Filarial Parasite Research Articles

Complete mitochondrial genome of Onchocerca lupi (Nematoda, Onchocercidae)

Onchocerca lupi, Rodonaja 1967, is an emerging, zoonotic filarial nematode parasite that causes ocular disease in dogs, cats, wild canids, and humans. It is the causative agent of ocular onchocercosis in canines with increasing incidence in both North America and the Old World during the early twenty-first century. We report the complete mitochondrial genome of an O. lupi isolate from a dog from Arizona, southwestern USA, and its genetic differentiation from related Onchocerca species. The whole mitochondrial genome was obtained from whole genome sequencing of genomic DNA isolated from an adult worm. This mitogenome is 13,766 bp in size and contains 36 genes and a control region. This mitogenome provides a valuable resource for future studies involving epidemiological surveillance, population genetics, phylogeography, and comparative mitogenomics of this emerging pathogen and other parasitic nematodes.

An Accessible Alternative to Melarsomine: "Moxi-Doxy" for Treatment of Adult Heartworm Infection in Dogs.

Canine heartworm infection, caused by the filarial parasite Dirofilaria immitis, represents a serious and expanding animal welfare concern that is expected to increase due to the effects of climate change and the COVID-19 pandemic. A body of evidence has emerged to support the use of a non-arsenical adulticide treatment protocol, using moxidectin and doxycycline to kill adult heartworms over a prolonged period. While a three-dose protocol using the arsenical drug melarsomine is currently the safest and most effective treatment for heartworm infection, this drug is not available in some countries and is inaccessible for many owners and animal shelters. Moxidectin-doxycycline (moxi-doxy) provides a viable alternative to no treatment at all, in cases where arsenical treatment is not possible. Based on current evidence, the most effective non-arsenical treatment regimen is doxycycline 10 mg/kg PO q 12 or 24 h for 28 days, combined with topical moxidectin at label dose. Moxidectin is repeated monthly until no antigen detected (NAD) status is confirmed. Sustained release injectable moxidectin, in combination with doxycycline, may provide an alternative in remote regions or in settings where significant compliance or accessibility concerns exist, but more studies are needed. In moxi-doxy protocols, doxycycline should be repeated annually until NAD. This review summarizes the safety and efficacy of moxi-doxy, addresses controversies surrounding this treatment approach, and provides detailed recommendations for treatment regimens and post-treatment testing.

Optimization of a Loop-Mediated Isothermal Amplification Assay as a Point-of-Care Tool for the Detection of Wuchereria bancrofti in Human Blood in Tana River Delta, Kenya.

Introduction Accurate detection of filarial parasites in humans and vectors is essential for the implementation and evaluation of Global and National Programs to eliminate lymphatic filariasis. Immunological methods to detect infection are available; however, cross-reactivity issues have been reported in most of them. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity and can be used to detect the infections. Methods In this study, we evaluated loop-mediated isothermal amplification (LAMP) tests to amplify Wuchereria bancrofti DNA in patients' blood. The amplicons were tested by both pH-sensitive dyes for enhanced visual detection and agarose gel electrophoresis. A closed-tube LAMP assay was also evaluated. Cohen's Kappa statistics was used for statistical analysis of the assays. 125 patients consented for blood sampling which were used for clinical analysis of LAMP assays with the PCR method used as the “gold standard.” Results The sensitivity of the evaluated Wuchereria bancrofti LAMP was 92.3%, with a specificity of 97.3% and kappa statistics value of 0.84, which is in a strong agreement. Conclusion In this study, LAMP assays coupled with fluorescence dye detection have been found to be suitable for diagnosis and monitoring of Wuchereria bancrofti infections in the Kenyan population.

Distribution of Culicoides biting midges (Diptera: Ceratopogonidae) in southern Australia and insight into the Culicoides victoriae morpho‐variants

AbstractCertain Culicoides species (biting midges) are important vectors globally of a range of viruses, protozoa and filarial parasites, imposing a significant economic and health burden. In a changing climate, understanding which Culicoides species occur in a region is important for biosecurity risk management. We examined the occurrence of predominant Culicoides species in south‐east Australia and provide insight into five Culicoides victoriae morpho‐variants. Culicoides were surveyed using Centre for Disease Control light traps and Yellow Sticky traps, with identification performed morphologically and molecularly. Two polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) assays based on mitochondrial cytochrome c oxidase subunit I were developed to differentiate morphologically similar C. victoriae, with a subset sequenced for nuclear carbomoylphosphate synthase. The structure of the five C. victoriae morpho‐variants was investigated through phylogenetic trees, haplotype networks and the Barcode Index Number system (BINs) in the Barcode of Life Data (BOLD) database. Twenty‐five different Culicoides species were identified in the sampled region with Culicoides austropalpalis, C. victoriae, C. marksi and C. molestus Gp sp No 2 being the most common. Two PCR‐RFLP assays were developed using either one or three enzymes, with a 90% or 99% success rate, respectively, of being able to differentiate the five C. victoriae morpho‐variants. Mitochondrial and nuclear sequence divergence supported by wing patterning allowed the recognition of multiple potentially new species of C. victoriae and identified species in uncharacterised regions.

Human and Animal Dirofilariasis in Southeast of France.

Dirofilariasis is one of the oldest known zoonotic infections of humans mainly caused by the filarial parasites of the species Dirofilaria immitis and Dirofilaria repens, which primarily infect dogs. A five-year survey (2017 to 2021) was conducted among the dog population to assess the molecular prevalence of Dirofilaria spp. in southeast France. Morphological and genetic analysis were performed on filaroids from dogs and one infected woman from the studied area. A total of 12 (13%) dogs scored molecularly positive for Dirofilaria spp. of which nine carried blood microfilariae. Ocular dirofilariasis was detected in a 79-year-old woman with no travel history. Both electron microscopy and molecular sequencing identified the worm in the human case as D. repens. Molecularly, D. repens isolates were identical in the human and dog cases, representing the only genotype reported so far in France. Despite the distribution of this genotype through all Europe, it was grouped separately with the other two European genotypes and with Asian ones. As in almost all previous human cases in France, D. repens parasites were mainly recovered from the ocular region of patients and were geographically concentrated in the southeastern regions. Data demonstrate the sympatric occurrence of D. immitis and D. repens with high risk of infection to human and dog populations in these investigated geographical areas, thereby underlining the urgent need to implement preventive chemoprophylactic strategies and vector control to reduce the risk of these filaroids in dog and human populations.

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.

#56: A novel test for diagnosis and surveillance of Wuchereria bancrofti infection

Abstract Background Elephantiasis or Lymphatic filariasis (LF) is a parasitic infection that causes significant morbidity and impacts hundreds of millions of people in 73 countries. Most LF is caused by the nematode Wuchereria bancrofti, but Brugia species cause LF in some areas of Southeast Asia. The global program to eliminate LF uses mass drug administration (MDA) of antifilarial drugs in endemic areas to kill the microfilaria (MF) stage of the parasite that is required for ongoing transmission by mosquitos. Better tools are needed for assessing the success of MDA, because of limitations of available diagnostic tests. MF testing is often not feasible, because it requires collection of blood at night in most endemic areas. Existing antibody and antigen tests remain positive long after effective treatment, and their results do not correlate well with current infectivity. The Brugia Rapid test detects antibodies to BmR1, a Brugia protein that is expressed by MF. These antibodies disappear 2–3 years after effective treatment, and that makes the Brugia Rapid test a useful marker for persistent infection in the few countries with brugian filariasis. We set out to develop a novel antibody test for W. bancrofti infection based on a BmR1 homologue in W. bancrofti. Methods We cloned, expressed and purified a Wuchereria bancrofti protein (provisional name WbN1) that is a homologue of the Brugia malayi protein BmR1. Sera from patients infected with Wuchereria bancrofti as well as sera from patients infected with other closely related filarial species were tested for IgG4 antibodies to WbN1 by indirect ELISA. Results The ELISA has a sensitivity of 90.7% for infection with W. bancrofti based on the 80 bancrofti patient samples tested thus far. Specificity was 91.5% with 59 sera samples from patients infected with Onchocerca volvulus or Loa loa, which are filarial parasites that are co-endemic with W. bancrofti in Africa. Specificity was 97.9% with North American control samples. ELISA with sera from a clinical trial in Sri Lanka demonstrated that antibodies to WbN1 decreased significantly faster after treatment than antibodies to the previously described filarial antigen Bm14. Similar declines in antibody to WbN1 occurred after patients in Papua New Guinea received a single dose of triple drug treatment for LF with Ivermectin Diethylcarbamazine and Albendazole, that is effective for clearing MF from the blood without clearing filarial antigenemia. Conclusions While additional studies are needed, this ELISA for IgG4 antibody to the recombinant protein WbN1 could be a promising new surveillance tool for assessing for ongoing transmission of LF following MDA.

Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function.

In Mali, cutaneous leishmaniasis (CL) and filariasis are co-endemic. Previous studies in animal models of infection have shown that sand fly saliva enhance infectivity of Leishmania parasites in naïve hosts while saliva-specific adaptive immune responses may protect against cutaneous and visceral leishmaniasis. In contrast, the human immune response to Phlebotomus duboscqi (Pd) saliva, the principal sand fly vector in Mali, was found to be dichotomously polarized with some individuals having a Th1-dominated response and others having a Th2-biased response. We hypothesized that co-infection with filarial parasites may be an underlying factor that modulates the immune response to Pd saliva in endemic regions. To understand which cell types may be responsible for polarizing human responses to sand fly saliva, we investigated the effect of salivary glands (SG) of Pd on human monocytes. To this end, elutriated monocytes were cultured in vitro, alone, or with SG, microfilariae antigen (MF ag) of Brugia malayi, or LPS, a positive control. The mRNA expression of genes involved in inflammatory or regulatory responses was then measured as were cytokines and chemokines associated with these responses. Monocytes of individuals who were not exposed to sand fly bites (mainly North American controls) significantly upregulated the production of IL-6 and CCL4; cytokines that enhance leishmania parasite establishment, in response to SG from Pd or other vector species. This selective inflammatory response was lost in individuals that were exposed to sand fly bites which was not changed by co-infection with filarial parasites. Furthermore, infection with filarial parasites resulted in upregulation of CCL22, a type-2 associated chemokine, both at the mRNA levels and by its observed effect on the frequency of recruited monocytes. Together, our data suggest that SG or recombinant salivary proteins from Pd alter human monocyte function by upregulating selective inflammatory cytokines.

Field study to investigate the effectiveness and safety of a novel orally administered combination drug product containing milbemycin oxime and lotilaner (Credelio\xae Plus) for the prevention of heartworm disease (Dirofilaria immitis) in client-owned dogs in the USA

BackgroundDirofilaria immitis, a globally distributed filarial parasite of dogs, is known to cause serious or fatal cardiopulmonary disease. Client-owned dogs were enrolled in a clinical field study in the USA to evaluate the clinical effectiveness and field safety of an orally administered combination investigational product (IP) containing milbemycin oxime and lotilaner (Credelio® Plus) as compared to a control product (CP) for the prevention of heartworm disease when administered monthly for 11 consecutive months.MethodsIn this 11-month field study, 319 dogs ≥ 8 weeks old confirmed to be heartworm-negative were enrolled from eight geographically distinct US veterinary clinics, including sites in the southern USA and Mississippi River Valley. The dogs were treated with either the IP combination product at 0.75–1.53 mg/kg milbemycin oxime and 20–41.5 mg/kg lotilaner (n = 159) or the CP (Sentinel® Flavor Tabs®; milbemycin oxime/lufenuron) at the label-recommended dose rate (n = 158.) On day 330, effectiveness was evaluated in each dog using antigen and microfilarial (modified Knott’s) testing to assess the establishment of any patent adult heartworm infections.ResultsAll dogs treated with the IP combination product and the CP tested negative (100% prevention) for heartworm infection on day 330. The IP combination product tablets containing milbemycin oxime and lotilaner were well tolerated based on the safety assessments in all treated dogs.ConclusionsThis multi-site clinical study using client-owned dogs demonstrated that monthly use of flavored, chewable tablets containing a combination of milbemycin oxime and lotilaner administered orally under end use conditions is safe for dogs. None of the enrolled dogs developed heartworm infections. Eleven consecutive monthly treatments of the IP provided 100% prevention of heartworm disease caused by D. immitis.Graphical

Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview

Acanthocheilonema reconditum is a filarial parasite transmitted by arthropods (fleas, lice, and ticks) that infect dogs. There is minimal published data available to date on potential haematological and biochemical changes associated with this parasitic infection. Study aims were (i) provide an overview of A. reconditum in Europe, (ii) define A. reconditum prevalence and risk factors in a specific dog population (hunting) from southern Italy, and (iii) assess the frequency of haemato-biochemical abnormalities associated with infection. Blood samples collected from 3020 dogs were tested by a modified Knott’s technique to count and identify microfilariae. Eighty-four dogs were infected by A. reconditum (2.78%; 95% CI 2.19–3.37%). Microfilariae ranged from 1 to 212/ml. Based on clinical examination, all but six dogs with non-specific symptoms were healthy. Haematological abnormalities included leucocytosis (n = 15), with eosinophilia (n = 14) and monocytosis (n = 13). Serum biochemical abnormalities included increased total serum proteins (n = 19), albumins (n = 7), total globulins (n = 14), ALT (n = 1), and ALP (n = 1); one dog was hypoalbuminemic, and BUN was mildly increased in 2 dogs. Risk factors included the province origin (Napoli, OR=5.4, 95%CI: 2.1–14.0; Caserta, OR=5.1, 95%CI: 2.5–10.6), hunting wild mammals (OR=2.8, 95% 95%CI: 1.6–4.8), and ectoparasite infestation (OR=1.9, 95%CI: 1.1–3.1). There was a negative correlation between microfilaraemic load and decreased albumin level (−0.37; p=0.021). Our results showed that A. reconditum circulates within the hunting dog population of southern Italy, with seemingly low pathogenic potential.

An Overview of the Management of Mansonellosis.

Mansonellosis is caused by three filarial parasite species from the genus Mansonella that commonly produce chronic human microfilaraemias: M. ozzardi, M. perstans and M. streptocerca. The disease is widespread in Africa, the Caribbean and South and Central America, and although it is typically asymptomatic it has been associated with mild pathologies including leg-chills, joint-pains, headaches, fevers, and corneal lesions. No robust mansonellosis disease burden estimates have yet been made and the impact the disease has on blood bank stocks and the monitoring of other filarial diseases is not thought to be of sufficient public health importance to justify dedicated disease management interventions. Mansonellosis´s Ceratopogonidae and Simuliidae vectors are not targeted by other control programmes and because of their small size and out-door biting habits are unlikely to be affected by interventions targeting other disease vectors like mosquitoes. The ivermectin and mebendazole-based mass drug administration (iMDA and mMDA) treatment regimens deployed by the WHO´s Elimination of Neglected Tropical Diseases (ESPEN) programme and its forerunners have, however, likely impacted significantly on the mansonellosis disease burden, principally by reducing the transmission of M. streptocerca in Africa. The increasingly popular plan of using iMDA to control malaria could also affect M. ozzardi parasite prevalence and transmission in Latin America in the future. However, a potentially far greater mansonellosis disease burden impact is likely to come from short-course curative anti-Wolbachia therapeutics, which are presently being developed for onchocerciasis and lymphatic filariasis treatment. Even if the WHO´s ESPEN programme does not choose to deploy these drugs in MDA interventions, they have the potential to dramatically increase the financial and logistical feasibility of effective mansonellosis management. There is, thus, now a fresh and urgent need to better characterise the disease burden and eco-epidemiology of mansonellosis so that effective management programmes can be designed, advocated for and implemented.

Investigation of Armigeres subalbatus, a vector of zoonotic Brugia pahangi filariasis in plantation areas in Suratthani, Southern Thailand

In recent years, children in Thailand have been infected with zoonotic Brugia pahangi. However, the local environment of rubber or oil palm plantations, which would increase their exposure to risk factors of the infection due to mosquito transmission, is unclear. The present study first sought to determine the extent to which variations in the local landscape, such as the elevated versus low-lying ecotope of rubber or oil palm plantations, in a 2-km radius of the geographically defined landscape in a rural area of Suratthani, Southern Thailand could influence the abundance of Armigeres subalbatus and its susceptibility to zoonotic filarial parasite infections compared to Mansonia, Aedes, and Culex, and Coquillettidia. Thereafter, the filarial larvae found in the infected mosquitoes were molecularly investigated. Ar. subalbatus plantation ecotype was not only found to outnumber the local mosquitoes, but was identified as the predominant species that adapted well to the elevated ecotopes of the rubber or oil palm plantations, which existed at altitudes of 60–80 m. The overall rate of zoonotic filarial parasite infections (L1, L2, or L3 larvae) of Ar. subalbatus was 2.5% (95% CI, −0.2 to 4.1), with an average L3 load of 2.3 larvae per infected Ar. subalbatus (95% CI, −0.6 to 13.0); this is because the infections were found to be concentrated in the elevated ecotopes alone. Based on filarial orthologous β-tubulin gene-specific touchup-nested PCR and sequence analysis using 30 L3 larva clones as representatives of 9 Ar. subalbatus infectious pools, Ar. subalbatus either carried B. pahangi or Dirofilaria immitis, or both species. Such findings suggest that Ar. subalbatus might have played an imperative role in the transmission of B. pahangi in the plantation areas infested with Ar. subalbatus.

The Secretome of Filarial Nematodes and Its Role in Host-Parasite Interactions and Pathogenicity in Onchocerciasis-Associated Epilepsy.

Filarial nematodes secrete bioactive molecules which are of interest as potential mediators for manipulating host biology, as they are readily available at the host-parasite interface. The adult parasites can survive for years in the mammalian host, due to their successful modulation of the host immune system and most of these immunomodulatory strategies are based on soluble mediators excreted by the parasite. The secretome of filarial nematodes is a key player in both infection and pathology, making them an interesting target for further investigation. This review summarises the current knowledge regarding the components of the excretory-secretory products (ESPs) of filarial parasites and their bioactive functions in the human host. In addition, the pathogenic potential of the identified components, which are mostly proteins, in the pathophysiology of onchocerciasis-associated epilepsy is discussed.

Disruption of redox homeostasis with synchronized activation of apoptosis highlights the antifilarial efficacy of novel piperine derivatives: An in vitro mechanistic approach

A series of novel piperine derivatives were synthesized with high yield and were evaluated for its antifilarial potential against the bovine filarial parasite Setaria cervi. Among 21 (3a-3u) compounds screened, three of them (3k, 3l, 3s) showed significant potential against all the developmental stages (oocytes, microfilariae and adult) of the filarial worm in time and dose dependent manner. 3l showed the highest efficacy among the selected three compounds. These three compounds were further evaluated for both in vitro and in vivo toxicity analyses which further fortified the benign nature of the selected compounds. The antifilarial activities they exhibited were clearly fuelled through disparity of the internal redox homeostasis as evidenced from the alterations in the enzymatic and non-enzymatic antioxidants level which ultimately shifted towards activation of pro-apoptotic signaling cascade eventually leading to the death of the parasites. The ability of the compound 3l to bind thioredoxin reductase and CED-3 protein are the key findings of this study. The present study supported with several biological experiments is therefore a maiden report on the antifilarial effectiveness of these novel piperine derivatives.

In-silico evidences on filarial cystatin as a putative ligand of human TLR4

Cystatin is a small molecular weight immunomodulatory protein of filarial parasite that plays a pivotal role in downregulating the host immune response to prolong the survival of the parasite inside the host body. Hitherto, this protein is familiar as an inhibitor of human proteases. However, growing evidences on the role of cystatin in regulating inflammatory homeostasis prompted us to investigate the molecular reasons behind the explicit anti-inflammatory trait of this protein. We have explored molecular docking and molecular dynamics simulation approaches to explore the interaction of cystatin of Wuchereria bancrofti (causative parasite of human filariasis) with human Toll-like receptors (TLRs). TLRs are the most crucial component of frontline host defence against pathogenic infections including filarial infection. Our in-silico data clearly revealed that cystatin strongly interacts with the extracellular domain of TLR4 (binding energy=-93.5 ± 10 kJ/mol) and this biophysical interaction is mediated by hydrogen bonding and hydrophobic interaction. Molecular dynamics simulation analysis revealed excellent stability of the cystatin-TLR4 complex. Taken together, our data indicated that cystatin appears to be a ligand of TLR4 and we hypothesize that cystatin-TLR4 interaction most likely to play a key role in activating the alternative activation pathways to establish an anti-inflammatory milieu. Thus, the study provokes the development of chemotherapeutics and/or vaccines for targeting the cystatin-TLR4 interaction to disrupt the pathological attributes of human lymphatic filariasis. Our findings are expected to provide a novel dimension to the existing knowledge on filarial immunopathogenesis and it will encourage the scientific communities for experimental validation of the present investigation. Communicated by Ramaswamy H. Sarma

Therapeutic Efficacy of Anti-Bestrophin Antibodies against Experimental Filariasis: Immunological, Immune-Informatics and Immune Simulation Investigations.

Lymphatic filariasis (LF) is a debilitating parasitic disease caused by filarial parasites and it is prevalent across the underprivileged population throughout the globe. The inadequate efficacy of the existing treatment options has provoked the conception of alternative strategies, among which immunotherapy is steadily emerging as a promising option. Herein, we demonstrate the efficacy of an antibody-based immunotherapeutic approach in an experimental model of filariasis, i.e., Wistar rat infected with Setaria cervi (a model filarial parasite). The polyclonal antibodies were raised against filarial surface antigen bestrophin protein (FSAg) in mice using the purified Wuchereria bancrofti FSAg. The adoptive transfer of anti-FSAg antibody-containing serum resulted in the significant reduction of parasite burden in filaria-infected rats. Intriguingly, anti-FSAg sera-treated animals also displayed a reduction in the level of proinflammatory cytokines as compared to the infected but untreated group. Furthermore, our in silico immunoinformatics data revealed eight B-cell epitopes and several T-cell epitopes in FSAg and these epitopes were linked to form a refined antigen in silico. The immune simulation suggested IgM and IgG1 as the predominant immunoglobulins induced in response to FSAg. Taken together, our experimental and simulation data collectively indicated a therapeutic potential of anti-FSAg sera against LF.

Canine ocular onchocerciasis in New Mexico: Risk factors for disease.

To determine whether dogs are at variable risk of developing canine ocular onchocerciasis based on coat color or size, factors that may influence feeding behavior of the putative vectors of Onchocerca lupi¸ the filarial nematode parasite causing this disease. One hundred twenty-five client-owned dogs diagnosed with confirmed or suspected onchocerciasis. 1255 dogs without signs of this disease were utilized for comparison. Dogs lacking signs of canine onchocerciasis were assessed for coat color and weight. Proportions of dogs with these characteristics were used to predict signalment in a group of dogs with this disease, if the investigated characteristics were unrelated to disease risk. Predicted values were compared statistically with observed values in the diseased dog population. Black fly color preference was assessed utilizing black, brown, and white traps and statistically assessing any differences in trapping based on trap color. Results suggest that large, black dogs are more likely to develop canine ocular onchocerciasis than was predicted by chance alone (p=0.012). Results for smaller dogs with other coat colors were not significant. An increased risk for black dogs was supported by trapping data, as black flies were trapped significantly more often in black traps (p<0.001). While factors other than size and color probably also influence the likelihood of O. lupi infection and disease development, our results may assist clinicians in their diagnosis of canine ocular onchocerciasis and provide them with a tool to help educate their clients as to their dogs' risk of developing this disease.

Inhibition of thioredoxin reductase (TrxR) triggers oxidative stress-induced apoptosis in filarial nematode Setaria cervi channelized through ASK-1-p38 mediated caspase activation

Inhibition of an imperative antioxidant enzyme with subsequent death is a victorious and widely accepted strategy to combat various infectious diseases. Among different antioxidant enzymes, thioredoxin reductase (TrxR) is an exclusive one. Studies have revealed that direct inhibition of TrxR by different classes of chemical moieties promptly results in the death of an organism. Especially the structural as well as biochemical modifications of the enzyme upon inhibition project serious threat towards the subject organism. Herein, an attempt was made to inhibit TrxR of filarial species by administering Auranofin, 1 chloro 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo dithioperoxo(thioate) derivative (4a). Our study has revealed that inhibition of TrxR resulted in the induction of the classical CED pathway of apoptosis along with the intrinsic and extrinsic pathways of apoptosis (Caspase mediated) routed through the ASK-1/p38 axis. Druggability analysis of filarial TrxR for the selected compounds was performed in silico through molecular docking studies. Therefore, this study attempts to decipher the mechanism of apoptosis induction following TrxR inhibition. The safety of those four compounds in terms of dose and toxicity was taken under consideration. Thitherto, the mechanism of TrxR mediated initiation of cell death in filarial parasite has remained undercover, and therefore, it is a maiden report on the characterization of apoptosis induction upon TrxR inhibition which will eventually help in generating effective antifilarial drugs in the future.

Mitochondrial Genome of Brugia malayi Microfilariae Isolated From a Clinical Sample

Lymphatic filariasis is a neglected parasitic disease that is a leading cause of long-term disability. Information obtained from genome sequencing of filarial worm can help us identify systems in the worm that are likely to be useful for novel drug design. Brugia (B.) malayi is still the only lymphatic-dwelling filarial parasite with a nearly complete, fully annotated, and published genome. However, most previous studies were based on the FR3 strain of B. malayi, which originally was isolated from a human patient, and was adapted to the rodent model, then maintained in laboratories for more than 60 years. It is uncertain whether genetic variation exists, thus, sequencing of clinical isolates of lymphatic dwelling filarial parasites is a high priority. Here, we report for the first time the complete mitochondrial genome of B. malayi microfilariae from clinical isolate. Complete mitochondrial (mt) genome of the microfilariae isolated from a blood sample taken from a Thai subject living in Narathiwat Province, which is an endemic area of brugian filariasis, was assembled with sequencing reads obtained by Illumina sequencing. Gene annotation, phylogenetic analysis and single nucleotide polymorphism (SNP) were deployed. A complete 13,658-bp mt genome of B. malayi microfilaria was obtained, and it shows 68x coverage. Based on gene annotation, the mt genome consists of 12 protein-coding, two rRNA, and 23 tRNA genes. Phylogenetic analysis using all protein sequences of DNA sequences of mt genome or cytochrome c oxidase subunit I (COX1) revealed a close relationship among three lymphatic filariae (i.e., B. timori, zoonotic B. pahangi, and Wuchereria spp.). The SNPs in the COX1 gene can differentiate microfilariae of B. malayi in human from those found in canine. Furthermore, the number, order and transcription, and direction of B. malayi microfilariae mitochondrial genes were the same as those found in the FR3 strain of B. malayi. The comparison on mitochondrial genome of B. malayi could have important implications on the development of a new intervention or vaccine to treat or prevent this disease in endemic areas/regions around the world.

A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus)

BackgroundThe Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent.MethodsJird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry.ResultsIn vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry.ConclusionsThese results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.Graphical