri Ash et Little, 1964; B. buckleyi Dissanaike et Paramananthan, 1961: B. malayi (Brug, 1927) Buckley, 1960; B. pahangi (Buckley et Edeson, 1956) Buckley, 1960; B. patei (Buckley, Nelson er Heisch, 1958) Buckley, 1960 and B. timori Partono et al., 1977. Hha l repeat sequences are 322 nucleotides long, highly repeated, tandemly arranged and unique to the nuclear genomes of the genus Brugia. Hha l repeat sequence data was collected by PCR, cloning and dideoxy sequencing. The Hha l repeat sequences were aligned and analyzed by maximum parsimony algorithms, distance methods and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the robustness of the different phylogenetic reconstructions. The data indicated that the Hha l repeat sequences are highly conserved within species yet differ significantly between species. The various tree-building methods gave identical results. Bootstrap analyses on the Hha l repeat sequence data set identified at least two clades: the B. pahangi-B. beaveri clade and the B. malayi-B. timori-B. buckleyi clade; the first clade includes parasites of carnivores from Asia and America; the second includes species from primates and lagomorphs from Asiatic region. It was also noted that the Hha l repeat sequences obtained from B. malayi were identical to those obtained from B. timori, indicating very recent speciation.

Inter and intra-specific diversity of parasites that cause lymphatic filariasis.

Lymphatic filariasis is caused by three closely related nematode parasites: Wuchereria bancrofti, Brugia malayi and Brugia timori. These species have many ecological variants that differ in several aspects of their biology such as mosquito vector species, host range, periodicity, and morphology. Although the genome of B. malayi (the first genome sequenced from a parasitic nematode) has been available for more than five years, very little is known about genetic variability among the lymphatic dwelling filariae. The genetic diversity among these worms is not only interesting from a biological perspective, but it may have important practical implications for the Global Program to Eliminate Lymphatic Filariasis, as the parasites may respond differently to diagnostic tests and/or medical interventions. Therefore, better information on their genetic variability is urgently needed. With improved methods for nucleic acid extraction and recent advances in sequencing chemistry and instrumentation, this gap can be filled relatively inexpensively. Improved information on filarial genetic diversity may increase the chances of success for lymphatic filariasis elimination programs.

[Discrimination of geographic strains of periodic Brugia malayi by the cuticular ornamentation of the male]. / Discrimination de souches géographiques de Brugia malayi périodique par l'ornementation cuticulaire des males.

A comparative study of five periodic human strains of Brugia malayi, originating from India, China, Korea, Malaysia and Indonesia, is given. This morphological analysis is based on males; the "standard" characters (oesophagus, papillae, spicules...) appear identical. On the contrary, the cuticular ornamentation of the posterior region--which is composed of the area rugosa and of a system of bosses and constitutes a secondary non-skid copulatory apparatus--differs following the geographical origin of the strain. A key is given, based on this character. 1(2) At 800-1,200 micron from the tip of tail, numerous cuticular bosses present on the right side of the body (fig. 2 and 8 B). 2(1) At 800-1,200 micron from the tip of tail, cuticular bosses absent or scarce on the right side of the body (fig. 8 D). 3(4) At 1,800-1,200 micron from the tip of tail (fig. 4), scarce and slightly projecting cuticular bosses on the dorsal side of the body contrasting with well projecting lateral cuticular bosses (fig. 9 E and F). Anterior extremity of the area rugosa made by a few stripes of tiny bosses linked transversally (fig. 9 A). 4(3) At 1,800-2,200 micron, numerous cuticular bosses on the dorsal side of the body (figs. 5, 6 and 7). Anterior extremity of the area rugosa made by the stripes of longitudinal rods (fig. 9C). 5(6) Oblong transversally stretched cuticular bosses on the dorsal and left sides of the body, anteriorly to the area rugosa (fig. 5); big oblong bosses on the left side (fig. 9 B). Transversal wrinkles and stripes of rods absent on the dorsal side of the body. 6(5) Round cuticular bosses on the dorsal and left sides of the body anteriorly to the area rugosa (figs. 6 and 7): no big oblong bosses on the left side. Transversal wrinkles or stripes of rods present on the dorsal side of the body (fig. 9 D). Nomenclaturally, such differences could be used in defining different taxa, but it could be useful to perform "blind determination" (material without labelling), to study conveniently the morphology of microfilariae (often an excellent indication for speciation in that group of Nematodes) and, evenly, to proceed to parallel studies on isoenzymes. However, whatever could be the taxonomical conclusion, the differences observed in Brugia malayi originating from different regions appear to the sufficient to consider the existence of four distinct diseases.

Use of restriction fragment length polymorphisms (RFLPs) to distinguish between nematodes of pathogenic significance.

The availability of restriction fragment length polymorphisms (RFLPs) would be useful for studying the extent of diversity among morphologically indistinguishable populations of filarial parasites. Such polymorphisms may be useful in correlating various physiological and clinical differences with parasite heterogeneity. In order to identify such RFLPs, we isolated DNA from microfilaria of 6 filarial species (Acanthocheilonema viteae, Brugia malayi, Brugia pahangi, Dirofilaria immitis, Litomosoides carinii and Setaria digitatum), digested the DNA with several restriction endonucleases, prepared Southern blots and probed with 32P-labelled DNA probes. The patterns of fragments generated using two restriction endonucleases, Mbo I and Taq I, in combination with two probes, rDNA from the free-living soil nematode Caenorhabditis elegans, and pBM103, an anonymous DNA probe from B. malayi, unequivocally distinguish between all 6 of the species. To ensure that the differences we observed between the species represent true interspecies variation rather than fortuitous individual variations we analysed DNA from several individual B. malayi and B. pahangi worms. The individual B. malayi worms demonstrated restriction profiles that were invariant, as did the individual B. pahangi worms, demonstrating that the differences we observed were true interspecies variations.