Molecular Characterization of a Reemergent Brugia malayi Parasite in Sri Lanka, Suggestive of a Novel Strain.

Sri Lanka achieved elimination status for lymphatic filariasis in 2016; still, the disease remains a potential public health issue. The present study is aimed at identifying a subperiodic Brugia sp. parasite which has reemerged in Sri Lanka after four decades via molecular-based analysis. Polymerase chain reaction performed with pan-filarial primers specific for the internal transcribed spacer region-2 (ITS-2) of the rDNA of Brugia filarial parasites isolated from human, canine, and feline blood samples yielded a 615 bp band establishing the species identity as Brugia malayi. Comparison of the ITS2 sequences of the reemerged B. malayi isolates with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi with similar phylogenetic evidence. However, the mean interspecies Kimura-2-parameter pairwise divergence between the generated Brugia sequences with B. malayi and B. pahangi was less than 3%. During the analysis of parsimony sites of the new ITS2 sequences, substitutions at A36T, A296G, T373A, and G482A made the sequences different from both B. pahangi and B. malayi suggesting the possibility of a new genetic variant or a hybrid strain of B. malayi and B. pahangi. Mosquito dissections and xenomonitoring identified M. uniformis and M. annulifera as vectors of this novel strain of B. malayi circulating among cats, dogs, and humans in Sri Lanka.

UDP-galactopyranose mutase, a potential drug target against human pathogenic nematode Brugia malayi.

Lymphatic filariasis, a vector-borne neglected tropical disease affects millions of population in tropical and subtropical countries. Vaccine unavailability and emerging drug resistance against standard antifilarial drugs necessitate search of novel drug targets for developing alternate drugs. Recently, UDP-galactopyranose mutases (UGM) have emerged as a promising drug target playing an important role in parasite virulence and survival. This study deals with the cloning and characterization of Brugia malayi UGM and further exploring its antifilarial drug target potential. The recombinant protein was actively involved in conversion of UDP-galactopyranose (substrate) to UDP-galactofuranose (product) revealing Km and Vmax to be ∼51.15 µM and ∼1.27 µM/min, respectively. The purified protein appeared to be decameric in native state and its 3D homology modeling using Aspergillus fumigatus UGM enzyme as template revealed conservation of active site residues. Two specific prokaryotic inhibitors (compounds A and B) of the enzyme inhibited B. malayi UGM enzymatic activity competitively depicting Ki values ∼22.68 and ∼23.0 µM, respectively. These compounds were also active in vitro and in vivo against B. malayi The findings suggest that B. malayi UGM could be a potential antifilarial therapeutic drug target.

Comparative analysis of ITS1 nucleotide sequence reveals distinct genetic difference between Brugia malayi from Northeast Borneo and Thailand.

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.

Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolution melting assay.

Human lymphatic filariasis is caused by filarial worms such as Brugia malayi for which the major reservoir is domestic cats. However, domestic cats or dogs also carry nonhuman filaria such as Brugia pahangi and Dirofilaria immitis. We have developed a single-tube, real-time PCR with a high-resolution melting (HRM) analysis assay for detection and identification of B. malayi, B. pahangi, and D. immitis in blood samples. The designated primer pair in the PCR can amplify a 114-bp region of mitochondrial 12S rRNA genes of these filarial worms. Subsequently, the HRM assay showed a specific melting temperature for each species. The assay showed the highest sensitivity and specificity in comparison with DNA sequences after assessment with 34 cat and 14 dog blood samples. This assay could be helpful for epidemiological studies of reservoirs and vectors.

Inventory and analysis of ATP-binding cassette (ABC) systems in Brugia malayi.

ABC systems are one of the largest described protein superfamilies. These systems have a domain organization that may contain 1 or more transmembrane domains (ABC_TM1F) and 1 or 2 ATP-binding domains (ABC_2). The functions (e.g., import, export and DNA repair) of these proteins distinguish the 3 classes of ABC systems. Mining and PCR-based cloning were used to identify 33 putative ABC systems from the Brugia malayi genome. There were 31 class 2 genes, commonly called ABC transporters, and 2 class 3 genes. The ABC transporters were divided into subfamilies. Three belonged to subfamily A, 16 to subfamily B, 5 to subfamily C, 1 to subfamily E and 3 to subfamilies F and G, respectively. None were placed in subfamilies D and H. Similar to other ABC systems, the ABC_2 domain of B. malayi genes was conserved and contained the Walker A and B motifs, the signature sequence/linker region and the switch region with the conserved histidine. The ABC_TM1F domain was less conserved. The relative abundance of ABC systems was quantified using real-time reverse transcription PCR and was significantly higher in female adults of B. malayi than in males and microfilaria, particularly those in subfamilies B and C, which are associated with drug resistance.

Differential detection of Brugia malayi and Brugia pahangi by real-time fluorescence resonance energy transfer PCR and its evaluation for diagnosis of B. pahangi-infected dogs.

A real-time fluorescence resonance energy transfer PCR combined with melting curve analysis was developed for differentiating Brugia malayi and Brugia pahangi DNA in host blood using one set of primers and fluorophore-labeled hybridization probes specific for HhaI repetitive DNA. The differentiation of both species was based on their melting temperatures (Tm). The mean Tm +/- SD of B. malayi and B. pahangi were 56.18+/-0.21 and 52.49+/-0.07, respectively. The method was used for the molecular detection of B. pahangi in infected dog blood samples. The diagnostic sensitivity, specificity, accuracy,and positive and negative predictive values of this method were 100%. The detected mean difference of the Tm might allow the simple discrimination of two related species. This method is fast, sensitive, allows for a high throughput, can be performed on very small volumes, and has potential for diagnosis of B. pahangi-infected dogs in endemic areas as well as for large epidemiological investigations.

Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1.

This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA sequencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host.

Intraspecies variation of Brugia spp. in cat reservoirs using complete ITS sequences.

The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites.

Detection of filarial parasites in domestic cats by PCR-RFLP of ITS1.

Lymphatic filariasis has been targeted by the World Health Organization (WHO) to be eliminated by the year 2020. In addition to chemotherapy and vector control, the control of reservoir hosts is necessary for the control program to succeed. Malayan filariasis, caused by Brugia malayi, is endemic in the South of Thailand where domestic cats serve as the major reservoir host. However, in nature, domestic cats also carry B. pahangi, Dirofilaria immitis and D. repens infections and it is difficult to distinguish the different filarial species from each other just by morphology. To assess the burden of filarial parasites, we performed a study on domestic cats in an endemic area of malayan filariasis in the Prasang district, of Surat Thani, a province in Southern Thailand. Together with Giemsa staining and acid phosphatase activity studies, we performed PCR-RFLP analysis on the first internal transcribed spacer (ITS1) region of ribosomal DNA (rDNA). PCR-RFLP with Ase I could clearly differentiate between B. malayi, B. pahangi, D. immitis and D. repens. Out of the 52 cats studied, filarial parasites were identified in 5 (9.5%) cats, of which 4 (7.6%) were B. pahangi and 1 (1.9%) D. immitis. This PCR-RFLP technique detected two additional cats that were not detected by microscopy. The domestic cats are not an important host of B. malayi in this region. We could develop the PCR-RFLP assay test for differentiating filarial nematodes which can be applied to survey human, animal reservoir hosts and mosquito vectors in endemic areas.

Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis.

Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method to detect and differentiate a broad range of filarial species in a single PCR. The first internal transcribed spacer 1 (ITS1) along with the flanking 18S and 5.8S ribosomal DNA (rDNA) were isolated and cloned from Wuchereria bancrofti, Brugia malayi, and Brugia pahangi. Sequence analysis identified conserved sites in the 18S and 5.8S rDNA sequence that could be used as universal priming sites to generate ITS1-distinctive PCR products that were useful for distinguishing filariae at the genus level. The addition of a digestion of the ITS1 PCR product with the restriction endonuclease Ase I generated a fragment profile that allowed differentiation down to the species level for W. bancrofti, B. malayi, B. pahangi, Dirofilaria immitis, and D. repens. The PCR-RFLP of ITS1 rDNA will be useful in diagnosing and differentiating filarial parasites in human, animal reservoir hosts, and mosquito vectors in disease-endemic areas.

PCR based method for identification of zoonostic Brugia malayi microfilariae in domestic cats.

The survey of 326 human blood samples in the endemic area of Surat Thani and Narathiwat, the provinces in the south of Thailand, revealed that 5 of them were infected with Brugia malayi. Similarly, 53 feline blood samples were also investigated and found that 15 of the domestic cats were also infected with B. malayi. Upon the examination of human and feline blood specimens, a pair of human and domestic cat stayed in the same house and region. The periodicities of human B. malayi and feline B. malayi were similar as well as the results of Giemsa and acid phosphatase stained blood films of microfilaria positive cases. Likewise, the PCR-RFLP profile of Hha I repeat genes and PCR amplification of Trans-Spliced Leader Exon I (SLX) demonstrated that 15 samples the feline B. malayi were the same as those of human B. malayi. The data indicated that domestic cat plays an important role as the animal reservoir for B. malayi in the endemic areas of Thailand.