Parasite-bacteria interrelationship.

Parasites and bacteria have co-evolved with humankind, and they interact all the time in a myriad of ways. For example, some bacterial infections result from parasite-dwelling bacteria as in the case of Salmonella infection during schistosomiasis. Other bacteria synergize with parasites in the evolution of human disease as in the case of the interplay between Wolbachia endosymbiont bacteria and filarial nematodes as well as the interaction between Gram-negative bacteria and Schistosoma haematobium in the pathogenesis of urinary bladder cancer. Moreover, secondary bacterial infections may complicate several parasitic diseases such as visceral leishmaniasis and malaria, due to immunosuppression of the host during parasitic infections. Also, bacteria may colonize the parasitic lesions; for example, hydatid cysts and skin lesions of ectoparasites. Remarkably, some parasitic helminths and arthropods exhibit antibacterial activity usually by the release of specific antimicrobial products. Lastly, some parasite-bacteria interactions are induced as when using probiotic bacteria to modulate the outcome of a variety of parasitic infections. In sum, parasite-bacteria interactions involve intricate processes that never cease to intrigue the researchers. However, understanding and exploiting these interactions could have prophylactic and curative potential for infections by both types of pathogens.

Microbe Profile: Wolbachia: a sex selector, a viral protector and a target to treat filarial nematodes.

Wolbachia is the most widespread genus of endosymbiotic bacteria in the animal world, infecting a diverse range of arthropods and nematodes. A broad spectrum of associations from parasitism to mutualism occur, with a tendency to drive reproductive manipulation or influence host fecundity to spread infection through host populations. These varied effects of Wolbachia are exploited for public health benefits. Notably, the protection of insect hosts from viruses is being tested as a potential control strategy for human arboviruses, and the mutualistic relationship with filarial nematodes makes Wolbachia a target for antibiotic therapy of human and veterinary nematode diseases.

Chronic filarial infection provides protection against bacterial sepsis by functionally reprogramming macrophages.

Helminths immunomodulate their hosts and induce a regulatory, anti-inflammatory milieu that prevents allergies and autoimmune diseases. Helminth immunomodulation may benefit sepsis outcome by preventing exacerbated inflammation and severe pathology, but the influence on bacterial clearance remains unclear. To address this, mice were chronically infected with the filarial nematode Litomosoides sigmodontis (L.s.) and the outcome of acute systemic inflammation caused by i.p. Escherichia coli injection was determined. L.s. infection significantly improved E. coli-induced hypothermia, bacterial clearance and sepsis survival and correlated with reduced concentrations of associated pro-inflammatory cytokines/chemokines and a less pronounced pro-inflammatory macrophage gene expression profile. Improved sepsis outcome in L.s.-infected animals was mediated by macrophages, but independent of the alternatively activated macrophage subset. Endosymbiotic Wolbachia bacteria that are present in most human pathogenic filariae, as well as L.s., signal via TLR2 and modulate macrophage function. Here, gene expression profiles of peritoneal macrophages from L.s.-infected mice revealed a downregulation of genes involved in TLR signaling, and pulsing of macrophages in vitro with L.s. extract reduced LPS-triggered activation. Subsequent transfer improved sepsis outcome in naïve mice in a Wolbachia- and TLR2-dependent manner. In vivo, phagocytosis was increased in macrophages from L.s.-infected wild type, but not TLR2-deficient animals. In association, L.s. infection neither improved bacterial clearance in TLR2-deficient animals nor ameliorated E. coli-induced hypothermia and sepsis survival. These results indicate that chronic L.s. infection has a dual beneficial effect on bacterial sepsis, reducing pro-inflammatory immune responses and improving bacterial control. Thus, helminths and their antigens may not only improve the outcome of autoimmune and allergic diseases, but may also present new therapeutic approaches for acute inflammatory diseases that do not impair bacterial control.

Corallopyronin A - a promising antibiotic for treatment of filariasis.

Lymphatic filariasis and onchocerciasis are diseases of severe morbidity that affect the poorest of the poor in the world. The diseases are caused by filarial nematodes that are transmitted by mosquitoes or biting blackflies and are endemic to more than 80 countries worldwide, mainly in the tropics and sub-tropics. Current control programs aim to eliminate the diseases by distributing antifilarial drugs. However, the primary effect of the drugs is to kill the microfilariae in the blood or skin, thus preventing uptake by the obligate insect vector. Since the adult worms live 10 years or longer, drug distribution requires many years of treatment, which is a heavy burden on the burgeoning health care systems. Sub-optimal response, possible resistance and inadequate population coverage lessen the chances for successful elimination in all endemic areas. The search for new drugs that could enhance elimination by permanently sterilizing or killing adult worms has identified the Wolbachia intracellular bacteria of filarial nematodes as a target. Depleting the obligate endosymbionts from the worms with doxycycline or rifampicin causes a permanent block in oogenesis, embryogenesis and development, and in slow death of the adult worms. These two antibiotics are suitable for individual drug administration, but caveats exist for their inclusion in broader drug administration programs. Here we review Wolbachia as targets for antifilarial drug discovery and highlight the natural product corallopyronin A as an effective drug that is currently being developed specifically for use against filarial nematodes.

Wolbachia filarial interactions.

Wolbachia pipientis is a widespread intracellular bacterial symbiont of arthropods and is common in insects. One of their more exotic and unexpected hosts is the filarial nematodes, notable for the parasites responsible for onchocerciasis (river blindness), lymphatic filariasis (elephantiasis) and dirofilariasis (heartworm). Wolbachia are only present in a subgroup of the filarial nematodes and do not extend to other groups of nematodes either parasitic or free-living. In the medically and veterinary important species that host Wolbachia, the symbiont has become an essential partner to key biological processes in the life of the nematode to the point where antibiotic elimination of the bacteria leads to a potent and effective anti-filarial drug treatment. We review the cellular and molecular basis of Wolbachia filarial interactions and highlight the key processes provided by the endosymbiont upon which the nematodes have become entirely dependent. This dependency is primarily restricted to periods of the lifecycle with heavy metabolic demands including growth and development of larval stages and embryogenesis in the adult female. Also, the longevity of filarial parasites is compromised following depletion of the symbiont, which for the first time has delivered a safe and effective treatment to kill adult parasites with antibiotics.

Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria.

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

Detection and characterization of vector-borne parasites and Wolbachia endosymbionts in greater one-horned rhinoceros (Rhinoceros unicornis) in Nepal.

Vector-borne parasite infections affect both domestic and wild animals. They are often asymptomatic but can result in fatal outcomes under natural and human-induced stressors. Given the limited availability of molecular data on vector-borne parasites in Rhinoceros unicornis (greater one-horned rhinoceros), this study employed molecular tools to detect and characterize the vector-borne parasites in rescued rhinoceros in Chitwan National Park, Nepal. Whole blood samples were collected from thirty-six R. unicornis during rescue and treatment operations. Piroplasmida infections were first screened using nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA gene. Wolbachia was detected by amplifying 16S rRNA gene, while filarial nematodes were detected through amplification of 28S rRNA, COI, myoHC and hsp70 genes. Our results confirmed the presence of Theileria bicornis with a prevalence of 75% (27/36) having two previously unreported haplotypes (H8 and H9). Wolbachia endosymbionts were detected in 25% (9/36) of tested samples and belonged to either supergroup C or F. Filarial nematodes of the genera Mansonella and Onchocerca were also detected. There were no significant association between T. bicornis infections and the age, sex, or location from which the animals were rescued. The high prevalence of Theileria with novel haplotypes along with filarial parasites has important ecological and conservational implications and highlights the need to implement parasite surveillance programs for wildlife in Nepal. Further studies monitoring vector-borne pathogens and interspecies transmission among wild animals, livestock and human are required.

CCL17 controls mast cells for the defense against filarial larval entry.

Filarial parasites have to trespass many barriers to successfully settle within their mammalian host, which is equipped with mechanical borders and complex weaponry of an evolved immune system. However, little is known about mechanisms of early local events in filarial infections. In this study, bone marrow-derived dendritic cells not only upregulated activation markers CD40 and CD80 upon in vitro stimulation with filarial extracts, but also secreted CCL17, a chemokine known to be produced upon microbial challenge. Mice deficient for CCL17 had an up to 4-fold higher worm burden compared with controls by day 10 of infection with the murine filaria Litomosoides sigmodontis. Also, numbers of mast cells (MCs) invading the skin and degranulation were significantly increased, which was associated with enhanced vascular permeability and larval establishment. This phenotype was reverted by inhibition of MC degranulation with disodium cromoglycate or by blockade of histamine. In addition, we showed that CCL17-mediated vascular permeability was dependent on the presence of Wolbachia endosymbionts and TLR2. Our findings reveal that CCL17 controls filarial larval entry by limiting MC-dependent vascular permeability.

Corallopyronin A specifically targets and depletes essential obligate Wolbachia endobacteria from filarial nematodes in vivo.

Doxycycline and rifampicin deplete essential Wolbachia from filarial nematodes that cause lymphatic filariasis or onchocerciasis, resulting in blocked worm development and death. However, doxycycline is contraindicated for children and pregnant/breastfeeding women, as is rifampicin in the latter group with the additional specter of possible resistance development in Mycobacterium spp. Novel antibiotics with a narrower spectrum would aid in eliminating filarial diseases. Corallococcus coralloides synthesizes corallopyronin A, a noncompetitive inhibitor of RNA polymerase ineffective against Mycobacterium spp. Corallopyronin A depleted Wolbachia from infected insect cells (1.89 Thus the antibiotic is effective against intracellular bacteria despite the many intervening surfaces (blood vessels, pleura, worm cuticle) and membranes (worm cell, vesicle, Wolbachia inner and outer membranes). Corallopyronin A is an antibiotic to develop further for filariasis elimination without concern for cross-resistance development in tuberculosis.

Filarial and Wolbachia genomics.

Filarial nematode parasites, the causative agents for a spectrum of acute and chronic diseases including lymphatic filariasis and river blindness, threaten the well-being and livelihood of hundreds of millions of people in the developing regions of the world. The 2007 publication on a draft assembly of the 95-Mb genome of the human filarial parasite Brugia malayi- representing the first helminth parasite genome to be sequenced - has been followed in rapid succession by projects that have resulted in the genome sequencing of six additional filarial species, seven nonfilarial nematode parasites of animals and nearly 30 plant parasitic and free-living species. Parallel to the genomic sequencing, transcriptomic and proteomic projects have facilitated genome annotation, expanded our understanding of stage-associated gene expression and provided a first look at the role of epigenetic regulation of filarial genomes through microRNAs. The expansion in filarial genomics will also provide a significant enrichment in our knowledge of the diversity and variability in the genomes of the endosymbiotic bacterium Wolbachia leading to a better understanding of the genetic principles that govern filarial-Wolbachia mutualism. The goal here is to provide an overview of the trends and advances in filarial and Wolbachia genomics.

Parasitism and mutualism in Wolbachia: what the phylogenomic trees can and cannot say.

Ecological and evolutionary theories predict that parasitism and mutualism are not fixed endpoints of the symbiotic spectrum. Rather, parasitism and mutualism may be host or environment dependent, induced by the same genetic machinery, and shifted due to selection. These models presume the existence of genetic or environmental variation that can spur incipient changes in symbiotic lifestyle. However, for obligate intracellular bacteria whose genomes are highly reduced, studies specify that discrete symbiotic associations can be evolutionarily stable for hundreds of millions of years. Wolbachia is an inherited obligate, intracellular infection of invertebrates containing taxa that act broadly as both parasites in arthropods and mutualists in certain roundworms. Here, we analyze the ancestry of mutualism and parasitism in Wolbachia and the evolutionary trajectory of this variation in symbiotic lifestyle with a comprehensive, phylogenomic analysis. Contrary to previous claims, we show unequivocally that the transition in lifestyle cannot be reconstructed with current methods due to long-branch attraction (LBA) artifacts of the distant Anaplasma and Ehrlichia outgroups. Despite the use of 1) site-heterogenous phylogenomic methods that can overcome systematic error, 2) a taxonomically rich set of taxa, and 3) statistical assessments of the genes, tree topologies, and models of evolution, we conclude that the LBA artifact is serious enough to afflict past and recent claims including the root lies in the middle of the Wolbachia mutualists and parasites. We show that different inference methods yield different results and high bootstrap support did not equal phylogenetic accuracy. Recombination was rare among this taxonomically diverse data set, indicating that elevated levels of recombination in Wolbachia are restricted to specific coinfecting groups. In conclusion, we attribute the inability to root the tree to rate heterogeneity between the ingroup and outgroup. Site-heterogenous models of evolution did improve the placement of aberrant taxa in the ingroup phylogeny. Finally, in the unrooted topology, the distribution of parasitism and mutualism across the tree suggests that at least two interphylum transfers shaped the origins of nematode mutualism and arthropod parasitism. We suggest that the ancestry of mutualism and parasitism is not resolvable without more suitable outgroups or complete genome sequences from all Wolbachia supergroups.

The bacteria Wolbachia in filariae, a biological Russian dolls' system: new trends in antifilarial treatments.

Filarial nematode species can host Wolbachia bacterial endosymbionts. To understand the symbiosis, a higher level of complexity should be considered, taking in account the tripartite association between Wolbachia, filariae and mammals. This overview article discusses the biology of Wolbachia in filariae, including their distribution and phylogeny, mechanisms of action, inflammatory consequences on mammal host and biological control implications for filariases. Potential directions for future research are also discussed.

The Wolbachia Symbiont: Here, There and Everywhere.

Wolbachia symbionts, first observed in the 1920s, are now known to be present in about 30-70% of tested arthropod species, in about half of tested filarial nematodes (including the majority of human filarial nematodes), and some plant-parasitic nematodes. In arthropods, they are generally viewed as parasites while in nematodes they appear to be mutualists although this demarcation is not absolute. Their presence in arthropods generally leads to reproductive anomalies, while in nematodes, they are generally required for worm development and reproduction. In mosquitos, Wolbachia inhibit RNA viral infections, leading to populational reductions in human RNA virus pathogens, whereas in filarial nematodes, their requirement for worm fertility and survival has been channeled into their use as drug targets for filariasis control. While much more research on these ubiquitous symbionts is needed, they are viewed as playing significant roles in biological processes, ranging from arthropod speciation to human health.

Diminutive, degraded but dissimilar: Wolbachia genomes from filarial nematodes do not conform to a single paradigm.

Wolbachia are alpha-proteobacteria symbionts infecting a large range of arthropod species and two different families of nematodes. Interestingly, these endosymbionts are able to induce diverse phenotypes in their hosts: they are reproductive parasites within many arthropods, nutritional mutualists within some insects and obligate mutualists within their filarial nematode hosts. Defining Wolbachia 'species' is controversial and so they are commonly classified into 17 different phylogenetic lineages, termed supergroups, named A-F, H-Q and S. However, available genomic data remain limited and not representative of the full Wolbachia diversity; indeed, of the 24 complete genomes and 55 draft genomes of Wolbachia available to date, 84 % belong to supergroups A and B, exclusively composed of Wolbachia from arthropods. For the current study, we took advantage of a recently developed DNA-enrichment method to produce four complete genomes and two draft genomes of Wolbachia from filarial nematodes. Two complete genomes, wCtub and wDcau, are the smallest Wolbachia genomes sequenced to date (863 988 bp and 863 427 bp, respectively), as well as the first genomes representing supergroup J. These genomes confirm the validity of this supergroup, a controversial clade due to weaknesses of the multilocus sequence typing approach. We also produced the first draft Wolbachia genome from a supergroup F filarial nematode representative (wMhie), two genomes from supergroup D (wLsig and wLbra) and the complete genome of wDimm from supergroup C. Our new data confirm the paradigm of smaller Wolbachia genomes from filarial nematodes containing low levels of transposable elements and the absence of intact bacteriophage sequences, unlike many Wolbachia from arthropods, where both are more abundant. However, we observe differences among the Wolbachia genomes from filarial nematodes: no global co-evolutionary pattern, strong synteny between supergroup C and supergroup J Wolbachia, and more transposable elements observed in supergroup D Wolbachia compared to the other supergroups. Metabolic pathway analysis indicates several highly conserved pathways (haem and nucleotide biosynthesis, for example) as opposed to more variable pathways, such as vitamin B biosynthesis, which might be specific to certain host-symbiont associations. Overall, there appears to be no single Wolbachia-filarial nematode pattern of co-evolution or symbiotic relationship.

Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum.

BACKGROUND: Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. METHODS: We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearman's correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. RESULTS: The detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10-1 to 1.5 × 10-4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98-1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively. CONCLUSIONS: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

In vivo efficacy of the boron-pleuromutilin AN11251 against Wolbachia of the rodent filarial nematode Litomosoides sigmodontis.

The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal-adult worm killing-drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10-14 days. Therefore, AN11251 represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis.

Litomosa chiropterorum Ortlepp, 1932 (Nematoda: Filarioidea) from a South African miniopterid: redescription, Wolbachia screening and phylogenetic relationships with Litomosoides.

69 Miniopterus notalensis, type host of the onchocercid Litomosa chiropterorum, were collected in caves in the Western Province and Gauteng Province, South Africa. The prevalence of these filariae was about 50 %. The microfilaria is folded, as in other Litomosa and an area rugosa composed of cuticular bosses is present in the male posterior region. L. chiropterorum is close to the species parasitic in other Miniopterus spp. and some Rhinolophus spp. from Africa, Madagascar and Europe; it is unique with the expanded anterior extremity and the four cephalic submedian bosses. The molecular analysis of L. chiropterorum, the first done with Litomosa species from a bat, supports the hypothesis that Litomosa and Litomosoides, which have an exceptionally large buccal capsule in common, form a group in which Litomosa has a basal position. Interestingly, L. chiropterorum does not harbour Wolbachia, as proved with immunohistological staining and PCR screening using the 16S rDNA gene as target. This is contrary to L. westi from rodents and the majority of the Litomosoides species parasitic in bats or rodents. The absence of Wolbachia in a filarioid group considered ancient based on traditional and molecular approaches opens interesting scenarios on the evolution of the endosymbionts spread through filarial lineages.

The Wolbachia Endosymbionts.

The Wolbachia endosymbionts encompass a large group of intracellular bacteria of biomedical and veterinary relevance, closely related to Anaplasma, Ehrlichia, and Rickettsia. This genus of Gram-negative members of the Alphaproteobacteria does not infect vertebrates but is instead restricted to ecdysozoan species, including terrestrial arthropods and a family of parasitic filarial nematodes, the Onchocercidae. The Wolbachia profoundly impact not only the ecology and evolution but also the reproductive biology of their hosts, through a wide range of symbiotic interactions. Because they are essential to the survival and reproduction of their filarial nematode hosts, they represent an attractive target to fight filariasis. Their abilities to spread through insect populations and to affect vector competence through pathogen protection have made Wolbachia a staple for controlling vector-borne diseases. Estimated to be present in up to 66% of insect species, the Wolbachia are probably the most abundant endosymbionts on earth. Their success resides in their unique capacity to infect and manipulate the host germ line to favor their vertical transmission through the maternal lineage. Because the Wolbachia resist genetic manipulation and growth in axenic culture, our understanding of their biology is still in its infancy. Despite these limitations, the "-omics" revolution combined with the use of well-established and emerging experimental host models is accelerating our comprehension of the host phenotypes caused by Wolbachia, and the identification of Wolbachia effectors is ongoing.

In vivo kinetics of Wolbachia depletion by ABBV-4083 in L. sigmodontis adult worms and microfilariae.

Depletion of Wolbachia endosymbionts of human pathogenic filariae using 4-6 weeks of doxycycline treatment can lead to permanent sterilization and adult filarial death. We investigated the anti-Wolbachia drug candidate ABBV-4083 in the Litomosoides sigmodontis rodent model to determine Wolbachia depletion kinetics with different regimens. Wolbachia reduction occurred in mice as early as 3 days after the initiation of ABBV-4083 treatment and continued throughout a 10-day treatment period. Importantly, Wolbachia levels continued to decline after a 5-day-treatment from 91.5% to 99.9% during a 3-week washout period. In jirds, two weeks of ABBV-4083 treatment (100mg/kg once-per-day) caused a >99.9% Wolbachia depletion in female adult worms, and the kinetics of Wolbachia depletion were recapitulated in peripheral blood microfilariae. Similar to Wolbachia depletion, inhibition of embryogenesis was time-dependent in ABBV-4083-treated jirds, leading to a complete lack of late embryonic stages (stretched microfilariae) and lack of peripheral microfilariae in 5/6 ABBV-4083-treated jirds by 14 weeks after treatment. Twice daily treatment in comparison to once daily treatment with ABBV-4083 did not significantly improve Wolbachia depletion. Moreover, up to 4 nonconsecutive daily treatments within a 14-dose regimen did not significantly erode Wolbachia depletion. Within the limitations of an animal model that does not fully recapitulate human filarial disease, our studies suggest that Wolbachia depletion should be assessed clinically no earlier than 3-4 weeks after the end of treatment, and that Wolbachia depletion in microfilariae may be a viable surrogate marker for the depletion within adult worms. Furthermore, strict daily adherence to the dosing regimen with anti-Wolbachia candidates may not be required, provided that the full regimen is subsequently completed.