Brugia malayi is one of the parasitic worms which causes lymphatic filariasis in humans. Its geographical distribution includes a large part of Asia. Despite its wide distribution, very little is known about the genetic variation and molecular epidemiology of this species. In this study, the internal transcribed spacer 1 (ITS1) nucleotide sequences of B. malayi from microfilaria-positive human blood samples in Northeast Borneo Island were determined, and compared with published ITS1 sequences of B. malayi isolated from cats and humans in Thailand. Multiple alignment analysis revealed that B. malayi ITS1 sequences from Northeast Borneo were more similar to each other than to those from Thailand. Phylogenetic trees inferred using Neighbour-Joining and Maximum Parsimony methods showed similar topology, with 2 distinct B. malayi clusters. The first cluster consisted of Northeast Borneo B. malayi isolates, whereas the second consisted of the Thailand isolates. The findings of this study suggest that B. malayi in Borneo Island has diverged significantly from those of mainland Asia, and this has implications for the diagnosis of B. malayi infection across the region using ITS1-based molecular techniques.
The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.
This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
Echinococcus granulosus is a worldwide zoonotic infection that causes human cystic echinococcosis (CE) or hydatid disease. The present study describes the isolation and production of a monoclonal antibody against recombinant AgB protein using the developed Human AntibodY Disease ENhanced (HAYDEN)-Filariasis library. The DNA sequences of the isolated clones were analyzed, followed by gene analysis and binding assays. Clone E1 showed a full-length sequence and represents the IgHV5-LV3 antibody gene family. The antibody protein yield was satisfactory, and it reacted specifically against rAgB. The novel E1 protein is potentially useful for the development of an antigen detection assay for CE. The ability of the Brugia malayi immune antibody library to isolate antibodies against Echinococcus granulosus antigens highlights the broad coverage of immune antibody libraries.
In the absence of a suitable Brugia malayi antigen detection assay, PCR remains one of the more sensitive alternatives to Giemsa-stained thick blood films for B. malayi detection. The need for refrigerated storage and transportation of blood has limited the use of PCR for large-scale epidemiology studies in remote endemic areas. Here we report simple finger-prick blood-spot collection, a one-tube DNA template extraction method and the development of a B. malayi-specific nested PCR assay. The assay was tested on 145 field samples and was positive for all 30 microscopy-positive samples and for an additional 13 samples which were microscopy-negative.
Random amplification of polymorphic DNA (RAPD) was used to analyse genomic DNA from virgin females and males of Brugia malayi, with a view to identifying sex-specific differences predicted by an XX/XY system of chromosomal sex determination. A product of 2338 bp, amplified with the arbitrary primer 5' GTTGCGATCC 3', was obtained exclusively from males. Primers based on the sequence of this product amplified a DNA fragment of the expected size from each of two independent isolates of B. malayi (from Malaysia and Indonesia) by PCR. No reaction product was obtained from the closely related species Brugia pahangi. In a genetic cross between B. malayi males and B. pahangi females, F1 hybrid microfilariae were PCR-positive, indicating that the locus is paternally-inherited. Southern blotting demonstrated that the target sequence resides in the high molecular weight fraction of genomic DNA, confirming that it is of chromosomal, rather than mitochondrial, origin. Sequencing of the locus revealed significant similarity with members of a family of reverse transcriptase-like genes in Caenorhabditis elegans. In-frame stops indicate that the gene is non-functional, but multiple bands of hybridisation in Southern blots suggest that the RT sequence may be the relic of a transposable element. Multiple repeats of the dinucleotide AT occurred in another region of the sequence. These varied in number between the two isolates of B. malayi in the manner of a microsatellite, surprisingly the first to be described from the B. malayi genome. Because of its association with the Y chromosome, we have given the locus the acronym TOY (Tag On Y). Identification of this chromosome-specific marker confirms the XX/XY heterogametic karyotype in B. malayi and opens the way to elucidation of the role of Y in sex determination.