Nodulisporic acid produces direct activation and positive allosteric modulation of AVR-14B, a glutamate-gated chloride channel from adult Brugia malayi.

Glutamate-gated chloride channels (GluCls) are unique to invertebrates and are targeted by macrocyclic lactones. In this study, we cloned an AVR-14B GluCl subunit from adult Brugia malayi, a causative agent of lymphatic filariasis in humans. To elucidate this channel's pharmacological properties, we used Xenopus laevis oocytes for expression and performed two-electrode voltage-clamp electrophysiology. The receptor was gated by the natural ligand L-glutamate (effective concentration, 50% [EC50] = 0.4 mM) and ivermectin (IVM; EC50 = 1.8 nM). We also characterized the effects of nodulisporic acid (NA) on Bma-AVR-14B and NA-produced dual effects on the receptor as an agonist and a type II positive allosteric modulator. Here we report characterization of the complex activity of NA on a nematode GluCl. Bma-AVR-14B demonstrated some unique pharmacological characteristics. IVM did not produce potentiation of L-glutamate-mediated responses but instead, reduced the channel's sensitivity for the ligand. Further electrophysiological exploration showed that IVM (at a moderate concentration of 0.1 nM) functioned as an inhibitor of both agonist and positive allosteric modulatory effects of NA. This suggests that IVM and NA share a complex interaction. The pharmacological properties of Bma-AVR-14B indicate that the channel is an important target of IVM and NA. In addition, the unique electrophysiological characteristics of Bma-AVR-14B could explain the observed variation in drug sensitivities of various nematode parasites. We have also shown the inhibitory effects of IVM and NA on adult worm motility using Worminator. RNA interference (RNAi) knockdown suggests that AVR-14 plays a role in influencing locomotion in B. malayi.

Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development.

Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.

Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans.

BACKGROUND: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. 'hongkongensis'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott's test (MKT). RESULTS: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. 'hongkongensis'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. CONCLUSIONS: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT' small and portable MinION™ means that such methods could be deployed for field use.

Mass Spectrometric and Glycan Microarray-Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens.

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host-parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.

Pharmacological Profiling of a Brugia malayi Muscarinic Acetylcholine Receptor as a Putative Antiparasitic Target.

The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor of B. malayi (Bma-GAR-3) that is highly expressed across intramammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Finally, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

Diethylcarbamazine elicits Ca2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles.

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals' lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.

Activation of mosquito immunity blocks the development of transmission-stage filarial nematodes.

Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.

Localization and RNA Interference-Driven Inhibition of a Brugia malayi-Encoded Interleukin-5 Receptor Binding Protein.

A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by ~50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.

Canine filaria species in selected lymphatic filariasis endemic and non-endemic areas in Sri Lanka.

Subperiodic brugian filariasis and dirofilariasis show a rising trend in Sri Lanka posing a threat to public health. As information was limited on canine filaria species in Sri Lanka, we studied the filaria parasites among dog populations in lymphatic filariasis (LF) endemic and non-endemic regions by microscopy and molecular methods. Thick blood smears (TBSs) were performed among 295 dogs presenting to veterinary clinics for surgical or sterilization procedures in Galle (LF endemic) and Mullaitivu (LF non-endemic) districts, of which 55.6% were positive for any microfilariae. We identified Dirofilaria repens (50.8%) and Brugia spp. (20.6%) by microscopy, which, included mono-infections (D. repens 35.3% and Brugia spp. 5%) and co-infections (15.6%). Infections in Galle and Mullaitivu were 61% and 44.9% respectively. The brugian filariasis rate was significantly higher among canines in LF endemic Galle district (29.9%) than in Mullaitivu (LF non-endemic) (1.1%) (P < 0.001), while D. repens infections were comparable in both districts. Genomic DNA extracted from 10% of microfilariae positive TBSs was amplified using pan-filarial primers targeting the internal-transcriber-spacer region-2 (ITS-2). Sequencing of amplicons confirmed the presence of D. repens (89.28%), Brugia pahangi (7.14%) and B. malayi (3.57%) infections. The phylogeny constructed and analysed in MEGA X indicated genetic variability among D. repens and B. pahangi isolates from Sri Lanka. With this study, we were able to report B. pahangi infections for the first time in Sri Lanka.

Cross-protective efficacy of immuno-stimulatory recombinant Brugia malayi protein HSP60 against the Leishmania donovani in BALB/c mice.

Coinfection of Leishmania with bacteria, viruses, protozoans, and nematodes alter the immune system of the host, thereby influencing the disease outcomes. Here, we have determined the immunogenic property and protective efficacy of the cross-reactive molecule HSP60 of filarial parasite B. malayi against the L. donovani in BALB/c mice. Parasitological parameters results showed a significant decrease in the parasite burden (~59%; P < 0.001) and also a substantial increase in the delayed-type hypersensitivity (DTH) response (P < 0.001) in mice immunized with 10 µg of rBmHSP60. Protection against L. donovani in mice immunized with rBmHSP60 resulted from activation of the T cells, which is characterized by higher levels of nitric oxide (NO) production, enhanced cell proliferation, higher levels (expression and release) of IFN- γ, TNF- α, and IL-12, also, higher production of IgG and IgG2a antibodies. This strong Th1 immune response creates an inflammatory domain for L. donovani and protects the host from VL.

Chemotactic responses of Brugia pahangi infective third-stage larvae to tris(hydroxymethyl)aminomethane-related compounds and amino acids.

Since the exogenous compound tris(hydroxymethyl)aminomethane (Tris) showed a potent chemoattractant activity for Brugia pahangi infective third-stage larvae (L3), it was assumed that, in natural infection to a host, filarial L3 can be expected to recognize an endogenous Tris-related compound. In addition, a few amino acids have been identified as water-soluble attractants for second-stage juveniles of Meloidogyne incognita, a plant parasitic nematode. Therefore, the present study assesses the in vitro chemotactic responses of B. pahangi L3 to Tris-related compounds and amino acids using an agar-plate assay. Among Tris-related compounds, 2-amino-1,3-propanediol (APD) and 2-amino-2-methyl-1,3-propanediol (AMPD) exhibited a potent chemoattractant activity for filarial L3 at a level similar to Tris. Furthermore, arginine (Arg) was identified as a potent attractant for filarial L3 among amino acids. In addition, filarial L3 were attracted to Arg, APD and AMPD in mild alkaline conditions rather than acidic conditions. The chemoattractant activity of the three compounds for filarial L3 was observed in concentrations between 6.3 and 200 mm. This is the first report to demonstrate that Arg, APD and AMPD are potent chemoattractants for B. pahangi L3. Endogenous Arg and APD, in particular, may be involved in the regulation of the chemotactic behaviour of filarial L3 in the infection to a host. The present results will help to elucidate the mechanism of filarial skin-penetrating invasion of a host.

Wuchereria bancrofti macrophage migration inhibitory factor-2 (rWbaMIF-2) ameliorates experimental colitis.

Immunomodulatory molecules produced by helminth parasites are receiving much attention recently as novel therapeutic agents for inflammation and autoimmune diseases. In this study, we show that macrophage migration inhibitory factor (MIF) homologue from the filarial parasite, Wuchereria bancrofti (rWbaMIF-2), can suppress inflammation in a dextran sulphate sodium salt (DSS)-induced colitis model. The disease activity index (DAI) in DSS given mice showed loss of body weight and bloody diarrhoea. At autopsy, colon of these mice showed severe inflammation and reduced length. Administration of rWbaMIF-2 significantly reduced the DAI in DSS-induced colitis mice. rWbaMIF-2-treated mice had no blood in the stools, and their colon length was similar to the normal colon with minimal inflammation and histological changes. Pro-inflammatory cytokine genes (TNF-α, IL-6, IFN-γ, IL-1ß, IL-17A and NOS2) were downregulated in the colon tissue and peritoneal macrophages of rWbaMIF-2-treated mice. However, there were significant increases in IL-10-producing Treg and B1 cells in the colon and peritoneal cavity of rWbaMIF-2-treated mice. These findings suggested that rWbaMIF-2 treatment significantly ameliorated the clinical symptoms, inflammation and colon pathology in DSS given mice. This immunomodulatory effect of rWbaMIF-2 appeared to be by promoting the infiltration of Treg cells into the colon.

Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics.

Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (Kb 13.9 nM), P-nAChRs activated by pyrantel (Kb 126 nM), and L-nAChRs activated by levamisole (Kb 0.96 µM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L- and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M- and/or N-receptors) did not.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.

Effect of Brugia pahangi co-infection with Plasmodium berghei ANKA in gerbils (Meriones unguiculatus).

Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.

Risk of Recrudescence of Lymphatic Filariasis after Post-MDA Surveillance in Brugia malayi Endemic Belitung District, Indonesia.

Belitung district in Bangka-Belitung Province, Indonesia with a population of 0.27 million is endemic for Brugia malayi and 5 rounds of mass drug administration (MDA) were completed by 2010. Based on the results of 3 transmission assessment surveys (TAS), the district is declared as achieving elimination of lymphatic filariasis (LF) in 2017. The findings of an independent survey conducted by the National Institute of Health Research and Development (NIHRD) in the same year showed microfilaria (Mf) prevalence of 1.3% in this district. In 2019, NIHRD conducted microfilaria survey in 2 villages in Belitung district. Screening of 311 and 360 individuals in Lasar and Suak Gual villages showed Mf prevalence of 5.1% and 2.2% with mean Mf density of 120 and 354 mf/ml in the respective villages. Mf prevalence was significantly higher among farmers and fishermen compared to others and the gender specific difference was not significant. The results of a questionnaire based interview showed that 62.4% of the respondents reported to have participated in MDA in Lasar while it was 57.7% in Suak Gual village. About 42% of the Mf positive cases did not participate in MDA. Environmental surveys identified many swampy areas supporting the breeding of Mansonia vector species. Persistence of infection is evident and in the event of successful TAS3 it is necessary to monitor the situation and plan for focal MDA. Appropriate surveillance strategies including xenomonitoring in post-MDA situations need to be developed to prevent resurgence of infection. Possible role of animal reservoirs is discussed.

Decreased nematode clearance and anti-phosphorylcholine-specific IgM responses in mannose-binding lectin-deficient mice.

Brugia malayi is a nematode that causes human lymphatic filariasis. Previously, we showed that mannose-binding lectin (MBL)-A is necessary for clearance of B. malayi microfilariae in mice and presence of MBL-A is linked with maximal levels of parasite-specific IgM. Common human MBL gene polymorphisms result in low MBL expression and lead to recurring bacterial infections. Furthermore, these low-expressing human MBL polymorphisms result in greatly increased susceptibility to lymphatic filarial infection. Indeed, gain of new filarial infections over a 30-year period are 10-fold higher in people with low, compared to high, MBL-expression phenotypes. Human MBL closely resembles mouse MBL-C, rather than MBL-A; therefore, we examined the role of mouse MBL-C in clearance of microfilariae. Absence of MBL-C alone, or both MBL-A and -C, resulted in delayed clearance of microfilariae and reduced parasite-specific IgM in mice. There were few profound changes in B cell sub-populations or in the ability of MBL-deficient mice to respond to T-dependent or T-independent antigens. However, absence of MBL-A and/or MBL-C resulted in reduced IgM to phosphorylcholine, a constituent of filarial and bacterial antigens, suggesting that inability to form proficient antibody responses to this moiety leads to lack of microfilarial clearance and overall susceptibility to filariasis.

Diagnostic Identification and Differentiation of Microfilariae.

The morphologic similarities of the microfilariae and their infrequency in clinical specimens in settings of endemicity present challenges to clinical laboratories in maintaining competence for accurate identification and differentiation. We present here a review of the primary filarial nematodes causing human infection, including an illustrated key, which we hope will improve the diagnostic capabilities of hematologists, microbiologists, medical technologists, and similarly qualified laboratorians.

Immunome and immune complex-forming components of Brugia malayi identified by microfilaremic human sera.

Adult Brugia malayi proteins with high potential as epidemiological markers, diagnostic and therapeutic targets, and/or vaccine candidates were revealed by using microfilaremic human sera and an immunoproteomic approach. They were HSP70, cytoplasmic intermediate filament protein, independent phosphoglycerate mutase, and enolase. Brugia malayi microfilaria-specific proteins that formed circulating immune complexes (ICs) were investigated. The IC-forming proteins were orthologues of hypothetical protein Bm1_12480, Pao retrotransposon peptidase family protein, uncoordinated protein 44, NAD-binding domain containing protein of the UDP-glucose/GDP-mannose dehydrogenase family which contained ankyrin repeat region, ZU5 domain with C-terminal death domain, C2 domain containing protein, and FLJ90013 protein of the eukaryotic membrane protein family. Antibodies to these proteins were not free in the microfilaremic sera, raising the possible role of the IC-forming proteins in an immune evasion mechanism of the circulating microfilariae to avoid antibody-mediated-host immunity. Moreover, detection of these ICs should be able to replace the inconvenient night blood sampling for microfilaria in an evaluation of efficacy of anti-microfilarial agents.

Therapeutic efficacy of doxycycline in domestic cats naturally infected with Brugia malayi in field condition.

OBJECTIVE: The aim of this study was to evaluate the efficacy of oral doxycycline treatment for Brugia malayi as measured by microfilarial and filarial DNA clearance in naturally infected domestic cats. METHODS: This study included 8 domestic cats that lived with families that resided in Tak Bai District of Narathiwat Province, which is located in Southern Thailand. The study area is a known B. malayi endemic area. All study cats received doxycycline treatment doses by their respective owners according to a previously described protocol. Briefly, doxycycline (VibraVet@) was given orally once a day during weeks 1-4, 10-11, and 16-17. Blood collections were performed at baseline before treatment, and then every month for 12 months after the initial dose of doxycycline to assess microfilaraemia by Giemsa stain, and filarial DNA detection by high-resolution melt (HRM) real-time polymerase chain reaction (PCR). RESULTS: One month after the start of doxycycline treatment, five of eight cats were negative for microfilaraemia, and 4 of those were negative for filarial DNA. All cats receiving doxycycline treatment were negative for microfilaria by Giemsa stain, and for filarial DNA by HRM real-time PCR within 8 months after receiving the initial dose of doxycycline treatment. CONCLUSION: Administration of oral doxycycline to domestic cats naturally infected with B. malayi in disease endemic areas can significantly reduce microfilaraemia at 1 month and filarial DNA was undetectable by 8 months after the initial dose of doxycycline treatment. No recurrence of microfilaraemia or filarial DNA was observed in study cats at 1 year after the start of doxycycline. Included cats appeared to tolerate doxycycline (VibraVet@) well, with no adverse drug reactions reported by any study cat owner.